ADVANCES I N CANCER RESEARCH VOLUME V
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ADVANCES IN CANCER RESEARCH Edited by
JESSE P. GREENSTEIN National Cancer Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland
ALEXANDER HADDOW Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London, England
Volume V
@
1958
ACADEMIC PRESS INC., PUBLISHERS, NEW YORK, N. Y.
COPYRIGHT 0 1958 BY
ACADEMICPRESSINC. 111 FIFTHAVENUE NEWYORK3, N. Y. All Rights Reserved N o part of this book may be reproduced in any form by photostat, microfilm, or any other means without written permission from the pztblishers. Library of Congress Catalog Card Number 52-13360
PRINTED IN THE UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME V I<. W. BEGG,Collip Medical Research Laboratory, University Ontario, London, Canada*
05 Western
CHARLES BERMAN, Consolidated Main Reef Mines and Estate Ltd., Maraisburg, Transvanl, South Africa
P. N. CAMPBELL, Courtauld Institute Medical School, London, England
05 Biochemistry, Middlesex
Hospital
FUMIKO FUKUOKA, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan
I ~ O B E R TE. GREENFIELD,Laboratory 0s Biochemistry, National Cancer Institute, National Institutes 05 Health, Public Health Service, U. S. Department 05 Health, Education, and Welfare, Bethesda, Maryland WARONAKAHARA, Cancer Institute, Japanese Foundation JOT Cancer Research, Tokyo, Japan
P. R. PEACOCK, Cancer Research Department, Royal Beatson Memorial Hospital, Glasgow, Scotland
VINCENTE. PRICE,Laboratory 05 Biochemistry, National Cancer Institute, National Institutes of Health, Public Health Service, U . S. Department 05 Health, Education, and Welfare, Bethesda, Maryland ELIZABETH K. WEISBURGER, Laboratory 05 Biochemistry, National Cancer Institute, National Institutes 05 Health, Public Health Service, U . S. Department 05 Health, Education, and Welfare, Bethesda, Maryland JOHNH. WEISBURGER, Laboratory 05 Biochemistry, National Cancer Institute, National Institutes 05 Health, Public Health Service, U . S. Department 05 Health, Education, and Welfare, Bethesda, Maryland
L. A. Z i m m , N . F. Gamaleya Institute Moscow, U.S.S.R.
05 Epidemiology and
Microbiology,
* Present address: Saskatchewan Research [Init, National Cancer Instilute of Canada, and Department of Cancer Research, Universily of Saskatchewan, Saskatoon, Saskatchewan. V
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CONTENTS CONTRIBUTORS TO VOLUME V . . . . . . . . . . . . . . . . . . . . . . .
v
Tumor-Host Relations R . W . BECG
I . Introduction. . . . . 1I.Enzymes . . . . . I11. Metabolism . . . . I V Nutrition . . . . . V.Hormones, . . . . V I . TheBlood . . . . . VII . Morphology . . . . VIII. Discussion . . . . . References . . . . .
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Primary Carcinoma of the liver CHARLES BERMAN
I. I1. I11. IV . V.
Introduction . . . . . . . . . . . . . . . Incidencc . . . . . . . . . . . . . . . . Clinical Manifestations . . . . . . . . . . Morbid Anatomy . . . . . . . . . . . . . Etiology: Environmental Factors . . . . . References . . . . . . . . . . . . . . . .
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56 57 65 71 79 90
Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N . CAMPBELL
I . Introduction . . . . . . . . . . . . . . . . . . . . . I1. Relationship between Mitotic Rate and Protein Structure I11. Metabolism of Tissue Proteins in uiuo . . . . . . . . . IV . Protein Synthesis in Whole Cells in vitro . . . . . . . . V. Incorporation of Amino Acids into Subcellular Particles . VI . Conclusions . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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98 99 102 117 131 147 148
The Newer Concept of Cancer Toxin WARONAKARARA A N D FUMIKO FUKUOKA
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 I1. Toxohormone Production a Universal Property of Cancer Cells . . . . . 158 I11. Isolation of Toxohormone from Materials Other than Cancer Tissue . . 160 vii
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CONTENTS
Vlll
. Normal Liver Catalase Level . . . . . . . . . . . . . . Chemical Nature of Toxohormone . . . . . . . . . . Mode of Action of Toxohormone . . . . . . . . . . . Toxohormone in General Tumor-Host Relations . . . . Concluding Remarks . . . . . . . . . . . . . . . .
IV V VI VII VIII
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Chemically Induced Tumors of Fowls P. R . PEACOCK
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 I1. Chemically Induced Sarcoma . . . . . . . . . . . . . . . . . . . . 180 I11. Chance Infection by Viruses . . . . . . . . . . . . . . . . . . . . 180 IV. Host Factors Influencing Transmissibility . . . . . . . . . . . . . . 181 V. Tests for Virus . . . . . . . . . . . . . . . . . . . . . . . . . . 184 VI . Evaluation of Negative Experiments . . . . . . . . . . . . . . . . 184 VII . Histogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 VIII . Influence of Solvents on Carcinogenesis with 1:2: 5: 6-Dibcnsanthracene . 185 I X. Methylcholanthrene-Induced Sarcoma . . . . . . . . . . . . . . . . 187 X . Immunological Evidence . . . . . . . . . . . . . . . . . . . . . . 187 X I . Attempts to Infect Chemically Induced Tumors . . . . . . . . . . . 188 XI1. Essential Diffcrences between Certain Fowl Tumors . . . . . . . . . 189 XI11. Fractionation of Tumor Homogenates . . . . . . . . . . . . . . . . 190 XIV . Selective Action of Radiations . . . . . . . . . . . . . . . . . . . 190 XV . Epithelial Tumors in Fowls . . . . . . . . . . . . . . . . . . . . . 191 XVI . Lymphosarcoma (GRCH 22) Induced by 2-Acctylaminofluorene . . . . 193 XVII . Species and Tissue Susceptibility . . . . . . . . . . . . . . . . . . 194 XVIII. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Refercnces . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Anemia in Cancer VINCENTE. PRICEA N D ROBERTE . GREENFIELD
I. Introduction . . . . . . . . . . . . . . . . I1. Incidence of Anemia . . . . . . . . . . . . I11. Role of Decreased Erythrocytc Formation . . IV . Role of Incrcased Erythrocyte Destruction . . Rcferences . . . . . . . . . . . . . . . . .
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199 201 . . . . . . . . . . . . 217 . . . . . . . . . . . . 226 . . . . . . . . . . . 284
Specific Tumor Antigens L . A . ZILBER
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 I1. Antigens of Filterable Tumors . . . . . . . . . . . . . . . . . . . 292 I11. Antigens of Nonfilterable Tumors . . . . . . . . . . . . . . . . . . 296
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IV Antigens of Human Tumors . . . . . . . . . . . . . . . . . . . . 300 V. Detection of Specific Tumor Antigens with the Help of Anaphylaxis Fol303 lowing Desensitization . . . . . . . . . . . . . . . . . . . . . . VI . The Reaction of Passive Anaphylaxis . . . . . . . . . . . . . . . . 311
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VII . Localization of the Specific Antigen in Tumor Cells . . . . . VIII. Time of Appearance in the Cell of the Specific A4ntigen . . . I X . The Nature of the Specific Tumor Antigen . . . . . . . . . X . Adsorption upon the Red Cells of the Specific Tumor Antigen XI. Discussion . . . . . . . . . . . . . . . . . . . . . . . . XI1. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Chemistry. Carcinogenicity. and Metabolism of 2-Fluorenamine and Related Compounds ELIZABETH I<. WEISBURGER A N D JOHN H . WEISBURQER
I . Introduction . . . . . . . . . . . . . . . I1. Historical . . . . . . . . . . . . . . . . 111. Chemistry . . . . . . . . . . . . . . . . IV. Carcinogenesis . . . . . . . . . . . . . . V. Metabolkm-Action of Agent on the Host . . VI . Metabolism-Action of the Host on the Agent V I I . Summary and Conclusions . . . . . . . . . References . . . . . . . . . . . . . . . .
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AUTHORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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SUBJECT I N D E X
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TUMOR-HOST RE LATlONS R. W. Begg Collip Medical Research Laboralory, University of Western Ontario, London, Canada'
I. Introduction.. . . . . . . . . . . . . 11. Enzymes. . . . .......................... .................. 1. Liver Catalase.. . . . . .............................. A. General ....... , . . . . . . . . . . . . . .................. B. Purification of Catalase.. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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B. Liver Xanthine Oxidase. . . . . . . . . . . . . . . . . . . . . . .
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111. Metabolism.. . . . . . . . . 1. Protein Metabolism. . . . . . . . .
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IV. Nutrition ...... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Experimental Approach to the Problem.. . , . . . . . . . . . . . . . . . 2. Caloric Requirements.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... 3. Protein Requirements. . . . . . . . . . . . . 4. Vitamin Requirements. . . . . . . . . . . . ' . ' . ' . . . ' . . . ... . f . . . . 5. Miscellaneous.. . . . . . . . . ....................................... 6. Summary ....................... ...... .............. . . . . . . . . .....
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* Present address: Saskatchewan Research Unit, National Cancer Institute of Canada, and Department of Cancer Research, University of Saskatchewan, Saskatoon, Saskatchewan, 1
R. W. BEGQ
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V. Hormones .......................................................... 1. The Adrenal
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............................... 35 3. Miscellaneous 4. Cancer Tests.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 36 36 VII. Morphology.. .............................. 36 .......................... 37 2. Spleen. . . . . . . . . . . ............. 37 3. Adrenal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4. Miscellaneous. . ..................... 37 5. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 VIII. Discussion. .. ............................. 1. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2. Metabolism.. . 3. Nutrition.. . . . .............
I. INTRODUCTION Tumor-host relations usually are regarded as those changes produced in the tissues of the host remote from the tumor and in which no evidence of metastatic malignant cells is found (Begg, 1955a). The older name of “systemic effects of tumors” is more precise, since tumor-host relations implies a two-way relation which does indeed exist. Increasing reference is being made t o the dependence of the tumor on the host; while this is better defined in terms of hormone dependent tumors (Furth, 1953; Noble, 1957), it has become evident also in the field of nutrition and other aspects that will be mentioned. Peyton Rous (1947), discussing the implications of the hormonal control of cancer of the prostate, wrote: “The significance of this discovery far transcends its practical application; for it means th a t thought and endeavor in cancer research have been misdirected in consequence of the belief that tumor cells are anarchic.” Autonomy of the cancer cell is a relative matter, and there is an effect of the host on the tumor. The present review will not be concerned with this aspect of the relationship, but will be confined t o the effect of the tumor on the host. The reader should con-
TUMOR-HOST RELATIONS
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sult the excellent chapter on the “Chemistry of the Tumor-Bearing Host” by Greenstein (1954). Tumor-host relations and ‘(cachexia” are confused a t times, with a tendency to discredit tumor-host relations as a consequence of the effects of hemorrhage, malnutrition, and infection (Willis, 1948). It is true that none of the effects of a tumor to date are specific for the presence of a cancer; no such claim is made. It is equally true that effects of a tumor may be noted when infection and hemorrhage are absent, invasion is minimal, and nutrition is adequate (Fenninger and Mider, 1954; Mider, 1955). A clinician’s view of malignant cachexia has been presented by Donovan (1954), who feels that it is an entity and favors the view that it is the result of a substance given off from the tumor. It adds nothing to knowledge or understanding to state that these effects are not confined to cancer. Tumor-host relations occur, they are part of the natural history of cancer and the host, and until an understanding of them is achieved only a partial knowledge of cancer will be attained. If a contribution can be made to the pathology of other diseases while studying cancer an additional advantage will be gained. A tumor can manifest its malignant properties only in the host. It can kill an animal without destroying vital organs; such a cancer death must be a “metabolic death.” The ultimate objective of tumor-host relations should be to define the effects, or summation of effects, that lead to the death of the host. The immediate objective should be the definition of individual effects, and a study of the mechanisms by which they are produced. We should proceed with caution in an attempt to explain all effects by any one mechanism. A tumor may produce its effects a t a distance both by the production of a chemical substance (“toxin”) and by the concentration of a material necessary for continued normal metabolism in the host. The author has been impressed by the number of systems involved and the problems arising from a study of tumor-host relations (Begg, 1955a). Whether or not one agrees with Greenstein (1956) that “the host-tumor relationship is the key to the cancer problem,” observation and consideration of the rodent or human with a tumor will convince one of the part such a study has to contribute to a knowledge of cancer. The author now is appalled that he accepted such a vast assignment as the general discussion of tumor-host relations. Any section of this review would be a fit subject for such an article, and indeed some have been completed (Fenninger and Mider, 1954; Haven and Bloor, 1956). The coverage of the literature is far from complete, particularly the continental literature which, in general, has been available only in abstract form, and thus difficult to evaluate. Degrees of emphasis are not necessarily a reflection of importance, but rather of available information, colored by a subjective
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element. The author has expressed his opinions, right or wrong, on the basis that it is better to state one’s views, and perhaps stimulate discussion, than to provide an annotated bibliography. However, in some sections the information is so incomplete that it is difficult to do more than report experimental results, and reasonable discussion will have to await additional information.
11. ENZYMES Progress in enzymology made available to the biochemist a tool to explore cancer research. The prime interest was in enzyme activity of the tumor, but this led to a consideration of the enzyme systems in the nonneoplastic tissues of the host animal. The net was flung widely in search for a particular enzyme pattern, and from this arose a more concentrated study of part,icular enzymes. The subject has been reviewed by Greenstein (1954), who proposed the thesis that the liver enzymes of a rodent bearing a tumor tend to resemble the enzyme activities of the tumor. 1. Liver Catalase A. General. The activity of liver catalase in the animal bearing a tumor has been studied more intensively than any other tissue enzyme in cancer. The initial observation of low catalase activity by Brahn (1916) was based on post-mortem material. This was confirmed in rodents by Greenstein et al. (1941b). Greenstein and his group produced the first thorough tissue enzyme study in tumor-host relations and gave subsequent investigators a base from which to work. The inevitable question from those outside the field is-“Why catalase?” The answer must be pragmatic, for there is doubt as to the role of liver catalase in the normal animal (Theorell, 1951), without attempting to rationalize the interest it has aroused in the tumor-host field. Material for study is plentiful, and a variety of methods are available for the assay of enzyme activity (Greenstein, 1942b; Greenfield and Price, 1954). Since the enzyme activity is halved by the presence of a tumor, some latitude in the sensitivity of the assay system may be allowed. The reason for the popularity of liver catalase in tumor-host studies appears to rest on the ready availability of something that can be measured, and thus provides an end point for experiments designed to give information on how the effect occurs. The emphasis has been on the production of the effect, not on the meaning of the low enzyme concentration to the host. Greenstein (1954) established the decrease in enzyme activity in a variety of strains of rats and mice, bearing several spontaneous and transplantable tumors. The degree of depression of enzyme activity bore a relation to the size of the tumor (Greenstein and Jenrette, 1941; Begg, 1951),
TUMOR-HOST RELATIONS
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and the activity returned to normal when the tumor was removed (Greenstein and Andervont, 1942a). The activity was not affected by rapidly growing non-malignant tissue such as an embryo (Greenstein and Andervont, 1943). Thus by 1945 the depression of liver catalase activity had been cataloged as an effect of a tumor, widespread in occurrence, related to tumor growth, and dependent on the continuous presence of the tumor. The loss of enzyme activity is not as marked from the mitochondria1 as from the supernatant fraction (von Euler and Heller, 1949). The depression of kidney catalase is much less than that of liver catalase, and blood catalase in the tumor-bearing animal is normal (Greenstein and Andervont, 1942b). The demonstration of different rates of synthesis of catalase in different tissues (Theorell et al., 1951) may explain the variation in tissue response, but this points up the peculiar selectivity of the effect. Not all liver enzymes are depressed by the presence of a tumor, and not all tissues are affected to the same degree (Greenstein, 1954). It must not be supposed that the catalase effect is specific for the animal bearing a cancer. Depression of catalase has been observed after radiation (Feinstein et al., 1950) and during the growth of a chronic granuloma (Dounce and Shanewise, 1950). As will be noted below, nutrition and hormonal status influence the level of liver catalase activity. While no claim for specificity is intended or implied, it is considered that the liver catalase effect is related to the presence of the tumor, and thus is a useful indicator in tumor-host studies. A malignant tumor produces a greater depression of catalase activity than a benign tumor of similar size and growth rate (Begg et al., 195313). The effect is not confined to the mammal, and has been reported in the frog bearing a renal carcinoma (Lucke and Berwick, 1954). Tumor growth on the chorioallantoic membrance produces a depression of catalase activity in the chick embryo liver (Stavinski and Stein, 1951). Adams and Berry (1956) have reported that the incubation of mouse liver slices leads to an increase in the activity of catalase in a homogenate prepared from the slices. They suggest, as one alternative, that catalase may be released from an inactive complex; this must be an active process as it requires the intact cells of the slice. The presence of a tumor has a greater effect on the initial level of catalase than on the increase during incubation, though with large tumors the total amount present after incubation is much less than in the control slices. The addition of a tumor homogenate had no effect on the activity of the slices during incubation. This is a refreshing approach to the catalase problem and may be related to the findings of von Euler and Heller (1949) on the greater sensitivity of catalase in the supernatant fraction of a homogenate to the presence of a tumor. another approach being investigated in the author’s laboratory is
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the use of liver perfusion (Burke and Miller, 1956) to study the response of the liver to blood from a tumor rat or perfusion from the tumor through the liver. Depressed liver catalase activity has not been demonstrated in clinical cases (Young et al., 1947). In the rodent, small tumors may lead to an increase in liver catalase activity, and the depression begins when the tumor is 5-10% of the body weight (Greenstein and Jenrette, 1941; Begg, 1951). Many patients with cancer die before this tumor mass is achieved, and rarely does clinical cancer grow at a rate observed in the transplantable tumor of the rodent, on which much of this work is based. Tumor-host studies would benefit from more frequent use of a transplantable or spontaneous tumor that grows slowly and produces its effects, leading to death, over a longer period of time. B. Purification of Catalase. Most of the work on the catalase effect has been done with crude or centrifuged homogenates. The results have to be expressed in terms of enzyme activity, and it is not known if the depression is the result of an inhibition of the normal number of enzyme molecules or a reduction in the total amount of enzyme. Catalase may undergo partial inactivation by modification of one or more of the four prosthetic groups on the molecule (Sumner et al., 1940). Methods are available for the preparation of crystalline catalase (Bonnichsen, 1947; Herbert and Pinsent, 1948) , but relatively large amounts of starting material are necessary. Price and Greenfield (1954) developed a chromatographic purification method applicable to the rat liver catalase. They suggest that the total amount of catalase is reduced in the liver of the tumor-bearing rat, with some additional evidence for the presence of an inhibitor. Progress in this field will depend partly on the development of methods such as this to permit a study of purified enzymes from small amounts of tissue. C. Nutrition and Catalase Activity. Studies on non-tumor animals have demonstrated that starvation or protein depletion may produce a loss of liver enzyme activity, with variation in individual enzyme response (Miller, 1948, 1950). Protein depletion can depress liver catalase activity (Appleman et al., 1950), and the presence of a tumor leads to a further loss (Appleman et al., 1951). A high-protein diet did not prevent t.he loss of enzyme activity in the tumor-bearing rat (Weil-Malherbe and Schade, 1948). These observations are pertinent as tumor-bearing rodents usually are in a state of inanition associated with a reduced food intake (Mider et al., 1948). Tumor-bearing rats may be force fed, and the body weight maintained, yet liver catalase activity is depressed (Begg and Dickinson, 1951). Dietary deprivation usually leads to small livers, as well as enzyme loss, presumably the result of competition for amino acid building blocks (Miller, 1948). The
TUMOR-HOST RELATIONS
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low liver catalase in the tumor-bearing rat is found in a liver that may be larger than normal (Yeakel, 1948) and is competing successfully for amino acids which should be available for enzyme synthesis. The loss of catalase is not a mere dilution factor (Klatt and Taylor, 1951) as it can occur in livers that are normal in size (Begg et al., 1953a). The relation of nutrition to liver enzymes in the tumor-bearing rat will be discussed further under xanthine oxidase, but it is apparent that the loss of catalase activity from the liver of a tumor-bearing animal is not explicable on the basis of nutrition alone. D. Hormones and Catalase Activity. A study of a possible relation between loss of liver catalase activity and altered hormone function was natural in view of the suggested effects of hormones on enzyme activity (Conference, 1951) and the hormonal imbalance in the tumor-bearing rat. The results have been conflicting, and additional work is necessary to clarify this problem. The pituitary (Gaebler and Mathies, 1951) and the thyroid (Zeckwer et al., 1954) influence liver catalase activity, but the adrenal has received the greatest attention in the tumor-bearing rodent.. Adams (1952a) has reported that adrenalectomy depresses liver catalase in the mouse, and the activity is restored to normal by cortisone. Begg, working with the rat, found that adrenalectomy did not affect liver catalase activity, contrary to a previous report (Begg and Reynolds, 1950) and that cortisone administration leads to loss of catalase activity from the liver (Begg et al., 1953a). This would be in keeping with the concept of the hyperfunctional adrenal of the rat with a tumor (Begg, 1953) and loss of liver catalase activity. The conflicting observations of Adams and Begg have not been resolved, unless the explanation lies in species difference (Kochakian, 1947). Adams (1952~)has expanded his observations to include the hormones of the gonads and the relation to nutrition (Adams, 1955), and feels that catalase depression is the result of interference with hormonal factors. E. Toxic Factors. The idea of a “cancer toxin” is not new (Ewing, 1934), and a diffusible substance produced by a tumor has been suggested as a possible explanation of the systemic effects of a tumor (Greenstein, 1954). Adams (1950) demonstrated that the injection of a tumor suspension into mice led to an early depression of liver catalase. The activity returned to normal, with a second drop when the tumor became established. From this, and subsequent experiments, he concluded that tumor tissue contains a factor, present in only low or negligible concentrations in normal tissue, that produces a fall in liver catalase activity (Adams, 195213; Adams, 1953). Depressed liver catalase in a nontumor rat in parabiosis with a tumorbearing rat has been reported (Lucke et al., 1952). Early in his work Greenstein studied the effect of a crude tumor extract
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on liver catalase, with negative results (Greenstein, 1943). Nakahara and Fukuoka (1948) prepared a fraction from human tumors by a crude concentration procedure on the premise that a toxin might be present but in low concentrations. This preparation depressed the activity of liver catalase in mice. The material was of a protein nature, active in doses of 25 mg. in vivo, but had no effect on catalase in vitro. A similar preparation from normal tissues caused very little change in liver enzyme activity. Subsequent work has confirmed their early observations, and the later protein fractions were active in l-mg. doses (Nakagawa et al., 1955). It was claimed that the toxic effect could be overcome by iron salts (Fukuoka and Nakahara, 1951), but this could not be confirmed by von Euler (1952). The report that the factor can be found in the urine of cancer patients (Nakagawa, 1952) has been denied (Carbone et al., 1955). In a later publication, Fukuoka and Nakahara (1952) noted that a single injection of the “toxohormone” material induced involution of the thymus in mice. This raises the question of adrenal activation and may fit in with the requirement for an adequate level of adrenal hormone in a permissive role (Adams, 1952a). Greenfield and Meister (1951) confirmed the early work of Nakahara and Fukuoka (1948) that the factor depressed the activity of catalase and demonstrated further that no effect was found on three other liver enzymes. Their material was heat stable and acid resistant, and preparations from normal tissues exhibited only low activity. An active material has been prepared from the spleen of mice (Day et al., 1954), suggesting that the material is not confined to tumors. Albaum and Potter (1943) investigated an inhibitor for the succinoxidase system which was present in necrotic tumor or autolyzed normal tissue, but not in healthy tumor. Hargreaves and Deutsch (1952) prepared a kochsaft from tumors which inhibited crystalline catalase, apparently by a loose chemical combination with the prosthetic groups. Previous failures to demonstrate such an activity were attributed to the presence in liver homogenates of an inactivator of the inhibitor, and the assay must be performed on a purified catalase. Seabra and Deutsch (1955) showed that a boiled aqueous extract of aged liver homogenate is capable of inhibiting catalase i n vitro. A report that such a tumor decoction inactivates the catalase of leucocytes (Frisch-Niggemeyer and Holler, 1955) would imply that the inactivator is not present in these cells. It is claimed that a factor present in the plasma of cancer patients can inactivate purified catalase (Cervigni, 1954), and a similar factor is present in the ultrafiltrate from the fluid of an ascites tumor (Hirsch and Pfutzer, 1955). A single injection of 3-amino-1,2,4-triazolet o a rat produced a sharp drop in liver and kidney catalase activity wit,h no effect on blood catalase or general toxic effects
TUMOR-HOST RELATIOKS
9
(Heim et al., 1955). The contrast between the i n vivo action of “toxohormone” and the in vitro activity of the kochsaft has been confirmed (Endo et al., 1955; Alexander, 1957), indicating that these are quite separate and distinct substances. The lack of in vivo activity by the kochsaft factor and the presence of an inactivator in liver homogenates suggest that it does not produce the loss of catalase from the liver of the cancerous rat. The evidence for a factor in tumors that does act in vivo is rather impressive, and it appears to have a selective action on catalase (Greenfield and Meister, 1951). The fact that a triazole (Heim et al., 1955) and a spleen extract (Day et al., 1954) are reported to have similar activity in no way detracts from the tumor factor, for the nonspecificity of the catalase depression has been noted. More information is required on the chemical nature of the tumor “toxin” and the mechanism whereby it produces the catalase effect. This discussion of the factors known to affect the activity of liver catalase brings into focus our ignorance of the mechanism whereby the normal level of a tissue enzyme is maintained and how the activity of such an enzyme is controlled (Knox et al., 1956). Our knowledge of the physiology of enzymes has not kept pace with advances in the chemistry of enzymes.
2. Enzymes of Protein Metabolism Against a background of alteration in the anabolism and catabolism of protein in the tumor-bearing animal, surprisingly little is known of changes in enzyme systems that might be involved. Maver et al. (1945, 1948) have reported some change in the catheptic activity of tissues of tumor-bearing mice and rats. Maver has warned against a danger of interpretation that is often ignored: the spleen and liver of tumor-bearing rodents may exhibit extramedullary hematopoiesis, and thus a new type of cell is introduced and contributes to enzyme activities. Purr (1934) observed an increase in tissue cathepsins during tumor growth. Feinstein (1950) noted an increase of catheptic activity in the liver and spleen of the rat bearing the Walker 256 carcinoma. Babson (1956) confirmed Maver’s hdings on cathepsins when the activity was assayed at a pH of 3.5, but the tissues of normal and tumor-bearing rats exhibited the same degree of proteolysis when the determinations were conducted at a pH of 7.5. Increased liver cathepsin in the host was found to be proportional to the growth of the tumor, and the serum of tumor rats stimulated the activity of liver cathepsin from control animals (Migliarese, 1956). Carboxypeptidase activities were reduced in the tissues of tumor-bearing rats and mice (Feinstein and Ballin, 1953), and an inhibitor for this enzyme was lacking from the red cells in the blood of the tumor-bearing animal.
10
R. W. BEGG
A decrease in a serum proteinase occurs during tumor growth and in pregnancy in the rat (Weil and Russel, 1938). Dillard and Chanutin (1949) have reported on elevated levels of a proteolytic enzyme and a trypsin inhibitor in the blood of cancer patients. The increase of antityrpsin level has been confirmed in the blood of a tumor-bearing rat (Waldvogel and Schmitt, 1950). Many of the observations on the proteinase have been based on the use of substrates such as hemoglobin or casein. More useful information and enzyme differentiation might be obtained from the use of model substrates (Smith, 1951). The level of D-amino acid oxidase is reduced in the livers of rats bearing tumors, the apoenzyme more than the coenzyme (Robertson and Kahler, 1942; Lan, 1944). An inhibitor for D-amino acid oxidase is present in tumors and may be a pyrophosphatase that splits flavin adenine dinucleotide (Reddy, 1957). Liver arginase is reduced slightly in the tumor-bearing rat (Greenstein et al., 1941a). Tryptophan peroxidase may undergo a biphasic response in the livers of tumor-bearing mice, and a similar effect can be produced by growth hormone and stress (Wood el al., 1956). An increase in aspartic-glutamic transaminase (White et al., 1954) and a decrease in threonine dehydrase (Greenberg and Sassenrath, 1955) have been reported in the livers of tumor-bearing rats. The observations on the activity of tissue cathepsins are difficult to evaluate lacking a knowledge of their physiological role. A more exact definition of the enzymes by current techniques might show a correlation with protein mobilization. The change in activity of the enzymes of amino acid metabolism may be a reflection of a decreased catabolism of amino acids in a liver undergoing hypertrophy. 3. Enzymes of Carbohydrate Metabolism
Though tumor-host relations have not been involved in the controversy over aerobic glycolysis in tumors (Weinhouse, 1956; Burk and Schade, 1956), several of the enzymes concerned with glycolysis are altered in the tumor-bearing host. Sibley and Lehninger (1949) reported a marked increase in the serum aldolase content of rats bearing tumors with some decrease of activity in the liver. They attributed the high serum values to release of enzyme from the tumor. A similar increase has been observed in the fluid from ascites tumors (Schade, 1953), and increased serum aldolase has been noted in some patients with cancer (Sibley and Fleisher, 1954). Removal or destruction of the tumor causes the serum level to fall to normal (Sibley el al., 1955). Mice bearing induced and transplantable tumors exhibit an increase in serum lactic dehydrogenase (Hsieh et al., 1956; Hill and Jordan, 1956). Bodansky and Scholler (1956) confirmed the increase in serum lactic
TUMOR-HOST RELATIONS
11
dehydrogenase in the tumor-bearing rat, and noted that phosphohexose isomerase was increased to even greater levels. It is considered that the increased serum level is a result of release from the tumor. Serum lactic dehydrogenase has not shown a consistent elevation in cancer patients, though i t has been suggested that more than one component may be involved (Sayre and Hill, 1957). Serum phosphohexose isomerase levels increase during the growth of metastatic carcinoma (Bodansky, 1954, 1957) and are elevated in chronic myelocytic leukemia, but not in chronic lymphocytic leukemia or in the leucocytosis of infection (Israels and Delory, 1956). A slight reduction of phosphorylase activity in the liver of the tumorbearing mouse has been reported (Goranson et al., 1954). Glucose-6-phosphatase is decreased in the liver of tumor-bearing mice, but not in the liver of a rat bearing the Novikoff hepatoma (Weber and Cantero, 1955). Betaglucuronidase is increased in the urine in cases of bladder cancer (Boyland et al., 1955). A serum inhibitor of hyaluronidase has been reported in human cancer (Hakanson and Glick, 1948). The similarity between the depression of liver catalase activity and that of formic acid oxidase has been noted (Stein and Mehl, 1955). The activity of cytochrome oxidase is increased in homogenates and in mitochondria from the liver of tumorbearing rats (Greene and Haven, 1957). Goranson (1955) has discussed the relation of liver enzymes to carbohydrate metabolism in the tumor-bearing animal. It should be noted that many of the increased serum enzymes are regarded as the result of a direct release from the tumor and not an effect produced a t a distance by the tumor. As such they might be excluded as true tumor-host relations. 4. Enzymes oj Lipid Metabolism Very few of the investigations on lipid metabolism in tumor-bearing animals have been supported by enzyme studies. Decreased esterase and lipase activity has been reported in the tissues and serum of tumor-bearing rats (Green, 1934; Edlbacher and Neber, 1935; Troescher and Norris, 1940; Stewart and Gauerke, 1955). The development of hyperlipemia in the tumor-bearing rat on a high fat diet suggested some fault in “clearing activity,” and this was observed using an optical system (Begg and Lotz, 1956). The decrease of plasma enzyme in the rat bearing a tumor was confirmed using a glycerol assay for lipoprotein lipase (Begg and Lotz, 1957). The situation is complex, as labeled fatty acids given by gavage resulted in equal counts in the respiratory COz and the tissue lipids in control and tumor-bearing rats despite the lipemia and demonstrable deficit in clearing activity (Lotz, 1957). It is interesting to note that Green (1934) considered, and rejected, a correlation-between serum esterase activity and lipemia in rats bearing tumors
12
It. W. BEGG
5. Miscellaneous Enzymes No attempt will be made to discuss every enzyme alteration that has been reported in an animal with a tumor, but two enzymes deserve special mention. A. Serum Phosphatase. Serum phosphatase may be increased in a variety of conditions (Greenstein, 1954), but frequent reference is made in the cancer literature to the increase in alkaline phosphatase activity in bone tumors (Woodward, 1942) and acid phosphatase in carcinoma of the prostate (Woodward, 1952). These serum changes appear to be the result of a release of enzyme into the blood from the tumor and, like serum aldolase, are not effects produced at a distance by the presence of a tumor. Contrary to earlier reports (Greenstein, 1942a) that serum alkaline phosphatase activity was normal in tumor-bearing rats, Frederick and Begg (1956) found a fall in the serum activity early in tumor growth. A return to normal values was noted when the tumors approached 20aJ, of the body weight. Enzyme changes in the host should be studied by sampling at more than one phase of tumor growth. B. Liver Xanthine Oxidase. In experiments on the relation of liver enzymes to nutrition, one of the most labile liver enzymes is xanthine oxidase (Miller, 1948, 1950). The maintenance of normal liver xanthine oxidase has been used as a criterion of the adequacy of protein in the diet (Williams and Elvehjem, 1949). If the chief factor in the loss of enzyme activity from the liver of the tumor-bearing animal were malnutrition, a loss of liver xanthine oxidase might be anticipated. Greenstein et al. (1941a,b) had reported normal xanthine oxidase in the liver of the tumorbearing rat. This was confirmed, and the further observation made that nontumor rats in a state of nutrition comparable to that of tumor-bearing rats, as judged by carcass weight, did show a loss of xanthine oxidase (Begg, 1954). Thus the presence of a tumor resulted in a maintenance of the liver xanthine oxidase despite the poor nutritional state. This action of the tumor is demonstrated in a more striking manner by the maintenance of normal xanthine oxidase activity in the liver of a tumor-bearing rat on a low protein diet which reduced the enzyme activity in the liver of the non-tumor rat to very low values (Begg, 1955a). This observation has been confirmed for a variety of tumors and rat strains, and the associated high excretion of allantoin noted, although a causal relation could not be proven (Burton, 1956). 6. Summary It is important t o remember that many enzymes in the tissues of tumorbearing animals are not affected by the presence of the tumor (Greenstein, 1954). In some manner the tumor produces a selective effect at a distance
TUMOR-HOST RELATIONS
13
with a resultant change in activity in some tissue enzymes. Nutritional or hormonal factors do not provide adequate explanations of the process, but evidence implicating a toxic factor is accumulating. Even if accepted, this provides only a partial explanat,ion, and the biochemical lesion produced by the toxin will require definition. A very great deal of work must be done in this field with emphasis on isolation of enzymes concerned. Progress will be limited until more information is available on the factors controlling enzyme synthesis and activity in normal animals.
111. METABOLISM 1. Protein Metabolism
A. General. The loss of tissue by the host, the search for blood changes in cancer, and the alteration of enzyme activity in the tumor-bearing animal have served to focus attention on protein metabolism. Only in recent years have carbohydrate and lipid metabolism received the attention merited. The interrelation of all components of metabolism is apparent in a study of tumor-host relations. The author has been led from a study of enzymes and protein metabolism to the influence of hormones on lipid and carbohydrate metabolism and back again to enzymes (Begg, 1955a; Begg and Lots, 1957). B. Nitrogen Balance. The excellent reviews of Mider (1951; 1955) and Fenninger and Mider (1954) on this subject make a detailed analysis here superfluous. I n general a tumor contains more nitrogen than is stored by the host during tumor growth (Mider et al., 1948); the difference has been obtained from the tissues of the host, chiefly from muscle (Sherman et al., 1950). The tumor-bearing animal restricts its food intake a t the very time additional protein and calories are needed for synthesis of the tumor tissue (Mider et al., 1948). Mider visualizes the tumor as a “nitrogen trap,” with the nitrogen lost to the host, as is the urinary nitrogen. A tumor-bearing rat must then ingest the equivalent of the urinary nitrogen, plus the tumor nitrogen, to be in nitrogen “balance.” Force-fed tumor-bearing rats are in a positive nitrogen balance by classical concepts (Begg and Dickinson, 1951). These animals do not have as much carcass nitrogen as the controls, and some has been lost, presumably to the tumor (Stewart and Begg, 195313). I n neither of the forced-feeding experiments were all three required factors measured: tumor nitrogen, urinary nitrogen, and dietary nitrogen. Calculation does suggest that the nitrogen lost to the tumor and urine was in excess of the dietary nitrogen, and presumably the host was in a negative nitrogen “balance,” according to the concept of Mider. The urinary nitrogen of a tumor rodent is frequently less than the dietary nitrogen (White,
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R. W. BEGG
1945), and the total nitrogen retained by the tumor rat may be more than the control (Bach, 1949). These determinations are not realistic unless the nitrogen retained by the tumor is considered. The advantage of a high protein intake in clinical cancer has been demonstrated (Bolker, 1953; Pareira et al., 1955). The validity of the “nitrogen trap” concept will be discussed below in Section D. The proportion of urinary urea-N and ammonia-N is similar in tumor and control rats (Bach, 1949). Proteinuria was observed only in rats bearing liver tumors (Rumsfeld and Baumann, 1955). A study of the amino acid pattern of the urine in cancerous and control subjects reveals a lower excretion of glycine and histidine by the cancerous group (Eades and Pollack, 1955). The administration of tryptophan to cancer patients results in a higher excretion of metabolic products than in control cases (Spacek, 1955; Boyland and Williams, 1956; Boyland et al., 1956; Price et al., 1956). A prominent tumor-host effect is the ability of the tumor to cause mobilization of host protein for the benefit of the tumor. Mice on a low protein diet and in negative nitrogen balance exhibited tumor growth at 75% of the rate in the control mice on an adequate protein intake (White, 1945; White and Belkin, 1945). This has been confirmed on hepatomabearing rats (Voegtlin and Thompson, 1949). The latent period, but not the growth rate, of the Walker 256 carcinoma is affected by nitrogen deprivation (Green et al., 1950). The growth rate of sarcoma R-1 and the FlexnerJobling carcinoma were both reduced with the host on a low protein diet (Babson, 1954), the growth of the Flexner-Jobling tumor being more dependent on dietary protein than sarcoma R-1. Supplementation of a 12% casein diet with methionine increased the carcass weight and decreased the tumor weight of sarcoma R-1 (Hilf, 1956). A negative nitrogen balance induced by cortisone does decrease the growth rate of the Walker tumor (Ingle et al., 1950). Thus a tumor can obtain nitrogen from the host that is in negative nitrogen balance, but not with the ease that pertains when the host is in positive balance. The results of amino acid excretion mentioned above suggest that a tumor alters the amino acid metabolism of the host in the process of obtaining nitrogen. The relation of dietary protein to tumor growth will be considered further under the section on nutrition. The ability to demand nitrogen for protein synthesis is not confined to cancer. The pregnant rat on a protein-free diet from the 11th day of gestation can produce viable, though small, young (Seegers, 1937). The growth of a tumor has little effect on the mean fetal weight during a pregnancy (Paschkis et al., 1956). Despite the demand for amino acids, pregnancy does not have the malignant effect on the host exhibited by a tumor. Liver regeneration in mice bearing an ascites tumor produced limitation
TUMOR-HOST RELATIONS
15
of tumor growth, and the rate of liver regeneration was less in the presence of a tumor, suggesting competition for a common amino acid pool (Straube and Hill, 1956). These results with the ascites tumor are in contrast to those of Paschkis el al. (1955) in rats with a hepatoma and the Walker 256 tumor; the growth of the tumor was stimulated by liver regeneration, and the presence of the tumor increased the regeneration. Here again rapidly growing normal tissue can compete with a tumor for amino acid building blocks. C. Plasma Proteins. Winzler (1953) has reviewed the position of plasma proteins in relation to cancer and indicated the wide interest the subject has demanded, frequently as a possible basis for a cancer diagnostic test. The changes described to date are nonspecific and have contributed more to a study of tumor-host relations than to cancer diagnosis. There has been general agreement that the patient or rodent with wellestablished cancer will exhibit a decrease of total plasma protein, a low plasma albumin, and an increase in a-globulins and fibrinogen (Seibert et al., 1947; Petermann and Hogness, 1948; Mider et al., 1950; Bernfeld and Homburger, 1955). The changes observed with the Tiselius apparatus may be detected by paper electrophoresis (Esser et al., 1953; Roboz et al., 1955). Alteration in components associated with t k plasma proteins have been reported, such as increase in plasma mucoprotein (Winzler and Smyth, 1948) and increase in blood proteose (Winzler and Burk, 1944). Blood and urine mucoprotein frequently are above normal in cancer, but similar elevations were noted in inflammatory and in the collagen diseases (Lockey et al., 1956). Simpson et al. (1957) suggest that alteration in serum mucoprotein may be a good index of response to tumor therapy. Accepting the nonspecificity, the interpretation of these plasma changes is not clear. The low plasma albumin may be a reflection of poor nitrogen balance, but is attributed usually to a defective synthesis of albumin by the liver (Abels et al., 1943). A change in the optical rotation of serum albumin from cancer cases has been described (Jirgensons, 1955). The optical rotation of rat’s serum has been shown to decline during tumor growth, and the suggestion has been made that this could be associated with the hypoalbuminemia, or with the appearance of an abnormal protein (Neish, 1956). The increase in plasma a-globulin of tumor-bearing mice is reported to be the result of the appearance of a new protein, immunologically different from the normal a-globulin (Bernfeld and Homburger, 1955; Nisselbaum and Bernfeld, 1957). A decrease in y-globulin in the serum of mice bearing spontaneous mammary tumors has been reported, the decrease preceding the appearance of the tumor (Johnson et al., 1954). A special and dramatic case is the elevation of a globulin fraction in the plasma in multiple myeloma. This is regarded as an abnormal protein,
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R. W. BEGG
synthesized by the tumor, and transferred to the blood (Dent and Rose, 1949; Miller et al., 1952; Putnam, 1955). A plasmacytoma in mice appears to be the source of an abnormal yglobulin in the serum (Fahey, 1957; Osserman, 1957). Parakeets with pituitary tumors have another abnormal plasma protein which is not present in birds bearing a variety of other tumors (Wall and Schlumberger, 1957). The recent papers raise again the question of a qualitative as well as a quantitative difference in the plasma proteins in cancer. In view of past experience this will require further documentation before it receives wide acceptance. D. Intermediate Metabolism. Variation in nitrogen balance and plasma proteins are manifestations of the manner by which amino acids are metabolized in the tissues. The tumor and the tissues of the host must. compete for amino acids in the metabolic pool. What is the ability of the t,umor to concentrate amino acids from the pool as compared to normal tissue? Does the presence of a tumor alter the capacity of the non-cancer tissue to concentrate amino acids? Amino acids, administered by a variety of routes, appear in higher concentrations in the free amino acid fraction of tumors than in normal tissues (Christensen and Henderson, 1952; Roberts and Borges, 1955). The presence or absence of glutamine has been correlated with progressive growth or regression in some ascites tumors (Roberts et al., 1956). Not only is the concentration of amino acids high in tumors, but tumors are among the tissues that exhibit rapid incorporation of labeled amino acids into protein. I n vitro the capacity may far exceed normal tissues (Zamecnik, 1950). I n vivo the uptake is at least equal to such tissues as liver and intestinal mucosa (Reid and Jones, 1948; Winnick et al., 1948; Nyhan et al., 1957), with the suggestion that activity is retained for a longer period by the tumor (Shemin and Rittenberg, 1944; Kremen et al., 1949). The specific activity of tumor proteins is higher than that of normal tissues after the injection of labeled plasma into tumor-bearing rats (Busch and Greene, 1955; Busch et al., 1956). Perfusion of labeled lysine through the livers of normal and azo-dye-fed rats demonstrates lower oxidative deamination by the precancerous liver and a greater incorporation of the lysine into liver and plasma protein (Burke and Miller, 1956). It is possible that not only does the tumor concentrate amino acids, but also reduces catabolism, thus setting the stage for synthesis. A tumor has a considerable capacity for the conversion of glucose to amino acids (Kit and Graham, 1956). The position of the tunior itself 11:~s been considered, for if the tumor does concentrate amino acid building blocks this may be an example of the production of a tumor-host effect by the second method postulated by Greenstein (1954). There is a tendency for the worker in the tumor-host
TUMOR-HOST RELATIONS
17
field to ignore the metabolism of the tumor and consider it as a mere tool for the production of the effect to be studied. Glycine was incorporated more readily intjothe plasnial liver, and spleen of tumor-bearing mice than control mice, but less readily into muscle (Norberg and Greenberg, 1951). This is in keeping with the enlargement of liver and spleen and loss of muscle tissue noted by Sherman et al. (1950). Histidine-2-CI4 behaved like glycine in respect to liver protein and also exhibited increased incorporation into the nucleic acid of host liver (Reid et al., 1956). The utilization of glutamic acid is impaired in the presence of a neoplasm (McHenry, 1955). The tissues of tumor-bearing rats did not show a n increase in free amino acids (Sassenrath and Greenberg, 1954) unless they were given massive doses. I n this instance there was a reduction in the maximum concentration attained by glycine as compared to controls, suggesting a more rapid metabolic turnover (Greenberg and Sassenrath, 1955). Plasma amino acids increase more rapidly in the eviscerate tumor-bearing rat than the control (Ingle et al., 1956). El Mehairy 1950) has reported an increase of a-amino nitrogen in the blood of tumorbearing mice and believes the tumor to be the source of the excess a-amino nitrogen. Free amino acids of the liver increase to a higher level after introduction of ethionine in the tumorous rat (Levy et al., 1955). Babson and Winnick (1954) have suggested that protein is translocated in the tumor rat without complete hydrolysis to amino acids. These abnormalities of amino acid metabolism are in keeping with the excretion and plasma protein studies, but the basic “biochemical lesion” has not emerged from experimentation to date. Lepage et al. (1952) demonstrated that after radioactive glycine was incorporated into the tissues of tumor-bearing rats, subsequent starvation led to a decrease in the total activity of the organs of the rats but to an accumulation of the radioactivity in the tumor. It was concluded th a t the proteins of the tumor were not available to the host, even during starvation, and the data supported the concept of the tumor as a ‘(nitrogentrap.” Greenless and Lepage (1955) extended the study to the early transplant generations of a mammary carcinoma of the mouse and found the accumulation of counts in the tumors during starvation only after the fifth transplant generation. Ascites tumor cells were labeled by the injection of radioactive glycine into the host. The labeled cells were harvested and transplanted to new hosts. Radioactivity declined in the tumor cells and appeared in the tissues of the host, the loss of radioactivity from the tumor being about 9% per day. Thus the tumor is a “nitrogen trap” in the relative sense only and does to some degree participate in dynamic exchange in the body. This does not deny the tendency for tumors to accumulate amino acids, but places it in proper perspective. The implication of the change in tumor properties a t the fifth trans-
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It. W. BGGG
plant generation is important, whether related to a change in growth rate or to some other biochemical mechanism. Much of the tumor-host work has been done with the transplantable tumor. There is, perhaps, a n unconscious extrapolation to spontaneous tumors. The transplantable tumors themselves vary in response to dietary protein (Babson, 1954). The uptake of labeled tyrosine in the tissues of the rat with a transplantable tumor was less than in the control (Winnick et al., 1948), but labeled glycine was higher in the tissues of mice bearing the 6 CIH ED tumor than in controls (Norberg and Greenberg, 1951). Further work is necessary to study tumorhost relations in spontaneous tumors and their subsequent transplant generations, and care must be taken to confine interpretation of experimental findings to the species and tumor under study.
2. Carbohydrate Metabolism I n contrast t o the large literature on the carbohydrate metabolism of tumors, this aspect of metabolism has received little attention in tumorhost studies. Cori and Cori (1925) were able to demonstrate an increased concentration of lactic acid in the venous blood from a tumor in the wing of a chicken. Norman and Smith (1956) found a n increase in blood lactic acid in tumor-bearing mice, but only immediately after the administration of a glucose load. This again is a tumor product, not a tumor effect. Busch (1955) has published a series of papers on the fate of injected acetate and pyruvate in tumor-bearing rats. There is a n extremely rapid conversion of these substrates to other metabolites, but the tumor is usually of low activity when compared to the tissues of the host. Unfortunately there are too few observations on non-tumor rats to compare for tumor-host effects. Haven et al. (1949) reported an increase in citric acid content in the tissues of tumor-bearing rats. A serum polysaccharide associated with the albumin fraction is increased in tumor-bearing rats and in cancer patients (Shetlar et al., 1950; Shetlar, 1952). The levels of liver glycogen in fasted control and tumor-bearing rats are essentially the same (Goranson et al., 1954), but the glycogen levels are so low in fasting that interpretation is difficult. Tumor-bearing mice and rats given a glucose load deposit less glycogen in the liver than control animals (Goldfeder, 1928; Young et aE., 1947b; Goranson, 1955). Pretreatment of tumor-bearing rats with insulin permits them to convert the same amount of glucose to glycogen as controls, but the tumor-bearing rat should not be regarded as diabetic (Begg, 1955a). Goranson (1954) could find no real difference in the phosphorylase activity of liver in control and tumor-bearing rats. The reason for the diminished ability to deposit glycogen by the tumor bearers is not apparent, but may be concerned with the demands of the tumor for the administered glucose.
TUMOR-HOST REL.4TIONS
19
The relation of diabetes to tumor growth presents an interesting problem. Before insulin became available, it was noted that cancer might have a modifying effect on diabetes (Braunstein, 1924). Carrie and Ham (1949) reported no effect of a tumor on diabetes in the rat and no effect on normal growth of the tumor. Alloxan diabetes reduced the number of takes of the Novikoff hepatoma and slowed the growth rate of the established tumors. The presence of the tumors reduced the blood sugar values (Goranson et al., 1954; Goranson and Tilser, 1955). Jehl et al. (1955) noted increased survival of hereditary obese-hyperglycemic mice bearing an ascites tumor and of alloxan diabetic Cb7BL mice with the same tumor. Depancreatized rats bearing the Walker tumor have less glycosuria than non-tumor depancreatized rats (Ingle, 1956). Some suppression of the growth of the tumor was noted. (See also Section V.3.) These observations tend to support the clinical view that the presence of a tumor does modify the course of diabetes; conversely they suggest also that the tumor is in some degree dependent on the carbohydrate metabolism of the host. All of the observations, with the exception of those of Carrie and Ham (1949), on the depancreatized or alloxan-diabetic animals suggest that the presence of a tumor reduces blood sugar levels and glycosuria. It is unlikely that the tumor does so by a hormonal mechanism since the increased activity of the adrenal cortex would tend to accentuate the diabetic state, while the tumor itself might be relatively immune (Burk and Woods, 1956). This provides some indirect support for the above suggestion that blood glucose may be shunted to the tumor, a suggestion that should not be difficult to test by further experiments’. 3. L i p i d Metabolism
The reader is referred to the comprehensive review of this subject by Haven and Bloor (1956) for detailed study. Some of the information will be examined from a different viewpoint, and recent work that has appeared in the past year included. A. General. The loss of lipid from the cancerous animal or human is apparent to anyone who has opened the abdominal cavity. Mesenteric and retroperitoneal fat have vanished, and the disappearance of subcutaneous fat is obvious. This has not excited interest and usually has been dismissed as part of the “wasting” associated with the disease. The author’s attention to lipid metabolism in the tumor-bearing rat was attracted forcibly by an accidental observation. Hemoglobin determinations could not be done accurately by the Evelyn method on tumor rats force fed a high-fat diet. Even a t a blood dilution of 1:500 the solution was too turbid to be read. The blood serum resembled cream, and analysis for total serum fatty acids yielded values as high as 15,000 mg. per 100 ml.
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R. W. BEGG
B. Loss of Carcass Lipid. This has been well documented by analysis, and the role of nutrition considered (Fenninger and Mider, 1954; Mider, 1955; Haven and Bloor, 1956). There is a loss of neutral fat from most tissues of the host which assume a lipid composition resembling the tumorthe “hydrolipotropic shift” of Boyd et al. (1956). Fat is an excellent source of energy, and a good correlation had been demonstrated between energy expenditure and fat loss in tumor-bearing rats (Mider et al., 1951). Thus the “mobilization for energy” concept was not an unreasonable explanation for the loss of lipid. This came under a more critical examination when considered in conjunction with lipemia (see Section C below and Haven and Bloor, 1956). Most theories concerning the depletion of the fat depots had considered some mechanism involving an excessive catabolism of fat. Fat depots participate actively in the synthesis of fat (Hausberger et al., 1954). Fat could disappear from the depots during normal catabolism and mobilization, if the rate of anabolism were depressed with a delay in replacement of lipid. Experiments to test this hypothesis were begun by studying the incorporation of labeled acetate into the fatty acids of an adipose tissue mince in vitro. The incorporation by retroperitoneal fat depots of tumor-bearing rats with relatively small tumors was much less than that from comparable tissue of control rats. A similar effect was noted using labeled glucose, and the difference was not explicable on nutritional grounds, using both caloric restriction and force-feedingtechniques (Trew, 1957; Begg and Trew, 1957). Jablonski and Olson (1955) reported that labeled glucose was incorporated into the carcass lipids of tumor rats in vivo at half the rate of control rats. The in vitro experiments demonstrate the lesion even when adequate substrate is available; this may be accentuated in vivo by competition for glucose by the tumor with reduction of available substrate for lipogenesis in situ. The exploration of this aspect of the problem has just begun, but it is possible that the fat disappears from the depots as a result of failure to replace, rather than excessive loss of, the depot lipid. The relative positions of fat depots and nonstorage tissues will require definition. C. Hyperlipemia. “A rat bearing a large Jensen sarcoma, but active and otherwise apparently healthy, was used as a control. The blood serum was noted to be very milky in appearance and its content of esterase was almost negligible.” Green (1934) observed blood lipid levels as high as 1764 mg. per 100 ml. of total fatty acids in tumor rats on a stock diet. Haven et al. (1949) found an abnormally high concentration of lipid in the blood of rats bearing the Walker 256 carcinoma. The lipemia was accentuated to a marked degree by force feeding a high-fat diet (Begg and Dickinson, 1951). The Ehrlich carcinoma causes a hypercholesteremia in the host resulting from an increased rate of sterol appearance (Kabara et al., 1967).
TUMOR-HOST RELATIONS
21
Fatty livers in starved tumor-bearing mice have been reported (Adams, 1950), and Begg and Dickinson (1951) found fatky livers in tumor rats on a high fat diet. The fatlty livers coiild be abolished by increasing the choline content of the diet, with no reduction of the lipemia (Stewart and Begg, 1953b). Thus the hyperlipemia occurred in rats without fatty livers, ketosis (Mider et al., 1951), or other evidence of abnormal fat metabolism. Several hypotheses were put forth to explain the hyperlipemia: that it was the result of a mobilization of lipid for energy or to meet the demand for unsaturated fatty acids (Haven el al., 1951), and that it depended on hormonal mechanisms (Begg, 1955b). Frederick and Begg (1956) cited evidence against both the energy and hormonal hypotheses, although insulin was demonstrated to modify the degree of lipemia. A review of the available information in the author’s laboratory (see Stewart and Begg, 1953b) indicates a correlation between the amount of fat ingested and the degree of lipemia. Stewart (1952) had shown that a fat load from a single intragastric administration of oil to a tumor-bearing rat led to a lipemia within an hour. This suggested that the lipemia was essentially an accentuated alimentary lipemia, and a study of clearing activity in the blood was undertaken. A tumor rat had a lower level of clearing factor activity in the bIood and a delay in the removal of .an intravenous oil emulsion from the blood (Begg and Lotz, 1956). The lipoprotein lipase of Korn (1955) was of low activity in the blood of tumor-bearing rats eating ad libitum, and this was not explained by starvation or anemia (Begg and Lotz, 1957). A good correlation between defective clearing activity and lipemia was apparent, but two factors indicated the dangers of correlation data: (a) If C14 labeled fatty acids were given b y gavage, the activity of respiratory C1402was as high in the tumor bearers as the controls; as early as five hours after administration, the total radioactivity in the carcass of the tumor rats was as great as the controls. The administered fatty acids were reaching the tissues despite the apparent low clearing activity. (b) The second factor was that the tumor-bearing rat force fed a high-fat diet had a high clearing activity, estimated as lipoprotein lipase. This may have been the result of the high concentration of substrate by the very presence of lipemia. Fat may stimulate a transfer of enzyme to the blood, but inadequate for the amount of lipid to be cleared (Lotz, 1957), or some of the “clearing” may take place in the tissues (Cleland and Iacono, 1957). The cause of the hyperlipemia remains to be defined, although the author feels that it is related to the amount of dietary fat and a n abnormality of the clearing mechanism. The suggestion of Haven and Bloor (1956) that several mechanisms are involved may prove correct although it is doubtful that the degree of hypoalbuminemia noted in tumor animals is sufficient to be the causative factor. Additional information on the lipoprotein lipase
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R. W. BEGG
system as the normal physiological response to a fat load, and on the mechanism of transfer of fatty acids from the albumin complex to the tissues, would contribute to a solution of the lipemia problem in the tumorbearing rat. D. Tumor Phospholipid. Mider (1955) made the very important observation that the inclusion of lyophilized Walker 256 carcinoma in the diet of the tumor-bearing rat maintained the appetite and body weight of the host. The experimental development of this factor is discussed by Haven and Bloor (1956), who demonstrated that the active principle is in a phospholipid fraction extracted from the tumor. The administration of the phospholipid to cachectic tumor-bearers led to a gain in body weight and activity. The high intestinal phospholipid concentration noted by Haven and Bloor in the anorexic tumor rat returned to normal levels. Anyone who has had the opportunity will have observed that on the death of a tumor-bearing rat the first tissue cannibalized by his cage mates is the tumor followed frequently by brain. This is the first demonstration of a tumor factor that counteracts tumorhost effects. It is possible that such effects are produced both by a “toxohormone” given off from the tumor and by substances such as the phospholipid, concentrated in the tumor, and denied to the host.
4. Nucleic Acid Metabolism Nucleic acids are regarded as playing a central role in metabolism, particularly in relation to protein and enzyme synthesis (Chargaff and Davidson, 1955). A change in the concentration and synthesis of nucleic acids in the tissues of the tumor-bearing animal might be anticipated in view of the known abnormalities in protein metabolism and enzymes. Cerecedo et al. (1952a; 195213) reported an increase in DNA, adenine, and guanine in the liver, kidney, and lung of tumor-bearing mice. This relation was not altered by hepatectomy (Rodriguez et al., 1954). Studies with labeled precursors support the analytical data by suggesting an increased rate of synthesis of nucleic acids in the tissues of the tumor host. Kelly and Jones (1950) and Payne et al. (1952a) found a n increase in the incorporation of P32into the DNA of liver, spleen, and kidney of tumor-bearing mice, with the same effect in pregnant mice and rats. Similar findings were noted by Payne et al. (1952b) using labeled formate and glycine, and in addition low specific activity was observed in intestinal DNA. Conzelman et al. (1954) observed increased incorporation of 4-amino5-imidazolecarboxamide into both the DNA and RNA of tumor-bearing mice. The increased nucleic acid turnover has been confirmed for the liver of the tumor-bearing rat, with the suggestion that it may represent the presence in the liver of substance derived from the tumor (Khouvine and Mortreuil, 1954). Administration of glycine-2-C14 and P32was followed by
TUMOR-HOST RELATIONS
23
an increase in the radioactivity of the DNA nucleotides in the livers of tumor-bearing rats as compared with normal livers (Tyner et al., 1953). Diphosphopyridine nucleotide synthesis is depressed in the liver of tumorbearing mice (Waravdekar et al., 1956). Adenine 8-C14 is incorporated into the adenine and guanine of tumorbearing mice more readily than controls, with higher activity in the DNA purines (Way et al., 1954). An increased incorporation of labeled adenine into nucleic acids was demonstrated immediately after the injection of a tumor homogenate. It was suggested that the effect was produced by a thermolabile substance given off by the tumor, and that the substance is rapidly inactivated by homogenates (Furlong et al., 1955b). The effect could be demonstrated in vitro (Furlong et al., 1955a; Griffin, 1956). High turnover of DNA and PNA in the livers of tumor-bearing mice has been noted with a labeled pyrimidine precursor (Anderson et al., 1955). Balk et al. (1955; 1956) have compared the incorporation of a series of precursors into the tissues of tumor-bearing hamsters and rats. Human tumor transplants were used in both instances, the hamsters receiving cortisone and the rats irradiation and cortisone to permit heterologous tumor growth. Controls were given appropriate treatment. The incorporation of labeled adenine, guanine, hypoxanthine, and glycine into pentosenucleic acid adenine and guanine was followed in tumor, liver, kidney, spleen, and intestine. Whereas the tumor showed a preference for de novo synthesis from glycine, the non-tumor tissues exhibited better incorporation from the purines. I n general the results confirm other tracer studies in that the presence of the tumors led to higher activity in the purine bases of the tissues, with the exception of intestine. This tissue was one of the most active, but the presence of the tumor sometimes depressed incorporation of precursors. The study indicates that each tissue has its own pattern of incorporation as well as species difference (Brown et al., 1949). There seems to be little doubt that the presence of a tumor causes an increased synthesis and concentraton of nucleic acids in the tissues of the host. The meaning is not so obvious. Morphological changes in the tumorbearing animal will be discussed in Section VII, and there is undoubtedly a relation to the altered nucleic acid metabolism. Whether the effect begins at the nucleic acid level or this is a later occurrence in the sequence of events will have to be established by experiment. I n the discussion on xanthine oxidase, the high excretion of allantoin by the tumor-bearing rat was mentioned (Begg, 1954; Burton, 1956). Bass and Place (1949) observed a high excretion of uric acid by tumor-bearing mice, but noted only a terminal increase in the excretion of allantoin. The urine collections were limited to a three-hour period from a pool of ten mice, and results expressed as the ratio of allantoin to creatinine. The injection of nuelcotides can produce morphological changes in the
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tissues similar to those observed in tumor-bearing mice (Parsons et al., 1947; Barakan et al., 1948). Release of adenine nucleotides has been considered in the causation of traumatic shock, but regarded as “not proven” (Green and Stoner, 1950), and adenine is toxic to mice (Barker et al., 1949). An increase in blood nucleotides in the later stages of tumor growth in mice has been reported (Albaum and Zahl, 1953). Nucleic acids, or their products, should be considered as possible causative factors in the production of tumor-host effects, although they do not appear to be a part of the ‘Lt,oxohormone’’substance (Greenfield and Meister, 1951).
5. Summary Manifestations of tumor-host relations may be found in all aspects of metabolism. A tumor is a relative “nitrogen trap” tending to place the host in a negative nitrogen balance with resultant tissue loss and alterations in plasma proteins. Glycogen storage is reduced in tumor-bearing rodents, and the presence of a tumor reduces the severity of diabetes. Large amounts of lipid are lost from the carcass of a tumor animal who may exhibit a massive lipemia on a high fat diet. The feeding of a tumor phospholipid preparation can reverse some tumor-host effects. Increased concentrations and rates of synthesis of nucleic acids have been observed in the tissues of the tumor host. The extent and severity of the metabolic lesions in an animal bearing a tumor are becoming more apparent, although our understanding of the observed effects is limited. IV. NUTRITION The food intake of a tumor-bearing animal usually is below normal. This single effect of a tumor will in itself produce the consequence of chronic malnutrition. Consideration of this factor should be given in the exploration of any particular tumor-host relation, and an attempt made to divorce the tumor effect from that of inanition. The fundamentals of the problem have been reviewed by McHenry (1955), and caloric and nitrogen requirements discussed by Fenninger and Mider (1954) , and Mider (1955). 1. Experimental Approach to the Problem
The investigator in the field of tumor-host relations would like to have as a control a non-tumor animal that had been subjected to the same degree of dietary deprivation as the cancerous animal. The obvious approach would be to pair feed the non-tumor individual to a comparable tumorbearer. On day A the consumption of a synthetic diet by the tumor-bearer is measured rn ith adequate precautions against spillage. The non-tumor member of the pair receives this amount of the diet on day B, when the
consumption of the tumor-bearer is again recorded and fed to the nontumor individual on day C. The relative magnitude of the one day lag is reduced a s the experiment proceeds. The voluntary intake of the tumor-bearer is reduced as tumor growth proceeds (Mider et al., 1948). A situation develops where the tumor member of the pair eats his ration over a twenty-four-hour period (usually during the night), and the nontumor individual consumes his few grams immediately. This difference can be reduced by multiple feedings, but this is limited by practical considerations, including the amount to be divided. A more serious obstacle to the use of pair-feeding in a study of tumorhost relations arises from a consideration of the objective of the technique. One assumes that each animal is given the same amount of the diet. This pertains if the tumor and the host are considered as one organism, but the purpose of the experiment is to study the effect of one on the other. The tumor partner has been forced to share his ration with the tumor, and obviously the host must suffer. If the objective of the study was the effect on carcass weight (total body weight minus tumor weight equals carcass weight), the tumor-bearer would be smaller, unless the ration had been utilized with greater efficiency. Carcass weight may be taken as the control criterion on the assumption that this is a valid measure of the nutrition of the host-a very limited assumption on several grounds, including the fact that the tumor-bearer has a n increase in water content of certain tissues (McEwen and Haven, 1941). Tumor weight can be calculated in vivo by Schrek’s formula with a workable accuracy after experience (Schrek, 1935). The carcass weight of the tumor-bearer may be determined by difference, and the body weight of the non-tumor animal restrained to this level by an empirical caloric restriction. The non-tumor member is fed less than the determined intake of the tumor-bearer, the difference being the amount required to maintain the carcass weight of the tumor-bearer and body weight of the control at the same level. This can be accomplished to a surprising degree. As a n example, a n experiment was attempted to demonstrate that the tumor rat should have a low liver xanthine oxidase activity, in view of the inanition, rather than the high activity found (Begg, 1955a). Caloric restriction of the control to a body weight equivalent to the carcass weight of the tumor rat depressed the activity of both liver xanthine oxidase and catalase; the presence of the tumor led to an increase in the activity of the former and a depression of the latter. It would be difficult to explain both results by inanition, and, further, the tumor had a greater effect on catalase than did caloric restriction. Many criticisms can be made of the caloric restriction technique, but i t is considered to be a closer approximation to the experimental situation than pair feeding.
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A third approach is the use of the method of force feeding to overcome anorexia (Begg and Dickinson, 1951; Talalay et al., 1952; Mider, 1953). The loss of carcass weight, nitrogen, and lipid by the tumor rat was overcome in large measure by this technique, although with a 70 calories per day intake, a small deficit was observed when compared with the controls (Stewart and Begg, 1953a,b). The better nutrition of the animals, as measured by carcass weight and analysis, did not prevent the loss of liver catalase activity (Begg and Dickinson, 1951). Force feeding has been used in metabolic studies (Samuels, 1947; Ingle, 1948), but has been subjected to the valid criticism that it is nonphysiological (McHenry, 1955; Haven and Bloor, 1956). Pair feeding, caloric restriction, and force feeding all have limitations as practical approaches to nutrition control in tumor-host studies. Caloric restriction permits body weight reduction of the non-tumor animal to the carcass weight of the tumor bearer. Force feeding maintains the carcass weight of the tumor rat near to the body weight of the control. 2. Caloric Requirements Mider et al. (1951) demonstrated an increased energy requirement in tumor-bearing rats, and Mider has reviewed the general subject (Mider, 1951, 1953, 1955; Fenninger and Mider, 1954). Due to experimental limitations in the determination of respiratory exchange in small animals, most of the information has been based on indirect methods. A recent abstract suggests that direct measurements are feasible with advances in methodology (Pratt, 1957). The increased energy requirement implies the necessity of an increase in caloric intake at the very time the tumor rat limits its consumption of food (Mider et al., 1948). Mider (1951) noted considerable weight loss in tumor rats force fed a t the 50-calorie level. Stewart and Begg (1953a,b) maintained carcass weight and lipid to a greater degree by the administration of 70 calories per day. Comparable non-tumor control rats eating ad libitum consumed 55 calories per day. Further studies will be necessary to determine whether a dietary allowance can be made for the additional energy requirement of the tumor rat; possibly the control and tumor rat should be force fed on calculated different caloric schedules. The experience of Stewart and Begg (1953a,b) suggests that no real difference in the maintenance of carcass composition results from the force feeding of high carbohydrate, fat, or protein diets to tumor-bearing rats, providing minimal dietary requirements are satisfied. 3. Protein Requirements Protein metabolism in the tumor-bearing animal has been discussed in Section II1,l and reviewed by Fenninger and Mider (1954) and Allison
TUMOH-HOST IiELATIONS
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(1956). The demand of the tumor for amino acids in protein synthesis has been mentioned and the ability of the tumor to husband the amino acids as a “nitrogen trap.” The tunior-bearing host, is subjected to a parasite which diverts available amino acids from the metabolic pool for its own use. At the same time the tumor produces n shift in the partition of amino acids among the host’s tissues, witness the enlargement of liver and spleen. The requirement for an abundant supply of protein is apparent, perhaps more than the additional mass of tissue would merit. Greenless and Lepage (1955) showed that the ability of the tumor to retain nitrogen may be accentuated during transplantation. Babson (1954) and Allison (1956) suggest that transplantable tumors may vary in their dependency on dietary nitrogen. Migliarese and Bly (1956) compared the growth of the Rutgers-1 and Walker-256 tumors in the rat. The Walker tumor was able to demand more of the host, based on competitive growth with the Rutgers tumor and depletion of the carcass of the host. The ability of the tumor to compete for the nitrogen pool varies, and the demand on the host for nitrogen will change as a result. It is possible that this demand may correlate with what we call “malignancy.” The protein demands of a series of spontaneous tumors in different species would be of value, with a determination of the protein balance of the host. Fenninger and Mider (1954) considered the protein requirement of the patient with cancer, the partition of amino acids between the patient and the tumor, and the effect on nitrogen balance of regression of the tumor. The clinical advantage of a high nitrogen diet for the cancer patient has been demonstrated (Pareira et al., 1955). The possibility that a tumor might have a particular demand for one or several amino acids has been considered, but the only demonstration of a sparing effect on the host by dietary supplementation is in the case of methionine (Allison, 1956; Allison et al., 1956; Hilf, 1956; Allison et al., 1957). This does not agree with the findings of Ghadially and Wiseman (1956) who suggest that methioiiine inhibits normal somatic growth while stimulating the growth rate of the RD3 rat sarcoma. This apparent discrepancy may be based on variation in the tumor and on the level of methionine supplementation.
4. Vitamin Requirements There is a considerable literature on the effect of nutrition on the genesis and growth of tumors (Burk and Winzler, 1944; Rusch, 1944; Tannenbaum and Silverstone, 1953), but references to a particular role of vitamins in the metabolism of the host are limited. Stern and Willheim (1943) reviewed the older literature which exhibits much conjecture and speculation and is based frequently on questionable assay methods. Low levels of plasma and liver vitamin A have been reported in patients
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with cancer (Abels et al., 1941, 1943). Riboflavin content of the liver is less in rats bearing a transplantable hepatoma than in the control (Robertson and Kahler, 1042). The concentration of ascorbic acid in the tissues of tumor-bearing mice depended on the strain and tumor studied, and no consistent pattern was noted (Leise et al., 1952). Shapiro et al. (1953, 1956) and Shils et al. (1956) have made a systematic vitamin assay of the tissues of mice bearing a transplantable tumor, and have reported values for vitamin Ba, riboflavine, thiamine, and coenzyme A. Figures for control mice under comparable conditions are not available, and the effect of the presence of the tumor is not clear. Woolley (1955) has presented evidence in a series of papers to suggest that there is an apparent synthesis of vitamin Blz by some mammary tumors in mice, and the tumor host may contain more BIZ than controls. It is not obvious that the additional BIZperformed any beneficial function. There is indirect evidence that the tumor-bearing rat may have a higher requirement for choline than the control. Begg and Dickinson (195l)'noted fatty livers in tumor-bearing rats fed a high fat diet, but not in control rats on the same diet. Stewart and Begg (1953b) demonstrated that additional choline in the diet abolished the fatty livers. Thus the choline in the original diet would prevent fatty livers in control rats, but not in the tumor rat who required additional choline to maintain normal liver lipid levels. Mider (1953) suggested that kidney lesions in cancerous rats might be the result of choline deficiency. 5. Miscellaneous No particular requirement for carbohydrate or lipid in the diet of the tumor host has been defined, although the interpretation of the work of Haven and Bloor (1956) on the phospholipid fraction suggests that a dietary lipid component may exist that will protect against some of the changes noted in the tumor-bearing rat. 6. Summary The chief dietary requirements of tumor-bearing animals, in excess of controls, are for adequate protein and caloric intake. There is slim evidence to suggest that additional vitamin A, ascorbic acid, and choline may be necessary to the tumor rat, but peculiar vitamin requirements of a host bearing a tumor have not been defined in exact terms.
V. HORMONES The role of hormones in tumorigenesis has been reviewed by Gardner (1957), and Noble (1957) has given a lucid discussion of hormone-dependent tumors. No adequate review of the response of the endocrine system of the
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host to the presence of a tumor is available. A preliminary survey indicated that a considerable hormone imbalance existed in the tumor-bearing rat even when allowance was made for the nutritional state of the host (Begg, 1955a). 1. The Adrenal
McEuen and Selye (1935) described enlarged adrenals, with myeloid infiltration, in tumor-bearing rats. Ball and Samuels (1938) found that the adrenal enlargement depended on the intact pituitary; the adrenals in their experiments did not exhibit any myeloid infiltration. Sure et al. (1939) reported adrenal hypertrophy, thymus at,rophy, and a fall in adrenal ascorbic acid in tumor-bearing rats. Dalton (1944) described a diminished osmophilia of the adrenals in mice bearing tumors. Savard (1948) noted a low level of ascorbic acid in the enlarged adrenals of tumor-bearing mice, and Haven et al. (1949) found low cholesterol in the adrenals of tumorbearing rats. Tumor-bearing rats exhibited an increase in adrenal size during tumor growth with a progressive fall in adrenal ascorbic acid, cholesterol, and sudanophilia; atrophy of the thymus was present in the rats (Begg, 1951). These were the expected findngs in a nonspecific activation of the adrenal mediated through the pituitary (Sayers and Sayers, 1948). The important point to establish was whether this represented a persistent hyperfunction, or an exhaustive hypofunction, of the gland. The early conclusions of Dalton (1944) and Begg (1951) favored a hypofunctional adrenal. It may be noted that Ashworth (1954) found that adrenal breis from rats with large tumors were less act)ive than normal in the conversion of deoxycorticosterone to corticosterone. A similar study in this laboratory (unpublished) led to inconclusive results. The involution of the thymus would have suggested persistent adrenal activity, but interpretation was complicated by a report that thymus involution in tumor-bearing mice was independent of the pituitary-adrenal axis (Savard and Homburger, 1949). The hypophysectomized mice in these experiments were not given maintenance doses of pituitary extract. It had been shown by Noble and Collip (1941) that a pituitary factor low in ACTH could increase the weight of the involuted thymus of the hypophysectomized rat. Begg (1953) demonstrated that adrenalectomy restored the thymus toward normal weight in tumor-bearing rats, suggesting that the adrenal was in a hyperfunctional state. However, the presence of a transplantable tumor in mice did not alter the day-night differences in blood eosinophiles rioted in controls, nor was there a significant difference in the eosinophile level between the control and tumor-bearing groups (Halberg et al., 1956).
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The deposition of injected glucose as liver glycogen may be related to the level of adrenal cortical hormones (Dorfman et al., 1946). The low deposition of glycogen in the livers of tumor-bearing rodents has been mentioned in Section 111,2, and this would be compatible with a low level of adrenal hormones. However, the defect was not correctable by adrenal hormones, but by insulin (Begg, 1955a). One theory of the control of adrenal activity is based on the sensitivity of the pituitary to utilization of adrenal hormones by the tissues (Sayers, 1950). Some concept of the additional demand of the tissues for adrenal hormones may be attained by determining the amount required to cause the hypertrophied adrenal to revert to normal size. Unpublished observations from this laboratory indicate that this amount is approximately 5 mg. daily of cortisone. The above are indirect methods to assess the activity of the adrenal cortex by determining peripheral factors known to be related to adrenal activity. These factors may be influenced by conditions other than the level of adrenal hormones as has been demonstrated for the thymus and liver glycogen. A more direct approach to the problem is necessary and is now possible. Studies on adrenal slices or breis (Ashworth, 1954) are one approach, but is it known that the activity of the adrenal in vitro is a measure of its activity i n vivo, particularly when a pre-incubation period is so commonly used? Determination of adrenal metabolites in urine, blood from the systemic circulation, or blood from the adrenal vein should give a more reliable index of adrenal activity. Southcott et al. (1956) have been able to demonstrate corticosterone in the pooled blood of mice after ACTH, and this could be repeated on the blood of tumor-bearing mice and rats. Bush (1953) demonstrated adrenal hormones in blood from the adrenal vein of rats, and this experiment is feasible on the tumor host. Nadel and Burstein (1956) have published an interesting paper on the urinary excretion of adrenal metabolites in the guinea pig during tumor growth. A threefold increase in urinary corticosteroids was noted in the later stages of tumor growth, approximately the increase resulting from the administration of 10 U of ACTH to non-tumor pigs. ACTH did not cause a further increase in the high excretion level of the tumor guinea pigs, suggesting that the adrenals were producing maximally. More direct experiments are needed, but it would appear a t the moment that the large adrenal of the tumor-bearing animal is hyperfunctional and is producing considerable quantities of adrenal hormones. Histological changes in the adrenals of cancer patients have been described (Sarason, 1943; Parker and Sommers, 195S), but they do not appear t o be a consistent feature in clinical cancer. Gordon et al. (1954) found a n adequate excretion of formaldehydogenic corticoids in cancer
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31
patients, and the response to ACTH was of the same order as the controls. Observations on adrenocorticosteroids in blood in cancer have been reported (Sweat, 1955) and the general significance of blood corticoicls reviewed by Gold (1957). Dobriner (1952) discussed steroid excretion in neoplastic disease and pointed out that qualitative as well as quantitative changes might occur. It is difficult to reach general conclusions from an examination of the literature on corticoids in clinical cancer. I n some instances there is a suggestion of an increase in adrenal activit.y; certainly the evidence does not point to hypofunction of the adrenal. 2. The Pituitary
Nonspecific histological changes in the pituitary of rats bearing tumors have been reported (McEuen et al., 1934; Haddow et al., 1957). Ball and Samuels (1938) found that adrenal hypertrophy in tumor rats was prevented by hypophysectomy. This suggested that the adrenal enlargement was the result of excessive production by the pituitary of the factor first described by Collip et al. (1933), and now known as ACTH. The adrena enlargement has been confirmed in several laboratories, and it is assumed to be a n index of a high level of ACTH production by the anterior pituitary. This should be confirmed by the demonstration of an increase of ACTH in the blood of the tumor-bearing animal (Sydnor et al., 1954). Dubnik et al. (1950) suggested that a high release of TSH by the pituitary, induced by goitrogen, led to a reduced production of gonadotropins. The same argument would lead one to suspect a limitation of the production of other pituitary hormones in view of the assumed high level of ACTH release. There is some experimental evidence to support this view. The gonads were observed to be smaller than normal during routine autopsy of tumor-bearing rats. This was confirmed by weighing testes, seminal vesicle, and prostate, and the atrophic gonads were found to be responsive to injected PMS (Begg, 1955a; Haddow et al., 1957). Smaller prostates and seminal vesicles were present when carcass weight was maintained by force feeding, suggesting that the effect was not the result of inanition. This fact, and the response to injected PMS, supports the view that the prime fault lies in deficient release of gonadotropic hormone from the pituitary. The theory would be strengthened by finding a low assay for gonadotropins in the pituitary of the tumor-bearing rat (Purves and Griesbach, 1955). Haddow et al. (1957) have considered the changes in the male accessory sex organs produced by the presence of a tumor and the relation to the pituitary and adrenal. They argue in favor of the change resulting from a nutritional deficiency, admitting the case not proven. Inconstant changes in the size and histology of the pituitary were observed in their series. Similar observations were made on the thyroid (Begg, 1955a), and in
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the course of the investigation it was found that the thyroid of the tumorbearing rat responded very poorly to goitrogens (Begg and White, 1956). The limited response was not related to nutrition, and the suggestion was made that the pituitary could not release sufficient TSH during the high level of ACTH production. Haven and Bloor (1956) found that growth hormone would revert the high ingestinal phospholipid concentration back toward normal values in tumor-bearing rats. The lipemia and loss of carcass lipid noted in the tumorbearing rat appeared to be independent of hormones of the pituitary (Frederick and Begg, 1956). In the course of this investigation i t was noted that the hypophysectomized tumor-bearing rat died at a very small tumor size unless supportive therapy such as cortisone or ACTH was given. The hormones of the posterior pituitary have not been studied in the tumor-host organism. The known alterations in water balance would suggest an investigation of antidiuretic hormone and the hypothalamus. A lesion of the hypothalamus will limit the response to a goitrogen (Greer, 1955; Greer and Erwin, 1956) as noted above in the tumor-bearing rat. 3. The Pancreas
The tumor-bearing rat may have a normal glucose tolerance curve, yet the faulty deposition of injected glucose as liver glycogen is correctable by insulin (Begg, 1955a) which also may moderate lipemia (Frederick and Begg, 1956). Thirty-five per cent of cancer patients have abnormal glucose tolerance curves (Glichman and Rawson, 1956). Patients with neoplastic disease have a decreased sensitivity to insulin associated with a slower rate of blood glucose disappearance after a test load (Marks and Bishop, 1957). The presence of a tumor tends to reduce the glycosuria of pancreatectomized rats (Ingle, 1956). (See Section III,2 for a discussion of carbohydrate metabolism.) The pancreas of a tumor-bearing animal might be studied by the histological procedures of Haist and Pugh (1948) and by an assay for insulin (Best et al., 1939) to gain further information on the effect of a tumor on the host pancreas. The activity of the pituitary and the adrenal must be considered as it is possible that the production of insulin by the pancreas may be adequate, but the utilization of insulin in the tissues antagonized. 4. The Thyroid
If the above suggestion of a low release of TSH from the pituitary of a tumor-bearing animal is correct, a hypothyroid state might be expected. Stevens et al. (1949, 1950) reported a lower concentration of radioactive iodine in the thyroid of tumor-bearers than in control rats and mice. Scott and Daniels (1956) have found increased levels of 1181 in the tissues of rats
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33
bearing f,umors, and this should be considered in the interpretation of radio-iodine studies. Mice bearing spontaneous tumors have a low thyroid activity based on a histological study of the gland (Jacobs and Lawrence, 1956). The information a t the moment favors a hypofunctional thyroid, but additional experimental evidence is required. 5. The Gonads Reduction in the size of the gonads in tumor-bearing rats has been reported (Begg and Stewart, 1952; Babson, 1954; Haddow et al., 1957). There is agreement that this may be associated with a low release of gonadotropins from the pituitary, but disagreement on the relation of nutrition to pituitary activity in the tumor-bearing rats (Begg, 1955a; Haddow et al., 1957). Haddow el al. (1957) have described histological changes in the seminal vesicles and testes, including an increase in sudanophilia of the Sertoli cells. They noted a return of the seminal vesicle to normal size after tumorectomy .
6. Summary The information available on the endocrine system of the host to a tumor indicates that the dominant feature is an activation of the pituitaryadrenal system. It is suggested that the relative deficiency of thyrotropic and gonadotropic hormones is the result of an excessive formation of the adrenocorticotropic hormone. There is some indirect evidence pointing to a deficiency of growth hormone and insulin, but the experimental data on the latter is conflicting.
VI. BLOOD h study of the blood has received considerable attention in cancer research, particularly in clinical cancer. Much of this effort has been devoted to the development of a diagnostic test for cancer, but information has accumulated that is of interest in tumor-host relations. It is proposed to discuss anemia and the white blood cells and to mention briefly cancer diagnostic tests. Plasma proteins have been considered under protein metabolism (Section II1,lC). Greenstein (1954) should be consulted for an extensive discussion of the chemical and enzymatic changes in the blood in cancer. 1. Anemia
A fall in the number of red blood cells and in hemoglobin has been associated with cancer. Anemia tended to be dismissed as the result of hemorrhage and infection or to be ascribed to a “toxic influence on the marrow”
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(Leitner, 1949). Until recent years it has been assumed that there was a depression of hemoglobin synthesis (Greenstein, 1954). Progressive anemia in the rodent bearing a tumor has been described by several investigators (Taylor and Pollack, 1942; Begg, 1951). A tumor in the yolk sac produced anemia in the chick (Armstrong and Ham, 1947.) Strong and Francis (1940) found that the hemoglobin levels of high cancer strain mice began to fall before the tumor appeared. The idea that the anemia of cancer might be associated with hemolytic phenomena received little attention until some ten years ago (Editorial, 1948). Hemolysis has received support from the work of Brown (1950) and Hyman (1954), the latter observing normal cellular activity in the bone marrow. Ponder and Nesmith (1952) could not find any difference in the hemolysins of mouse tumors and normal mouse tissue. Adelsberger and Zimmerman (1954) found the red blood cells from tumor-bearing mice more resistant to hemolysis than those from normal mice. However, Ponder and Ponder (1954) reported the demonstration of an antibody in tumor-bearing mice that induced a hemolytic anemia in the mouse. Chromium-labeled erythrocytes in tumor-bearing rabbits had a normal life span, but the hosts exhibited only a mild anemia (Ultmann et al., 1956). Studies with labeled erythrocytes and hemoglobin indicated a shorter life span of the red cells in tumor-bearing rats and suggested that the loss of red blood cells and iron was chiefly to the tumor (Greenfield and Price, 1956). An increase in blood protoporphyrin in tumor-bearing animals has been reported (Sugimura et al., 1956). Reilly et al. (1956) reported no increase in whole-blood volume in cancer, but an increase in plasma volume and decrease in red cell mass in patients. Tumor-bearing rats had an increase in whole-blood volume, the result of an increase in plasma volume with a slight increase in red cell mass. Blood folic acid activity falls in tumor-bearing mice, but a wide range of activity was found in patients with cancer (Toennies et al., 1956). Two recent papers provide strong evidence that in many instances the anemia of cancer is the result of excessive hemolysis and that a normal or increased rate of hemoglobin production does not provide sufficient replacement. Hyman and Harvey (1955) demonstrated considerable hemolysis by the Ashby technique and found increased erythropoiesis using labeled iron. Miller et al. (1956) also found decreased red cell survival and normal or increased erythropoiesis and considered the “functional inadequacy” of erythropoiesis to be the fundamental mechanism of anemia in cancer. Red blood cells from cancer patients, and patients with other chronic illness, have an increased glycolytic rate. This may be associated with a younger red cell population, resulting from an increase of marrow activity (Ultmann
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et al., 1957). The sole cause of anemia in a few cases was deficient red cell production even when there was no evidence of metast,atic tumor growth in the marrow (Miller ~t al., 1056). The authors report a hypoferrcmia with normal quantities of storage iron. Greenstein (1954) has mentioned the ample tissue iron in the face of low hemoglobin and liver catalase; the irou is there, but is not incorporated into active components. 2. Leucocytosis Tumor-bearing mice and rats exhibit a leycocytosis with an increase in neutrophiles (Parsons et al., 1947; Babson, 1954; Begg, 1955a). Non-tumor rats were subjected to chronic bleeding to produce a marked anemia, but no extramedullary hematopoiesis was noted in the liver as the result of the fall in hemoglobin (Begg el al., 1953a). The tumor-bearing hamster has a leucocytosis, and again the observation has been made that chronic bleeding does not stimulate extramedullary hematopoiesis in the liver (Sherman and Patt. 1956). It is possible that the extramedullary hematopoiesis is a myelopoiesis and concerned chiefly with the production of white cells which would be in keeping with observations on tumor-bearing hamsters (Kelsall, 1952). It has been suggested that the spleen exerts a n inhibitory function on the bone marrow in mice with spontaneous tumors (Parfentjev, 1957). 3. Miscellaneous I n over half of a series of cancer patients, an increase in the blood and plasma volume was observed (Kelly et al., 1952). The use of P32to determine total erythrocyte volume indicated a low volume in a third of a series of cancer cases (Berlin e2 al., 1955). Several papers have appeared demonstrating that the antibody response in both rats and humans is inhibited in cancer (Bogden and Aptekman, 1953; Geller, 1953). The position of immunochemistry in cancer research has been considered by Plescia (1956). Homografts of human cancer cells into volunteers resulted in greater quantity and duration of growth in cancer patients than in controls (Southam et al., 1957). 4. Cancer Tests The development of a cancer diagnostic test is a lively issue as may be seen from the perusal of any abstracting service, e.g. Ezcerpta Medica (Cancer). No attempt will be made to discuss the principles or details of such tests as several adequate reviews are available (Hill et al., 1952; Dunn and Greenhouse, 1953; Sprunt et al., 1955; Evaluation of Tests, 1957). The fact that a reaction is evoked by a tumor brings the tests into the field of tumor-host relations. No adequate test is available to date.
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5. Summary
In general the blood of a cancer patient or animal tends to a hypoalbuminemia and anemia, and animal cancers in particular may be associated with a leucocytosis. A n increased blood volume has been noted, and an inhibition of antibody response reported. None of these factors are inevitable in, or confined to, cancer, and none have formed the basis of an adequate cancer test. It is not known how the presence of a tumor induces the effect in the host, though more information is available on the pathogenesis of the anemia in cancer.
VII. MORPHOLOGY More effort should be devoted to the evaluation of morphological changes that occur in tumor-bearing animals. Many of these changes are obvious to those who have done routine post-mortem examinations, but have not received the attention given to the functional aspects of tumorhost relations. Studies of structure and function should be complementary, and the biochemist should have available facilities for the microscopic examination of tissues under study. Apparently similar samples of tissues from normal and tumor-bearing animals may show marked variation in cell type when examined under the microscope (Maver et al., 1945; Allard, 1955; Begg, 1955a). 1. Liver
Yeakel (1948) documented the increase in weight, of the liver in the tumor-bearing rat. Sherman et al. (1950) reported that the large liver in the tumor rat lost mass and nitrogen in the pre-agonal phase; the demonstration of an increase in nitrogen content in the early phase of tumor growth indicated a real increase in liver substance and not merely the increase in water content that had been noted (McEwen and Haven, 1941). Increase in liver weight was prominent in force-fed animals (Stewart and Begg, 1953a),but was reduced by limitation of protein intake (Begg et al., 1953a). Annau et al. (1951) noted an increase in mitotic activity of the liver in rats and mice bearing tumors. Malmgren (1956) was able to produce an increase in mitosis of the livers of mice by the injection of a tumor brei, or of a cell-free extract from the tumor. The extramedullary hematopoiesis that may be found in the livers of tumor-bearing mice and rats has been mentioned (Maver et al., 1945; Begg, 1955a). Kelsall (1952) has described an infiltration of plasmacytes and lymphocytes in the liver of the tumor-bearing hamster. Amyloid infiltration and focal necrosis have been found in the liver of tumor-bearing mice (Parsons et al. , 1947).
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Personal inquiry among investigators and observations in the laboratory indicate that these microscopic findings are not, universal in the livers of all tumor-bearing animals. The fact that they have been reported places the burden of proof of their absence on anyone working on the biochemical aspects of the liver in the tumor-bearing animal. Otherwise, reported changes may be the result of altered or new cell types, not biochemical alteration per se. 2. Spleen
Splenomegaly is obvious in tumor-bearing rodents and may be tenfold (Stewart and Begg, 1953a; Antopol et al., 1954). Marked alteration in the histological picture has been reported (Maver et al., 1945; Parsons et a/., 1947; Kelsall, 1949). The splenomegaly may diminish in the later stages of tumor growth (Rodriguez and Cerecedo, 1955). Some radioactive material is taken up to a lesser degree by the liver and spleen of tumor-bearing rodents than by controls. Autoradiograms indicate that the material is concentrated in the phagocytic reticuloendothelial cells, and the reduced uptake may be a reflection of decreased phagocytosis in tumor-bearing animals (Argus et al., 1956, 1957).
3. Adrenal Gross and histological changes in the adrenals of tumor-bearing animals have been mentioned in Section V,1. Selye (1955) has discussed the appearance of myeloid infiltration and PAS-positive granules in the adrenal. An increase in nuclear size has been reported by Kasten (1955). 4. Miscellaneous
Morphological changes in the thymus and gonads of tumor rodents have been noted in Section V. Albert et al. (1954) have found a greater increase in lymph node weight after the growth of homologous tumors than of isologous, and suggest that the changes may be an antigenic response. Sarcoma 180 contains a protein factor which stimulates the growth of nerve fibers from chick ganglia in vivo and in vitro (Cohen and LeviRlontalcini, 1956). The Ehrlich ascites tumor produces a nuclear growthstimulating and a mitosis-stimulating agent, and it is claimed that each acts on host cells independently (Chu, 1957). Replacement of pancreatic alveoli by fat has been reported in tumor-bearing hamsters (Kelsall and Crabb, 1956). 5. Sumrrinrg I n the study of tumor-host relations it should be remembered that the host may have made gross and microscopic morphological adjustments to
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the presence of the tumor. These changes could affect a study of function and should be the subject of experiment for their intrinsic interest. Alterations in the morphology of the liver, spleen, adrenal, thymus, lymph nodes, and gonads have been described, but further exploration undoubtedly would reveal other morphological effects of the presence of a tumor.
VIII. DISCUSSION An attempt should be made to gather together into one general thesis the diverse information on tumor-host relations that has been presented. The attempt may be futile, and, as the gaps in our knowledge of the subject are so great that many connecting links could be missing, premature. We may be looking for order where very litt.le exists and searching for the “overriding single cause” when the real explanation lies in a summation of many factors. 1. Enzymes
The tissue enzymes of the host exhibit varying response to the presence of a tumor; some are depressed in activity and others unchanged. This is not unique, as tissue enzymes may show varying degrees of lability to a low-proCein diet (Miller, 1950; Prigmore et al., 1955). The pattern of change is not the same in the response to a tumor and a low-protein diet. Xanthine oxidase activity is normal in the liver of the tumor rat (Greenstein et al., 1941b), but one of the most labile to dietary restriction (Miller, 1948). The normal level of liver xanthine oxidase, and the low liver catalase activity despite the maintenance of good nutrition by force feeding (Begg and Dickinson, 1951), argue against malnutrition as the explanation of altered tissue enzyme activity in the tumor-bearing rat (Begg, 1955a). I n most instances we know only that the activity of an enzyme has been depressed and have no information as to the actual amount of enzyme present. More studies along the lines of Price and Greenfield (1954) are indicated demonstrating an actual change in the amount of an enzyme. Such studies will require the development of enzyme methods for dealing with relatively small amounts of tissue in isolation and purification procedures. The finding of a lower concentration of liver catalase strengthens the original assumption based on the presumed shortage of building blocks for protein synthesis and the correlation with a low level of another hemeprotein, hemoglobin. It should be remembered that low catalase occurs in a liver that is increasing in mass and competing successfully with the tunior for amino acid substrate in protein synthesis. Recent work indicates that the synthesis of hemoglobin is normal or increased in the tumor-bearer (Miller et al., 1956), the anemia resulting from excessive destruction of
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hemoglobin. The low catalase may be the result of excessive destruction rather than depressed synthesis, and this has been suggested by Appleman et al. (1950), from indirect evidence. The whole question of the turnover rate of catalase in the liver of the tumor rat requires investigation by an isotopic method based on Price and Greenfield (1954) and Theorell et al. (1951). Where enzyme activity has been altered by the presence of a tumor, the possible role of activators and inhibitors must be considered (Swartz et al., 1956), and the kochsujt factor of Hargreaves and Deutsch (1952) could be an example. Hormones could play a major role in the control of enzyme activity, but experimental demonstration has been disappointing (Renold et ul., 1956). The evidence for a hormonal effect on catalase in the tumor-bearing rodent is conflicting (Adams, 1952a; Begg, 1955a) and must be the subject of further experiments. Wood et al. (1956) have suggested that growth hormone and stress can reproduce the findings on liver tryptophan peroxidase in tumor-bearing rats. A most interesting development in tumor-host relations has been the “toxohormone” of Nakahara and Fukuoka (1948), the tumor fraction described by Greenfield and Meister (1951), and the work of Adams (195213, 1953). If substantiated by further experiment, it will mean that the catalase effect is produced by a substance given off by a tumor and, when developed, should be able to replace the tumor in the production of the effect. Several questions will have to be answered: (a) how does the substance produce the effect; (b) will it produce other effects; (c) what is the structure; and (d) does it occur in other than tumor tissue? Furlong et al. (1955a) have suggested that a tumor factor may increase the incorporation of labeled adenine into nucleic acids, and Malmgren (1956) reproduced the increased mitosis in the liver of tumor-bearing mice by injection of a saline extract from the tumor. If the present trend is supported by future experiments, we will have a t hand a cancer “toxin,” and the validity of a direct effect of the tumor on the host will no longer be in doubt. Liver catalase has been the “model system” in the study of the enzyme aspects of tumor-host relations and has been subjected to considerable experimentation. Relatively little is known of the production of other enzyme effects. An attempt to relate the sustained liver xanthine oxidase of the tumor rat to the high excretion of allantoin, on the basis of induced enzyme formation, did not succeed (Burton, 1956). 2. Metabolism
The impression gained from ti consideration of the metabolism of the tumor host is of a great demand for energy and protein and a basically
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normal intermediate metabolism. The difference between the metabolism of the tumor rodent and the control are quantitative rather than qualitative. Mider et al. (1948) suggested a partition of nitrogen by the tumor into a separate metabolic compartment which did not exchange with the general pool of the host. This was described as the “nitrogen trap,” and Mider considered that in a determination of the nitrogen balance of the tumor-host organism, the nitrogen in the tumor must be regarded as lost to the host. Thus “nitrogen balance” implied : dietary nitrogen = urinary nitrogen increase in tumor nitrogen. This has been a useful concept and suggests, for example, that the data of Begg and Dickinson (1951) lack the figures for tumor nitrogen, and that of Stewart and Begg (1953b), the urinary nitrogen determinations required for an adequate assessment of nitrogen balance in the host. Early experiments with labeled amino acids supported the “nitrogen trap” idea (Lepage et al., 1952), but Greenless and Lepage (1955) found that the trap did not apply to the early generations of transplanted tumors, and ascites tumors could exchange amino acids with the host. The tumor is a nitrogen trap in the relative sense only, and the degree may be related to the degree of “malignancy” of the tumor. White (1945) demonstrated only a slight retardation of tumor growth when the host to a transplantable mouse tumor was placed on a nitrogen deficient diet. Babson (1954) has shown a difference in the response of Sarcoma R-1 and the Flexner-Jobling carcinoma to a low-protein diet. Allison (1956) referred to degrees of “nutritional malignancy.” These observations are in keeping with the varied response of transplantable tumors in the segregation of labeled amino acids (Greenless and Lepage, 1955). Primary tumors vary in their growth rate and in the time required to kill the host. The effect of tumors appears to be based on mass rather than number. Goodman (1957) has demonstrated in both transplanted and spontaneous tumors of mice that the total tumor mass at death is the same for single and multiple tumors. For purposes of simplification, we tend to regard all tumors as highly autonomous and malignant neoplasms which grow very rapidly at the expense of the host. The classic experiments of White and Mider support this, although Mider was careful to specify that his results applied only to the Walker tumor. In addition to variation in the properties of the primary neoplasm, transplantation may well impose a selection factor where the most vigorous and “malignant” cells thrive and displace others. Thus after several transplant generations, the tumor mag have quite different quantitative properties from the tumor of origin. Some experimental data is available to support this concept (Greenstein, 1954). but it must be subjected to further study.
+
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The laboratory investigator in cancer research, particularly if he lacks a biological or medical background, must be aware of the great variation within the definition of cancer and malignancy. Variability in the properties of tumors has been presented by Karnofsky (1956) and Law (1956), and Engelbreth-Holm (1955) has discussed some of the difficulties encountered in the classification of tumors. Possibly we can evolve a workable ‘(biochemical Broders” classification to aid in the orientation of effects produced by a particular tumor under certain experimental conditions. A fault in glyconeogenesis is the most prominent abnormality in the intermediate metabolism of carbohydrate in the tumor host (Young et al., 1947b; Begg, 1955a). This has not been explained in enzymatic or hormonal terms and may be the result of a diversion of glucose to the tumor rather than a defect in the glyconeogenic pathway. Lower blood sugar values and diminished urinary glucose have been reported in the diabetic rat bearing a tumor (Goranson and Tilser, 1955; Ingle, 1956). The loss of carcass lipid in the tumor rat has been well documented, and a lipemia of varying degree may be noted (Haven and Bloor, 1956). The fat content of the liver is normal, if adequate choline is supplied (Stewart and Begg, 1953b), and ketonuria does not appear (Mider, 1951). Ketonemia is less than in the controls in starved tumor-bearers, and the utilization of ketone bodies by the tissues from tumor rats tends to be higher than in the tissues from controls. The tumor has an ability to utilize ketone bodies equal to that of the heart (Trew, 1957). Labeled fatty acids given to tumor rats and controls appear equally in the respiratory COZ of both groups (Lotz, 1957). The tumor rat has an adequate metabolic system for the complete oxidation of fat and a ‘‘normal” lipid metabolism except for the low tissue and high blood lipid. Mider (1951) demonstrated an energy deficit in the tumor-bearing rat and pointed out the good correlation between the amount of lipid and energy lost. This led to the idea that the lipemia was a manifestation of mobilization of lipid for energy purposes. Stewart and Begg (1953b) disagreed with the theory, and Frederick and Begg (1956) could not find evidence in support of it; nor could they support the thesis that the lipemia was the result of a hormonal imbalance. Gross lipemia could be observed in the hypophysectomized tumor-bearing rat. It is possible that the loss of carcass lipid represents an inability to replace lipid in the depots rather than an excessive mobilization. Adipose tissue from tumor rats has a poor capacity for the incorporation of acetate into fatty acids in vitro (Trew, 1957; Begg and Trew, 1957). Jablonski and Olson (1955) demonstrated that labeled glucose was incorporated into the carcass lipids of the tumor rat a t half the rate of the control. A causal relation between the loss of lipid from the depots and the
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development of lipemia has been assumed. If lipid loss is not the result of excessive mobilization, the relation may be a coincidental time factor. There is a good correlation between the amount of dietary fat and the degree of lipemia (Begg, unpublished), and gross lipemia can be produced in two hours by a single intragastric fat load in the tumor-bearing rat (Stewart, 1952). Tumor-bearing rats have a defective clearing activity (Begg and Lotz, 1956, 1957)) and the lipemia may be the result of a low level of lipoprotein lipase. This is not a complete explanation of a complex situation-despite the low clearing activity in vitro and in vivo, fatty acids do get to the tissues and depots in the same time as controls (Lotz, 1957). Haven et al. (1951) suggested that lipemia might be the result of mobilization of carcass lipids to meet the demands of the tumor for unsaturated fatty acids. This is a n extension of the idea that tumor-host effects may be the result of an excessive demand of the tumor for a normal metabolite which is concentrated and utilized to the detriment of the host. The nitrogen trap theory and the possibility that carbohydrate is diverted to the tumor are in the same category, Some evidence in support of the Haven hypothesis has come from a very important study in tumor-host relations. Mider (1955) fed tumor-bearing rats a synthetic diet in which lyophilized tumor replaced protein. The rats maintained weight and appetite and survived for longer periods. Something in the lyophilized tumor was able to prevent a prominent effect of a tumor. Haven and Bloor (1956) report that the factor is in the phospholipid fraction of the lyophilized tumor. When fed as a dietary supplement to preterminal tumor rats with marked anorexia, it restored appetite and led to a weight gain and a n increase in activity. These observations rank with the toxohormone experiments in demonstrating two types of mechanisms that may operate in the production of systemic effects. Both need further experimental confirmation, but they have opened new approaches to the study of tumor-host relations. Tissues of tumor-bearing rodents have an increased concentration and turnover rate of nucleic acids (Cerecedo et al., 195213; Way et al., 1954; Balis et al., 1956). The central role that nucleic acids are assuming in biology, and in particular the control of protein and enzyme synthesis (Davidson, 1953)) would suggest the possibility that some of the observed effects of the tumor on the host might be a result of a prime effect on nucleic acid metabolism. Several years ago the author prepared to study the distribution of purine bases in the nucleic acids isolated from organelles of the liver of control and tumor rats. Fortunately the extramedullary hematopoiesesis in the liver was noted and the experiment suspended, or a very great deal of work might have produced chemical confirmation of what is obvious under the microscope. With advances in technique and proper biological material, such studies might be fruitful in an exploration of the control of metabolism.
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3. Nutrition The important factor in the nutrition of the tumor host, with present information, is an adequate intake of calories and protein. The lack of special requirements for vitamins, particular amino acids, etc. may be a reflection of ignorance rather than an immunity of the host to such deficiencies. There is indirect evidence to suggest a special requirement for choline (Stewart and Begg, 1953b). Allison (1956) has described a protective effect of methionine, and Burk and Winzler (1944) mention other possible factors. A great deal more information on this aspect of tumor-host relations is required. Neither the protein nor the caloric requirement of the host has been defined. Mider (1951) noted weight loss in tumor-bearing rats force fed 50 calories per day. This was the voluntary intake recorded by Stewart and Begg (1953a) who force fed a t the 70-calorie level, but still noted a slight deficit in carcass nitrogen and lipid. It would be reasonable to feed the tumor rat more than the control if it is required that provision be made for tumor growth and for an increase in carcass weight that is comparable to the increase in body weight of the control. This argument holds if the control rat is to be limited to the nutrition of the tumor rat and body weight accepted as a criterion of nutrition. The control should be given less to eat than the cancerous rat, since no provision must be made for the tumor. These are practical problems facing the investigator who wishes to do a controlled study of the nutrition of the tumor-bearing rat and to which no completely satisfactory answer is available. 4. Hormones
Current information suggests that the stimulated adrenal of the tumorbearing animal produces excessive amounts of adrenal hormones. Nadel and Burstein (1956) demonstrated a sharp increase in urinary corticosteroids during tumor growth in guinea pigs. The in vitro studies of Ashworth (1954) indicated an initial stimulation and subsequent depression of the conversion of precursors to corticosterone, but such experiments are subject to the usual difficulties of interpretation in terms of the intact animal. The information needed is the level of adrenal hormones in systemic and adrenal vein blood (Gold, 1957) and the utilization of adrenal cortical hormones by the tissues. The postulated low levels of gonadotropic and thyrotropic hormone production in the pituitary of the tumor-bearing rat (Begg, 1955a) should be supported by pituitary assays, and the position of nutrition in pituitary function of the tumor-bearing animal defined (Haddow et al., 1957). The endocrine status of the pancreas and the peripheral function of insulin during tumor growth require clarification. The endocrine aspects of tumor-host relations need the attention of the
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trained endocrinologist with his techniques, assays, and background to assess the findings in terms of the function of the animal. It is to be hoped that such interest can be aroused, for it should not be difficult to introduce the animal or patient with a tumor into a particular investigative pattern and facilities. 5. Blood The anemia in cancer did not stimulate research interest until recent years. It was found that the assumed depression of hemoglobin synthesis did not exist, and many cases of anemia were the result of excessive destruction rather than defective synthesis of hemoglobin (Miller et al., 1956). There is now a considerable interest in the anemia of cancer, and it is possible that a study of the white cells in cancer patients and animals might be as rewarding. No real explanation is available for the hypoalbuminemia (Winder, 1953) or for the defects in antibody response (Geller, 1953; Southam et al., 1957). Possibly some of the attention given to the evolution of an empirical cancer test should be diverted to a study of fundamental questions, although the urgency of a workable test for cancer cannot be overlooked. 6. Morphology The study of morphology in the host to a tumor should constitute a tissue control in biochemical investigations (Begg, 19554. More important would be morphological studies for their intrinsic value , exploiting the newer microscopic and manipulative techniques. The work on liver mitosis should stimulate further study (Annau et al., 1951; Malmgren, 1956). We must turn to the anatomist, as well as to the endocrinologist and hematologist, for assistance in the study of tumor-host relations which should be based on structure as well as function. Sufficient observations on morphological abnormalities in liver, spleen, gonads, etc. are available to suggest that these studies should be expanded and a search made for other tissues which may exhibit an effect of the tumor at the morphological level.
7 . General The wisdom of attempting a general thesis on tumor-host relations at the present time was questioned. The attempt is premature if anything resembling the complete picture is required. However, the thesis could be based on preliminary sketches which have the following outlines: (1) A tumor produces effects on a host that are not the result of invasion by malignant cells, displacement by secondary growths, hemorrhage, infection, or malnutrition. (2) The magnitude of the effects is related to the growth of the tumor, and the host reverts to normal when the tumor is
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removed. (3) The effects could result from a concentration by the tumor of metabolites required by the host (amino acids, lipids) as well as the liberation of substances with a systemic effect on the host (toxohormone, liver mitosis factor). (4)The effects are not specific for cancer, and practically any one can be reproduced by other states. Pregnancy leads to a n accumulation of metabolites in the uterus and may proceed during poor nutrition; catalase in the liver can be depressed by a variety of factors; anorexia is marked in some cases of tuberculosis; chronic diseases may produce comparable changes in the endocrine system; liver mitosis is stimulated by hepatectomy ; patients with idiopathic hyperlipemia are in comparative good helath. To deny the importance of any of the effecbs on the grounds that it is not specific is unreasonable-death is very final and very nonspecific. (5) The production of the effects, and the eventual death of the animal, represent the summation of many factors. The lethal component in cancer may be the ability to produce widespread metabolic and anatomic lesions; the host is overwhelmed. We are now in possession of information to aid in the design of experiments that will permit this preliminary sketch to be converted, over the years, into a more complete picture of tumor-host relations.
8. Summary and Conclusions The presence of a tumor in the host produces biochemical and morphological changes in tissues which do not contain any malignant cells. These changes may be noted in the absence of hemorrhage, infection, and malnutrition, and collectively constitute one aspect of tumor-host relations. The effect of the host on the tumor has not been considered. Examples of tumor-host relations may be found in alteration of enzyme activity, metabolism, nutrition, hormone balance, composition of the blood, and structure of tissues. Our information on the extent of the tumor effect is incomplete, but it is known to be considerable. It is suggested that the death of the animal with a tumor, in many instances, is the result of the widespread alteration in metabolism. It would appear that a tumor both concentrates metabolites to the detriment of the host and releases substances into the circulation which produce structural and functional changes in the tissues of the host. We urgently require further information on the mechanism whereby the effects of a tumor are produced. Much work remains to be done both on the scope of tumor effects on the host and on an intensification of research on particular effects; their dependence on the tumor, relation to nutrition, anemia, hormones, and other factors that may be involved. As in all cancer research, tumor-host relations will benefit greatly from advances in our knowledge of normal and abnormal structure and function.
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ACKNOWLEDGMENTS The author is indebted to the National Cancer Institute of Canada for generous support over the past ten years.
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Sherman, C. D., Morton, J. J., and Mider, G. B. 1950. Cancer Research 10, 374-378. Sherman, J. D., and Patt, D. I. 1956. Cancer Research 16, 394-401. Shetlar, M. R. 1952. Texas Repts. Biol. and Med. 10, 228-238. Shetlar, M.R., Erwin, C. P., and Everett, M. R. 1950. Cancer Research 10, 445-447. Shils, M. E., Friedland, I. M., Fine, A. S., and Shapiro, D. M. 1956. Cancer Research 16, 581-584. Sibley, J. A., and Fleisher, G. A. 1954. Proc. Staff Meetings Mayo Clin. 29, 591-603. Sibley, J. A., and Lehninger, A. L. 1949. J . Natl. Cancer Inst. 9, 303-309. Sibley, J. A., Fleisher, G. il., and Higgins, G. M. 1955. Cancer Research 16, 306-314. Simpson, W. L., Goodman, M., and Brennan, M. J. 1957. Proc. Am. Assoc. Cancer Research 2, 250. Smith, E. L. 1951. I n “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. I, Part 11, pp. 793-872. Academic Press, New York. Southam, C. M., Moore, A. E., and Rhoads, C. P. 1957. Proc. Am. Assoc. Cancer Research 2, 251-252. Southcott, C . M., Bandy, H. E., Newson, S. E., and Darrach, M. 1956. Can. J . Hiochem. and Physiol. 34, 913-918. Spacek, M. 1955. Can. Med. Assoc. J . 73, 198-201. Sprunt, D. H., Hale, W. H., Chang, F. C., Richmond, S. C., and Erikson, C. C. 1955. Science 122, 273-274. Stavinski, E. R., and Stein, A. M. 1951. Cancer Research 11, 768-771. Stein, A. M., and Mehl, J. W. 1955. Federation Proc. 14, 286. Stern, K.,and Willheim, R. 1943. “The Biochemistry of Malignant Tumors.” Chemical Publ., Brooklyn, New York. Stevens, C. D., Meinken, M. A., Quinlin, P. M., and Stewart, P. H. 1950. Cancer Research 10, 155-161. Stevens, C. D., Stewart, P. H., Quinlin, P. M., and Meinken, M. A. 1949. Cancer Research 9,488-497. Stewart, A. G. 1952. Ph.D. Thesis. University of Western Ontario, London, Canada. Stewart, A. G., and Begg, R. W. 1953a. Cancer Research 13, 556-559. Stewart, A. G., and Begg, R. W. 1953b. Cancer Research 13, 56Ck565. Stewart A. G., and Gauerke, L. 1955. Proc. Am. Assoc. Cancer Research 2, 48. Straube, R. L., and Hill, M. S. 1956. Proc. A m . Assoc. Cancer Research 2, 150-151. Strong, L. C., and Francis, L. D. 1940. A m . J . Cancer 38, 399-403. Sugimura, T., Umeda, M., and Ono, T. 1956. Gann 47, 87-90. Sumner, J. B., Dounce, A. L., and Frampton, V. L. 1940. J . Biol. Chem. 136, 343-356. Sure, B., Theis, R. M., and Harrelson, R. T. 1939. A m . J . Cancer 36, 252-256. Swartz, M. N., Kaplan, N. O., and Frech, M. E. 1956. Science 123, 50-53. Sweat, M. L. 1955. J . Clin. Endocrinol. and Metabolism 16, 1043-1056. Sydnor, K. L., Sayers, G., Burgess, L., and Heiselt, L. 1954. Endocrinology 66,621-636. Talalay, P., Takano, G. M. V., and Huggins, C. 1952. Cancer Research 12, 838-843. Tannenbaum, A., and Silverstone, H. 1953. Advances i n Cancer Research 1, 451-501. Taylor, A., and Pollack, M. A. 1942. Cancer Research 2, 223-227. Theorell, H.1951. I n “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. 11, Part I, pp. 426-427. Academic Press, New York. Theorell, H., Beznak, M., Bonnichscn, R. K., Paul, K. G., and Akeson, A. 1951. A d a . Chem. Scand. 6,445-475. Toennies, G., Frank, H. G., and Gallant,, D. L. 1956. Cancer 9, 1053-1058. Trew, J. A. 1957. Ph.D. Thesis. University of Western Ontario, London, Canada. Troescher, E. E., and Norris, E. R. 1940. J . Biol. Chem. 132, 553-557.
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PRIMARY CARCINOMA
OF THE LIVER
Charles Berman Consolidated Main Reef Miner and Estate Lld., Marairburg, Tranrvaal, South Africa
.................................
.................................
Page 56 57
phic Distribution of Primary Liver Cancer 1. Geographic and D A. Western Races ............................................ B. African Races.. . . . . . . . . . . , . . . . . . . . . , . . . . . . . . . . . . . . . . C. Oriental Races . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Asian Immigrants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .,............ E. American Negroes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Primary Liver Cancer in Animals and Birds.. . . . . . . . . . . . . . . . . . . . . . . . , . , 3. Limitations of the Available Primary Liver Cancer Statistics 111. Clinical Manifestations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Sex and Age Incidence. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Onset ........................................... ... 3. Clinical Classification.. . . . , . . . . . . . . . . . . . . . . . . . . , . . . . . . , . . . . . . . . , , 4. Symptomatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Diagnosis of Primary Liver Cancer Through Laboratory and Other Aids.. A. Liver Function Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Liver Aspiration Biopsy. . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Peritoneoscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. D. Duodenal Aspiration, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. E. Roentgenological Diagnosis. . . a. PortalSplenic Venography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Abdominal Aortography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Radioactive Materials.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Suggested Treatment, . . . . . . . . . . . . B. Radiotherapy. . , . , . . . . . , , . . . C. Radioactive Isotopes. . . . . . . . , . D. Surgery . . . . . . . . . . . . . . . . . . . . . IV. Morbid Anatomy. . . . ............................................. 1. Gross Pathology: Macroscopic Classification. . . . . . . . . . . . . . . . . . . . . . . . . . . .......................... A. Nodular Carcinoma. . . . . . . . . . B. Massive Carcinoma.. . , , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . , . . . . . 2. Histopathology . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ .. ...._..... A. Hepatocellular Carcinoma. . . . . . . . . . . . . . . . . . . B. Cholangiocellular Carcinoma. . . . . . . . . . . . . . . . . . ...... . . . . . . . . .
57
63 63 64
66 66 66 67 67 68 68 68 68 69 69 69
,
............................
A. Intrahepatic Metastases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
71 71 71 71 71 72 73 74 74
56
CHARLES BERMAN
B. Extrahepatic Metastases.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Histogenesis.. ...... A. Hepatocellular a ancer ....................... B. Vesicular Cancer.. . . . . . . . . . . C. Anaplastic Cancer... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
74 75
76
F. Embryology and Histogenesis. .......................... V. Etiology: Environmental Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
79
......................
80
A. Hepatic Cirrhosis a. Post-Mortem I b. Hepatic Cirrho
I. INTRODUCTION This communication concerns the problem of primary liver cancer mainly as it affects mankind. It is divided into four sections: the first is devoted to geographic and demographic distribution, the second to clinical manifestations, the third to pat,hology, and the fourth to the possible role of environmental factors in the etiology. Perhaps the most extraordinary feature of this disease is its distribution. Of rare occurrence among the populations of Europe and North America, it is on the contrary, remarkably frequent in the native inhabitants of tropical and subtropical Africa and certain parts of Asia, among whom it is often the most common form of internal cancer affecting relatively young male adults. As with other forms of malignant disease, the true cause of primary liver cancer remains unsolved. From clinical observations, however, and from the striking evidence that cirrhosis and primary liver cancer can be readily induced in experimental animals by deficient diets and by means
57
PRIMARY CARCISOMA O F T H E LIVER
of specific carcinogens, it is becoming increasingly apparent that preexisting liver damage (including hepatic cirrhosis) is closely associated with the malignant process, and that chronic malnutrition and other as yet undet#ermined environmental factors may play a decisive role in the etiology.
11. INCIDENCE 1. Geographic and Demographic Distribution of Primary Liver Cancer A survey of the world’s available post-mortem statistics has indicated that the incidence of primary liver. cancer varies strikingly among the different races of mankind (Berman, 1951). Of rare occurrence among the peoples of Western Europe and North America, it is by contrast remarkably common among the inhabitants of Africa and certain parts of Asia (Table I). TABLE I Geographic and Demographic Incidence of Primary Liver Cancera Population
No. of Autopies
European United States Bantu (African) Oriental Chinese Filipino Indian Japanese Javanese (Indonesian)
248,053 108,632 8,068 76,196 23,764 13,876 14,768 15,565 8,253
a
Percentage of Percentage of primary Cases of Cancer primary Liver Cancer of a**Organs Liver Cancer 0.14 0.27 1.1 0.76 0.90 0.44 0.32 0.97 1.31
24,537 5,602 2,796 4,364 456 275 222 3,149 262
1.2 2.5 50.9 13.9 33.0 22.2 17.5 7.5 41.6
After Berman, 1951.
Geographically, the areas of known or suspected high prevalence of the disease extend from the West African coast and Central Africa south of the Sahara Desert, along the southeastern and eastern portions of Africa, across eastern and southeastern Asia, including particularly Indonesia, the Philippines, China and Japan (Fig. 1). A. Western Races. Primary carcinoma of the liver is rare among all Western people, irrespective of whether they live in Europe, America, Africa, Asia, or elsewhere. The post-mortem rate averaged 0.14% in Europe, and o.27y0in the United States of America, and the relative frequency of primary liver cancer to all other carcinomata was 1.2% in Europe and 2.5y0in the United States of America (Table I). B. African Races. Primary liver cancer, on the other hand, is extra-
58
CHARLES BERMAN
b
ordinarily common among the Bantu and other indigenous people inhabiting the vast territorial regions of Africa south of the Sahara Desert, including French West Africa, Sierra Leone, Gold Coast, Nigeria, French Equatorial Africa, Belgian Congo, Uganda, Kenya, Tanganyika, Southern Rhodesia, Portuguese East Africa, Bechuanaland, and South Africa (Fig. 2 and Tables I1 and 111). No information is available for Angola and Southwest Africa. According to recorded statistics, the post-mortem rate varied between
59
PRIMARY CARCINOMA OF THE LIVER
FIQ.2. Reported areas of high primary liver cancer frequency in Africa (after Berman, 1957).
TABLE I1 Post-Mortem Incidence of Primary Liver Cancer Among Africans Source City Deep Mine Hospital, Johannesburg, South Africa Non-European Hospital, Johannesburg, South Africa Government Hospital, Salisbury, Southern Rhodesia Research Laboratory, Kenya Mulago Hospital, Kampala, Uganda
Authors
Primary of Liver Autopsies Cancers '0.
Per Cent
Fischer (1929, 1932)
1,963
16
0.8
Strachan (1934); Becker (1954) Gelfand (1949)
6,395
154
2.4
2,000
19
0.9
1,100 3,822
8 51
0.7 1.3
Vint (1935) Muwazi et al. (1942); Davies (1952)
60
CHARLES BERMAN
TAQLE I11 Relative Frequency of Primary Carcinoma of the Liver Compared with Carcinoma of all Organs in African Racesa*L
Observer(s)
Locality
Union of South Africa Pirie (1921); MacFarlane (1924); MacVicar (1925, 1935);Watkins-Pitchford (1925); Beyers (1927); Orenstein (1927); des Ligneris (1927, 1936); Med. Officer of Health, Johannesburg Health Report (1914-33); Rosset (1936); Berman(t) (1951) Portuguese East Africa Gentil (1936) Southern Rhodesia Gelfand (1949) Kenya Vint (1935) Tanganyika Tanganyika Territory Health Report (1925) Uganda Davies (1948) Mouchet and Gerard (1919, Belgian Congo 1926)
French West Africa
Denoix and Schlumberger
French Equatorial Africa, Togoland, and Cameroons
Surmont and Sava (1927); Nogue (1920); Ledentu (1934); Denoix and Schlumberger (1957) Nigeria Annual Medical and Sanitary Report (1927); Smith and Elmes (1934); Elmes and Baldwin (1947); Findlay (1950) Adler and Cummings (1923)
(1957)
Nigeria
Sierra Leone 4
b
Cases of Ctw33 of Primary Carcinoma Carcinoma Per of the Cent of all Organs Liver
2,565 63 65 16
1,336 23 19 8
51 36 29 50
11 85
3 29
27 34
28
15
53
4,077
1,455
35
1,474
257
17
861 5
156 2
18 40
Pathological, autopsy, clinical and official records. After Berman. 1951.
Note(?) : Include 507 primary liver cancer cases in workers from Portuguese East Africa out of the total 670 cases in all African mine workers.
0.770 in Kenya and 2.4% in South Africa; and the relative frequency of in the primary liver cancer to all carcinomata ranged between 17 and different localities (Table 111). In such widely separated areas as Lourenpo Marques in Portuguese East
PRIMARY CARCINOMA OF THE LIVER
61
Africa (Prates, 1940, 1943) and Dakar on the French West African Coast (Payet et al., 1953), primary liver cancer is indeed the most frequent form of internal cancer and occurs more especially in relatively young adult males. In Dakar, e.g., 60 cases (representing 75% of all cancers) are treated annually in the medical section of the Hopital Le Dantec (Payet et al., 1956), while 325 new primary liver cancers were sectioned a t the Dakar Pasteur Institute during the four years ending in 1954 (Camain, 1954). For French West Africa as a whole during the 16-year period 1940-1955, Denoix and Schlumberger (1957) documented 1455 cases of primary liver
il
Sex: Male
w
.-cm 0
5
r
Sex: Female
a0 l 0 m
20
nG .
0 Year of registration
Year of registration
(4 (b) FIQ.3. Annual relative frequencies of primary liver cancer compared with all other cancers in French West Africans. (a) Males. (b) Females. (After Denoix and Schlumberger, 1957.)
cancer, or 35% of all cancers (Table 111): these included 1227 cases in males, and 228 cases in females, among whom primary liver cancer averaged 48.3y0 and 14.7% of all cancers respectively, with annual ranges of 16 to 65% in males (Fig. 3a), and 5 to 18% in females (Fig. 3b). These remarkable statistics from a single geographical region recorded over a relatively short period are in striking contrast with the total 1391 cases of primary liver cancer which Carnahan (1950) was able to compile from the entire world’s literature. In South Africa, among the migratory Bantu laborers of the Witwatersrand gold mines-a specially selected and fit group of young men who hail
62
CHdRLES BERMAN
from various tropical and subtropical regions of Central and Southern Africa-primary liver cancer is also extraordinarily frequent. This tumor was responsible for 670 out of 772 cases (or 87y0) of all carcinomata (Berman, 1951). It is significant that these liver cancers were found to occur almost six times more frequently in Bantu miners from Portuguese East Africa (East Coast natives) than from South Africa (Fig. 4). 35
1
'
East coast
507
cases
163
cases
FIQ.4. Comparison of primary liver cancer incidence in Portuguese East African and South African Bantu employed on the Witwatersrand gold mines (after Berman, 1951).
Among the young British West African native soldiers in Nigeria, similarly, primary liver cancer accounted for almost 70% of deaths from carcinoma during the war years 1940-1945. On the other hand, not one of the deaths from malignant disease in the European troops stationed in British West Africa during the same period was due to liver cancer (Findlay, 1950). C. Oriental Races. There must be some factor which is common for the livers of African and Eastern races but which is absent or less operative in Western races, since the frequency of primary liver cancer is sometimes as high among certain Oriental people as in Africans. A high autopsy rate for primary liver cancer has been recorded among Indonesians (Javanese), Filipinos, Chinese, Indians, and to a lesser extent among Japanese (Table I). Scattered reports of an enhanced incidence of the disease have also come from French Indochina (Degorce, 1913; Bablet, 1932), Korea (Yun, 1949), and from the Chinese island Formosa (or Taiwan) (Usudu and Uchida, 1941; Yeh and Cowdry, 1954).
PRIMARY CARCINOMA OF THE LIVER
63
The post-mortem rate for Asia, according to available statistics, was 0.76y0 and the percentage frequency of primary liver cancer to all other carcinomas was 13.9y0 (Table I); the highest frequency has been recorded in Indonesia (Java), where t.he post-mortem rate among the Javanese was 1.3% (Table I), and the percentage frequency of primary liver cancer to other forms of malignancy was 79.3% for male Javanese, and 6.2% for females (Kouwenaar, 1932). Few cancer statistics are available from China. In Peking, however, Liu (1953) found 19 primary liver cancers among 2424 autopsies, i.e., O.8Y0 of all autopsies and 11.4% of all carcinomata. Moreover, Hou (1955) discovered 170 primary liver cancers among 2400 post-mortem examinations at the Queen Mary Hospital in Hong-Kong, or 7y0 of autopsies and 35% of all malignant tumors. D. Asian Immigrants. Mention must be made of the striking number of primary liver cancers that have been found at autopsy in different parts of the world on immigrants from Oriental countries. These include Chinese immigrants in Singapore (Tull, 1932; Shanmugaratnam, 1956), Indonesia (Snijders and Straub, 1923; Kouwenaar, 1932; Bonne, 1937), Netherlands West Indies (Hartz, 1945), New York (Gustafson, 1937), San Francisco Wilbur et al., 1944), and British Columbia (Strong and Pitts, 1930, 1932; Strong et al., 1949); Japanese and Filipino immigrants in Los Angeles (Edmondson and Steiner, 1954) ; and Japanese, Chinese, and Filipino immigrants in Hawaii (Mamie, 1953; Quisenberry et al., 1954). E. American Negroes. Primary liver cancer appears to be as infrequent in the American Negro as it is in the white population (Gustafson, 1937; Kennaway, 1944; Rosenberg and Ochsner, 1948; Edmondson and Steiner, 1954). Quinland and Cuff (1940) found 4 liver cancers among 300 Negro cases of carcinoma autopsied a t Meharry Medical College in Nashville, Tennessee, a relative frequency of 1.3%. In Chicago, Robinson (1951) saw only one primary liver cancer among 72 cancer cases in Negro males and none among 230 cancers in Negro females. Webb (1945), on the other hand, recorded the higher relative frequency rate of 7.2y0 in Negro autopsies a t Freedman’s Hospital in Washington. At the Los Angeles County Hospital, however, Steiner (1954) found 6072 malignant neoplasms including 83 cases of primary liver cancer of which 63 were in white individuals and 4 were in Negroes; the relative frequencies were 1.2y0 for whites and 1.4y0 for Negroes. Dorn and Cutler (1955) showed similarly that the relative frequency of primary liver cancer to all other cancers was 1.6% in non-whites (predominantly Negroes) and 1.3% in whites of both sexes, although it was slightly higher and occurred somewhat earlier in non-white males, 2.4y0 as against 1.6%.
64
CHARLES BERMAN
2. Primary Liver Cancer in Animals and Birds
Primary liver cancer is not unusual in lower animal life and has been found in a frog (Willis, 1948). Spontaneous liver cancers have been described in domestic animals and birds, including bovines, dogs, ducks, sheep, cats, fowl, horses, pigs, and in a few wild mammals and birds. Berman (1951) tabulated a total of 254 such cases from the literature; these included 120 cases in aged cattle observed in a single year by Trotter (1904, 1905) at a Glasgow abattoir.
3. Limitations of the Available Primary Liver Cancer Statistics The foregoing statistics indicate that primary liver cancer, though rare in Europe and in the United States of America, is by contrast relatively frequent in Africa and certain parts of Asia. The exact frequency of the disease in the different population groups, however, is uncertain for almost all the available statistics in current use are from post-mortem data, and the observations are necessarily expressed as relative frequencies rather than as incidence rates. A. Post-Mortem Datu. Autopsy data concerning primary liver cancer, expressed as percentages of all autopsies or as relative frequencies of all other forms of cancer, have for long been the chief means of comparing the geographic and demographic prevalence of the disease. The main advantage of post-mortem statistics is that they are available even in hospitals serving the more remote and primitive areas. Their use, however, is limited for they cannot provide an absolute basis for judging the true incidence of the disease in different population groups, chiefly because cases coming to autopsy a t individual hospitals cannot be representative of all deaths. Moreover, autopsy statistics for the greater part of Asia and Africa are largely lacking owing to scarcity of medical centers where routine post-mortems are carried out, and also because of religious restrictions or popular prejudice against autopsies. B. Cancer Mortality Statistics. National mortality statistics are unsuitable for estimating primary liver cancer incidence as the disease is not listed separately in the International Classification of Disease, but is included with tumors of the gall bladder and the extrahepatic bile ducts. C. Cancer Incidence Rates. A more valid method for calculating primary liver cancer frequency is through cancer incidence rates which are obtained by assessing the total number of primary liver cancer cases diagnosed during a specified period of time related to the population at risk, and expressed as age- and sex-specific ratios. This method requires an exact statistical knowledge of the population, including its composition by age and sex groups, and the reporting of all malignancies (verified by biopsy
PRIMARY CARCINOMA OF THE LIVER
65
or autopsy) discovered during the period under survey. Thus far, a few localities in the United States of America (Dorn and Cutler, 1955) and Denmark (Clemmesen, 1952) are the only ones where modified studies of this nature have been carried out. Cancer incidence rates for the vast undeveloped African and Asian regions are perhaps the most difficult to obtain, mainly because medical facilities generally are poor and vital statistics rudimentary or absent. In Africa, moreover, few Bantu know their own age with any degree of accuracy. However, preliminary results from the first inquiries in Africa on cancer incidence rates recently held in the neighborhood of Johannesburg and in Uganda have indicated that the rates are in accord with the known postmortem findings, and that the high incidence of primary liver cancer is absolute and is not due to a relative absence of other tumors, as suggested by Gilliam (1954). In the Johannesburg Bantu, for example, primary liver cancer was found to occur 10 times more frequently in males and five times more frequently in females when compared with the expected incidence rates of corresponding white populations in the United States of America and 50 to 100 times those of a Danish population (Oettle and Higginson, 1956); in Uganda the highest incidence rate was obtained in males of the age group 16 t o 45 years, among whom primary liver cancer occurred 30 times more frequently than in the corresponding Danish male population, although in females the differences were only slight (Davies, 1956). No cancer incidence rates are available for Asia.
111. CLINICALMANIFESTATIONS 1. Sex and Aye Incidence Primary liver cancer occurs predominantly in males (Gustafson, 1937; Davies, 1948; Berman, 1951), although there seems to be an enhanced frequency of the disease in female Africans (Fig. 3b) when compared with those of other races. Among Africans (Berman, 1951; Payet et al., 1953) and Indonesians (Kouwenaar, 1932; Bonne, 1935), the disease is preeminently one of youth, occurring mainly under the age of 40 and very often before the age of 20 or 30 years; in other races it is commonest at or after middle life, and is rare before the age of 40 (Yamagiwa, 1911; Dorn, 1956). No age, however, is exempt for the tumor has been discovered in children of all ages; Wilbur et al. (1944) reported a case in n 3-day-ultl infant. Steiner (1938), Tomlinson and Wolff (1942), Bigelow and Wright (1953), and others a t different times have tabulated and reviewed almost 100 reports of primary liver cancers in children that had been recorded in the literature.
66
CHARLES BERMAN
2. Mode of Onset
The disease makes steady silent progress over an indefinite period and the patient is often unaware of the ailment until the malignant liver has reached alarming proportions. The prodromal symptoms are vague and indefinite and are usually attributed to gastric disturbances, including lack of appetite, nausea, vomiting, constipation, diarrhea, abdominal pressure, and a sense of fullness in the upper abdomen after meals.
3. Clinical Classification From a study of 75 Bantu cases of primary liver cancer and from an extensive review of t,he literature, Berman (1951, 1956) found that the clinical course is by no means uniform. Symptomatology is dependent upon: (a) the degree of associated cirrhosis, (b) the rate of tumor growth, (c) complications, and (d) the development of metastases. Accordingly, Berman has classified primary liver cancer into five clinical groups (one typical, four atypical) as follows. Group I: “frank” or typical cancer (63Y0 of cases), where the symptoms indicate primary liver involvement from the outset; Group 11: “acute abdominal” cancer (BYoof cases), where the disease is ushered in with dramatic suddenness as an acute abdominal catastrophe resulting from rupture of a necrotic nodule or erosion of a blood vessel on the free surface of the liver; Group 111: “febrile” cancer (8% of cases), where the disease may reveal itself equally abruptly as a rapidly growing tender liver accompanied by fever and may simulate an amebic liver abscess; Group I V : “occult” cancer (16Y0 of cases), where the liver tumor may remain latent until discovered accidentally or only a t autopsy; Group V : “metastatic” cancer (5% of cases), where symptoms due to extrahepatic metastases in remote organs or parts of the body are the first to direct attention to a n otherwise symptomless primary tumor in the liver, e.g., paraplegia due to secondary growths in vertebrae, pathological fractures, etc. 4. Symptomatology (Table IV)
In established primary liver cancer the more constant symptoms are progressive muscular weakness, loss of weight, emaciation, and abdominal pains of dull aching character localized to the right hypochondrium and unrelated to food. The liver, more especially the right lobe, is almost always enlarged, often to a remarkable degree. The surface is nodular and tender, and its firm lower outline may be visible as well as palpable and extend to the level of the umbilicus or even lower; upward enlargement into the thorax is less marked.
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PRIMARY CARCINOMA O F THE LIVER
TABLE IV Percentage Frequency of Symptoms and Signs in Primary Liver Cancers
Symptoms and Signs
Asthenia Abdominal pain Dyspnea Loss of Weight Enlarged liver Tender liver Jaundice Ascites Dilated abdominal veins Edema of legs Anemia Fever 0
31 Cases at Cases the Mayo 75 Bantu 134 Chinese 37 Cases at Reported Clinic (Hoyne Cases Cases Cincinnati in the and (Berman, (Tull, 1932) (Warvi, 1945) Literature Kernohan, 1951) (Warvi, 1945) 1947) 89 9
68 15 34 47 52 83 38 28
94 71 94 91 15 80 37
88 52 88 68 17 34 46
87 87 71 58 58 77 26
86 90 25 83 100 90 45 55 19
40 97 52
60 68 34
74 42 39
30 100 38
After Warvi, 1954.
Jaundice develops in approximately half the cases, as also ascites with straw-colored or blood-stained fluid. Secondary anemia is almost invariably present. Signs pointing to portal hypertension, including dilatation of the superficial abdominal veins and edema of the lower extremities, occur in approximately one-third of the cases, and hematemesis from rupture of esophageal or gastric varices which may terminate fatally. The temperature is normal or moderately elevated and the pulse rate may be raised even in cases without fever.
5. Diagnosis of Primary Liver Cancer Through Laboratory and Other A i d s On account of its rarity, primary carcinoma of the liver is seldom diagnosed clinically in Europe and America. In Africa and in the Orient, on the other hand, the disease occurs with such great frequency that clinicians readily accept it as a definite entity and one, moreover, which can be recognized with a certain degree of confidence. A. Liver Function Tests. It would appear that the many liver function tests available today are useful only insofar as they indicate the presence of general diffuse liver damage (Table V). They are not capable of pointing t o the actual cause of the disease (Galuzzi et al., 1953). Neither is there any single test which is diagnostic for primary liver cancer. Research is necessary, therefore, to discover liver function tests which would make
68
CHARLES DERMAN
TABLE V Results of Liver Function Tests in 15 Bantu Cases of Primary Liver Cancer" Test Thymol turbidity Thymol flocculation Colloidal red Cephalin cholesterol flocculation (24-hour reading) Takata Ara reaction (Ucko's modification) Zinc sulphate turbidity Serum alkaline phosphatase Van den Bergh reaction
Normal Range 0-2 units
negative negative negat,ive
15 15
13
9 9
9 9 9
negative
15
15
12.5 units 4-13 King Arm-
8 15
8 8
strong units negative
15
12
15
3
8 8
8
Total bilirubin 0-1.2 mg.% Albumin-globulin ratio 1.3-1.5 Gamma globulin 0.8-1.25 gm.% 0
No. of No. with Patients Abnormal Tested Results
8
Range of Abnormal Results 3.5-14.5 units 14 3-5 1-4
+ + +
1-3
+
27.8-74 units 14.1-45.2 King
Armstrong units delayed, weak or prompt direct; and indirect reactions 1.5-2.1 mg% 0.4-0.9 3.4-5.6 gm.%
After Berman, 1966.
it possible to differentiate specifically not only between primary and secondary liver cancers, but also between these neoplasms and hepatic cirrhosis or other forms of liver damage. B. Liver Aspiration Biopsy. Liver puncture biopsy by means of the apparatus designed by Silverman (1938), Iversen and Rohlm (1939), or Gillman and Gillman (1945) is invaluable in establishing the diagnosis of many obscure hepatic enlargements, including primary liver cancer. The procedure, however, is not entirely without risk, for fatal intraperitoneal hemorrhage (Terry, 1952) and biliary peritonitis (Gallison and Skinner, 1950) are among the occasional complications. C. Peritoneoscopy. Peritoneoscopy introduced by Ruddock (1937) for visualizing the peritoneal cavity and its contents has proved useful in the diagnosis of primary liver cancer, especially in conjunction with liver biopsy through the peritoneoscope (Beling, 1941; Narancio et al., 1945). D. Duodenal Aspiration. By using the Papanicolaou (1946) cytology technique, exfoliated neoplastic cells can be demonstrated in the aspirated duodenal contents of patients with carcinoma of the liver, the bile ducts, or the pancreas (Lemon and Byrnes, 1949). E. Roentgenological Diagnosis. In a liver distorted by cancer, roent-
PRIMARY CARCINOMA O F THE LIVER
69
genography may help the diagnosis by demonstrating the shape and size of the liver, elevation of the diaphragm, and restriction of its movements. Pulmonary or other metastases may also be discovered. Roentgenography after a barium meal may reveal esophageal varices secondary to the associated cirrhosis (Schatzki, 1941) and displacement or distortion of the stomach and the duodenum by the liver tumor. a. Portal-Splenic Venography. By injection of radio-opaque Diodone percutaneously into the spleen followed by immediate roentgenography of the liver, Bergstrand and Ekman (1955) were able to diagnose primary liver tumors in patients wiOh enlarged livers. The portal vein and its branches were clearly outlined in the unaffected portions of the liver, whereas in the cancerous areas they were either distorted or devoid of the contrast medium. b. Abdominal Aortography. The injection of contrast media (e.g., Urokon) into the aorta through the back at the level of the 11th thoracic vertebra followed by rapid roentgenography demonstrated primary and secondary liver cancers as areas of diminished vascular density (Milanes et al., 1953; Rigler and Olfelt, 1954). c. Radioactive Materials. Stirrett et al. (1953) were able to signify the presence of metastatic liver cancers by the intravenous injection of radioactive iodinated human serum albumin which appears to have a special affinity for the tumor cells. Application to the liver region of a scintillation counter 24 hours after the injection demonstrated abnormal radioactivity in the cancerous areas which could be outlined accurately in this way. Research on similar lines is necessary to determine whether this procedure, which is claimed to be safe and reliable, could be adopted also in the diagnosis of primary liver cancer. 6. Suggested Treatment
A. Chemotherapy. There seem to be no chemotherapeutic agents beneficial for the treatment of primary liver cancer. Owing to the hopelessness of the disease, medical treatment is essentially palliative and is directed toward the relief of pain and discomfort. Nitrogen mustard (NH2) has been tried by Pack and Miller (1953) for the control of diffuse and inoperable primary liver cancers and the more radioresistant metastatic lesions, but the results have been questionable. The blood supply of liver tumors is primarily arterial (Breedis and Young, 1954). Nitrogen mustard is injected, therefore, either directly into the hepatic artery a t the time of laparotomy or by means of a catheter introduced into the celiac axis through the external carotid artery and the aorta. This procedure is not without danger, as it may be followed by thrombosis of the hepatic artery and hemorrhage from trauma a t the time of catheter-
70
CHARLES BERMAN
ization. Selective chemotherapeutic agents which could be administered without harm to the patient should be developed. B. Radiotherapy. It is possible that radiotherapy may prove of value in treating early stages of the disease. But usually by the time the patient presents himself the condition seems so far advanced that it is apparently beyond cure; indeed, palliative irradiation at this stage appears to produce added discomfort and hasten deterioration. More information on the effects of roentgentherapy in primary liver cancer is necessary for, contrary to general belief, liver parenchyma seems to tolerate large doses of radiation, and primary as well as secondary liver tumors appear to regress rapidly, although this effect is only temporary (Cohen et al., 1954; Phillips et al., 1954; Ariel, 1956). C. Radioactive Isotopes. The use of radioisotopes in the treatment of primary liver cancer has been suggested by the irradiating action in experimental animals of labeled anhydrous chromic phosphate which is taken up largely by the reticulo-endothelial system including that of the liver (Jones el al., 1944). This compound gave temporary benefit to a patient with melanosarcoma and extensive metastases in the liver (Low-Beer et al., 1942). Stirret et al. (1953) have shown that radioactive iodinated human serum albumin appears to have a specific affinity for the tumor cells in the liver when injected intravenously into patients with metastatic liver cancers. Investigations on the therapeutic effects of radioisotopes in primary liver cancer are highly desirable, and these could be advantageously carried out at hospital centers in Africa. D. Surgery. Analyses of the case reports in the world’s literature (Berman, 1951; Hauch and Lichstein, 1954) have indicated that operation results in primary liver cancer are generally discouraging. As a rule, the tumor recurs, and life is prolonged in exceptional cases only. In the series of cases recorded by Berman (1951) all attempts at radical cure had to be abandoned as metastases in other parts of the liver were already present at the time of operation. Surgical treatment must be considered where the tumor is found to be solitary, small, and circumscribed, and particularly if it affects the left lobe of the liver. The tumor is then excised by partial lobectomy, due care being taken to control hemorrhage which can be alarming. Removal of the more frequently involved right lobe of the liver was regarded until recently as too hazardous an undertaking. This attitude is changing, for, thanks to current advances in surgical and anesthetic techniques, a number of total right lobectomies for primary liver cancers and other liver tumors have already been successfully carried out (Quattlebaum, 1953; Pack and Baker, 1953; Schottenfeld, 1955; Brunschwig, 1955). Considerable experience will be required before the exact place of
PRIMARY CARCINOMA OF THE LIVER
71
radical surgery in primary liver cancer can be determined, and this could more readily be obtained in the large African hospitals serving those primitive native populations known to be prone to this form of cancer.
IV. MORBIDANATOMY 1. Gross Pathology: Macroscopic Classification
For practical and descriptive purposes, primary liver cancer can be divided into two macroscopic groups : “nodular” carcinoma, a tumor formed by numerous discrete nodules of varying size, and the less frequent “massive” carcinoma, consisting mainly of a single large dense mass located in one lobe of the liver, usually the right lobe. A sharp demarcation between the two groups is scarcely possible. Many tumors originally “nodular” in character tend to transform into “massive” cancers (the so-called L‘pseudo-tumoral” form of Payet et al., 1953); and conversely, some “massive” cancers become surrounded by secondary smaller discrete tumor foci and present the appearance of “nodular” cancers. A. Nodular Carcinoma. The liver is large, hard, scarred, deformed, and studded with closely grouped or scattered irregular nodular masses more often involving the right lobe than the left. On section, much of the liver substance is seen to be replaced by firm, scattered, or closely grouped nodules which are mostly white to gray in color, although they may also be reddish or soft and friable when they are found occupying portal or hepatic veins as tumor emboli. The intervening liver tissue usually is the site of well marked fibrosis. B. Massive Carcinoma. The liver is enlarged and distorted by a uniform, smooth swelling with occasional secondary irregularities in its vicinity. On section, the tumor mass may be found to be occupying practically the whole of the right lobe to which it is confined, or there may be additional smaller growths localized especially a t the periphery. Depending upon secondary changes, the consistency of the tumor varies greatly: it may be soft, cystic, hemorrhagic, honeycombed, or sometimes cartilaginous, and the tumor masses may be white, gray, yellow, red, or opaque. The portal or hepatic radicles may contain tumor thrombi which, as in nodular cancer, may extend as a cancerous thrombosis into the vena cava and the right auricle. A varying degree of cirrhosis is also almost always present. 2. HISTOPATHOLOGY
Primary cancer of the liver occurs in two forms, “hepatocellular” carcinoma and “cholangiocellular” carcinoma. It is generally assumed, but not positively proved, that hepatocellular cancer originates from the cells
72
CHARLES BERMAN
of the liver lobules and cholangiocellular cancer from the cells of the intrahepatic bile ducts. A. Hepatocellular Carcinmna.* In this, the most frequent form of primary liver cancer, the microscopic structure is characteristic. a. The Tumor Cells. The tumor cells are usually larger than normal liver cells which they resemble; they are polygonal in shape with distinct borders, and when stained with hemat.oxylin and eosin, they show an abundant eosinophilic or moderate basophilic granular cytoplasm. The bizarre nuclei reach extraordinary dimensions and frequently occupy almost the entire cell; they are oval to spherical or irregular in shape, and are markedly hyperchromatic with sharply defined membranes. They are usually single, but some cells may have two or more such nuclei. The nuclei usually contain a large, single, purple-staining nucleolus which is either centrally or eccentrically situated. Giant nucleoli are a prominent feature. Multiple nucleoli are occasionally present. Mitotic figures in diverse phases are frequent. Giant cells containing either a single large, dark nucleus or many small nuclei are often observed. These giant cells sometimes dominate the microscopic field, although more usually they are scattered in a haphazard manner throughout the tumor. b. Cellular Arrangement. The malignant cells are characteristically arranged as compact trabeculae, varying from 2 to 30 cells in thickness and devoid of intercellular stroma. These cell columns either anastomose with each other, or are arranged as long parallel columns which terminate freely by means of blunt, rounded extremities. Their margins are bordered by a single layer of endothelium resembling Kupffer cells which adhere closely to the peripheral tumor cells forming, in this way, a pronounced vascular stroma containing reticulum. Numerous vascular spaces running between adjacent cancer columns are thus formed, and they vary in width from mere slitlike crevices to markedly dilated sinuses, many of which are engorged with blood. Bile pigment is often seen in the tumor cells and in the vascular spaces. In the more anaplastic tumors, however, this trabecular arrangement is less distinct or even absent. c. Intrahepatic Metastases in the Cirrhotic Tracts. Bands of fibrous tissue of varying widths are almost always found traversing the normal tissue which, in fact, they frequently replace. Here numerous newly formed bile ducts are to be seen, as well as many poorly formed blood vessels of varying sizes. The latter are often crowded together and engorged with blood containing free tumor cells, small tumor emboli, or organizing nodules. These emboli are the main sources of metastases.
* Synonyms: ‘‘malignant hepatoma,” “liver-cell carcinoma,” “carcinoma simplex,” “carcinoma solidum,” “trabecular carcinoma.”
P R I M l R Y C..ZRCINOMA O F THE LIVER
73
The tumor also grows by direct infiltration of the liver and through hematogenous metastasis of the sinusoidal channels. Fatty and scirrhous changes are often encountered in the tumor cells. d. Necrosis of the Cancer and Hemorrhage. Regressive changes in the tumor cells appear with almost unerring regularity, and, whereas they are usual in columns composed of many cell layers, they are absent in thin cords. The earliest effects are upon the most centrally situated cells, and as the necrosis spreads the advance occurs in a centrifugal manner, until finally the whole thickness of the cancer column becomes involved. A constant sequel to these necrotic changes is hemorrhage, when lakes of blood of varying sizes are frequently seen. This is important clinically, for ruptured superficial cancer nodules account for hemorrhagic peritoneal effusions and for the large quantities of blood in the peritoneal cavities of rapidly fatal cases. B. Cholangiocellular Carcinoma.* Cholangiocellular carcinoma occurs far less frequently than hepatocellular cancer. It is characterized by a glandular structure and an alveolar pattern of arrangement. a. The Tumor Cells. The cells are columnar to cuboidal in shape and show some resemblance to bile-duct epithelium. The cytoplasm is nongranular and faintly basophilic. Their prominent nuclei are round, oval, or spindle-shaped, are proximally situated, and a t times extend along the whole axis of the cell. They are particularly hyperchromatic, contain a fine granular network, stain intensely with hematoxylin, and are confined by a distinct nuclear membrane. The majority of cells contain a single nucleus, but occasionally double nuclei are present. Each nucleus has one or more prominent, round, dark-staining nucleoli. Mitotic figures are fairly frequent, but giant cells are rare. b. Cellular Arrangement. As a rule, the cells are arranged into alveoli varying in shapes and sizes, a characteristic which distinguishes them from the solid trabecular formation of the hepatocellular tumors. The usual histological picture is that of irregular tubules or multiloculated cystlike spaces formed by one or several layers of cuboidal cells which lie in a moderate or pronounced fibrous tissue matrix containing rudimentary blood sinuses and lymphocytes. On the other hand, they may be separated from each other by open spaces formed by a peripheral endothelial investment. The lumina of the tubules and the cystlike spaces may be clear or may contain degenerating tumor cells and a homogeneous pink-staining amorphous material or mucus. Bile is absent. As the result of inward growth of the lining cells, the lumina may be further subdivided and may become obliterated. It is on this account that solid columns of tumor cells are often encountered side by side with typical
* Synonyms: bile-duct carcinoma, alveolar carcinoma, adeno-carcinoma.
74
CHARLES BERMAN
glandular tumors or with elongated cords lying in a dense fibrous tissue matrix. The cells proliferating into the cystic spaces, however, may assume a pronounced papilliferous structure, and this, indeed, may become the salient feature of the tumor. The remaining liver is usually the seat of well marked cirrhosis. 3. Metastases
Primary liver cancer possesses vigorous metastatic powers and dispersal may often be widespread. A. Intrahepatic Metastases. Metastases in the liver commence at an early stage when the tumor characteristically penetrates the wall of a portal vein. This becomes occupied by proliferating groups of tumor cells which grow steadily until the whole vessel and its branches are occluded by a continuous organizing thrombus. With the invasion of the portal system, dissemination to other parts of the liver readily occurs. In this way, a single primary focus may give rise to widespread metastases in different parts of the liver. B. Extrahepatic Metastases. Secondary deposits outside the liver occur relatively frequently. They are often multiple and may occur at a stage when the primary growth is still symptomless. According to different observers, extrahepatic metastases are to be found in over 50% of cases, e.g., Eggel (1901), 66%; Herxheimer (1930), 58y0; Wilbur et al. (1944), 53%; Hoyne and Kernohan (1947), 68%; Berman (1951), 60%; Kohn (1955), 75%; Edmondson and Steiner (1954), recorded an incidence of 47% in 100 cases. Extrahepatic metast,asis begins with the invasion of the hepatic veins. Dislodged tumor cells or cancerous thrombi reach the inferior vena cava (which itself often escapes infiltration) and are conveyed via the heart to the terminal arterioles in the lungs. Here they proliferate, invade the surrounding lung tissue, and by penetrating the pulmonary veins become the source of systemic dissemination, although in exceptional cases the tumor cells pass directly into the general circulation without involving the lungs. It is for this reason that the lungs are the most common site for metastases. To a lesser extent, metastatic spread occurs also by the lymphatic vessels. There is a close resemblance in histological structure between the metastases and the parent hepatocellular and cholangiocellular liver tumors. In their new environment they may become highly organized and may even show evidence of unmistakable bile production in the lungs, in skeletal structures, and in other distant parts of the body A comprehensive survey of the literature (Berman, 1951) has shown that metastases from liver cancer may be located in most organs or parts of the body, buto more particularly in the lungs and the regional lymph glands. To a lesser
PRIMARY CARCINOMA OF THE LIVER
75
extent, metastases occur in the heart, brain, adrenal gland, vertebrae, skull, and other osseous structures.
4. Histogenesis A. Hepatocellular and Cholangiocellular Cancer. Primary liver cancer is said to originate either from the polygonal liver cells (“hepatocellular” carcinoma) or from the cuboidal to columnar epithelium of the intrahepatic bile-ducts (“cholangiocellular” carcinoma). All observers are agreed that hepatocellular cancer is more common than cholangiocellular cancer, but there is a remarkable divergence of opinion as to their relative frequency. In Europe, Eggel (1901) found the proportion approximately 2 : l ; Herxheimer (1930), 3 : l ; Kohn (1955), 5 : l ; in the United States of America, Smith (1933), 3 : l ; Wilbur el al. (1944), 1 1 : l ; H o p e and Kernohan (1947), 2 : 1 ; Edmondson and Steiner (1954), 4 : 1 ; in Asia, Snijders and Straub (1922), 19 : 1 among Javanese; Tull (1932), 4 : 1 among Chinese; Yamagiwa (1911), 1.5:1 among Japanese; in Africa, Pirie (1921), 9 : l ; Berman (1951), 1 4 : l among South African Bantu; Prates (1940), 85:O among Portuguese East African Bantu; Davies (1948), 2 : l in Uganda; and Payet et al. (1956), 34: 1 in French West Africa. Differentiation between hepatocellular and cholangiocellular cancer has offered many difficulties. The criteria employed have been based upon (a) the property of hepatocelular cancer to imitate the solid trabecular architecture of liver tissue, and the property of cholangiocellular cancer to mimic the alveolar formation of bile ducts; also, the resemblance of the tumor cells t o the cells of origin, i.e., polygonal cells in hepatocellular cancer and cuboidal or cylindrical cells in cholangiocellular cancer (Eggel, 1901); (b) the presence of bile pigment in the tumor cells (Ribbert, 1909) or in the metastases (Loehlein, 1908) of hepatocellular cancer and its absence in cholangiocellular cancer; (c) the delicate capillary stroma in hepatocellular carcinoma in contrast to a relatively abundant fibrous stroma in cholangiocellular cancer (Wegelin, 1905; Yamagiwa, 1911 ; Adelheim, 1913; Firminger, 1955); (d) the presence of mucus in cholangiocellular carcinoma and its absence in hepatocellular cancer (Firminger, 1955). B. Vesicular Cancer. With these criteria as a basis, most primary liver cancers can be divided into one or other of the above groups. The tumors which occasion difficulties in classification include those cancers with well defined trabeculae which tend to form acinar spaces containing bile or other material, producing in this way the appearance of glands or ducts. Such tumors have been referred to as a “vesicular” form of hepatocellular carcinoma (Payet et al., 1956), although other observers regard these as “adeno-carcinoma,” or cholangiocellular carcinoma.
76
CHARLES BERMAN
C. Anaplastic Cancer. Again, there are those rapidly growing, undifferentiated liver cancers composed of small, or large, and often irregular cells with numerous mitotic figures and frequent giant cells. These tumors have no definite architectural pattern but are grouped together into irregular broad sheets. Since they have neither the trabecular and sinusoidal characteristics of hepatocellular cancer nor the acinar structure of cholangiocellular cancer, they do not fall into either classification and are often referred to as “anaplastic” cancers. D. Concepts of Histogenesis. The histogenesis of primary liver cancer has not been clarified. Ewing (1940) and others claim that hepatocellular cancer originates from the hypertrophic liver cords (so often seen in cirrhosis) which are stated to proceed to nodular hyperplasia, adenoma, and finally carcinoma. Cholangiocellular carcinoma is thought to begin as a proliferating “cholangiocholitis” in a cirrhotic liver (Milne, 1909; Yamagiwa, 1911) or as “cholangiofibrosis” (Opie, 1944b; Firminger, 1955), and is succeeded by adenoma of the bile ducts to end as adenocarcinoma. On the other hand, Stewart and Snell (1956), from their extensive experience with experimentally induced liver cancers, are not convinced that the origin of any liver cancer can be traced to a single cell or groups of cells. E. “Cholangiohepatoma,” “Hepatobiliary,” OT “Mixed” Liver Cancer. Tumor tissue, however, has a wide range of reactivity, and it is not entirely unexpected that human as well as experimentally induced primary liver cancer can exhibit a variety of morphological patterns in different parts of the same tumor. Thus, in one part of a liver cancer the predominant pattern may be that of characteristic hepatoma whereas in another the cells may adopt a tubular pattern. Consequently, even within the same histological section, all gradations between these two divergent patterns may be detected. Such tumors have been regarded by some observers including Pirie (1921), Gustafson (1937), Feasby (1945), Willis (1948), Allen and Lisa (1949), Lemmer (1950), Schupbach and Chappell (1952) as “mixed,” “combined,” “intermediate,” or “duplex” tumors; as “cholangiohepatomas” by Warvi (1945) ; and as “hepatobiliary carcinoma” by Edmonson and Steiner (1954), thereby implying a dual origin. The histogenesis of primary liver cancer, on this account, has become the subject of controversy. Fischer (1903) believed that all primary cancers of the liver arise from bile-duct epithelium, even where the tumor cells have the features of liver cells. Kinosita (1937, 1940), in his experimental studies of early lesions, on the other hand, maintains that all rat-liver tumors induced by the azo-compounds are derived from hepatic cells, regardless of whether a tumor develops into a typical hepatic-cell carcinoma or a cholangioma. This latter opinion has been supported by Edwards and White (1941) who, in their experimentally induced liver tumors, demon-
PItIMARY CARCINOMA OF THE LIVER
77
strated ductlike adenomatous growths which were predominantly hepatocellular in type. F. Embryology and Histogenesis. Rigid classification into hepatocellular and cholangiocellular cancer, therefore, can be contemplated only for those tumors possessing a uniform morphology and not for the “mixed” or so-called “hepatobiliary” cancers. Either the liver cells, even in carcinoma, possess a wide range of reactivity, or a dual origin must be postulated to reconcile the transitions in structure of the “mixed” tumors. Strongly favoring the former possibility are the embryological studies of Horstmann (1939) who showed that intrahepatic bile duct epithelium arises as a direct transformation from primitive liver cells. Horstmann also stressed the importance of the presence of mesenchymatous tissue for this transformation. In this connection, the observations of Doljanski and Roulet (1933) are significant: they noted that when pure chicken embryo cultures of epithelial cells and fibroblasts are grown together the resulting epithelial growths tend to assume a .tubular formation. Since liver cells and the smaller intrahepatic bile ducts may originate from the same embryonal source, it is reasonable to assume that a single source may also provide those cell replacements in the fully formed liver which are necessitated by physiologic or pathologic activities within the organ. The various cell elements in the liver may in this way preserve their inherent tendencies to metamorphose, so that regenerating liver cells, especially if they are in contact with abundant connective tissue, may on occasion become transformed into the low cuboidal cells of lining ducts. This often occurs in those forms of cirrhosis commonly associated with increase in the small bile ducts, the so-called “pseudo-bile canaliculi” (Hanot, 1896; Rao, 1935). I n the postcirrhotic regeneration of the hepatic parenchyma, likewise, some observers favor the origin of hepatic cells from bile ducts (MacCallum, 1902; Herxheimer, 1930). If such a metamorphosis can occur in one type of liver disease, then it is not entirely unexpected that a similar reaction may also become manifest in carcinoma. Theoretically, a cancer may arise from the epithelium of the liver or from that of the intrahepatic ducts. Such a tumor may exhibit a morphology similar t.0 the tissue from which it originated, or it may proceed t o differentiatein such a manner that it resembles either liver parenchyma or the parenchyma of its duct system. I n cancers with both types of morphology in the same tumor, the precise derivation may be impossible to determine. Kohn (1955), in his recent monograph, therefore, recognizes only one histological form of human liver cancer, namely, hepatocellular cancer. He has discarded the cholangiocellular form on the assumption that all liver
78
CHARLES BERMAN
cancers stem from a common pluripotential and undifferentiated liver cell ancestor which, however, has the power to differentiate into (1) “mature” hepatocellular cancers, (2) “immature” anaplastic cancers, (3) more tubular forms of liver cancers containing fibrous stroma, or (4)mixtures of all these varieties. The presence of “mixed” forms of liver tumors in experimental animals has also occasioned much controversy concerning their origin and histological classification. Price et al. (1953) have likewise concluded that the accepted classification of induced liver cancers is arbitrary on the grounds that the “mixed” tumors show a gradual transition from one type of structure to the other, and that all tumor types (whether classified as hepatoma or cholangioma) appear to have a common origin in areas of cholangiofibrosis. From these considerations, it is evident that while the classification of primary liver cancer into hepatocellular and cholangiocellular carcinoma is useful for descriptive purposes, this division cannot be taken to mean that because a tumor assumes a particular pattern, it is necessarily derived from the tissue which it simulates. More information on histogenesis is necessary, and this could perhaps be best supplied by experimental embryology and pathology. G. Multicentric Versus Unicentric Theories of Origin. The initial mode of origin of primary liver cancer has been the cause of much debate and has not yet been settled. Two views have been formulated: one claims that the tumor is multicentric, the other that it is unifocal in origin. According to the supporters of the multicentric theory of origin, simultaneous or successive independent tumor foci appear within the liver. Van Heukelom (1894) described what he thought to be transitions of liver cells to cancer cells at the periphery of numerous nodules, which nodules he claimed arose from preexisting liver cells in the area affected. Cholangiocellular cancer, according to Yamagiwa (1911), arises as the result of inflammatory hyperplasia of bile ducts. Van Heukelom and Yamagiwa concluded that primary cancer of the liver is multicentric in origin, in that there is progressive metamorphosis of liver or bile duct cell epithelium to tumor cells at the periphery of the various growths. These conclusions have been adopted by Loehlein (1907), Muir (1908), Milne (1909), Goldzieher and Von Bokay (1911), McIndoe and Counsellor (1926), and others. The experimentally induced liver tumors in rats with azo-compounds are held by many to be multicentric in origin. The theory of multicentric origin has been vigorously opposed by Ribbert (1909) who, on the contrary, held the view that there is only one primary focus within the liver and that the rest of the tumors are secondary; they are due to spreading within the liver from the original focus either as
PRIMARY CARCISOMA O F THE LIVER
79
a single, continuous tumor thrombus of a large portal vein, or by multiple metastases via the entire portal system. Ribbert showed that once the portal system became involved, venous spread to distant portions of the liver was the rule. He maintained that most tumor nodules in the liver are sections of tumor cords which represent the portal vein dilated and filled with tumor thrombi. Ribbert’s view has been supported by Winternitz (1916), Snijders and Straub (1922), Wilbur et al. (1944), Berman (1951), and Steiner (1956). Willis (1948) was of the opinion that both modes of origin may occur. V. ETIOLOGY: ENVIRONMENTAL FACTORS The true cause of primary liver cancer, like that of cancer in general, is unknown. From experimental and clinical evidence, however, it is becoming increasingly apparent that environmental factors play a vital role in the etiology. 1. Induced Liver Cancer in Experimental Animals
A. T h e Azo-Compounds. Few fields of experimental cancer research have yielded more striking results than the production of primary liver cancers in laboratory animals with compounds of known composition which, moreover, are specifically carcinogenic for the liver. Outstanding examples of these are the azo-compounds, o-aminoazotoluene (Sasaki and Yoshida, 1935) and p-dimethylaminoazobenzene or “butter-yellow” (Kinosita, 1940), the essential effects of which are destructive changes in the liver parenchyma followed by nodular cirrhosis of the Laennec type and areas of cholangiofibrosis, progressive hyperplasia of the regenerating liver and bile-duct cells, leading ultimately to cancer (Orr, 1940; Opie, 1944b). Histological appearances of the induced liver cancers are strikingly similar to the human counterpart insofar as cellular structure and architectural arrangement into hepatocellular and cholangiocellular cancer are concerned, except that in induced liver cancers there is a greater incidence of ‘Lmixed’’(or “hepatobiliary”) cancers. Metastases occur relatively frequently, particularly into the lungs and the mesentery. The hepatomata and cholangiomata experimentally induced in rats and mice are easily transplantable within the same species, and their production can be modified by diet. A low protein, high fat or rice diet, or one deficient in riboflavin, methionine, or cystine, increases the incidence of the tumor, whereas a high protein diet or one containing vitamin B-complex or yeast has the opposite effect (Opie, 1944a). It is interesting to note in this connection that factors which modify the incidence of experimental liver cancer have the same effects in experimental nutritional cirrhosis.
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Cancer development, however, is not necessarily connected with the seriousness of the existing liver cirrhosis (Warren and Drake, 1951), and, although cirrhosis usually precedes the liver tumor (Orr, 1940; Sugiura and Rhoads, 1942), it is not necessarily a precursor of the lesion (Maruya, 1939; Miller et al., 1941; Opie, 1944a). Nevertheless, all are agreed that the liver tumors appear earlier in rats fed on a cirrhosis-producing diet. Sex hormones seem to influence the development of experimental liver tumors; androgen stimulates, whereas estrogen appears to inhibit, carcinogenesis. In spontaneous mouse liver tumors and in rat liver tumors induced with dimethylaminoazobenzene or with aminofluorene, the incidence is much higher in males, but with castration is lowered to approximately the incidence level in females (Gorer, 1940; Andervont, 1950; Cantarow, 1956). The subject of carcinogenesis with aminoazo compounds, including pathogenesis, metabolism, carcinogenicity mechanism, and the effects of diet on tumor formation, has been comprehensively dealt with in a recent review by Miller and Miller (1953). B. Other Carcinogenic Compounds. The following are among the other hepatotoxic agents which have been used to induce hepatic tumors in experimental animals : (a) the carcinogens dibenzcarbazole (Boyland and Brues, 1937) , dibenzanthracene (Andervont and Lorenz, 1937), benzanthracene (White and Eschenbrenner, 1945), and methylcholanthrene (Strong, 1944) ; (b) acetylaminofluorene and its related compound 2-aminofluorene (Wilson et al., 1941; Bielschowsky, 1944; Cantarow et al., 1946; Harris, 1947; Kirby, 1947), although these compounds are not specific for the liver since they also produce simultaneous tumors in the bladder, the renal pelvis, the pancreas, and the lungs; (c) selenium (Nelson et al., 1943) ; (d) carbon tetrachloride (Edwards and Dalton, 1942) ; (e) ethyl urethane (Jaffe, 1947); (f) tannic acid (Korpassy and Mosonyi, 1950); and (g) senecio alkaloids (Cook et al., 1950; Schoental et al., 1954); Campbell (1956) induced benign and malignant liver tumors in fowls by the addition of senecio alkaloids to their diets and by their introduction into the intravenous route. 2. Carcinogenic Factors from Human Livers, Tissues, and Excretions The discovery that the carcinogenic hydrocarbon, methylcholanthrene, is chemically related to naturally occurring biological products, e.g., bile acids and sterols, has given rise t o t,he speculation that carcinogenic compounds may actually be formed in the human body. Investigations for such compounds in cancerous and noncancerous human livers have resulted in the isolation of nonsaponifiable lipid extracts which, when tested in the subcutaneous tissues of mice, yielded malignant
PRTM.4RT C.\RCIYOM.\
OF THE
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tumors a t the site of injection (Shabatl, 1937; des Ligneris, 1940; Hieger, 1940; Steiner, 1943) as well as in dist,ant organs including the liver, mammary gland, stomach, pancreas, and lungs (Kleinenberg et al., 1940; Sannie et al., 1941). Similar tumors a t the site of injection have also been obtained with bile (Neufach and Shabad, 1938), urine (Steele et al., 1941), and with extracts from lungs, kidneys, and other tissues obtained from cancerous as well as from noncancerous subjects (Kleinenberg et al., 1941; Hieger, 1946). Although des Ligneris (1940) found carcinogenic factors only in the livers of Negroes (Bantu) and none in those from Europeans (whites) in South Africa, Steiner et al. (1947) did not observe these racial differences in the United States. The carcinogenic factor of the iionsaponifiable fractions, according to Hieger (1946), is a crystalline material of which 85% consists of cholesterol and possibly other closely related steroids which have not been identified. It is interesting to note in this connection that Hieger (1949) and Hieger and Orr (1954) were able to induce sarcomata in mice at the site of injection by means of commercial cholesterol. 3. Primary Liver Cancer in Humans In mankind, it is highly improbable that a common genetic factor is responsible for the enhanced incidence of primary liver cancer among such diverse and widely separated people as the indigenous races of Africa and the Orient. That this high incidence is due to environmental rather than racial factors is indicated by the fact that the Negroes of North America are no more subject to this cancer than the white population among which they live (Kennaway, 1944). Moreover, there is strong presumptive evidence that frequency of the disease varies with environment even in the same race; on the Witwatersrand gold mines, for example, primary liver cancer was found to be almost six times more frequent in Bantu miners from Portuguese East Africa (East Coast natives) than in those from South Africa (Fig. 4), although there is little, if any, genetic difference between them (Berman, 1951). A. Hepatic Cirrhosis and Primary Liver Cancer. Most observers regard the frequent coincidence of hepatic cirrhosis with primary liver cancer as an important intermediate stage in the carcinogenic process. That this relationship is not merely an accidental one may be gathered from the fact that cirrhosis has been discovered in a large proportion of liver cancers throughout the world. In Africa and Asia, indeed, it is exceptional to encounter liver cancer without cirrhosis (Berman, 1951 ; Bonne, 1935). Moreover, the clinical features of established primary liver cancer usually are those of the malignant process in intimate association with progressive cirrhosis (Table IV) , including hematemesis which sometimes terminates
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fatally from ruptured esophageal and gastric varices secondary to portal hypertension. Hepatic cirrhosis appears to be more frequently associated with hepatocellular cancer than with cholangiocellular cancer. According to Ewing (1940), cirrhosis is present in 85y0 of hepatocellular cancers and in 5oy0 of cholangiocellular cancers. For both tumor forms, the association between the two diseases averaged 67y0,with a range of 53 to 1OOyo in a large series of cases compiled from the literature (Berman, 1951). a. Post-Mortem Incidence of Cirrhosis. In those geographic regions where primary liver cancer is infrequent, hepatic cirrhosis likewise is relatively uncommon. The post-mortem rate in European countries, for example, was 1.8% in Germany (Kohn, 1955) and 2.1% in Great Britain (Stewart, 1931), while in the United States of America it varied between 2.1% (Counsellor and McIndoe, 1926) and 3.2% (Rosenthal, 1932). Among the people of Africa and Asia, who are so prone to primary liver cancer, cirrhosis occurs relatively frequently. I n East Africa, Vint (1931) encountered cirrhosis in 6.7% of autopsies on Africans, and in the Southern Rhodesia Bantu the incidence was 8.7% (Gelfand, 1949). I n the young mine workers of South Africa, multilobular cirrhosis was found in 10% of the autopsies on the Portuguese East African Bantu and in 2.5% of the South African Bantu (Fischer, 1929). This is significant in view of the finding (Berman, 1951) that primary liver cancer occurs almost six times more frequently in the Portuguese East African (East Coast) natives. I n Indonesia, the autopsy rate was 6.9y0 for Malay males and 5.8% for Chinese males (Bonne et al., 1931). This relationship, however, is not absolute, for although a high incidence of infantile hepatic cirrhosis has been reported in certain parts of India (Rao, 1933; Wahi, 1949), in Ceylon (Tyagoraja, 1937; Fernando et al., 1948), and in Jamaica (Waterlow, 1948; Bras et al., 1954), liver cancer is held to be relatively uncommon in these localities. b. Hepatic Cirrhosis Developing into Carcinoma. Most observers agree that cirrhosis precedes the onset of liver cancer. Although it is difficult to assess the exact percentage of cirrhotic livers which become cancerous, available statistics indicate that the malignant transformation occurs more readily in Africans and Orientals than in Western people. I n Europe, Blumenau (1920) found primary liver cancer in 3.5y0 of deaths from hepatic cirrhosis, De Jong (1931) in 6%, and Kohn (1955) in 7Y0. I n the United States of America, the incidence varied between 3.4% (Berk and Lieber, 1941) and 10% (Rosenthal, 1932). Hemochromatosis associated with cirrhosis, however, is considered to give rise to liver cancer more frequently than cirrhosis alone (Sheldon, 1935; Berk and Lieber, 1941).
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I n French West Africa, on the other hand, primary liver cancer was associated with 43% of deaths from cirrhosis (Roulet, 1951). In Uganda, the corresponding figure was 170/, (Davies, 1956); and in the South African Bantu it was 32y0, in contrast to 5.2% in European (white) subjects, despite the fact that in this particular post-mortem series the incidence of cirrhosis in each race (Bantu 8.2y0, Europeans 7.6y0) was approximately equal (Becker, 1955). Liu (1953) observed malignant change in 12.670 of cirrhosis cases in China. For the Javanese of Indonesia, Kouwenaar (1932) estimated that neoplastic transformation occurs in 20-25y0 of all cirrhotic livers, and among the Chinese immigrants in Malaya Shanmugaratnam (1956) found that malignancy was present in 25.9y0 of cirrhosis. c. Cirrhosis-Producing Agents and Liver Cancer. Because of its frequent association with primary liver cancer and its high incidence among the very races known to be prone to this tumor, cirrhosis is, therefore, commonly regarded as an important precursor of the malignant process. On this account, a number of known cirrhosis-producing factors sometimes found in close association with human liver cancer have a t different times been credited with possessing carcinogenic properties or acting as co-carcinogens on already damaged livers. These factors include: (a) intestinal parasites (Vint, 1931), and (b) liver flukes (Hou, 1955) which are said to elaborate substances that are toxic for the liver; (c) schistosomiasis (Pirie, 1921; Van der Horst and Eerkens, 1939) which produces fibrosis around embolized schistosome ova in the liver; (d) hydatid cysts (Muller, 1932; Plazy and Damany, 1933); (e) hepatic syphilis (Favre, 1932; Montpellier and Loubeyre, 1933) ; (f) infectious hepatitis (Walshe and Wolff, 1952; Payet et aZ., 1953); (g) hemochromatosis and siderosis (Sheldon, 1935; Berk and Lieber, 1941; Gillman and Gillman, 1951; Warren and Drake, 1951); (h) alcohol; (i) spiced foods; and (j) a keloid diathesis in Negroid races, among whom there is often a tendency toward excessive connective tissue hyperplasia even after seemingly unimportant wounds. Not one of these factors, however, is common to all communities known for their high incidence of primary liver cancer. Thus, liver fluke infestation, though common in China, Japan, and other Oriental countries, is unusual in Africa; and although schistosomiasis is prevalent in many parts of Africa and Asia, primary liver cancer is rare in Egypt where schistosomiasis is estimated to affect 70-90% of the Nile population (Girges, 1934; Afifi, 1948), and it is rare also among Western people living in bilharziainfested areas. Moreover, infectious hepatitis appears to be no more frequent among Africans and Orientals than in Europeans or North Americans. Although highly spiced foods may conceivably help increase liver dam-
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age in Oriental countries, the dietaries of Africans in Uganda and South Africa, for example, do not as a rule contain spices. On the other hand, the incidence of primary liver cancer is relatively low in Mexicans, yet their dietaries are notoriously rich in condiments (Hall and Sun, 1951). d. Malnutrition and Cirrhosis. From clinical and experimental observations and from a recent inquiry by the International Society of Geographic Pathology, it would appear that chronic malnutrition, (so prevalent in Africa and certain parts of Asia) is one of the more important causes of cirrhosis, and perhaps also of primary liver cancer; conversely, the general good state of nutrition prevailing in Europe and North America may perhaps account for the relative infrequency of these liver lesions (Berman, 1955). The monotonous diet of the African, is a notoriously deficient one. Depending upon geographic and climatic conditions, the staple foods are: maize (corn) in Southern Africa, and plantains, yams, potatoes, and cassava in tropical Africa. Cattle do not thrive as a rule in Central Africa and are seldom slaughtered in southern Africa because they represent wealth to the rural Bantu; moreover, their milk yield is poor. I n Indonesia and other parts of Asia known for their high liver cancer rates, the staple diet is rice. The supply of meat, milk, and other animal products is inadequate because most of the available soil in these densely populated and underdeveloped countries is used for the production of rice and other predominantly carbohydrate foodstuffs which offer a more generous caloric yield. The common defects of all these high starch staple African and Asian foods are deficiencies in animal and other first-class proteins, in fats, in calcium, and in vitamins of the B-complex group. Carbohydrates, however, are in excess (Berman, 1955). For this reason, and also because of the seasonal fluctuations in food supplies among Africans during the dry winter and early summer months, chronic malnutrition is rife and manifests itself as an acute (and sometimes fatal) form of “Kwashiorkor,” a nutritional syndrome that was originally reported from West Africa by Williams (1935). This syndrome has also been described in many parts of the world under a variety of names, including “malignant malnutrition” in Uganda (Trowell, 1944) and in Indonesia (Oomen, 1953), “infantile pellagra” in South Africa (Gillman and Gillman, 1951), “fatty liver disease” in Jamaica (Waterlow, 1948), and “mbuaki” and “diboba” in Belgian Congo (Van Daele, 1938; Doucet, 1946). It is significant that this form of malnutrition is currently referred to as “protein malnutrition” (Brock and Autret, 1952). Kwashiorkor (a West African term referring to the reddish tint of hair in African children during the time of weaning) is mainly due to a lack in the diet of animal proteins and of vitamins of the B-complex group. It
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affects children during the late breast-feeding, weaning, or post-weaning periods, i.e., between the ages of six months and three or four years. The main clinical features include : edema, (associated with low plasma proteins and sometimes with ascites), retardation in growth and muscular wasting; diarrhea, various dermatoses, angular stomatitis, cheilosis, and glossitis, dyspigmentation of the skin, depigmentation and loss of texture of the hair, lethargy or irritability, anemia, atrophy of the pancreas, and enlarged fatty livers, which in later life may become fibrotic and culminate as a profound form of portal (Laennec) cirrhosis. Liver changes following kwashiorkor: The etiological relationship between kwashiorkor and hepatic cirrhosis is supported by the geographical distribution of the two conditions, although the regions in which kwashiorkor and cirrhosis occur do not overlap completely. Deposition of fat in kwashiorkor begins at the periphery of the liver lobule and may spread until practically every cell is affected. As the condition improves, the fat disappears and fibrosis may develop. Frank cirrhosis usually manifests itself after adolescence in Africans, and is but rarely seen in children. Complete recovery without visible liver d’amage, however, may occur even in severe cases unless toxic or infective factors intervene. Subclinical kwashiorkor cases, on the other hand, may develop fibrotic liver changes and cirrhosis which may account not only for the frequent incidental findings of these lesions in later life on biopsy or autopsy, but may also be responsible for the gross changes in liver metabolism, as is evidenced by the abnormal liver function tests so often encountered in seemingly healthy Bantu children and adults (Walker and Arvidsson, 1954). The architectural pattern of the fibrotic livers associated with kwashiorkor is not uniform and appears to vary to some extent in different localities. (a) I n the more pronounced cases, the hepatic lobules are distorted, hyperplastic, and separated by broad bands of fibrous tissue with or without cellular infiltration, although in other cases the liver lobules appear to be more intact with fine strands of fibrous tissue radiating from the portal tracts, producing in this way the “stellate fibrosis” observed by Davies (1954) in Uganda African children and adults. (b) Sometimes there is severe fatty infiltration of the liver with little fibrosis; a t other times, there is marked fibrosis with a minimum of fatty infiltration. This anomaly was thought by Davies (1954) to be due to two separate etiologic factors, the one giving rise to fatty infilt,ration, the other to fibrosis, which may operate either together, as in Uganda and South Africa, or individually, as in Gambia, where only the fibrogenetic factor appears to function. Walters and Waterlow (1954) found extreme portal cirrhosis in Gambia infants and children which was not associated with the extreme fatty infiltration characteristic of kwashiorkor elsewhere, and for this they postulated a combined
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etiology of malnutrition and malaria. (c) In the South African Bantu cases of pellagra occurring in adults but not in infants and children under the age of 7 years, the fibrosis is often accompanied by well marked hemosiderosis which is part of a widespread deposition of iron pigment throughout the body (Gillman and Gillman, 1951). (d) In Jamaica, the main emphasis of the fibrotic reaction is often on branches of the hepatic veins which become obliterated, and this leads to well marked cirrhosis with distortion of the lobular architectural pattern, thereby producing the so-called “veno-occlusive disease of the liver” described by Bras et al. (1954). The histological appearances of the fatty and fibrotic livers associated with kwashiorkor generally resemble those produced in rats fed on the staple Bantu diet of corn (maize) meal and sour milk (Gillman et al., 1945), and also those of rats fed on experimental diets deficient in the lipotropic factors, methionine, or choline (Gyorgy and Goldblatt, 1942; Himsworth and Glynn, 1944). Moreover, liver cancers, some with metastases in the lungs, have been experimentally induced in special strains of rats (Copeland and Salmon, 1946; Staub et al., 1948; Engel, 1952), in mice (Buckley and Hartroft, 1955), and in chickens (Schaefer et al., 1944) by the sole means of choline deficient diets unsupplemented by cai cinogenic agents. Chronic hepatic lesions and liver tumors have also been induced in rats after prolonged administration of ethionine, the synthetic analog of methionine (Popper and Bruce, 1955; Farber, 1956). 4. Commentary From the foregoing, it will be seen that primary liver cancer, so remarkably frequent among the malnourished populations of Africa and Asia, originates almost invariably in an organ that is the seat of preexisting hepatic fibrosis or cirrhosis, and that the geographic and demographic distribution of both diseases is to a large extent identical. The relevance of these observations lies in the influence of dietary factors upon the production of cirrhosis and of primary liver cancers in experimental animals. Diets low in protein or in riboflavin not only induce cirrhosis, but also facilitate hepatic tumor formation in rats fed these diets supplemented with dimethylaminoazobenzene (“butter yellow”) , whereas high protein or riboflavin diets under similar conditions have the opposite effect. If the results obtained in animal experiments can be paralleled in man, it is reasonable to assume that human populations existing on the deficient diets responsible for kwashiorkor can develop cirrhosis and other liver lesions similar to those induced in rats on somewhat identical dietary deficiencies, and that accessory hepatotoxic or carcinogenic agents may likewise produce liver cancer.
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No known external agents, however, have yet been isolated to account, for human liver cancer. The question arises whether primary liver cancer is initiated by chemical agents similar to the am-compounds, including dimethylaminoazobenxene. If this is so, whcre do these carcinogens originate? Are they to be found in the food and beverages of populations registering high liver cancer rates? They may perhaps be products of faulty metabolism derived from deficient diets, since potent carcinogens, possibly of metabolic origin (including cholesterol, which is closely related to methylcholanthrene) ,have been extracted from human livers and various other tissues and excretions, and these, strangely enough, were obtained from noncancerous as well as from cancerous subjects. Since latent carcinogenic factors are to be found in the presumably healthy human body, would cancer eventually develop if a person lived long enough, or are there factors in the body which inhibit the carcinogenic process? If so, what are the circumstances that destroy the equilibrium presumably existing between the carcinogenic factors and the inhibitors and so allow cancer to develop? In the liver, is the loss of this equilibrium the result of an inherited weakness of the hepatic parenchyma, the result of generations of malnutrition; or is it due to the effects of abnormal metabolic products on the liver cells during phases of acute and chronic malnutrition, which enhance the activity of latent carcinogenic substances, or to the absence from the diet of protective foods (e.g., proteins essential as a defense mechanism against cancer)? Perhaps there are carcinogenic substances with specific effects on the liver in the diets of populations known to be prone to liver cancer which either act directly on their already impaired liver cells or destroy the inhibitors, thereby securing for themselves and for latent metabolic hepatotoxins full freedom of action. 5. Indications for Research
It must be remembered that much of the evidence pertaining to etiology in human liver cancer is circumstantial and that the actual cause for the remarkable sensitivity of the liver to cancer in African and Asian races has not yet been established. Neither the evolution of cirrhosis from kwashiorkor nor the exact mode of neoplastic transformation from active cirrhosis is as yet understood. A great deal of research is obviously necessary. A. Accessory Hepatotoxic and Carcinogenic Agents. In cirrhosis, environmental factors additional to malnutrition are perhaps required to effect carcinogenesis, for there is as yet only scanty evidence that primary liver cancer can be induced experimentally by the sole means of a deficient diet. In Africa or Asia, it is possible that a susceptible liver already deranged by the ravages of malnutrition may in this way become more vulnerable to other external stimuli which may then have the effect of initiating the can-
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cerous process. These adventitious agents may include endemic parasitic infestation, acute infections, tropical diseases, and irritation by known toxic substances, or perhaps some as yet undiscovered compounds with carcinogenic potentialities. Absence of one or other such additional toxic factor may possibly explain the apparent lack of correspondence between cirrhosis and liver cancer on the one hand and malnutrition on the other in Jamaica, Central America, and other localities where kwashiorkor-like syndromes are notably prevalent. B. Epidemiologic Research. I n order to determine whether primary liver cancer is a preventable disease (as seems likely) and in order to search for naturally occurring carcinogenic substances analogous to the azocompounds or other chemical substances used in the experimental induction of liver cancers, local research should be conducted into the dietaries, customs, and habits of those African and Asian populations showing the highest susceptibilities to liver cancer. Present day knowledge indicates that French West Africa, Portuguese East Africa, South Africa, and Indonesia are the more likely regions for these investigations, but the actual localities could be chosen with greater accuracy by comput,ing cancer incidence rates. The inquiries should include determinations of the selenium contents of maize, wheat, and other cereals grown in these localities as well as that of the soil on which these crops are raised. Searches should likewise be made for senecio alkaloids and other hepatotoxins in the wild fruits, berries, weeds, and grasses which the indigent African and Asian villagers gather for food, beverages, and medicine from their immediate surroundings. C. Cyclical Fluctuations in Diets. Observations should be undertaken on the question whether the additional burdens of periodic fluctuations in the nutritional states of chronically undernourished populations play a part in the genesis of liver cancer. In tropical and subtropical Africa, the worst food shortages occur during the dry winter and early summer months, i.e., before the seasonal rainfalls, when fluctuations in the daily caloric intake of individuals can be extreme and may vary from 3,000 calories daily in the good seasons to less than 1,000 calories per day during the so-called “hungry months’’ (Davies, 1955). D. Sex Hormones. Primary liver cancer occurs more readily in males than in females. Gynecomastia and other signs of feminization, said to be due to failure of the liver to detoxicate endogenous hormones and frequently found in malnourished Africans, has led Davies (1952) and others to support the concept that a state of hyperestrogenism exists in subjects with cirrhosis. Investigations should, therefore, be carried out on the question of whether sex hormone patterns in malnourished populations influence the
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development of liver cancers, as seems to be the case in some experimentally induced liver tumors. E. Pathology Studies: Endogenous Carcinogenic Factors. The abundant biopsy and autopsy material available in Africa could be used for comprehensive comparative studies on the pathogenesis and histogenesis of human cirrhosis and liver cancer as there is still considerable controversy on the exact relationship between the fatty liver of kwashiorkor and hepatic fibrosis or cirrhosis (Dible, 1951) and liver cancer. The autopsy material should also be used for the purpose of intensifying and extending the original researches of Shabad, Hieger, and Steiner on the nature of the cholesterols and other endogenous carcinogenic factors which have already been isolated from the nonsaponifiable lipid fractions of human tissues. F. Clinical Research. Intensive clinical studies should be undertaken and should have as their main objective the recognition and treatment of primary liver cancer in its earliest stages. This could be effected to some extent by evolving more specific diagnostic liver function tests. Many early liver cancers could then be discovered and gathered together in regional medical centers where attempts could be made to initiate and carry out the more elaborate cancer curative methods which hitherto have not been generally available outside the Western countries; these should include likely chemotherapeutic agents, radioactive isotopes and high voltage radiotherapy. Bold surgical intervention planned by specially trained teams should also feature prominently in order to bring some relief to this otherwise hopeless form of malignancy. 6. Conclusions
I n primary liver cancer, there is little doubt that long periods of time may intervene between the initiation of irreversible liver damage and the first manifestations of malignancy. Subjective symptoms, as has been indicated, often appear for the first time only after the liver tumor has attained sizeable dimensions, or has produced catastrophic intraperitoneal hemorrhage through erosion of a blood vessel, or even only after extra-hepatic metastases have already become widely disseminated. These latent characteristics may perhaps explain the relatively high post-mortem frequency of primary liver cancer in many immigrants from poorly nourished localities in Asia where the incidence of the disease and of hepatic cirrhosis is known to be high. They may have left their native soil many years before the onset of the disease in their new environments, as witness the frequent reports on liver cancer concerning the Chinese, Japanese, and Filipino immigrants to Malaya, Indonesia, North America, and Hawaii.
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Again, i t is inconceivable that cancerization of the liver in the Bantu mine workers begins at the South African gold mines where the dietary arrangements are excellent, and where they rarely stay longer than one year; moreover, primary liver cancers in Bantu workers are frequently discovered within the first month of their arrival a t the mine (Berman, 1951). On this account, and because of the remarkable early age incidence of the disease in Africa and Indonesia, it can be reasonably assumed that the cause of the malignancy in susceptible populations is one which may begin to operate a t an early stage of life. The salient features, therefore, which emerge from a consideration of the etiological factors presented are that environmental factors (including cirrhotigenic and carcinogenic agents) may be responsible for precancerous and cancerous states of the liver, and that in man these probably act on a favorably prepared soil, the nature of which is determined largely on a nutritional basis. The possibility that environmental and social factors may cause cancer has aroused considerable interest in many parts of the world, and has been the main topic of the deliberations a t various recent cancer conferences sponsored by the International Society of Geographic Pathology of the World Health Organization International Union Against Cancer. Moreover, at the latest conference of the Society held during August, 1956 in Kampala, Uganda, the entire proceedings were devoted to the problem of primary liver cancer. Intensive research on the many facets of this unique problem in Africa, Asia, and elsewhere is clearly indicated, for it is felt that the solution of the mystery surrounding primary carcinoma of the liver may have profound effects on the whole problem of cancer.
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PROTEIN SYNTHESIS WITH SPECIAL REFERENCE TO GROWTH PROCESSES BOTH NORMAL AND ABNORMAL P. N. Campbell Courtould Institute of Biochemistry, Middlesex Hospitol Medico1 School, London, England
Paye
I. Introduction. . . . . 11. Relationship between Mitotic Rate and Protein Structure.. . . . . . . . . . . . . . . . . 99 1. Specificity of Protein 2. Effect of Growth on t 3. Differences between Proteins of Normal and Neoplastic Tissuc 111. Metabolism of Tissue Pr 1. General Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 102 2. Rate of Incorporation of Amino Acids.. ............................... A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 B. Normal Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 105 C. Tumor Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3. Degradation of Tissue Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 ....................... 107 B. Tumors .............................. C. Liver Slices.. ......................... ....................... 108 4. Plasma Protein as a Precursor of Tissue Protein.. ...................... 109 5. Intermediates in Protein Synthesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 A. Peptides ..... .................... 111 B. Interconversion of Proteins.. ..................................... 112 6. Synthesis of Bence-Jones Proteins. . . . . . . . . . . . . . . . . . . IV. Protein Synthesis in Whole Cells in vilro.. . . . . . . . . . . . . . . 1. General Considerations. ........................... 2. Ability of Cells to Concentrate Amino Acids.. . . . . . . . . . . . . 3. Cell Suspensions and Cells in Tissue Culture. . . . . . . . . A. Ascites.. . . . . . . . ............................... 119 B. Reticulocytes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 C. Other Types of Cell.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 D. Tissue Culture.. .... ..................................................... 123 ue Suspensions, . . . . . . . . . . . . . . . . . . . A. General.. . . . . . . . . . . B. Serum Albumin.. . . . . . . . . . . . . . . . . . . . . . 125 C. Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 .................................................. 128 E. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 F. Experiments of Anfinsen el al.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 6. Eggs, Larvae, etc.. . . . . . . . ......................... 130 V. Incorporation of Amino Acids into Subcellular Particles.. . . . . . . . . . . . . . . . . . . 131 1. Characterization of Subcellular Constituents. .......................... 131 97
98
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A. Homogenates. . . . . . .
.............. ..............
............... References. . . . . . .
...............
I. INTRODUCTION By the application of a wide variety of methods, ways are at last being found to study the biosynthesis of proteins. Among the methods used, those employing radioactive isotopes remain preeminent. On the basis that results obtained by the study of the synthesis of protein in one organism may be applied to other biological systems, reviews on protein synthesis have in the past covered such phenomena as enzyme adaptation, antibody production, metabolism of microorganisms, and tumor growth. However, owing to the increasing number of publications, such a coverage is no longer possible or even desirable so that at the outset some limitations must be admitted. As the title of this review indicates, attention will be directed to those aspects of the subject that more directly concern tumors and rapidly growing tissues. Thus protein synthesis in bacteria and yeast will not be discussed directly, and the whole subject of enzyme adaptation in microorganisms will be left out. The other major omission is a general discussion on the relationship between protein synthesis and the synthesis of nucleic acid. This has been the subject of numerous reviews, and it is felt that little of value can be added to the discussion a t the present time. I n order to overcome some of the difficulties that arise as a result of these limitations in scope, a list of general references has been added to the bibliography. There has, in the past, been a tendency t o regard ‘(growth” as the fundamental difference between normal and neoplastic tissue. Hence the metabolism of such tissues has often been compared with that of fetal tissue. It seems more likely that the growth of tumor tissue is a reflection of the loss of the control which the body exerts over normal tissues. Nevertheless if an agent could be found that specifically inhibited tumor growth, it would be immaterial whether it was directed against a fundamental property of neoplastic tissue or merely against a secondary manifestation of such a property. Since all tissue growth involves the synthesis of protein, it is therefore logical that attempts should be made to establish differences between protein synthesis in normal and tumor tissue. In such a comparison it is, of course, just as important to study the normal as the abnormal. In
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
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the case of protein synthesis, tumor tissues have not been neglected; indeed, the fact that some neoplastic tissue grows a t a fast rate has meant that such tissue offers real advantages experimentally. Before discussing protein synthesis, it is first necessary to det,ermine to what extent the proteins present in tumors and in growing tissues differ from those present in normal “adult” tissues.
11. RELATIONSHIP BETWEEN MITOTICRATEA N D PROTEIN STRUCTURE 1. Specificity of Protein Structure
Proteins may be characterized in a number of ways which may for the present purpose be grouped under three headings; physical prope1ties (e.g., migration in a n electric field, sedimentation under gravity), amino acid composition, and immunochemical behavior. By the application of such methods, it is now clear that, for the most part purified proteins show many of the attributes of a homogeneous molecular species. Thus in the now classical case of insulin, the protein, which is isolated from different individuals of the same species, is always identical, but insulins obtained from different species show characteristic differences (Harris et al., 1956). I n some cases protein specificity extends to individuals within a species. Thus Aschaffenburg and Drewry (1955) have shown differences between the P-lactoglobulins obtained from different breeds of cow, and similar differences were found by Evans et al. (1956) in sheep hemoglobins. A further recent example is that of the human haptoglobins whose genetic distribution is being studied by Smithies and Walker (1956). Even in an individual there appears to exist a degree of organ specificity of protein structure. Thus Schlamowitz ( 1954) found immunological differences between the alkaline phosphatases obtained from different organs of the dog. More recently similar differences have been found in phosphorylase obtained from rat tissues (Henion and Sutherland, 1957). It appears, therefore, that each type of cell possesses enzymatic mechanisms capable of exact control of the amino acid composition and sequence of all its proteins. It is now necessary to see whether the incidence of growth, be it normal or abnormal, has any general influence on the nature of the proteins produced by the tissues. 2. Eflect of Growth o n the Nature of Proteins in Normal Tissues
Probably the most clear-cut case of a difference in the structure of a protein occurring in the growing and adult animal is that of hemoglobin, although the change in this case is more likely to be the effect of environment than of growth. It has long been known that the hemoglobin of the fetus and the newborn child differs from the hemoglobin of adults in many respects. By four months after birth, the small amount of fetal hemoglobin
100
P. N. CAMPBELL
present in the blood of the newborn child has almost disappeared. Recently Van der Schaaf and Huisman (1955) have shown that in addition to physical differences between Hb-F and Hb-A, their amino acid composition differs in many respects. Darcy (1955, 1957), using immunochemical methods, has compared the plasma proteins of rats bearing various tumors with those from normal rats. He found many differences between the two types of plasma but, in particular, found that one component was markedly raised in the plasma from the tumor rats. By immunoelectrophoresis he showed that this component had a mobility similar to an a-globulin, and other evidence suggested that it might be a glycoprotein. He has found that not only is the plasma level of this substance raised by the presence of a tumor but also high levels are found in pregnancy and after partial hepatectomy. From this and other evidence, he suggests that the concentration of this substance in the blood may be directly associated with the total mitotic activity of the body. A glycoprotein has also been isolated from the plasma of t.umor-bearing mice (Nisselbaum and Bernfeld, 1956) but., like Darcy, these workers consider it likely that this substance is a normal constituent of the plasma which increases in concentration during tumor growth. Petermann and her colleagues have also found differences in the nucleoproteins of the cytoplasm during either normal or abnormal growth. These results will be discussed in Section V,1. The detection of a component.of the plasma which reflects mitotic activity is a finding of some importance and raises many interest,ing questions as to the origin and function of the substance. Miller (1955) using his perfused liver technique with lysine-6-C1* and S36-labeledsulfate has shown that the liver is responsible for the synthesis of most of the glycoproteins in the rat, but whether the glycoprotein studied by Darcy is synthesized by the liver is not yet clear. That substances in the blood can initiate mitosis is suggested by the results of Bucher et al. (1951). In these experiments with rats, either one partner of parabiotic twins or two partners of parabiotic triplets were subjected to partial hepatectomy. The animals were killed 48-72 hours later, and it was shown that the rate of mitosis in the unoperated animal had increased, the increase being greater with the t'riplets than the twins. Partially hepatectomized rats provide an excellent experimental system for the study of tissue growth for the whole cycle from initiation to cessation of growth may be observed. Harkness (1957) has recently reviewed the subject. 3. Diferences between Proteins of Normal and Neoplastic Tissue
If protein from tumors could be shown either to contain amino acids that do not occur in normal tissue protein or to have an amino acid
m o r E I N SYNTHESIS WITH REFERENCE
GROWTH PROCESSES
101
composition quantitatively different from normal tissue, a rational basis for chemotherapy might exist. With this aim in view, much work has been directed to show such differences. Before accurate methods were available for the estimation of amino acids, Kogl and Erxleben (1939) reported that the optical rotation of some amino acids which had been isolated from tumor tissue differed from the corresponding amino acids from normal tissue. The differences were particularly marked in the case of glutamic acid. Controversy as to the significance of this finding has gone on ever since. Wiltshire (1953) has studied the hydrolyzates of four tumor proteins and found that the percentage of D-glutamic acid was in all cases very small and not more than would be formed by inversion of the L-isomer during hydrolysis. Wiltshire also reviews the evidence obtained from various sources (see also Miller, 1950). Since the introduction of rapid procedures for the analysis of amino acids, there have been many attempts to compare the overall amino acid composition of tumor protein with that from normal tissues. So far no striking differences have been found between the amino acid composition of tumor protein and that obtained from normal tissues. Owing t o the importance of devising a simple diagnostic method for the presence of tumor growth, extensive investigations have been made on the nature of the plasma proteins in cancer patients. This subject has been well reviewed by Winzler (1953). He concludes that “the hope that specific abnormalities in plasma proteins might be of diagnostic significance in det,ection of cancer has not been substantiated. Significant abnormalities are, indeed, associated with this disease, but thus far no specificity has been apparent.” Since then the position has changed little except for the progress made by Putnam with myeloma. Although it is probable that cryoglobulins (serum globulins which precipitate, crystallize, or gel upon cooling) are not confined to myeloma, they are most often found in this disease. Putnam and Miyake (1956) have studied eight highly purified cryoglobulins from different sources by chemical and physical methods. They found that all of the cryoglobulins differed from normal serum proteins in two or more respects and that all but two of the cryoglobulins differed from each other. They therefore consider that cryoglobulins are truly unnatural proteins formed only in disease. The excretion of BenceJones proteins in certain cases of myeloma appears t o be quite characteristic of these cases, the properties of the BenceJones proteins differing between individual patients (Putnam and Stelos, 1953). Putnam (1955) suggests that BenceJones proteins are incomplete y-globulins. Certainly the immunological evidence showing crossreactions with 7-globulins is consistent with this idea (Deutsch et al., 1955; Korngold and Lipari, 1956). Thus here again. there is a fundamental change in protein synthesis.
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P. N. CAMPBELL
Finally, there are the immunological studies of Weiler (1956). He has shown by Coons’s fluorescent ant,ibody technique that hepatoma cells contain very little of an antigen which is specific for the liver and has detected a series of changes in the livers of rats fed with the carcinogenic dye. These changes involved the progressive loss of a protein. Thus there are some differences between the proteins of the growing and adult animal and, in the case of myeloma, between tumor patients and normals. Since it has now been shown that the specificity of protein structure extends to the organs within the animal, it seems possible that such specificity may be found to extend to tumors. The findings by Putnam that the cryoglobulins in cayes of myeloma are in fact “foreign” proteins supports this idea, but in order to detect such differences between tumors and normal tissues it will be necessary to study the properties of specific proteins.
111. METABOLISM OP TISSUE PROTEINS in vivo 1. General Considerations
Studies on protein metabolism in the living animal can be grouped under three headings as follows. First, determination of the rate a t which proteins are synthesized and degraded. For this purpose isotopically labeled amino acids have been extensively employed to measure rates of incorporation and also so-called “turnover rates.” Secondly, the nature of the intermediates in protein synthesis between the free amino acids and the proteins has been studied. In particular, attention has been directed to the role that peptides might play as such intermediates. Finally, the possibility that one protein might be converted into another without first being broken down to free amino acids has been investigated. The first study of this type was by Heidelberger et al. (1942) who followed the fate of Nls-labeled antibody in passively immunized rabbits. 2. Rate of Incorporation of Amino Acids
A. General. With the possible exception of hydroxyproline (Stetten, 1949), it is now accepted that the ultimate precursors of the proteins are the amino acids. Thus by injecting an animal with a radioactive amino acid and determining the radioactivity of the tissue proteins after different intervals of time, it should be possible to determine the rate of incorporation of amino acid into the proteins. However, before useful conclusions can be drawn from such an experiment, various factors must be taken into consideration. First, it is obvious that differences in the amino acid composition of the
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
103
various tissue proteins could lead to differences in the radioactivity of the tissue proteins if a single amino acid were injected. Now that it is relatively simple to isolate amino acids from proteins this difficulty is usually overcome by comparing the activity of the isolated amino acids. A much greater difficulty in the interpretation of results arises from a lack of knowledge of the radioactivity of the amino acids a t the site of protein synthesis. For example, Gaitonde and Richter (1955) pointed out in their studies on protein synthesis in brain tissue that the failure of a tissue to incorporate an injected radioactive amino acid does not necessarily imply a low rate of synthesis of the proteins in that tissue; a knowledge of the radioactivity of the free amino acid is necessary before any deduction can be drawn. Loftfield and Harris (1956), during the course of some experiments on the synthesis of ferritin in rat liver, injected rats intravenously with large quantities of L-leucine-C14 at 10-minute intervals. They then compared the radioactivity of the injected leucine with that in the liver and found that the activity of the intracellular leucine did not exceed 40% of the injected activity even after 80 minutes. They therefore concluded that more than half of the leucine required for protein synthesis was derived from the endogenous protein. This finding has important implications for it means that intracellular amino acids are not in equilibrium with the circulating amino acids, so that following the injection of a radioactive amino acid, the intracellular amino acids in the different tissues may vary greatly in radioactivity. That this is so, was shown by some experiments of Gaitonde and Richter (1955) who estimated the radioactivity of methionineS35in seven different tissues after injecting rats with ~-methionine-S~~ and found great variations between the tissues. Similar results were obtained by Campbell and Halliday (1957) following the injection of a rat bearing a liver tumor with L-1y~ine-C'~. The results obtained one hour after injection are shown in Table I TABLE I Radioactivity of Amino Acids in the Supernatant Fraction of Homogenates of Rat Tissues 1 Hour after Injection of Lysine-C" asb Tissue
Protein Lysine (P)"
Free Lysine (F)c
Ratio F/P
Liver Liver tumor Kidney
980 1100 829
5978 10300 2900
6.1 9.4 3.5
0
From Campbell and Halliday, 1957.
The supernatant was treated with trichloroacetic acid and the lysine was isolated from the protein precipitate and from the filtrate. e Radioactivity expressed as counts/min./cm.z at infinite thickness. b
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P. N. CAMPBELL
It is clear from the above that if the rate of incorporation of amino acids into proteins is to be deduced from the injection of a radioactive amino acid, some account must be taken of the radioactivity of the amino acids in the tissues. Campbell and Halliday (1957) have proposed that, following the injection of a radioactive amino acid into an animal, the rate of protein synthesis in the tissues should be related to the ratio of the radioactivity of the intracellular amino acid (F) and the protein-bound amino acid (P) (see Table I). Thus, provided F > P, the lower the ratio the greater the rate of protein synthesis. From the results shown in Table I, the rate of protein synthesis in the three tissues would be in the descending order kidney, liver, liver tumor. Such a conclusion is, of course, strictly valid only if the same specific protein is being studied in each tissue. The possibility exists that the proteins with a rapid turnover in one tissue may be rich in a certain amino acid whereas in another tissue the reverse may be true. Further work is required to clarify this point. B. Normal Tissues. With the above considerations in mind, the evidence that there is a difference between the rate of incorporation of amino acid into normal and non-neoplastic growing tissue must be reviewed. Tissue growth takes place as a result of the growth of individual cells and may or may not involve cell division. For the purposes of protein synthesis these two processes are very closely related, and it is not very practical to attempt to differentiate between them. A further cellular activity which is likely to have a profound effect on amino acid incorporation is the secretion of protein. Thus if it is one of the functions of the cell to synthesize an enzyme or a protein such as casein or serum albumin, then, quite apart from growth, such a cell will be expected to rapidly incorporate amino acids. Freidberg et al. (1948) attempted to compare the rate of incorporation TABLE I1 Rate of Incorporation of Methionine-,386 into the Tissue Proteins of the Dogasb Tissue Intestinal mucosa Pancreas Spleen Kidney Lung Liver Thymus Muscle a
From Freidberg el al., 1948.
Specific Activity X 100 X wt. of Animal in kg. /(Dose Administered) 0.80 0.39 0.38 0.37 0.24 0.23 0.19 0.04
* Tissues were obtained 6 hours after intravenous injection of the amino acid.
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105
of amino acids into the various tissues by injecting a dog with methionine-Sa6 and measuring the radioactivity of the tissue proteins a t different times. The results in Table I1 show that intestinal mucosa was most active, followed by pancreas and then kidney and liver. No doubt the higher activity of intestinal mucosa is associated with the rapid replacement of cells, shown by the high mitotic rate of this tissue, and that of pancreas is due to the active secretion of a number of enzymes. An example of the effect of age on the rate of incorporation of amino acid into the tissues is provided by the work of Neuberger et al. (1951) and into Neuberger and Slack (1953) on collagen. They injected g1y~ine-C'~ rats of varying ages and determined the radioactivity of the glycine in the collagen obtained from various sites. In rats over 14 months old, the activity of the glycine samples from all the collagen fractions was very low. However, with young rats, the collagen fractions were much more active and there was an inverse relationship between the activity and age. The incorporation of glycine-N16 into regenerating rat liver proteins has been studied by Eliasson et al. (1951), Hammarsten et al. (1956), and Rqvist and Anderson (1956). They found that there was a good correlation between the rate of incorporation and rate of protein synthesis, but the peak of the latter came a little later than the peak of mitotic activity. Of a slightly different nature is the work of Borsook et al. (1952) on the rate of incorporation of radioactive amino acids into reticulocytes. The mature erythrocyte incorporates amino acids only very slowly but if reticulocytosis is induced in rabbits with phenylhydrazine, then the reticulocytes rapidly incorporate amino acids. Similarly, although leucocytes in the human incorporate amino acids in vivo, the immature cells present in cases of acute leukemia are much more active in this respect (Weisberger and Levine, 1954). From these results, there appears to be a reasonable correlation between the rate of incorporation of amino acid into the protein and the rate of growth of the tissues. Results obtained with tumor tissue must now be considered to determine whether a similar correlation exists. C. Tumor Tissue. While it is true that tumor tissue grows a t a faster rate than normal tissue from adult animals, this difference often tends to be greatly exaggerated. Thus, while a transplanted tumor such as the Walker carcinoma does grow at a fast rate, the mammary carcinoma of the August strain of rats is very slow growing. Spontaneous tumors in the human are also relatively slow growing, a t least in the early stages. Busch and Greene (1955) have injected rats bearing implanted Walker carcinoma and also Jensen sarcoma with glycine-2-C14. Figure 1 shows the change in the radioactivity of the tissue proteins with time. While the increase in radioactivity of the rapidly growing Walker tumor was equal
106
P. N. CAMPBELL
256
3
6
Time after injection (hr.)
FIG. 1. Rate of incorporation of radioactivity into tissue proteins after injection of tumor-bearing rats with glycine-2-Cl4 (from Busch and Greene, 1955).
to that of the liver, that of the Jensen sarcoma, a more slowly growing tumor, was lower than that of liver or kidneys. In these experiments, no account was taken of the radioactivity of the free amino acids in the tissues. Since these tumors were transplanted, it seems possible that this may be an important consideration. Nevertheless, even when account was taken of the activity of the precursor amino acid, as in the experiments of Campbell and Halliday (1957) already cited (Table I), the conclusion is reached that the rate of uptake of amino acid by liver tumor is slightly less than that of the liver. Thus, as was previously pointed out by Shemin and Rittenberg (1944) and Norberg and Greenberg (1951), the uptake of amino acids is not significantly faster in tumor tissue than in normal tissue if these tissues be considered as a whole. However, as is seen in Fig. 1, if the tumor tissue is compared with its tissue of origin, e.g., Jensen sarcoma with connective tissue, then the tumor tissue is the more active. I n the comparison of liver tumor with normal liver, account must be taken of the role of the liver in the synthesis of serum albumin. While it seeme probable that liver tumor still retains the ability to synthesize serum albumin (Campbell and Stone, 1957a), it is considerably reduced in the tumor compared with the normal tissue. The amino acid demands of the tumor compared with the normal liver for growth may, therefore, be more than counterbalanced by the extra demands of the liver for albumin synthesis. Forssberg and Klein (1954) have shown that in ascites tumors there is a good agreement between the relative growth rate of tumor cells with vary-
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
107
ing physiological age and the rate of C14-amino acid incorporation in vivo into the protein. Therefore, as with normal tissues, there is a correlation of the rate of incorporation of amino acid with the rate of tumor growth. 3. Degradation of Tissue Proteins
A. General. Borsook and Keighley (1935) were the first to suggest that there is a continuous and high rate of protein synthesis even in the adult animal. Later, Schoenheimer (1942), as a result of his work and that of his collaborators with NI6-labeled glycine, put forward his concept of the dynamic state of the tissue proteins. It was implied that in the adult organism there is, in addition to new protein synthesis necessary for secretion and cell division, a constant breakdown and resynthesis of all protein molecules a t varying rates. Recently, as a result of their work with bacteria, Hogness et al. (1955) have questioned whether the protein molecules within the cells of mammalian tissues are in a dynamic state. They think it possible that proteins are only lost from tissues as a result of cell lysis and secretion. This may indeed be true for the structural proteins, but there can be no denying that plasma proteins are not only secreted by tissues but are also degraded to free amino acids. Thus Whipple and Madden (1944) showed that an animal could be maintained in N equilibrium by the infusion of plasma protein, and this type of experiment has since been amply confirmed. The finding by Loftfield and Harris (1956) that the radioactivity of free leucine-C14 in the liver never rose above 40% of that of the injected leucine also indicates that in the liver there is an extensive degradation of protein. It is not clear whether this concerns secreted or nonsecreted protein. B. Tumors. On the basis that Schoenheimer was correct in his hypothesis, it has been argued that a tissue may grow either by synthesizing protein or by reducing the rate of degradation of protein. Thus it was thought that in tumors compared with normal tissue the rate of degradation relative to the rate of synthesis might be reduced and that this might lead to growth. As a result of his studies on the nitrogen balance of tumor-bearing rats, Mider et al. (1948) put forward the concept of the tumor as a nitrogen “trap.” Support for this idea was provided by the work of LePage et al. (1952). I n these experiments, glycine-2-C1* was fed to rats with FlexnerJobling tumors. The radioactivity of the tumor increased whether the animals were fed or fasted, whereas under fasting conditions little of the glycine went to the noncancerous tissues. Various methods have been used to determine whether the degradation of tumor tissue proteins is slower than that of normal tissues. The activity of the proteolytic enzymes in the tissues was studied, but, since the role of these enzymes in the intact cell is not a t all clear, it is difficult to interpret
108
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the results of such experiments. Another approach has been to study the release of radioact,ivity from a labeled transplant. Babson and Winnick (1954) transplanted into rats a Walker carcinoma which had been labeled with radioactive amino acid in vivo. They determined the loss in radioactivity of the transplanted tumor a,nd found that this was appreciable. Greenless and LePage (1955) have carried out a similar experiment with labeled ascites cells injected into a mouse. They found a continuous and regular loss of radioactivity to the organs, urine, and COZ. If the labeled ascites cells are placed in cellophane membranes and are implanted into the peritoneal cavity of mice, t,he cells divide but there is no growth (Christensen, 1955; Moldave, 1956), and over a period of two days the specific activity of the ascites cellular protein decreases. This loss of activit.y is associated with the nucleic acid, lipid, and proteins of each subcellular fraction except for the nucleic acid fraction of the nuclei which continues to increase in radioactivity (Table 111) (Moldave, 1957). TABLE I11 Release of Radioactivity from Cellular Fractions of Ascites Cells Labeled with Leucine-C14and Incubated in vivoasb Time of Incubation (hr.) 0 24 48
Nuclei
Mitochondria
Microsomes
Supernatant
L N A P
L N A P
L N A P
L N A P
3270 1130 1845 132 58 2625 1130 466 2080 2900 1500 2350 3150 1480 1580 41 20 2050 600 392 1735 1180 530 2120 1200 1480 1295 55 1950 780 345 1690 1035 440 1400
From Moldave, 1967. Results are expressed as total counta per. min. in lipids (L).and nucleic acids (NA). and as specific activity (oounts per min. per mg. of protein, P). a
b
These results are, of course, only valid if there were no necrosis of the labeled tissues and cells. The authors consider that there was little evidence of necrosis. If this is so, then the results suggest that in tumor tissue, at least, tissue proteins are in a dynamic state since the secretory proteins would not be expected to account for the loss in radioactivity in such tissues. Greenless and LePage (1955) have concluded that “Tumor per se is a nitrogen trap in the relative sense only. When the growth rate of a tumor has increased to such an extent that the tumor requires all the available building blocks for protein synthesis and the degradation products have little time to escape, it approaches this character.” C. Liver Slices. Recently some interesting experiments have been carried out on the mechanism of protein degradation. If liver slices are obtained from a rat which has been injected with radioactive amino acids, it is possible to follow the rate at which the proteins are degraded to amino
GROWTH SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
109
acids when the slices are incubated. As shown in Table IV, conditions which limit the release or utilization of energy such as dinitrophenol or anaerobiosis also depress the liberation of labeled amino acid (Simpson, 1953). The effect of reincorporation of the released radioactive amino acids is reduced by flooding the system with inactive amino acid. Simpson suggested that the two processes of synthesis and catabolism were interrelated. More recently Steinberg and Vaughan (1956) have carried out similar experiments and come to the same conclusions. These authors also studied the effect of amino acid analogs and found that analogs which inhibited synthesis also inhibited breakdown. TABLE IV Effect of Various Conditions on Release of Leucine-3-C14from Proteins of Liver Slices4v b
Added Components
Zero time None NrCOlr 3.0 X 10-3M NaCN 1.0 x 1 0 - 4 ~D N P ~ 5.0 x 1 0 - 4 ~DNP
Total c.p.m." in Nonprotein Fraction
c'p'm'c Re'eased
8 330 40 149 148 44
322 32 141 140 36
C.p.m.c Released as per cent of Total c.p.m. Protein 6.3 0.6 2.8 2.7 0.7
Per cent Inhibition
90 56 57 89
~~~~
From Simpson, 1953. Slices were obtained from the liver of a rat which had received leucine3-C14. The slices were incubated for 4 hours. c Counta per min. d 2,4-dinitrophenol. b
Roberts and Kelley (1956) have studied the metabolism of plasma proteins labeled with phenylalanine-3-C14 by rat liver slices. They found that these proteins, especially albumin, were readily utilized for the production of energy and gluconeogenesis. These results, showing that energy is required for the degradation of the cellular proteins, raise the question of the role of the proteolytic enzymes in protein degradation in the intact cell since the activity of such enzymes does not require an energy source. It would be interesting to extend the experiments on the degradation of cellular proteins to tumor tissue, for it still seems possible that the inability of the body to control tumor growth may be associated with the breakdown of protein.
4. Plasma Protein as a Precursor of Tissue Protein As has been pointed out, there is no doubt that secretory proteins are rapidly degraded to amino acids by the intact animal. Since the composi-
110
P. N . CAMPBELL
tion of the plasma proteins undergoes some marked changes in cancer patients, it was thought that tumors might be particularly effective in the utilization of these proteins for the synthesis of their own tissue proteins. Busch and Greene (1955) have recently studied this possibility in rats with transplanted tumors. Figure 2a and b shows a comparison of the results obtained by Busch and Greene when they determined the radioactivity of the tissue proteins in rats bearing the Walker carcinoma and Jensen sarcoma after receiving either g1y~ine-C'~ or plasma protein labeled with glycine-C14. It will be a
b
FIG.2. Comparison of incorporation of radioactivity into tissue proteins of tumorbearing rats from free amino acid and labeled plasma protein. (a) Glycine-2-0' was injected and the rats killed after 3 hr. The values for the Jensen sarcoma and Walker 256 carcinosarcoma (see Fig. 1) are pooled. Specific activities of protein of heart, testis, thymus, and brain ranged between those of muscle and kidney. (b) Plasma protein labeled with gly~ine-2-C~~ was injected and the rats killed after 3 hr. The values for the two tumors are pooled. The activities of protein of intestine, brain, testis, and lung ranged from 1.7-8.2 ct./min./mg. and fall betwcen those of muscle and pancreas (from Busch and Greene, 1955).
seen that the uptake by the tumor compared with the other tissues was much greater in the case of the labeled plasma than the labeled amino acid. Both tumors gave very similar results with t,he labeled plasma. These authors also determined the distribution of radioactivity in the subcellular fractions of the tumors and normal tissues after injection of the labeled plasma. They carried out a control experiment to determine the extent of t,he adsorption of plasma protein to such particulates by incubating the radioactive plasma protein with tissue homogenates and again determining the intracellular distribution of radioactivity. From such studies they concluded that the tumors were not only more effective in trapping radioactive plasma but were also more effective than normal tissues in its utilization. In a later paper (Busch et al., 1956), they have extended these experiments and studied the uptake of fractionated labeled plasma protein.
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
11 1
They found that the differences between tumor and normal tissues were much more marked in the case of albumin than of globulin when the radioactivity of whole tissue homogenates was compared. When the activity of the particulates was determined, the differences between the two types of tissue were greatest with microsomes after injection of albumin. It is interesting t h a t next to the tumors the tissue which concentrated plasma proteins best was the lung. The results quoted above are in agreement with those of Babson and Winnick (1954) in which the uptake of radioactivity by implanted Walker carcinoma, liver, and kidney was determined two hours after injection of either 1e~cine-C'~ or plasma protein labeled with the same amino acid. Similar results were obtained by Bauer et al. (1955), who injected rabbits with P1-labeled rabbit albumin and studied the uptake of radioactivity into the liver, in which had been implanted Brown-Pearce liver tumors. Since, after 1-5 days, the tumor was very much more radioactive than the liver, they concluded that the tumor takes up more homologous protein than the liver. However, the fact that the liver synthesizes large quantities of serum albumin seems not to have been considered in the interpretation of the results. The finding by Babson (1956) that the rate of disappearance of infused C14-labeled plasma protein in rats bearing the Walker carcinoma was much greater than in normal rats may well be associated with the rapid uptake of plasma protein by tumors. These results clearly demonstrate that implanted tumors incorporate amino acids from radioactive plasma prot,ein at a more rapid rate than normal tissues. Two important questions arise. First, is this a characteristic of their neoplastic state or merely a reflection of their growth rate? It would for instance be interesting to repeat the experiment with rats which had been partially hepatectomixed. Secondly, are the plasma proteins first degraded to free amino acids before being incorporated into tumor protein? Experiments bearing on this question will be considered in the next section. 5 . Intermediates i n Protein Synthesis
A. Peptides. One of the perennial problems of protein synthesis has been whether the amino acids are assembled in a stepwise manner through peptide intermediates or whether there are no intermediates before all the amino acids required for the synthesis of a complete chain are assembled together. A complete review of this aspect of the subject will not be attempted, but some points are particularly pertinent to the problems of growth. An argument against the role of peptides as intermediates is the failure in the past t o detect such peptides in tissue extracts. Until recently gluta-
112
.'1
N. CAMPBELI,
thione was the only peptide to be isolated from tissue extracts in reasonable amounts. However, Waley (1956) has found a considerable number of peptides in calf lenses as have Ramachandran and Winnick (1957) in the hog pituitary gland and Sampath Kumar et al. (1957) in mammary gland. Peptides which are metabolically active have also been found in yeast (Turba and Esser, 1955) and in Pseudomonas (Connell and Watson, 1957). Thus it may be that the previous failure to detect. peptides was one of technique. Many attempts have been made to study the problem of peptide intermediates with labeled amino acids. Work and hie collaborators (e.g., Askonas et al. 1955; Godin and Work, 1956) have studied the synthesis of milk proteins in goats. They found no evidence for peptide intermediates in the synthesis of casein and P-lactoglobulin from free amino acids. Simpson and Velick (1954), Heimberg and Velick (1954), and Simpson (1955) have studied the synthesis of various muscle enzymes and have come to the same conclusion as have Loftfield and Harris (1956) in their work on ferritin already referred to. On the other hand, Anfinsen and his collaborators, first in in vivo studies on the synthesis of ovalbumin in the chick oviduct, and later in a series of in vitro studies on the synthesis of ovalbubumin, ribonuclease, and insulin, obtained evidence of peptide intermediates in the synthesis of these proteins (Steinberg and Anfinsen, 1953; Vaughan and Anfinsen, 1954; and Flavin and Anfinsen, 1954). (This work will be referred to again in Section IV,5,F). Shimura et al. (1956) have obtained similar evidence from their work on the synthesis of silk fibroin in silkworm larvae as have Gehrmann et al. (1956) who studied collagen synthesis in rats. Much has been written in an attempt to decide whether peptides do play a role in protein synthesis (e.g., Dalgliesh 1953, 1957; Steinberg et al., 1956) without any definite conclusions having been reached. It seems unneccessary therefore to repeat the arguments again here. B. Interconversion of Proteins. a. Normal tissue. Another way in which peptides might act as intermediates in protein synthesis is in the interconversion of two different proteins. Thus when plasma proteins are utilized for the synthesis of tissue proteins is it necessary for the plasma proteins first t o be completely degraded to free amino acids? Yuile et al. (1951) were the first to study the problem by injecting dogs with plasma protein labeled with 1y~ine-C'~. They found that the amount of amino acid released to the circulation from the breakdown of the plasma protein was very small and argued that the interconversion of protein took place intracellularly and probably did not reach the amino acid level. That injected plasma proteins are able to enter a wide variety of cells without prior degradation was shown by Coons et al. (1951) using fluorescent antibody. However, as
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
1 13
already pointed out, Loftfield and Harris (1956) have shown that a considerable proportion of the amino acids required for tissue protein synthesis is supplied by the degradation of protein intracellularly and that the amino acids released do not equilibrate with the circulating amino acids. Thus, the results of Yuile et al. (1951) may be explained without postulating the interconversion of proteins a t the polypeptide level. The possibility that peptides derived from the plasma proteins might be used for the synthesis of the milk proteins has been extensively investigated in rabbits by Campbell and Work (1952) and in goats by Barry (1952) and Askonas et al. (1954, 1955), Askonas and Campbell (1953), and Godin and Work (1956). Similar experiments on the precursors of the proteins of rat pancreatic juice have been reported by Junqueiia et al. (1955). I n none of these experiments was any evidence found that partial degradation products of plasma proteins were used for the synthesis of new protein. A similar conclusion was reached by Walter et al. (1957) from their experiments on the metabolism of S35-and 1l3’-labeledserum albumin in rabbits. However, the possibility remained that in rapidly growing tissue the situation might be different. Thus Ebert (1954) has shown that when transplants of radioactive chick embryo kidney, liver, or spleen are made into chick embryos the radioactivity appears predominantly in the corresponding organ of the embryo. The embryonic transplant was labeled with methionine-F5. Control experiments with free methionine-Sa6 showed that the transfer did not take place at the level of free amino acids. Other experiments showed that the results could not be explained on the basis of the transfer of whole cells so that it was concluded that a “selective incorporation from grafts into homologous host tissues of tiseue-specific proteins or specific constituents of proteins larger than amino acids took place.” Ebert’s results for kidney and spleen are summarized in Table V. Francis and Winnick (1953) studied the amino acid and protein balance in cultures of chick embryonic hearts. They used either radioactive amino TABLE V Incorporation of Radioactivity from Proteins of Labeled Spleen and Kidney Grafts“ Specific Activity of Host Tissue Protein /Specific Activity of Graft Tissue Protein ~~~
Host spleen Host liver Host kidney ~~
4
From Ebert, 1954.
~~~~
~
Spleen Graft
Kidney Graft
0.14 0.04 0.04
0.038 0.038 0.098
114
P. N. CAMPBELL
acids in the medium or labeled protein in the form of embryonic extract, derived from eggs which had been incubated with radioactive amino acids (EE protein). In experiments with doubly labeled EE protein, they found that the presence of large quantities of nonradioactive amino acid partially depressed the incorporation of radioactive amino acids into the tissue. The value of such a metabolic trap is rather doubtful in view of the known lack of equilibration of intra- and extracellular amino acids, but the use by these authors of amino acid analogs seems to overcome these objections. Thus as shown in Table VI, fluorophenylalanine which inhibited the incorporaTABLE VI Effect of o-Fluorophenylalanine on Protein Synthesis and on the Incorporation of u~~phenylalanine-3-CS4 into Protein of Heart Cultures"vb Concentration of o-Fluorophenylalanine
Form of Labeled Phenylalanine
None None 6 X lO"M
EE protein Free amino acid Free amino acid EE protein Free amino acid Free amino acid
1.2 x IO-JM 1.2 x 10"M 3 x 10-3~ 4
Incorporation Phenylalanine per mg. of Protein pg.
0.145 1.1 0.7 0.140 0.5 0.1
Per cent Increase in Protein Content
100 75 55
From Francis and Winnick, 1953.
* The cultures were incubated for 6 days. tion of free amino acids did not inhibit the transfer of C14from EE protein. These results could be due to the transfer of complete unmodified protein from the EE protein to the embryonic heart culture. This explanation is supported by the failure of dinitrophenol to inhibit the transfer of radioactivity from EE protein to the culture, for it is now known that the intracellular breakdown of protein would probably be inhibited under such conditions (see Section 111,3C). Walter et al. (1956) have studied the utilization of various proteins by the chick embryo. Hen eggs were injected with a variety of labeled amino acids, peptides, and proteins. Since the difference in the uptake of radioactivity from the free amino acids compared with the proteins, in the 5-9 day period following injection, was not as great as would be expected if the amino acids were true intermediates in the synthesis of embryonic proteins, it is concluded that proteins are the preferred precursors. It is particularly interesting that egg albumin was not used to any greater extent than serum albumin. The possibility that. these results are due to the transfer of whole protein has not yet been excluded. Thus the role of intermediates in the utilization of protein precursors for embryonic growth is not at all clear at present.
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
115
b. Tumor tissues. As has been shown, plasma proteins are particularly efficient precursors of tumor tissue protein so that the interconversion of these two types of protein has received particular attention. Babson and Winnick (1954) have followed the uptake of radioactivity from labeled plasma protein by the tissues of rats implanted with the Walker carcinoma. They showed that whereas in the control experiments, in which free leucine-C14 was injected, the injection of inactive leucine reduced the radioactivity of the tissue proteins, there was no such reduction when labeled plasma protein was injected. Before conclusions can be drawn from this type of experiment, two points must be considered. First, how much of the unmodified radioactive protein which was injected is present in the tissues when the animal is killed. Secondly, as has been stressed before, account must be taken of the radioactivity of the free amino acids within the tissues, for the degree of dilution of the activity of the amino acids released by the degradation of protein will be different for each tissue. I n an attempt to overcome some of these difficulties, Campbell and Stone (1957b) have studied the utilization of albumin labeled with 1y~ine-C'~ by the tissues of a rat bearing a liver tumor. The radioactive albumin was intravenously injected together with some a1a11ine-C'~. After 155 hours, the rat was killed, and the radioactivity of the lysine and alanine in the soluble proteins of the tumor, liver, kidney, and spleen was determined together with that of the free amino acids in these tissues. The amount of serum albumin in these tissues was determined by an immunochemical method. The radioactivity of the lysine in the serum albumin circulating a t the end of the experiment was also determined. A determination of the amount of blood in the tissues allowed an estimation of the contribution of the unmodified serum albumin present in the tissues to the radioactivity of the tissue protein lysine t o be made. On the assumption that the rate of incorporation of different amino acids into the tissue proteins will be the same (Campbell and Halliday, 1957), it was thus shown that there was no evidence for the direct transfer of serum albumin to tissue protein a t other than the free amino acid level. As predicted by Loftfield and Harris (1956), the effect of even small amounts of the injected serum albumin on the radioactivity of the tissue protein extracts was very considerable. There is, therefore, at present no clear-cut evidence that tumor tissue differs from normal tissue in the way in which it converts plasma proteins for its own requirements. 6. Synthesis of Bence-Jones Protein
As was pointed out in Section II,3 the protein excreted into the urine in certain cases of myeloma, the so-called Bence-Jones protein, is characteristic of this malignant disease. The biochemical source of this urinary pro-
116
1'. N. CAMPBELL
tein is, therefore, of especial interest and has been the subject of an intensive study by Putnam and his colleagues. The excretion of Bence-Jones protein may or may not be accompanied by hyperglobulinemia, and the latter may be the result of abnormalities in the p-globulin fraction or may be a result of the presence of cryoglobulins. Putnam has used myeloma patients with different protein abnormalities for his studies on the synthesis of Bence-Jones protein. Thus Putnam and Hardy (1955) fed g1y~ine-C'~ to a patient with an abnormal 8-globulin. They found that the activity of the Bence-Jones protein rose rapidly and then fell rapidly, which was quite unlike the activity of the p-globulin. Thus, they concluded that the synthesis of the two proteins was independent. Hardy and Putnam (1955) studied a patient with a cryoglobulin in the serum and used glycine-NIS.In this case the excret.ion of the glyche-NI6 in the Bence-Jones protein closely paralleled the decline in the N16 the
'200[.
. E
.-E
*Urinary amino acids 0 Bence-Jones Dratein
Time after administration (hr.)
FIG. 3. Specific activity-time curves of the Bence-Jones protein isolated from the urine and of the BaCOs obtained by reaction of the urine with ninhydrin (from Putnam et al., 1956).
urinary urea and ammonia. More recently Putnam et aZ. (1956) have used CI4-labeledamino acids in a patient who had no hyperglobulinemia. Figure 3 shows the time-act,ivity curve for the Bence-Jones protein after the patient had received L-1y~ine-C'~ by intravenous injection. As expected from the previous results, the activity rose rapidly and then fell off steeply. From these results in which various isotopic labels were used in patients with different abnormalities in protein synthesis, the same conclusions can be drawn. Thus, there was no apparent metabolic relationship between the abnormal protein of the plasma and that of the urine, and in each case the Bence-Jones protein was synthesized rapidly and excreted rapidly and appeared to be formed de novo from free amino acids rather than by degrad-
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
117
ation of serum or tissue proteins. As judged by the first appearance of radioactive protein in the urine, tJhe maximal time lag for synthesis a n d excretion of BenceJones protein is 30-60 minutes. I t appears that the protein is not withheld appreciably more by the kidney than is lysine and that the protein synthesized in any given interval is excreted for the most part within the ensuing 12 hours.
IV. PROTEIN SYNTHESIS I N WHOLECELLSin vitro 1. General Considerations
Apart from bacteria which are not specifically included in the present review, in vitro naturally implies a degree of abnormality in the conditions under which the cells are being studied. In the case of a complex process such as is involved in protein synthesis, the experimenter has to be cautious in the interpretation of his results and to indicate to what extent they are :t reflection of the synthesis of whole protein. This is often not easy, for it has been shown on numerous occasions that incorporation of radioactive amino acid into a protein fraction of a tissue is not synonymous with the synthesis of a complete protein. Three general methods of demonstrating the synthesis of a complete protein under in vitro conditions have been used: (1) The demonstration of an increase in the amount of a specific protein during the courFe of incubation. Failure t o show such an increase does not imply that protein synthesis did not take place, for the rate of degradation may have equaled the [ate of synthesis. The increase in protein may be followed by means of a n antiserum prepared against the specific protein or, for example, by measuring the increase in enzyme activity. In this case it is best to follow the activity of more than one enzyme to avoid the possibility that merely the removal of an inhibitor is being studied. (2) The incorporation of a radioactive amino acid into a specific protein. (3) The necessity to have a full complement of all the essential amino acids. This condition is based on the assumption that the amount of peplide intermediate is small and that all the constituent amino acids of a protein must be available at the same time. This condition can seldom be applied except in bacteria, for it is not usually possible to reduce the supply of amino acids to a low level. Before discussing the various systems used for the study of protein synthesis, the methods by which the cell is able to accumulate the amino acids must be mentioned. 2. Ability of Cells to Concentrate A m i n o Acids
If the concentration of the various amino acids in the tissues of an animal is determined, great differences are found between the different tissues. This is well illustrated by the work of Tallan et al. (1954) on the
118
P. N . CAMPBELL
cat. Since the concentration of amino acids in the tissues differs from that in the fluid which bathes them, it follows that the cells are able to actively concentrate amino acid. The fact that the concentration of amino acids in fetal tissues compared with those of the mother is much higher (Christensen and Streicher, 1948) and the concentration in regenerating liver reaches a maximum which is much higher than in normal liver, when hepatic restoration is most rapid (Christensen et al., 1948), was thought to indicate an enhanced ability of the growing tissue to concentrate amino acids. If the amino acids of tumor tissue are studied, it is found that regardless of their origin tumors show a very similar qualitative pattern which is in marked contrast to the characteristic patterns shown by each type of normal tissue (Roberts and Frankel, 1949). Kit and Awapara (1953) have made a quantitative comparison of the amino acid content of lymphosarccmas and normal lymphatic tissues of the mouse, rat, and rabb't. The tumor tissues differed markedly f om the normals acd had much higher levels of alanine, glycine, and praline but less asFartic acid, ethanolamine phosphoric acid, and glutamine. There seems to be no evidence that tumor tissue, in general, has a higher concentration of free amino acid than normal tissue. The ability of tissues t o concentrate amino acids can be tested by a more direct method than a comparison of the level of amino acids in the tissues. Thus, by feeding large quantities of glycine and alanine to mice bearing an ascites tumor, Christensen and Henderson (1952) showed that the ascites cells were much more effectively able to concentrate these amino acids than were liver or muscle. If such ascites cells be incubated in vitro, they concentrate glycine so effectively that the intracellular concentration may reach a value of 90 mM compared with that of 30 mM in the medium (Christensen and Riggs, 1952). The ability of such cells to concentrate amino acids is not confined to those amino acids present in proteins. Thus the D-isomers may be concentrated (Christensen et al., 1952a) and such amino acids as a-y-diaminobutyric acid. The latter amino acid is concentrated so effectively that most of the potassium of the cell is displaced (Christensen et al., 195213). Heinz (1954, 1957) has studied the kinetics of the influx and exflux of glycine into ascites cells and concludes that most of the intracellular glycine is free and that the concentration is the effect of a metabolically linked active transport mechanism. The nature of this mechanism is being studied by comparing the effect of a variety of substances on the ability of the cells to concentrate many different amino acids and their analogs (Riggs el al., 1954, and Christensen and Riggs, 1956). A knowledge of the mechanism by which cells concentrate amino acids in the body and an understanding of the way in which this mechanism is controlled, would clearly be of great interest for the problem of the control
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
119
of tumor growth. Since the tumor continues to grow even in the starving animal, it is clear that tumor cells must possess some particular ability in this direction. 3. Cell Suspensions and Cells in Tissue Culture
A. Ascites. Although ascites cells have obvious advantages over other mammalian cells available for the study of protein synthesis, they have one serious disadvantage in that it is difficult to follow the synthesis of a specific protein in such cells. However, this has not prevented some most interesting work being done with ascites cells by Greenberg and his collaborators. If intermediates larger than amino acids play a relatively unimportant role in the synthesis of proteins, then the inhibition of the incorporation of one amino acid would be expected to inhibit the incorporation of other amino acids. On this basis it should be possible to determine whether amino acid incorporation under in vitro conditions represents synthesis of protein de novo or an exchange process. Rabinovitz et al. (1954) therefore studied the incorporation of valine, leucine, and lysine into the protein of Ehrlich ascites cells incubated in vitro under conditions in which the uptake of phenylalanine was severely inhibited by the antagonists o-fluorophenylDL-alanine and @-thienyl-~~-alaniiie.They found that the phenylalanine antagonists inhibited the incorporation of phenylalanine competitively but did not inhibit the incorporation of the other amino acids noncompetitively. As these authors point out in a later paper (Rabinovitz et al., 1956), the effect of an antagonist will depend on whether the antagonist enters the protein in place of its metabolite. It if does, then inhibition of incorporation of the natural amino acid would represent a competition among substrates, and incorporation of other amino acids into the unnatural protein thus formed would not be inhibited. This may well be the explanation of the results with the phenylalanine antagonists. Rather differentresults were obtained by Rabinovitz et al. (1955a) with 0-methylthreonine. This substance inhibited the incorporation of its analog isoleucine competitively but its inhibition of leucine incorporation was noncompetitive; since both inhibitions were relieved by isoleucine, the results indicate that the block in leucine incorporation arose from an inhibition of protein synthesis. Similar results were obtained with methionine sulfoximine (Rabinovitz et al., 1956) which acted as a noncompetitive inhibitor of amino acids other than of its analog glutamine. However, glutamine relieved the inhibition of all the amino acids. The results of these studies on amino acid antagonists are not, therefore, inconsistent with the idea that incorporation of amino acids into the protein of ascites cells incubated in vitro represents protein synthesis de novo.
120
1'. N. CAMPBELL
TABLE VII Oxidation-Linked and Anaerobic Glycolysis-Linked Incorporation of Amino Acids into Protein of Ehrlich Ascites Carcinoma0.b Aerobic
Anaerobic
Without Glucose With Glucose, 0.015M Added Labeled IncorporaAmino Acid OPUptake" tion IncorporaIncorporaCOa con tion tion (MI.) (M mole/g. protein/hr.) Evolved (P mole/g. Evolved (M mole/g. (P'*) protein/hr.) (P1') protein/hr.) Leucine Valine Lysine Phenylalanine Methionine
142 207 167 170 193
6 8 6 2 5.1 2.7 2 0
33 37 33 35 39
0.0 0.0 0.0 0.0 0.0
659 682 65 1 644 690
4.9 4.5 3.4 1.8 1.2
From Rabinovitz e l al., 1955b. The cells were incubated for 1 hour. c Oxidation of endogenous substrate.
S .
6
Rabinovitz et al. (1955b) have also studied the energy requirements for the incorporation of amino acids into ascites cells. They found, as shown in Table VII, that in the absence of glucose, anaerobic conditions completely abolished the incorporation of amino acids. The incorporation appeared to be linked to glycolysis. Incorporation was then studied in the presence of dinitrophenol in order to reduce the energy supply to suboptimal levels. Dinitrophenol inhibited oxidation-linked incorporation almost t o completion in concentrations a t which it stimulates oxygen uptake. It is concluded that dinitrophenol does not inhibit the incorporative process directly but only depletes its required energy supply. Further discussion of the energy requirements for amino acid incorporation must await the detailed discussion of the recent work on incorporation of amino acids into microsomal preparations. Finally, Rabinovitz el al. (1956) found that glutamine enhanced the incorporation of amino acids into ascites cells incubated in vitro. They found that the cells rapidly destroyed glutamine. The glutamine amide bond did not furnish energy for amino acid incorporation under anaerobic conditions. Since glutamine-C14 was rapidly incorporated into the cellular protein, they interpret their results on the basis that glutamine acts as a limitingcomponent amino acid in protein synthesis. (It has been shown that glutamine is incorporated into casein without prior hydrolysis of the amide bond, Barry, 1956; Sheldon-Peters and Barry, 1956.) These results are of interest in connection with the low levels of glutamine found in tumor tissue (Kit and Awapara, 1953). Moreover Roberts and Borges (1955) have
PROTEIN SYNTIIESIS WITH REFERENCE TO GROWTH PROCESSES
121
found that as regression sets in with leukemia tumors in mice, both in the solid and ascites form, free glutamine, whieh is absent from the growing tumor, appears. B. Reliculocytes. Borsook and his colleagues have studied the synthesis of hemoglobulin in isolated leucocytes. In many ways this system meets the requirements of the ideal experimental material for studying protein synthesis. Thus a specific protein is synthesized by cells which can be incubated under controlled conditions. Further, in glycine there is a common precursor for the synthesis of the globin moiety of hemoglobin and the porphyrin. Borsook et al. (1952) produced reticulocytosis in rabbits with phenylhydrazine. After these reticulocytes had been incubated with radioactive amino acids, they were able to isolate radioactive hemoglobin, which suggested that protein synthesis de novo was being studied. However since the results obtained with hemoglobin and the total proteins were essentially the same, they restricted their further studies to the total proteins. They found that the incorporation of radioactivity was greater when the reticulocytes were incubated in plasma than when they were incubated in saline. (A similar result is obtained when ascites cells are incubated in ascitic fluid instead of saline medium; Littlefield and Keller, 1957.) The effect of plasma was not due entirely to its content of amino acids-evidence being obtained that an accelerating nonprotein fraction was involved. Liver seemed to be the best source of this factor. The effect of the amino acid composition of the incubating medium on the rate of incorporation of amino acids was studied. Some of the results are shown in Table VIII. This shows that a limitation of one amino acid limits the rate of the whole process. TABLE VIII Effect of Amino Acid Composition of the Medium on the Incorporation of Amino Acids into Reticulocytes in vitro0-b Labeled Amino Acids Incorporatedb Unlabeled Amino Acids Added Glycine
Histidine
Leucine
Lysine
67 100 46 89 75 95 95 75
77 100
58 100 64
51 100 62
78 98 90 61
91 86 88 85 63
__-
None Complete mixture of amino acids Complete mixture Less histidine Complete mixture Less leucine Complete mixture Less phenylalanine Complete mixture Less tryptophan Complete mixture Less tryosinc Complete mixture Less valine
96 66 92 97 71
~
0
b
From Borsook et al., 1952. Results exprerraed as per cent of value with total amino acid mixture.
122
P. N. CAMPBELL
TABLE IX Partial Reconstitution of Stimulation of Leucine Incorporation into Reticulocytes by Known Constituents of Liver Extract'** Added to Reaction Mixture
None
None Mixture of amino acids Mixture of amino acids, plus O.Bpg.Fe/ml. Mixture of amino acids, plus S.Opg./Fe/ml. 0
b
100 160 210 290
Fructose Amino Acids Liver Extract Alanine Glutamic Glycine 170 350 350 350
100 160 290 300
100 160 260 300
100 160 285 300
From Borsook el al.. 1955. Results expressed an per cent of control.
Later, Borsook et al. (1955) studied the nature of the stimulating factors in the liver extract in greater detail. They found that the presence of iron in the extract had a stimulating effect on the incorporation of amino acids but even more interesting was the effect of fructose-amino acids (l-deoxy-l-amino-2-ketohexoses) . They isolated such substances from liver and also prepared a number of them synthetically. The effect of these on the uptake of leucine is shown in Table IX. Although the addition of iron and fructose-amino acids stimulates the incorporation of leucine, the effect is not quite as great as that of the liver extract. They showed that when fru~tose-C~~-~-leucine replaced 1e~cine-C'~ in the incubation mixture, there was no incorporation of radioactivity. The role of these amino acid derivntives is not yet clear. Later, Kruh and Borsook (1956) attempted to determine whether the synthesis of the globin and heme moiety of hemoglobin went in parallel. Iron, amino acids, fructose-amino acids, and plasma filtrate accelerated both heme synthesis and the incorporation of each of four amino acids into globin. It is argued that heme must have incorporated radioactivity from glycine as a result of synthesis since it could not have become labeled by an exchange process. Since heme synthesis and amino acid incorporation took place a t nearly the same rate, it seems probable that amino acid incorporation was the result of the synthesis of hemoglobin de novo from iron and free amino acids. Finally, Koritz and Chantrenne (1954) have studied the relationship between the ribonucleic acid content of reticulocytes and their ability to incorporate amino acids in in vitro. They found that the peak in the incorporating ability occurred before the ribonucleic acid content of the cells became maximal, but the hemoglobin and enzyme content of the cells became maximal at approximately the same time as that of the ribonucleic acid. C. Other T y p e s of Cell. When attempts are made to prepare suspensions
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
123
of whole cells from normal tissues such preparations, even if the cells appear to have remained intact, usually have very limited biochemical activity. Thus suspensions of liver cells may be prepared by the method of Anderson (1953), but such cells possess properties that differ markedly from those exhibited by liver slices (Longmuir and ap Rees, 1956; Laws and Stickland, 1956; Kalant and Young, 1957). However, suspensions of bone marrow cells may be readily obtained, and Borsook et al. (1950a) have studied the incorporation of radioactive amino acids into such suspensions obtained from rabbits. The amino acids were rapidly incorporated; the process being inhibited by anaerobiosis and a number of metabolic inhibitors. Similar experiments with very similar results were carried out by Farber et al. (1951) and Kit and Greenberg (1951a) using suspensions of the Gardner lymphosarcoma tissue obtained from mice. Kit and Greenberg (1951b) also compared the rate of incorporation of CI4-labeled glycine and alanine into the cell suspensions of the lymphosarcoma and normal spleen. They found no difference between the two types of tissue. In both these experiments and those with bone marrow cells, it would be interesting to determine whether such cell suspensions are able to synthesize antibody. Ranney and London (1951), and Askonas and White (1956) have carried out such experiments with tissue slices. D. Tissue Culture. At first sight systems involving the culture of tissues under in vitro conditions appear to offer many advantages for the study of protein synthesis. However, it is the common experience that the more closely such systems are investigated the less desirable they appear. First, there are the immense technical difficulties to be overcome before sufficient material can be obtained, and, second, there are many difficulties of interpretation mainly due to the long-term nature of the experiments. The experiments of Francis and Winnick (1953) using embryonic chick cultures have already been referred to. Previously, Winnick and his colleagues had studied the uptake of radioactive amino acids by a variety of chick embryonic tissues (Cerarde et al., 1952). They found that growth and protein increase were most rapid with lung tissue cultured in a n extract of chick embryo. The incorporation of amino acids was more rapid under conditions of tissue autolysis than under conditions favorable to growth. 4 . Perfusion
Miller and his colleagues have developed a technique for perfusing rat livers with blood. By adding radioactive amino acids to the blood and following the incorporation of radioactivity into the plasma proteins, it is possible t o study the synthesis of plasma proteins (Miller et al., 1954; Miller and Bale, 1954). Jensen and Tarver (1956), using lysine-CY4, have determined the rate of synthesis of new protein by the perfused liver, on
124
P. N. CAMPBELL
the assumption that incorporation is due to new protein formation, and they found that the rate of synthesis agrees well with the values they obtained from in vivo studies. Burke and Miller (1956) found that the incorporation of L-1y~ine-C’~ into both liver and plasma proteins was two to three times as great when precancerous livers were perfused compared with normal liver. Precancerous liver was obtained from rats which had been fed 3’-methyl-dimethylaminoazobenzenebut which had not quite developed tumors. Miller has applied his technique to the nonhepatic tissues by perfusing the “carcass” of the rat (Miller et al., 1954). He was thus able to show that into the y-globulin fraction of such a preparation incorporated 1y~ine-C’~ the plasma proteins. 5 . Slices and Tissue Suspensions
A. General. As was pointed out previously, attempts to obtain suspensions of whole cells from mammalian tissues which retain the biochemical functions of the original tissue have, for the most part, failed. Thus, while slices and minces have their disadvantages compared with whole cell suspensions, there is, at present, no alternative if the metabolism of most tissues is to be studied at the cellular level. An example of the use of tissue slices for comparing the synthesis of protein in tumors with that of normal tissues is the work of Zamecnik et al. (1948). They compared the rate of incorporation of radioactivity from alanine-C14into the protein of rat liver and liver tumor slices. They found that the uptake of activity into the tumor slices greatly exceeded that into the normal liver slices and was also greater than that into slices prepared from the nonmalignant portion of the tumor-containing livers. The tumors were induced by feeding the rats with 4-dimethylaminoazobenzene. They also compared the incorporation of radioactivity into fetal rat liver and later (Zamecnik and Frantz, 1949) extended their observations to include regenerating liver. A summary of their results is shown in Table X. The results with fetal and regenerating tissue are in accord with similar in wivo experiments (e.g., Schreier et al., 1956), but the higher rate of incorporation of amino acids into the liver tumor compared with the normal liver is not found in in wivo experiments (Zamecnik and Frantz, 1949). I n seeking an explanation for this difference, Zamecnik et aE. (1951) obtained some evidence that the circulation to the tumor in vivo is impaired. It is very difficult to draw conclusions concerning the rate of protein synthesis from studies on the rate of incorporation of amino acids into the proteins of tissue slices. Before such conclusions can be drawn, it is essential to have some knowledge of the radioactivity of the precursor amino acid. For example, if two different tissues are being compared, it is most
PROTEIN SYNTHESIS WITH REFEltENCE TO GROWTH PROCESSES
123
TABLE X Rate of Incorporationof Alanine-C14into Various Types of Hepatic Slices from the Rat*** Tissue
Radioactivity (counts per min.)
Normal liver Control liver tumor Liver tumor Regenerating liver Fetal liver From Zamecnik and Yrantz, 1949. Fetal liver was obtained from Ibday-old letu9e.r Control tumor bearing a liver tumor.
38 91
255 91
178
a
b
IR
the nonmallgnant part of a liver
unlikely that the amount of free amino acid present in the tissues a t the htart of the incubation, or arising from the degradation of tissue protein during the course of the incubation, will be the same in both tissues. The specific radioactivity of the added amino acid will therefore be diluted to a different extent in the two tissues. Unless account is taken of these factors in interpreting the results of such experiments, it is meaningless to compare the performance of slices from different tissues by this method. B. Serum Albumin. A very much more satisfactory approach to the problem of protein synthesis in tissue slices was made by Peters and hnfinsen (1950a,b). They first found that when chick liver slices were incubated in a bicarbonate medium with radioactive amino acids, a protein identical with serum albumin became labeled. Moreover, the radioactivity of the serum albumin was many times greater than that of the mixed tissue proteins. They then used an immunochemical method to determine the amount of serum albumin in the slices a t various times during the incubation. They were thus able to show that there was a net increase in the amount of serum albumin over a period of as long as 4 hours. Table XI shows the effect of various conditions during the incubation on the synthesis of serum albumin. Later Peters (1953) made a more detailed study of the rate of incorporation of amino acids into the serum albumin itnd into tissue proteins. He found that there was a lag in the incorporation of radioactivity into the albumin compared with the tissue proteins. A later report shows that this lag is associated with the release of the albumin from the microsomes (Peters, 1957). The incorporation of radioactivity from g1uc0se-C'~into the serum albumin and tissue proteins during the incubation of chick liver slices was later studied by Campbell (1955). All these results show clearly that liver slices are capable of synthesizing a specific protein, and it seems probable that the demonstration of the incorporation of a radioactive amino acid into a specific protein is a good indication of protein synthesis de novo.
126
P.
S . CAMPBELT,
TABLE XI Effect of Ions and Inhibitors on Net Production of Serum Albumin in Chick Liver Slicesn~* Synthesis (per cent of control)
Condition
0,replaced by NP Cyanide 0.002 M 0.0002 M Azide 0.005 M Arsenite 0.001 dT o.Ooo1 M Iodoacetate 0.001 M 0.0001 M
23 0 53 6 10 27 10 82 2 54 26 25 44 94
Dinitrophenol 0.0005 M 0.0001 M Arsenate 0.01 M 0.001 M Fluoride 0.005 M Malonate 0.01 M a
From Peters and Anfinsen, 1950b.
* The slices were incubated for 4 br. in a bicarbonate medium in 95% 015% COz.
The above studies taken together with the work of Miller et al. (1951) with perfused liver leave no doubt that synthesis of serum albumin is a characteristic of the liver cell. I n view of this, it seemed possible that tumor cells derived from liver cells might retain an ability to synthesize serum albumin. Campbell and Stone (1957a) have, therefore, applied the methods of Peters and Anfineen to the study of protein synthesis in slices of liver tumor obtained from rats which had been fed 4-dimethylaminoazobenzene. A summary of the results obtained is shown in Table XII. The liver tumor slices on an average synthesized 30-50% as much albumin as did a similar weight of liver slices. Parallel experiments were carried out in which the TABLE XI1 Synthesis of Albumin during the Incubation of Slices of Liver from Tumor-Bearing Rats and Slices of Liver TumorsaSb Experiment No.
I I11 VII XI
Liver
Tumor
0 hr.
2 hr.
4 hr.
0 hr.
2 hr.
4 hr.
1.98 1.05 1.50 1.29
2.82(43) 1.87(78) 1.82(21) 2.22(72)
2.77(40) 2.63(150) 1.82(21) 2.34(82)
0.74 0.55 0.41 0.49
1.12(52) 0.74(34) 0.68(66) 0.69(40)
1.35(84) 1.19(115) 0.70(71) 0.88(78)
From Campbell and Stone, 1957% Rats bad been fed 4-dimetbylaminoarobenrene. Results are given in percentage increase is shown in brackets. a
b
mg.
of albumin/g. wet tissue;
127
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
incorporation of g1y~ine-C'~ into the proteins was studied. Incorporation of radioactivity into the serum albumin and tissue proteins took place. The tumors used in the init,ial experimenh were found by histological examination to consist of hepatomas and cholangiomas. Since the latter consist of cells of bile duct origin they would not be expected to synthesize serum albumin. The results summarized in Table XI11 show that compared with the hepatomas, the cholangioma slices synthesized very little serum albumin. As a result of these experiments, a system is now available in which the synthesis of the same specific protein may be studied in slices of normal and tumor tissue. Such systems offer the best opportunity for detecting differences, if they exist, between the way in which proteins are synthesized in normal and tumor tissue. TABLE XI11 Synthesis of Albumin and Incorporation of Radioactivity into Albumin and Soluble Proteins of Liver, Hepatoma, and Cholangiomaa*b Incubation, 4 hours
Incubation, 0 hours Tissue
Radioactivity Wt,. of Albumin TCA (mg.) Albumin Protein
Wt. of Albumin (mg.)
Radioactivity TCA Albumin Protein
~~
Liver Hepatoma Cholangioma
0.82 0.68 0.96
0 0 0
17 11 15
1.11(+35) 1.13(+67) 0.90(-6)
37530 18252 6696
4524 4935 4274
From Campbell and Stone, 1957a. Slices were incubated with glycine-Cl4. Results are given in mg. of albumin/g. wet tissue; percentage change is shown in brackets. Radioactivity ia expressed as counts/min./cm.Z at infinite thickness. a
b
I n these experiments with liver slices, no requirement was found for a complete complement of amino acids to be present in the medium; presumably sufficient amino acids for the synthesis of albumin were obtained by the degradation of tissue proteins. However, if the chickens, from which the liver slices are obt.ained, are first starved for 48 hours, there is no net synthesis of albumin. In this case a mixture of amino acids added to the medium restores the synthesis of albumin (Severina, 1955). C. Amylase. This is another protein the synthesis of which has been extensively studied under in vitro conditions. The synthesis of this enzyme by slices of pigeon pancreas was greatly increased by the addition of a mixture of amino acids to the medium (Hokin, 1951a,b). Instead of slices, whole mouse pancreas may also be used (Hokin, 1956). I n these experiments, the synthesis of protein is followed by measurements of enzyme activity, a procedure which as previously pointed out may give erroneous
128
P. N. CAMPBELL
results. Amylase synthesis in pigeon pancreas slices may be inhibited by p-fluorophenyl-DL-ahnine ; the effect being reversed by DL-phenylalanine (Younathan and Frieden, 1956). D. Antibody. The synthesis of antibody may also be studied in slices of tissue. Thus Askonas and White (1956) and Askonas et al. (1957) have studied the synthesis of antiovalbumin in slices of tissue from guinea pigs immunized with ovalbumin. The lymph nodes, spleen, and bone marrow actively incorporated radioactivity from g1y~ine-C’~ during the period of incubation. In addit.ion to antiovalbumin, which could be precipitated by the addition of ovalbumin, incorporation into the remainder of the 7-globulin and into the insoluble protein was also studied. Table XIV shows that, whereas incorporation into the antiovalbumin and y-globulin ceased when the tissues were damaged by teasing with needles, incorporation into the insoluble protein was unimpaired. These results raise doubts as to the significance of studies of isotope incorporation into undefined tissue proteins. TABLE XIV Effect of Cell Damage on Uptake of Glycine-C14into the Proteins of Lymph Glands from an Immunized Guinea Pigap*
Antiovalbumin y-Globulin Insoluble protein
Sliced Gland
Damaged Cells
Homogenatec
762 266 220
8 22 435
2
0
-
a From Aakonas et al., 1957. *The tksue waa incubated aerobically for 3 hours at 37°C. Radioactivity is expressed in arbitrary figurea as counts/min./mg. protein at infinite thickness. 0 Whole cells and nuclei removed by centril’upation.
E. Insulin. Since so much is now known about the structure of insulin, it is an obvious choice for studies of protein synthesis. Vaughan and Anfinsen (1954) were able to show that radioactive amino acids were incorporated into insulin during the incubation of slices of calf pancreas. After incubation, inactive insulin was added and then re-isolated. Methods for the isolation of insulin on a microscale involving paper chromatography have now been devised by Light and Simpson (1956) and Grodsky and Tarver (1956). The last authors have shown that when slices of fetal beef pancreas were incubated in a bicarbonate medium, there was an increase in the amount of insulin. This increase took place under aerobic but not under anaerobic conditions. Light and Simpson using calf pancreas slices found that leucine-l-C14 in the medium was incorporated into the insulin and that this incorporation was inhibited by concentrations of dinitrophenol which had no effect on respiration. These results suggest that phosphorylation processes are involved directly or indirectly in the incorporation.
I’HOTEIK SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
129
F. Experiments of Anfinsen et al. As was previously mentioned in Section III,5A, Anfinsen and his colleagues have studied the synthesis of several proteins under i n vitro conditions in order to determine whether protein synthesis proceeds by a stepwise mechanism involving free or conjugated peptide intermediates. Thus ovalbumin was studied in coarse minces of chicken oviduct (Anfinsen and Steinberg, 1951; Steinberg and Anfinsen, 1952; Flavin and Anfinsen, 1954), and ribonuclease and insulin in calf pancreas slices (Vaughan and Anfinsen, 1954). I n each case the tissue was incubated with radioactive amino acids and the required protein isolated, with or without addition of carrier, and purified by crystallixation. The main purpose of the experiments was to determine whether the radioactivity of a given amino acid was uniform throughout the polypeptide chain. The radioactive proteins were, therefore, partially hydrolyzed, peptides isolated, and the radioactivity of the amino acid determined in each peptide. I n every case they found that the proteins were not uniformly labeled. It is not intended to discuss here the interpretation of these results regarding intermediates in protein synthesis, but they do raise another question. Gale and Folkes (1955) have shown that, when disrupted staphylococcal cells are incubated with glutamic acid containing C14, in the absence of other amino acids some of the glutamic acid residues in the protein exchange with the C14-glutamicacid. This exchange process requires energy but is not indicative of protein synthesis de novo. Since this exchange involves only some of the glutamic acid residues in the protein, it would be expected to lead to nonuniform labeling of the glutamic acid in the protein. The question arises, therefore, as to whether the results of Anfinsen are due to such a n exchange mechanism operating in slices and minces. Although in Anfinsen’s experiments, no evidence of the net synthesis of a specific protein was demonstrated, it seems probable that, a t least in the case of insulin, such a net synthesis did take place (Grodsky and Tarver, 1956). It might be possible to choose between the two alternatives of peptide intermediates and exchange if the change in the radioactivity of the free amino acids were to be determined during the incubation of the tissue preparations. Thus, if the extent of unequal labeling of the protein-bound amino acids exceeded the change in radioactivity of the free amino acids, an exchange mechanism would appear to be the most likely explanation of the results. TOsum up the experiments described involving tissue slices and minces, it seems reasonably certain that such preparations are capable of synthesizing complete proteins. When radioactive amino acids are used and specific proteins are isolatted, the evidence suggests that incorporation of radioactivity is a good measure of protein synthesis. If, however, specific
130
P. N. CAMPBELL
proteins are not studied, then incorporation studies are more difficult to interpret. 6. Eggs, Larvae, etc.
A review concerned with protein synthesis and growth should certainly deal with the large amount of work which has been carried out on the synthesis of proteins in eggs, larvae, and various small organisms. However, since space does not permit a complete review a brief survey will have to suffice. Experiments employing eggs and larvae have been directed to three main objectives: (1) The mechanism whereby the protein stored in the egg is utilized for the synthesis of the specific proteins required by the embryo; (2) the role of proteolytic enzymes in protein synthesis; and (3) the role of the nucleic acids in protein synthesis. Mention has already been made of the work of Ebert (1954) and Francis and Winnick (1953) on the utilization of proteins b y the chick embryo in grafts and tissue culture. Flickinger and Rounds (1956) have made an in vivo study in which hens were injected with Na2HPa20r,and the radioactivity of the phosphoproteins of the liver, blood, and egg yolk was determined. The results shown in Table XV indicate that the radioTABLE XV Incorporation of Radioactive Phosphorus into Phosphoproteins of Liver, Blood, and Egg Yolk in Laying Hcnse’b Time after Injection Organ
a b
6 hr.
12 hr.
184 38 0.G
101 45 4
From Flickinger and Rounds, 1958. 100 pc. of P8*was injected. Activity is expressed a8 counts per min. per pg. of phosphate.
activity of the egg yolk phosphoprotein was very much smaller than that of the liver and blood. They, therefore, conclude that the phosphoproteins of the egg are maternally synthesized. Using immunochemical techniques, they came t o a similar conclusion regarding phosphoprotein synthesis in the frog’s egg. Kavanau (1954) has determined the changes in free amino acids, peptides, and proteins that take place in the early development of the sea urchin Paracentrotus lividus. He found that the yolk-protein degradation and the synthesis of new embryonic proteins took place a t more or less
PROTEIN SYXTHESIS WITH REFERENCE TO GROWTH PROCESSES
131
different times so that the changes in the protein and nonprotein amino acids occurred in complementary cyclic waves. Hultin (1952) has followed the incorporation of N15-labeled glycine and alanine into developing sea urchin eggs. Autoradiography has been used by Sirlin and Waddington (1956) to determine the site of protein synthesis in the early chick embryo. Using labeled methionine, they found three loci of intense protein metabolism: the cytoplasm, nucleolus, and the chromatin associated with the nucleolus. The nucleolus was also found to be the site of most rapid incorporation of phenylalanine-C14 when developing Asterias oocytes were incubated with this amino acid (Errera and Ficq, 1955). Now that very sensitive methods are available for autoradiography and amino acids labeled with S35or C14 can be used, great developments in the use of this technique may be expected. Weber and his colleagues have used the regeneding tail of the Xenopus larvae to study the correlation between proteolytic enzymes and protein synthesis (Jensen et al., 1956). The interest in this work is that it is thought, by some that these enzymes play a role in protein synthesis by catalyzing transpeptidat*ion reactions. These authors found that catheptic activity increased after amputation and that this increase was unaffected by agents which retarded regeneration. They think that catheptic activity is more likely to be concerned with the breakdown than with the synthesis of proteins. The changes in the individual amino acids in embryos of Xenopus larvae which are under development have been studied by Deuchar (1955). Chen (1956) has carried out similar studies with embryos and larvae of Alpine newts. Brachet (1955) has recently reviewed the role of nucleic acid in morphogenesis and protein synthesis. Brachet himself has carried out some most interesting experiments on the inhibition of protein synthesis by ribonuclease in living root tips and ameba (Brachet, 1956a,b).
V. INCORPORATION OF AMINOACIDSINTO SUBCELLULAR PARTICLES 1. Characterization o j Subcellular Constituents
In the first section of this review, it was shown that the specificity of protein structure is such that it seems probable that each type of cell possesses mechanisms for controlling the structure of the proteins synthesized-by it. It is natural, therefore, to look for some organization within the cell which is responsible for controlling the structure of the synthesized protein. Claude (1946a,b) was the first to devise a scheme for the separation of cell components by differential centrifugation. The main fractions
132
P. N. CAMPBELL
thus obtained have been labeled nuclei, mitochondria, microsomes, and soluble protein (Hogeboom el al., 1948). De Duve and Berthet (1954) have most excellently discussed the value of such fractionations and pointed out the difficulties which may arise. For a long time such preparations of cell components had to remain as cytochemical concepts, for there was no microscopic method available for their study. However, more recently the development of the electron microscopy of ultrathin sections has enabled the structure of the cell t o be studied
FIG.4. The figure shows an array of nine elongated profiles (el, es) of the rough surfaced variety and five mitochondria1 profiles (m) in the cytoplasma of a parenchymatous liver cell. The elongated profiles are disposed parallel t o one another a t more or less regular intervals. In three dimensions, the array corresponds to a pile of cisternae. The outside surface of the membrane limiting these profiles bears numerous attached particles of small size and high density. A few similar particles appear freely scattered in the intervening cytoplasm. The arrows indicate continuity between rough surfaced and smooth surfaced profiles. X49,OOO (from Palade and Siekevitz, 1956a).
in greater detail. The problem now is to correlate the nature of the particles isolated by the ultracentrifuge with the structures revealed by electron microscopy. Progress has recently been made in this direction by Palade and Siekevitz (1956a) who have studied the microsome fraction from rat liver. One of their plates of a parenchymatous liver cell is shown in Fig. 4. It will be seen that the cytoplasm contains, in addition to the mitochondria, endoplasmic reticula with attached electron dense particles. Such struc-
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
133
tures are described as rough endoplasmic reticulum to distinguish them from the smooth endoplasmic reticulum, also known as the Golgi apparatus. The cytoplasm also contains another type of electron dense particle which is freely scattered. (For a better understanding of the structure of the endoplasmic reticulum, the reader is referred to an earlier paper by Palay and Palade, 1955, and also a review by Palade, 1956.) According to Palade and Siekevitz (1956a), the microsome fraction obtained by ultracentrifugation of liver homogenates is identical with the rough surfaced elements of the endoplasmic reticula. This fraction may also contain the freely scattered dense particles and some of the smooth surfaced endoplasmic reticula. Treatment of the microsomes with ribonuclease results in appreciable losses of ribonucleic acid and in partial or total disappearance of the attached particles. On the other hand, treatment with deoxycholate produces a partial clarification of the microsome suspensions and on centrifugation yields a small pellet of electron dense particles containing a high proportion of ribonucleic acid. Thus microsomal ribonucleic acid appears to be associated with the small particles of the rough endoplasmic reticula, whereas most of the protein and nearly all the phospholipid, hemochromagen, and DPNH-cytochrome-c reductase activity, is associated with the membranes. Kuff et al. (1956) have also examined the fractions obtained from rat liver homogenates by electron microscopy. Similar studies have been made by Watson and Siekevitz (1956) and Siekevitz and Watson (1956) on rat liver mitochondria and by Palade and Siekevitz (195613) on guinea pig pancreatic microsomes. Reid (1955) has shown that pituitary and adrenal hormones can influence the yield and composition of the fractions derived from liver cytoplasm. Parallel with these studies with the electron microscope, has been the work of Petermann and her colleagues using analytical ultracentrifugation and electrophoretic analysis. When cytoplasmic extracts of mouse spleen were prepared by centrifugation in 0.88 M sucrose, a number of well-defined boundaries were seen in the ultracentrifuge (Petermann and Hamilton, 1952). For convenience these were called components A, B, C, D, and E respectively. Similar components have been observed in extracts of liver, pancreas, and several tumors (Petermann et al., 1953). Electrophoresis of the extracts gave four peaks which are tentatively identified with the components observed in the ultracentrifuge (Petermann et al., 1954). They were thus able to show that component B contains equal amounts of ribonucleic acid and protein and is unaffected by treatment with deoxycholate; it probably corresponds therefore to the electron dense particles of Palade and Siekevitz (1956a). In the Jensen sarcoma of the rat, rat liver tumors, Ehrlich ascites tumor of the mouse, and regenerahg rat liver, component
134
P. N. CAMPBELL
C is particularly prominent, and so it is suggested that this component is connected in some way with cell division (Petermann, 1954). Later the livers of pregnant rats were also found to cont,ain elevated levels of component C (Petermann et aE., 1956). Attempts to purify nucleoproteins have been foiled by the extreme instability of these substances. However, some of the difficulties have been overcome (Petermann and Hamilton, 1955), and the purification of a nucleoprotein from rat liver in which the principal component is B has now been described (Petermann and Hamilton, 1957). 2. Incorporation in vivo
It has already been shown that there is a reasonable correlation between the rate of protein synthesis in a tissue of the body and the rate at which the proteins of that tissue become labeled after the injection of a radioactive amino acid. Such studies have now been taken further and applied to the rate at which the subcellular constituents become labeled. Solid curve
Dotted curve
1
Cell fluid
0.4 1
(TCA
sol. N fluid Protein N
La
9
- 4.0
Microsomes Protein N
?''-ra's
0.3
- 3.0
0.2
- 2.0
0
n
=
-
& 0.1 n 0
I
2
3
4
5
1.0
-0
Time after injection (hr.)
FIQ.5. Isotope concentrations in different chick liver fractions found at vanom timen after the intravenous administration of glycine-N" (5 mg. N, 61% N16 excess) (from Hultin, 1950).
Thus Hultin (1950) injected glycine-N" intravenously into young chicks, killed the animals at times varying from 10 minutes to 5 hours, and fractionated homogenates of the livers by differential centrifugation. Figure 5 shows the results he obtained when he determined the NI6 content of the protein in the fractions. It will be seen that the incorporation was most rapid in the microsomes and slowest in the nuclei. Similar results were obtained by Borsook et al. (1950b) using C14-amino acids in guinea pigs. In mouse pancreas and kidney, the rate of uptake of glycine-N16into the proteins of the microsome fraction is also greater than into the other
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
135
cell fractions (Allfrey et al., 1953). By comparing the uptake of isotope in liver, kidney, and pancreas, they showed that there was a correlation between the incorporation of isotope into the whole tissue protein and into the microsomal protein. Finally, Keller et al. (1954) injected various amounts of radioactive amino acid into rats and studied the incorporation into the subcellular fractions of the liver during the first few minutes after injection. They found a straight-line rate of incorporation of amino acid into the microsome fraction which greatly exceeded the incorporation into the other fractions.
c
46
0
5
10
15
20
25
Time (rnin.)
FIO.6. Incorporation of a small dose of leucine-C" into the two components of the microsomes and into the soluble protein of the cell after intravenous injection into the rat. The per cent RNA by weight of each deoxycholate-insoluble sample is indicated. The per cent RNA averaged 2.1 in the deoxycholate-soluble fractions of the microsomes and 1.7 in the soluble fractions of the cell (from Littlefield et al., 1955). The apparent importance of the microsome fraction in protein synthesis has led to a closer examination of the incorporation of amino acid into this fraction. Allfrey et al. (1955) extended their experiments with mouse pancreas. If the microsomes obtained a t a time after injection (1.5 hours) when the incorporation was maximal were treated with ribonuclease, a protein was released which contained twice the concentration of N16found in whole microsome protein. Littlefield et al. (1955) obtained a fraction rich in the electron dense particles of Palade and Siekevitz (1956a) from the livers of rats, which had received le~cine-c'~,by treating the microsomes with deoxycholate. They found that the incorporation of radioactivity was more rapid in these particles than in the other fractions and that the maximum
136
P. N. CAMPBELL
activity was reached only a few minutes after injection. The results are shown in Fig. 6. Since the particles contained approximately equal quantities of ribonucleic acid and protein and contained most of the ribonucleic acid of the cytoplasm, they are generally referred t.0 as the ribonucleoprotein fraction. Hultin (1955) used various salt solutions to fractionat,e the microsomes from the livers of chicken and rats which had received C14-aminoacids and again found the greatest incorporation into the fractions containing the major part of the ribonucleoprotein. Recently Hultin (1957) has given further details of the extraction procedures employed. Simkin has also fractionated guinea pig microsomes and obtained similar results (Simkin, 1955; Simkin and Work, 1957) after injecting the animals with a complete mixture of C14-aminoacids. As a warning that. the microsomes in all tissues may not play the same role in the incorporation of amino acids, Simpson and McClean (1955) found that when rats were injected with ~~-leucine-l-C~*, the rate of incorporation of radioactivity in the mitochondria1 fraction of the muscle was not appreciably lower than into the microsomes. At very short time intervals, the mitochondria were actually somewhat more active than the microsomes. Khesin (1954) determined the radioactivity of the serum albumin in the subcellular fractions of rats which had received methionine-Sa6and found that the mitochondria contained the albumin with the highest radioactivity. This may indicate that the microsomes are not the site where the protein takes on its final configuration. It may be concluded from these experiments in the intact animal that the initial site of incorporation of amino acid in many tissues is in the microsomes and that the ribonucleoprotein particles in the rough endoplasmic reticulum are particularly associated with this activity. However, there are suggestions that it may be premature to generalize too far about all tissues until more evidence has accumulated. 3. Incorporation into Whole Cells
A . Ascites and Reticulocytes. If mice bearing Krebs I1 ascites tumor cells are injected with g1y~ine-C'~ and the distribution of radioactivity in the subcellular fractions is determined, the microsomes are again found to be the most active fraction (Bennette et al., 1957). The results obtained six days after implantation of the tumor are shown in Fig. 7. It may be observed that compared with liver the nuclear fraction is particularly active. If similar ascites cells are incubated i n vitro with g1y~ine-C'~ in a bicarbonate medium, the initial site of incorporation is the microsomes but if the incubation is continued, the nuclear fraction then becomes the most active fraction (Campbell et al., 1957). These results are shown in
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
137
Fig. 8a and 6. Thus the nuclear fraction from the ascites tumor appears to be relatively more important than in t3hecase of liver. 200-
1
Microsoms
In
f
.L
.-
Nuclei Mitochondrio Supernatont
f
-.-
..0)
c C
c
f
0
g
N
100-
tE
.\
e
0
15
30
Time after injection (min.)
FIG.7. Intracellular distribution of radioactivity in Krebs I1 ascites tumor cells after injection of the mouse bearing the cells with glycine-C14. The injection took place 6 days after implanting the cells in the mouse. The cells were disrupted by ultrasonic disintegration, and the components fractionated in a sucrose medium (from Rennette el al., 1957). a
Y)
0)
b
5 80
zz
Nuclei
0)
c .-
2 .-
60
L
0
"E
2 .-c
40
E
\
i
:
t .> .c
20
0
.-
0
0 a
0
5
10
0
30
40
50
Time of incubation (min.)
FIG.8a and b. Intracellular distribution of radioactivity in Krebs I1 ascites tumor cells after incubation in vitro with glycine-C14. The washed cells were incubated in a bicarbonate medium under aerobic conditions a t 37°C. After incubation, the cells were disrupted by ultrasonic disintegration and the components fractionated in a sucrose medium (from Campbell el al., 1957).
138
P. N. CAMPBELL
Littlefield and Keller (1957) have followed the incorporation of L-Valine-C14 into Ehrlich ascites cells incubated in fortified ascitic fluid. After lysis of the cells, the microsome fraction was treated either with deoxycholate or sodium chloride. The results obtained are shown in Fig. 9. As with liver, this shows that the ribonucleoprotein particles are the initial site of incorporation. When reticulocytes are incubated with ~-leucine-C’~, the initial incorporation of radioactivity is into the microsomal protein (Rabinovitz and Olson, 1956). The microsomes had a content of 20% ribonucleic acid. After treatment with deoxycholate, the residue had a content of 30% ribonucleic acid and the protein contained more radioactivity than that of the untreated microsomes.
r
Deoxycholate- sol.
6 50E
0
5
10
15
20
25
30
Time of incubation (min.1
FIQ.9. Time curve of incorporationof ~-valine-Cl~ into proteins of ascites tumor cells incubated in aacitic fluid fortified with glucose. The curve for the “pH5 enzyme” fraction was almost. identical with that of the whole cell. The per cent RNA of the deoxycholate-insoluble particles is indicated on the chart for each sample. The per cent RNA averaged 8 for the whole cells, 4 for the “pH5 enzyme,” and 11 for the deoxycholatesoluble fraction of the microsomes (from Littlefield and Keller, 1957).
B. Slices and Brei. When rat liver slices are incubated in a bicarbonate medium under aerobic conditions in the presence of glycine-C14,the most active cell fraction is associated with the microsomes, as under in vivo conditions (Campbell et al., 1957). Typical results are shown in Fig. 10. If regenerating liver taken 40 hours after partial hepatectomy, when the rate of protein synthesis is maximal, is used, the incorporation into all the fractions is greatly enchanced compared with the normal liver as shown in Fig. 10. If, however, slices of liver tumor are incubated under the same conditions, the incorporation into the microsomes, supernatant fraction, and mitochondria is similar to normal liver; but the incorporat.ioninto the nuclear fractions of the tumor greatly exceeds that into the same fraction
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
139
of normal liver. Therefore as with ascites, the nuclear fraction appears to be relatively more important in the tumor than normal tissue. Since the incorporation into the nuclear fraction is not preferentially enhanced in the regenerating liver, this effect does not appear to be associated with growth. Hendler (1956) fractionated coarse minces of hen oviduct which had been incubated in a medium containing radioactive bicarbonate. He found a
In
b
0)
Normol Liver
x
.-
5
Microsomes
Liver Tumor Microsomes
160
120 Mitochondria I00 80
._ E
60
0
40
+
.-
0
Nuclei
10
30
50
40 20
0
10
30
50
U
Time of incubotion (min.)
lx
t 600u ._
Regeneroting Liver
f
2 500-
.a
c .-
>
0
10
20 Time of lncubatlon ( m i d
60
FIG.10. Intracellular distribution of radioactivity in tissue slices after incubation in a medium containing glycine-Cl*. The liver tumor (b) was obtained from rats fed 4 dimethylaminoazobenzene and the regenerating liver (c) from rats 40 hr. after partial hepatectomy. The slices were incubated in a bicarbonate medium under aerobic;conditions at 37°C. (from Campbell et al., 1957).
140
1'.
ti. CAMPBXLL
that, when the usual method of fractionation was applied, the most active fraction centrifuged down with the cell debris. This fraction, contrary t.0 that found in other tissues, contains most of the ribonucleic acid and light and electron microscopy has shown it to contain the microsomes (Hendler, 1957). Thus under these conditions, the initial site of incorporation of radioactivity is also the ribonucleoprotein rich fraction. On the other hand when rat pituitary glands are incubated in a medium containing methionineSas, the most active fraction after 1 and 2 hours incubation was the supernatant followed by the microsomes and then the mitochondria (Ziegler and Melchior, 1956). It seems possible that if shorter incubation times were used the microsomes might then be the most active fraction. I n a previous discussion, it was concluded that incorporation of radioactive amino acids into specific proteins was a good indication of de novo protein synthesis under in vitro conditions. From the experiments just described, i t will be clear that the distribution of radioactivity in the subcellular constituents under these in vitro conditions is very similar to that taking place in vivo. These experiments, therefore, support the idea that protein synthesis can be studied'under in vitro conditions. I t now remains to be seen what can be learned about. this process from incorporation studies in subcellular fractions.
4. Incorporation into Subcellular Fractions A. Hornoyenates. The first report of the incorporation of a radioactive amino acid int.0 a tissue homogenate was by Winnick et al. (1948). This showed that g 1 y ~ i n e - Cwas ~ ~ incorporated into homogenates of rat tissue under aerobic conditions and that incorporation appeared to be enzymatic in nature. Under these conditions, the incorporation of amino acids was faster with homogenates of fetal rat liver and mouse mammary carcinoma than with adult rat or mouse liver (Winnick, 1950). Our knowledge of the mechanism of the incorporation of amino acids by homogenates was advanced considerably by the work of Siekevitz (1952). He found that when rat liver homogenates were incubated aerobically with alanine-C14, the addition of a-ketoglutarate greatly increased the uptake of labeled alanine into the protein. The distribution of radioactivity into the subcellular constituents is shown in Fig. 11. If each cell fraction was incubated alone, there was no uptake of radioactivity. However, when the microsomal fraction was added to the mitochondria1 fraction, uptake occurred. It was also found that factors necessary for the optimal oxidation of a-ketoglutarate (Le., adenosine-5-phosphate1 MgC12, and phosphate) were necessary for optimal uptake of alanine. Since the presence of dinitrophenol reduced incorporation, it appeared that the uptake of alanine was coupled with phosphorylation and not oxidation per se.
PROTElN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
141
As the result of such experiments, it became clear that the incorporation of amino acids into the protein of homogenized tissues depended on an energy source and appeared to be enzymatic. Moreover, since the requirements were similar to those found for incorporation into the protein of tissue slices, it was tempting to consider the process as being a step in protein synthesis. However, since the homogenates possessed very much less activity for incorporation than the slices, objections were raised that incorporation of radioactivity represented adsorption or coupling of the amino acids with protein by bonds other than peptide. Such objections were gradually overruled, but so far the synthesis of a specific protein has not been demonstrated in a cell-free system. 120 105
d
tEE
.-
> c 0
90
Microsomes
75
6ol /
45
Mitochondria
15
0
10
20
30
40
Time of incubation ( m i n )
FIG.11. Incorporation of radioactivity into the proteins of the various fractions of a rat liver homogenate. The homogenate was incubated under aerobic conditions in the
a-ketoglutarate and adenosine-5-phosphate (from Siekevitz, presence of ~~-alanine-l-C14, 1952)
On the basis that the incorporation of radioactive amino acids represents a step in the path of the synthesis of protein, the results obtained with isolated fractions of subcellular constituents will now be reviewed. B. Nuclei. Although the incorporaOion of radioactivity into the nuclear fraction of liver homogenates is very small, this may be due to damage to the nuclei during the process of homogenization. Thus Stern and Mirsky (1953) in a comparison of the nuclei of calf thymus and liver and of rat liver found t ha t there was a much greater loss of protein from the liver than from the thymus nuclei. When such nuclei were incubated with
142
1’. N. CAMPBELL
alanine-C14 and a-ketoglutarate, there was quite a subst,antial incorporation of alanine into the nuclear protein (Allfrey, 1954). These studies on isolated nuclei have been extended by Allfrey et al. (1957). I n particular the role of deoxyribonucleic acid has been studied, and the nuclear proteins have been fractionated. The quantitatively small protein fraction which is strongly bound to deoxyribonucleic acid was the most active fraction. The role of the nucleus in protein synthesis has recently been reviewed by Brachet and Chantrenne (1956). There seems no doubt that under certain conditions isolated nuclei can actively incorporate amino acids. C. Microsomes. I n view of the rapid uptake of radioactive amino acids into the microsome fraction of liver tissue both in vivo and in vitro, the immediate objective was to find conditions under which incorporation would t’ake place in isolated suspensions of microsomes. Siekevitz (1952) had shown that microsomes plus mitochondria were effective. Zamecnik and Keller (1954) next showed that the mitochondria could be replaced by the presence of ATP plus a substrate such as phosphoenolpyruvate (PEP) which would maintain the level of ATP. Under these conditions, aerobic conditions were no longer necessary. Nevertheless, in order for incorporation into microsomes to take place, a soluble, heat labile, nondialyzable fraction from the supernatant of the liver homogenate (known as the cell sap) was required. The activity of this fraction was present in a precipitate obtained by lowering the pH to 5.2 (called the “pH5 enzyme”) provided GDP or GTP were added (Hoagland el al., 1956). A summary of the fractionations described up to this point is shown in Fig. 12. If supernatant A is incubated at 37°C. with radioactive amino acid plus ATP plus PEP, incorporation into the protein precipitated with trichloroacetic acid takes place. If, however, the microsome pellet is mixed with a small amount of cell sap, i.e., the concentration of microsomes in the medium is increased, the efficiency of the incorporation increases. I n either case if the microsomes are separated from the medium after incubation, the activity in the microsomal protein is found to be many times greater than that of the cell sap protein. In favor of the view that the incorporation of amino acids into isolated microsomes is connected with protein synthesis in vivo are the results obtained with deoxycholate. When C14-amino acid-containing microsomes were treated with deoxycholate, the activity of the insoluble fraction was up to twenty-two times that of the soluble fract.ion (Littlefield el al., 1955). Against the view are the results of Simkin (1957) who found that when the C“-amino acid-containing microsomes were fractionated by salt extraction, the pattern of incorporation was different under in vivo and in vitro conditions. If the relative rates of incorporation of amino acids into the microsomal
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
143
Ti iue Homogenize in wcrose Spin 12,OOO g
I
Cell debris Nuclei Mitochondria
Supernatant A
Spin 105,000 g
Rlicrosome pellet Treat with deoxycholate
1
Insoluble (ribonucleoprotein)
Soluble
pH 5.2 spin 12,000 g
1
Supernatant B
“pH5 enzyme”
FIG.12. Fractionation of subcellular particles.
fraction of tissue slices and isolated microsomes are compared, conflicting results are again obtained. Thus, from the experiments with slices, microsomes from regenerating liver would be expected to incorporate amino acids more rapidly than microsomes from normal liver. That this is so is shown in Table XVI (Campbell and Greengard, 1957). On the other hand, whereas normal liver and liver tumor microsomes had similar activities in slices, the tumor microsomes have only very low activity when isolated. Littlefield and Keller (1957) also found that the activity of microsomes from ascites was less than those from liver, whereas the reverse was true under in vivo conditions. Prom ultracentrifugal studies, they found that component C of Petermann was rapidly lost on incubating the microsomes, and this may account for the lower activity of the ascites mkrosomes. They also found that, whereas the ribonucleoprotein particles from liver obtained by treating the microsomes with deoxycholate failed to incorporate amino acid on incubation, similar particles from ascites microsomes did so.
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P. N. CAMPBELL
TABLE XVI Incorporation of Glycine-C14into Microsome Preparations from Normal Rn.t Liver, Livcr Tumor, and Regenerating Liver"** Normal liver Liver tumor Regenerating liver
Microsomes Cell sap Microsomes Cell sap Microsomes Cell sap
Radioactivity of proteinc
+- +- -- + - + - +
+
10
69
236
+ + -34
+ + -10
++ -
+ --
-
+
135 110
From Campbell and Greengard, 1957. Similar amounts of the microsome pellet suspended in cell ERP from the three types of tissue were incubated for 50 min. at 37°C. with glycine-C", ATP, and phosphoenolpyruvicacid. * Radioaotivity is expressed ae counts per min. at infinite thickness. 4
b
In their work with isolated liver microsomes, Zamecnik and Keller (1954) found that the incorporation of a radioactive amino acid was unin-
fluenced by the presence of a full complement of amino acids in the medium. This is a rather surprising result., for the amount of free amino acid in the microsomes would not be expected to be very large and if complete polypeptide chains were undergoing synthesis, a requirement for additional amino acids would be expected. Recently Zamecnik and his colleagues have made two important discoveries concerning the mechanism of the incorporation of amino acids into
Ad 0-
I HO-PII 0
0-
0
0
- IIPI - 0 - P I 0
I
9-
6
II 0
0-
R I HC-NH:
I
HO-P-0-P-0'
I
-O-i
0
0%-
C,
H~-NH:
I : I P - 0 AC 4 \ II
0
0-
0
FIG.13. Schematic representation of amino acid carboxyl activation by ATP and the "pH5 enzyme". Ad = adenosine. 0 indicates the attacking carboxyl oxygen which would remain with the nucleotide moiety upon subsequent splitting of the activated compound (dash line) (from Hoagland et al., 1956).
PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES
145
liver microsomes. First, they found that i n tlhe presence of “pH5 enzyme” the amino acids were activated before being incorporated into the microsomes (Hoagland et al., 1956). The reaction in which ATP, amino acid, and enzyme take part is illustrated in Fig. 13. The evidence for the activation reaction is mainly based on two observations. First, if radioactive pryophosphate is added to the system, there is an exchange of P32with ATP. Secondly, in the presence of high concentrations of hydroxylamine, hydroxamic acids and pyrophosphate accumulate. Since the “pH5 enzyme” is needed both for the activation step and for the incorporation of amino acids into microsomes, it seemed likely that amino acid activation was a step in the overall incorporation reaction. That this is so is made more likely by the fact that hydroxylamine inhibits the incorporation of amino acids into the microsomes. The degree of inhibition roughly parallels the aminohydroxamic acid formation with increasing concentration of hydroxylamine. Evidence that such an amino acid activation occurs with other biological systems is accumulating. Thus Littlefield and Keller (1957) have found it in ascites, and Cole et al. (1957) have tested a large number of tissue extracts from different animals and found it in many of them. They found the best source was guinea pig pancreas. DeMoss and Novelli (1956) have also demonstrated the presence of amino acid activation systems in many microorganisms and, moreover, have shown that leucyl adenylate behaves like an intermediary in this reaction (DeMoss et al. , 1956). Scarano and Maggio (1957) have found the system in unfertilized sea urchin eggs. The question arises as to whether there is a different enzyme responsible for the activation of each individual amino acid or whether one enzyme catalyzes the activation of all amino acids. Preliminary fractionation of the “pH5 enzyme” suggested that the former was the case (Hoagland et al., 1956). The isolation of an enzyme specific for tryptophan by Davie et al. (1956) and Cole et al. (1957) also suggests that there is one enzyme for each amino acid. If the kinetics of the incorporation of radioactive amino acids into the protein of the isolated liver microsomes is studied, evidence is obtained of a two-step reaction (Hultin, 1956; Hultin and Beskow, 1956). The explanation of this finding is provided by Hoagland el al. (1957) and concerns the presence in the “pH5 enzyme” fraction of ribonucleoprotein. This is a low molecular weight substance which contains only a very small quantity of protein relative to ribonucleic acid. It therefore differs from the ribonucleoprotein of the microsomes and may be denoted S-RNA. It has now been found that when 1e~cine-C’~ is incubated with ATP and “pH5 enzyme,” the S-RNA rapidly becomes labeled. The amino acid linkage is acid stable, but alkali labile. However, when this labeled S-RNA is incubated
140
P. N. CAMPBELL
with hydroxylamine, leucine hydroxamic acid is formed. Moreover, if the labeled S-RNA is incubated with microsomes, the label is transferred to the microsomes provided GTP is present. The incorporation of amino acid into the S-RNA, unlike the amino acid activation reaction, is sensitive to ribonuclease. The results on which these observations are based are shown in Table XVII. TABLE XVII Transfer of Leucine-C14from Prelabeled Activating Enzyme Fraction to Microsomal Proteina Total Counts in RNA Protein Complete system (before incubation) Complete system (after incubation) Complete system, minus GTP* Complete system, minus generating system Complete system, minus both GTPband generating system Complete system, minus generating system but with 5 X GTPb Complete system, CTPb replacing GTPb Complete system, UTPb replacing GTPb Complete system, plus 0.005 M C12-leucine a
489 180 111 72 23 145 96 101 183
30 374 40 155 30 129 44 53 314
From Hoagland el al., 1957
* ATP, GTP, CTP, UTP are the triphosphates of adenosine, guanosine, cytidine, and uridine respectively.
That the S-RNA plays an important role under i n vivo conditions is shown by results obtained with ascites tumor. I n these experiments, in which ascites cells were labeled i n vivo, the S-RNA fraction became labeled more rapidly than the ribonucleoprotein particles of the microsomes and fulfilled the requirements of a precursor. Sachs and Waelsch (1956) previously showed that if labeled microsomes were incubated with pyrophosphate, some of the microsome fraction was solubilized, and this fraction has a very much higher radioactivity than the untreated microsomes. These two findings of Zamecnik and his colleagues suggest, therefore, that the incorporation of amino acids into microsomes takes place in three steps as summarized in Fig. 14. One further aspect of these results is of interest, for it suggests a possible link between nucleic acid and protein synthesis. If ATP-C14 is incubated with “pH5 enzyme,’’ then the S-RNA becomes labeled (Zamecnik et aZ., 1957). This reaction is sensitive to ribonuclease and may be connected with the finding of Holley (1957) that a fraction obtained from “pH5 enzyme” catalyzes the conversion of adenylic acid-C14to ATP-C14 in the presence of alanine.
PROTEIN SYNTHESIS WITH I~EFERENCE TO GROWTH PROCESSES
Amino acid
I
147
+ enzyme + ATP Activation Stage
Amino acid-AMPenzyme I
+ PP
.1
Amino acid-AMP-(SRNA)
Ribonucleoprokin particle in microsomes
FIG.14. Steps in the incorporation of amino acids into liver microsomes.
VI. CONCLUSIONS Owing to the intriguing nature of the subject of the biosynthesis of proteins, there has been a tendency in the past to expect progress to be made on a rather dramatic scale, and for the lack of such progress to be counted as a failure. An attempt has been made in this review to assemble the known facts concerning protein synthesis within the limitations set out in the introduction. It is hoped that it will be apparent that steady progress has been made to elucidate the problems involved. In particular, much is now known about the struct.ure of proteins and the ways in which protein synthesis may be followed; both aspects of the subject which are essential preliminaries to an understanding of the way in which proteins are synthesized. Both in carbohydrate metabolism and fat metabolism, the most rapid advances had to await the development of methods involving cell-free systems. This knowledge has acted as an incentive to develop similar methods for the study of protein synthesis, but so far it has not been possible to demonstrate the synthesis of a specific protein in a cell-free system. It is, a t least, possible th at the integrity of the whole cell is required for such a synthesis. If this is so, then progress will indeed be difficult. It seems more likely that many subcellular components play a part in the synthesis of a complete protein. The isolation of such components and the understanding of their function will require the close collaboration of the electron microscopist and the biochemist. It is encouraging that progress in this direction has recently been reported by Palade and Siekevitz and others.
148
P. N. CAMPBELL
I n the meantime Zamecnik and his group have shown that even if the synthesis of a complete protein cannot be demonstrated in cell-free systems, the incorporation of amino acids into proteins may be studied under these conditions. To what extent this incorporation is connected with protein synthesis is a debatable point, but the correlation between the cell-free systems, in vitro systems (slices, etc.), and in viz~ostudies is sufficiently close t o suggest that steps in the synthesis of complete protlein are being studied. Among other things, the work of Zamecnik has clearly shown that there are various types of ribonucleic acid in the cell with different biochemical functions. This may explain why many of the attempts to correlate the synthesis of protein and nucleic acid have been rather inconclusive. The trend a t the moment is to replace theoretical discussions by practical experiments, and the fact that these are now possible is a measure of the progress of the subject in recent years. A comparison between the synt,hesis of protein in tumor and normal tissue has had to await developments in the field as a whole. I t is clear that in certain diseases the tumor cells synthesize proteins which differ from those synthesized by normal cells and that some tumor cells have an enhanced ability to utilize plasma protein for the synthesis of their tissue proteins. It is not known, at present, whether the mechanism of protein synthesis in tumor cells differs from that in normal cells. The need now is for careful comparisons between normal and tumor tissue to be made employing the techniques which have been developed in recent years. ACKNOWLEDGMENTS
I would like to thank Professor F. Dickens, F.R.S., Dr. I. M. Roitt, Mrs. 0. Greengard, and Mrs. N. E. Stone for many helpful discussions and Miss B. Kernot for preparing the figures.
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GENERALREFERENCES Borsook, H. 1950. Protein turnover and incorporation of labeled amino acids into tissue proteins in zivo and i n vilro. Physiol. Revs. 30, 206-219.
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Borsook, H. 1952.Enzymatic peptide syntheses: Their bearing on protein biosynthesis and thermodynamic considerations. Congr. intern. biochim. 2nd Congr. Paris, 196.2, pp. 3749. Borsook, H. 1953. Peptide bond formation. Advances in Protein Chem. 8, 127-174. Borsook, H. 1956. Biosynthesis of peptides and proteins. Proc. 3rd Intern. Congv. Biochem. Brussels, 1966, pp. 92-104. Borsook, H., and Deasy, C. L. 1953. Biosynthesis of Proteins. I n “Biochemistry and Physiology of Nutrition” (G. H. Bourne and G. W. Kidder, eds.), Vol. I, pp. 188211.Academic Press, New York. Burnet, F. M. 1956.“Enzymc, Antigen and Virus.” Cambridge UNv. Press, London and New York. Burton, K. 1957. Interrelationships of nucleic acid and protein in the multiplication of bacteriophage. Biochem. SOC.Symposia (Cambridge, Engl.) 14, 60-74. Campbell, P. N., and Work, T. S. 1953.Biosynthesis of Proteins. Nature 171, 997-1001. Dounce, A. L.1953.Duplicating mechanism for peptide chain and nucleic acid synthesis. Enzymologia 16, 251-258. Fenninger, L. D., and Mider, G. B. 1954. Energy and Nitrogen metabolism in Cancer. Advances in Cancer Research 2, 229-253. Fisher, R. B. 1954. “Protein metabolism”: Methuen’s Monographs on biochemical subjects. Wiley, New York. Fruton, J. S. 1952. Enzymatic Synthesis of peptide bonds. Cmgr. intern. biochim. 2nd Cmgr. Paris, 1962, pp. 5-18. Gale, E. F. 1953.Assimilation of amino acid? by Gram-positive bacteria and some actions of antibiotics thereon. Advances in Protein Chem. 8, 286-391. Gale, E. F. 1955. From amino acids to Proteins. I n “Amino acid Metabolism” (W. D. McElroy and B. Glass, eds.), pp. 171-192. Johns Hopkins, Baltimore, Maryland. Gale, E. F. 1957. Nucleic acids and the incorporation of amino acids. Biochem. Soc. Symposia (Cambridge, Engl.) 14, 47-59. Gale, E. F., and Folkes, J. P. 1954. Effect of nucleic acids on protein synthesis and amino acid incorporation in disrupted Staphylococcal cells. Nature 173, 1223-1227. Greenberg, D.M., Friedberg, F., Schulman, M. P., and Winnick, T. 1948. Mechanism of protein synthesis with radioactive carbon-labeled compounds. Cold Spring Harbor Symposium. Quant. Biol. 13, 113-117. Haurowitz, F. 1950. “The Chemistry and Biology of Proteins.” Academic Press, New York. Haurowitz, F. 1952. Synthesis of normal serum proteins and of antibodies. Cmgr. intern. biochim. 2nd Cmgr. Paris, 1962, pp. 56-61. Haurowitz, F. 1952. Mechanism of the immunological response. Biol. Revs. Cambridge Phil. SOC.27, 247-280. Huggins, C. 1949.Serum Proteins in Cancer: Cancer Research 9, 321-327. Lipmann, F. 1954. Mechanism of some ATP-linked reactions and certain aspects of protein synthesis. I n “Mechanism of Enzyme Action” (W. D. McElroy and B. Glass, eds.), pp. 599-607. Johns Hopkins, Baltimore, Maryland. McFarlane, A. S. 1952. Use of isotopes in the study of plasma protein metabolism. Brit. Med. Bull. 8, 213-218. Simkin, J. L.,and Work, T. S. 1957.Biochemical Approaches to the problem of Protein Synthesis. Nature 179, 1214-1219. Spiegelman, S., Halvorson, O., and Ben-Ishai, R.1955.Free amino acids and the enzymeforming mechanism. In “Amino Acid Metabolism” (W. D. McElroy and B. Glass, eds.) , pp. 124-170. John8 Hopkins, Baltimore, Maryland.
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Steward, F. C., and Thompson, J. F. 1954. Proteins and protein metabolism in plants. In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. IIA. pp. 513-594. Academic Press, New York. Tarver, H. 1954. Peptide and Protein Synthesis. Protein turnover. In “The Proteins” (13. Neurath and K. Bailey, eds.), Volume 1113, pp. 1199-1296. Academic Press, New York. Waelsch, H. 1951. Glutamic acid and Cerebral Function. Advances in Prolein Chem. 6, 299-341. Zamecnik, P. C., Keller, E. B., Littlefield, J. W., IIoagland, Pvl. B., and Loftfield, R. 1956. Mechanism of incorporation of labeled amino acids into protein. J . Cellular COTILP. I’hysiol. 47, Suppl. 1, 81-101.
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THE NEWER CONCEPT OF CANCER TOXIN W a r 0 N a k a h a r a a n d Fumiko Fukuoka Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Toxohormone Production a Universal Property of Cancer Cells.. . . . . . . . . 111. Isolation of Toxohormone from Materials Other than Cancer Tissue. . . . . IV. Normal Liver Catalase Level. . . V. Chemical Nature of Toxohormon VI. Mode of Action of Toxohormone VII. Toxohormone in General Tumor-Host Relations . . . . . . . . . . . . . . . . . . . . . . . VIII. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Referenccs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION What are the characteristics of cancer cells as distinct from normal cells? This is a n important question, to which the present state of our knowledge can give only a partial answer. Classical pathology has long since established atypia, as to the cancer cell itself, and heterotopia, in relation to other structural elements of the body, as characterizing cellular malignancy. These terms are descriptive of the fundamental phenomena of malignancy, though subject to the limitation inherent in the nature of morphology itself. A great stride was made in the understanding of the properties of cancer cells when Otto Warburg, some thirty years ago, demonstrated a high aerobic and anaerobic glycolysis as an outstanding biochemical feature of these rells. In spite of t,he fact that the greatly increased glycolytic capacity has since been found to be not strictly specific to cancer cells but t,o be shared also with certain normal cells, such as embryonic and placental cells, Warburg’s original observations stand to this day as the most salient, single contribution to cancer cell biochemistry. During the past several years an interesting phase of this problem has been uncovered in a somewhat unexpected manner. This newer knowledge stemmed from studies on the systemic effect of cancer growth instead of from direct attacks on t.he cancer cell itself. We refer to the discovery of a characteristic toxic substance which is produced by cancer cells and which is responsible for certain systemic changes constantly occurring in cancer bearing hosts. The toxic substance in question is toxohormone (Nakahara 157
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and Fukuoka, 1948), and the systemic change it produces is represented by bhe marked decrease of liver catalase activity. It can be said that the demonstration of what may truly be regarded as cancer toxin opened up a new vista of promise, although some of the more important implications of the cancer toxin concept do not seem to be as generally recognized as they should be. Indeed, there has been an extremely cautious attitude in some quarters toward the toxohormone theory as the explanation of the marked decrease of liver catalase activity in cancer bearing animals. Accumulating evidence, however, seems to suggest that such extreme caution may serve only to hinder progress by blinding one to a patent fact which could otherwise be profitably exploited. I n the present article, we will attempt to outline the current status of the problem, to indicate the points which require further elucidation, and to emphasize the prospect of the future development of the subject. To a great extent, these grounds have already been covered by Greenstein in the second edition of his “Biochemistry of Cancer” (1954), and an effort will be made to minimize unnecessary duplications in this article. Newer experimental results will be considered more fully, especially those originating from Japanese laboratories where much work of interest has been done of late. The term “toxohormone” will be used throughout for the characteristic substance which is produced by cancer tissue and which is nssayable by its marked action in decreasing liver catalase activity in vivo. The substance is frequently referred to in the literature by various terms such as ‘(liver catalase inhibiting tumor factor,” and “tumor agent depressing liver catalase activity.” These terms, however, not only fail entirely to connote the basic significance of this unique substance, but they are also inconveniently long. Even for this latter reason alone, the term “toxohormone” may be useful, a t least until such a time as when the substance can be designated by its proper chemical name.
11. TOXOHORMONE PRODUCTION A UNIVERSAL PROPERTY OF CANCER CELLS Blumenthal, Brahn, and Ilosenthal early reported that liver catalase is markedly reduced in tumor bearing humans and animals (Blumenthal and Brahn, 1910; Rosenthal, 1912; Brahn, 1914, 1916). The significance of this fact was not fully realized until Greenstein and co-workers, thirty years later, (Greenstein, 1942, 1943; Greenstein and Andervont, 1942, 1943; Greenstein et al., 1941, 1942), demonstrated conclusively that the observed decrease of liver catalase activity is specifically attributable t o the presence of the growing tumor itself, and not to any secondary cause to which the tumor’s presence may give rise. Von Euler and Heller (1949) showed, by means of centrifugal fractionation of liver homogenates. that catalase
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activity is normally about equally divided between mitochondrial and microsomal supernatant fractions and that it is mostly in the latter fraction that the enzyme activity becomes greatly reduced in tumor bearing animals; decreased activity in the mitochondrial fraction is also observed after a rapidly growing tumor has reached a certain size. A new milestone in the progress of this study was established when a potent chemical fraction, capable of strikingly decreasing liver catalase activity when injected into normal animals (Nakahara and Fukuoka, 1948), was isolated from cancer tissues. The isolation of the active fraction was first attained by a simple method of extracting human tumor tissues with water under heat and precipitating with alcohol; this yielded crude material active for mice in 50-100 mg. doses. This alcohol precipitate was later purified by reprecipitation with one of the conventional protein precipitants, and the most active of such fractions produced a n unequivocal effect in mice in 5-mg. amounts. In these early studies, all the samples of cancer tissue examined, without exception, yielded the active material. They included 14 gastric carcinomas, 5 rectal carcinomas, 2 carcinomas of the sigmoid region, 3 mammary carcinomas, and 1 each of carcinoma of the transverse colon, lymph node metastasis of liver carcinoma, bladder carcinoma, mammary fibrosarcoma, and lymphosarcoma, all human surgical materials CLf confirmed histological diagnosis. Necrotic portions of cancer tissue yielded no more active fraction than fresh, nonnecrotic portion, nor did decomposition in vitro of cancer tissue increase the activity or yield of the fraction. Similar fractions from 14 samples of normal human tissue were uniformly inactive. It is the active substance of these cancer tissue fractions that we tentatively designated “toxohormone” in the hope of indicating its singular biological status of being a cell-borne substance which is released into circulation and which produced a clearly definable biochemical lesion (namely, decreased catalase activity) in the target organ. It may be conceived of as a pathological counterpart of hormones in that its presence produces a pathological condition, and its withdrawal restores the normal state. We did not call it “cancer toxin” in order to avoid possible confusion with miscellaneous alleged “cancer toxins’’ of the past literature. Our original experimental results have since received full confirmation from all sides. An active alcohol-precipitable fraction was isolated by Greenfield and Meister (1951), in Greenstein’s laboratory, from mammary carcinoma 3CHb of mouse and from Walker sarcoma and fibrosarcoma AXCMCA2 of rat; by Okushimu (1952a) from several gastric, rectal, and mammary carcinomas of man; and by Kawamorita et al. (1951) from Brown-Pearce carcinoma and Kato sarcoma of rabbit, to mention the earlier reports. Kuzin et al. (1955) duplicated some of these findings. Osawa
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(1954) isolated alcohol precipitable fraction from the Chiba strain of chicken sarcoma, as well as from the original Kous No. 1, and found them to be active for mice in 50-mg. doses. Sat0 and Yunoki (1951) reported separating a similar liver catalase reducing fraction by adsorbing an extract of human gastric carcinoma with kaolin a t pH 5.4, eluting with ammonia water (pH 9.0), and finally precipitating the eluate with acetone. This fraction was found to be active in 30-mg. amounts for mice. I n a series of experiments, Adams (1950b, 1951a) did not attempt to separate the active substance, but found that injections of whole homogenate of mouse sarcoma 37 or of mouse carcinoma 63 produced a marked depression of the liver catalase in normal mice, and he inferred that some chemical components of the cancer tissue must be responsible for the enzymatic change. According to the recent study by Nakagawa and Nakagawa (1956), centrifugal fractionation of tumor homogenates, using Yoshida sarcoma and ascites hepatoma of the rat, yields a supernatant fraction consisting of soluble protein and microsome which seems to be the main carrier of toxohormone, whereas the nuclear or mitochondria1 fraction failed to show clear evidence of the liver catalase depressing action. I n later studies, not only have many of the animal tumors already mentioned been tested butothers also, and all have been found to be good sources of toxohormone. Umeda’s rhodamine sarcoma of the rat, Murphy’s lymphoma, Ehrlich’s carcinoma (both ascitic and solid forms) Nakahara and Fukuoka’s N F sarcoma and N F carcinoma of the mouse are now being used successfully for the purpose of separation and concentration of this interesting substance. It should be noted that in all these experiments no malignant tumor was encountered, carcinoma or sarcoma, which, upon experimentation, did not yield active toxohormone fraction. This is in keeping with the fact that greatly decreased liver catalase activity has been demonstrated in all the cases of malignant tumors, so far as these have been investigated, and suggests very strongly that the production of toxohormone may be a universal property of all the malignant tumors.
111. ISOLA4TION O F TOXOHORMONE FROM MATERIALS OTHER THAN CANCERTISSUES As may perhaps be expected of a substance elaborated by cancer cells and thence thrown into circulation, toxohormone has been demonstrated t o occur in several types of body fluid in cancer bearing humans and animals. The first of this group of data emerged when Nakagawa (1952) reported the isolation of the liver catalase depressing fraction from the urine of
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cancer patients. By means of the benzoic acid adsorption method followed by precipitation with alcohol, this author obtained a material which was found to reduce markedly liver catalase activity in normal mice. The amount in terms of dry weight of the material necessary to produce the positive effect was not given, but it was stated that the yield from 50-200 cc. of urine was injected per mouse. The urine used in these experiments came from 42 different cancer patients of confirmed diagnosis. Fractions similarly prepared from urine of patients with diseases other than cancer did not show the catalase depressing effect in comparable doses. These findings were confirmed by Sat0 el al. (1953) who separated a similarly active fraction from cancer patients’ urine by the kaolin adsorption method which they had previously used for the isolation of toxohormone from cancer tissues. The yield from 200 cc. of the urine was injected per mouse. Both Nakagawa and Sat0 and the latter’s co-workers failed to detect any property which distinguishes the urinary factor from toxohormone of cancer tissue. More recently, Fuchigami et al. (1956), in our laboratory, reexamined the subject and confirmed the excretion of toxohormone in the urine of cancer patients. According to their results, however, the concentration of the active substance in the urine is generally very much lower than the two previous reports had given to underttand. Cancer ascites is a source from which toxohormone may reasonably be expected t o be recovered. Hirsch and Pfutzer (1953) reported that not only the cells but also the cell-free fluid of Ehrlich ascites carcinoma and of Yoshida sarcoma are active in decreasing liver catalase activity when injected into normal mice. There seems to be no doubt that toxohormone occurs in cancer ascites in man, but the amount present may be expected to vary greatly according to the case. Miyajima (1955) isolated the alcohol precipitable fraction from 46 cases and in doses of 25-100 mg. (mostly 50 mg.) found the fraction indisputably active for mice only in 28 of these cases. Gastric juice of patients with gastric cancer is another material which may be expected to contain toxohormone. The first evidence of the fact was brought forward by Kawamorita et al. (1951) working in the laboratory of Nakagawa. They used gastric juice from 10 gastric cancer cases and prepared the toxohormone fraction by alcohol precipitation; injecting the yield from 30-40 cc. gastric juice per mouse, they discovered that the fraction from 4 of these cases produced an indisputable effect. The urine from these cases also yielded a highly active fraction. None of the similarly prepared fractions from the gastric juice of 11 patients suffering from diseases other than gastric cancer showed any activity. These results were confirmed by Iwatsuru et al. (1945) who, in addition, also obtained active
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material by precipitation from concentrated cancerous gastric juice with the same volume of 10% trichloroacetic acid solution. It would seem quite certain that toxohormone is present in gastric juice of gastric cancer patients. Unfortunately, however, the actual amount of the active fraction injected was not given by any of these authors. The presence of toxohormone in the blood of tumor bearing hosts is to be expected, even without the experiments of Lucke et al. (1952) on parabiotic rats, but we are somewhat doubtful that it may occur in sufficient concentration to be demonstrable by the usual in v i m test. Okushima (1952b) prepared the alcohol precipitable fraction from serum of cancer patients and found it to be without effect. However, Plaza de 10s Reyes et al. (1953) reported that fresh plasma of cancer patients injected into normal rats reduced liver catalase activity, and Kuzin et al. (1955) also found that the alcohol precipitable toxohormone fraction separated from the blood of tumor bearing animals exerted a similar effect. With refinement of technique, the toxohormone in blood, even very small in amount, must eventually become demonstrable. It may be recalled that Greenstein el al. (1942) early noted that kidney catalase activity is lowered relatively little and blood catalase activity not at all in tumor bearing animals. Possibly the apparent relative insusceptibility of kidney catalase may be due to a rapid excretion of toxohormone, since we now know that toxohormone occurs in cancer urine in demonstrable amounts. The failure of the blood catalase to be affected may be explained by the insufficient concentration of toxohormone in the blood. However, according to Theorell et al. (1951) and Schmid et al. (1955), erythrocyte catalase has a far longer life-span than liver catalase, and it is possible that the apparently different behavior towards toxohormone of the two types of catalase may be accounted for by this fact. Also, if we follow the view that the liver constitutes the major site of catalase synthesis in an organism, blood catalase may be expected to be affected differently from liver catalase by the inhibition of the synthetic mechanism which is localized in the liver. There are obvious reasons for assuming that the attempts to demonstrate toxohormone in blood or urine were made with an eye to the possible utilization of the findings for the clinical diagnosis of cancer. Theoretically, the anticipated toxohormone test as a means of detecting the presence of cancer has the rationale not usually found in the numerous so-called cancer reactions that have been proposed in the past. Practically, however, since the production of toxohormone is a function of cancer cells, small cancer nodulen, such as are the objects of early diagnosis, cannot be expected to yield a ltirge amount of the substance. The most urgent need for diagnostic purposes, therefore, may be the discovery of some method whereby toxohormone in a very low concentration can be detected.
I\’. NORMAL LIVEHC r r r \ L . \ m LEVEL Thc possibility may be pointed out licrc that toxohormone, simply as a chemical substance, may not be strictly specific to cancer cells, but may occur in normal tissues in negligible amounts-negligible because, even if it occurs, it can serve a t most merely to maintain the normal level of catalase where it is. Although, as abundantly demonstrated, normal tissue fractions prepared by methods similar to those yielding potent toxohormone from cancer tissue fail to depress the liver catalase in comparable dosage, it is not impossible that they may do so in larger amounts. Greenfield and Meister (1951) stated in the summary of their paper that fractions obtained from normal tissues “possessed considerably less ability to lower liver catalase,” apparently implying that a small amount of active material may be present in such fractions. Obviously granting that small amounts of toxohormone may occur in normal tissues, Greenstein (1955) stated in his recent review that “that which is a toxin in a tumor due to abnormal production may be only a normal regulator of enzyme levels in a normal tissue.” It must be admitted, however, that there is nothing in our present knowledge to show that toxohormone in normal tissues actually plays a role in the regulation of the normal catalase level. The idea is merely conceivable. I n this connection, observations by Day et al. (1954) showing depression of liver catalase activity in mice injected with homogenate of normal mouse spleen may be referred to. This observation is a t variance with the previous results of Adams (1950b, 1951s) who injected homogenates of a variety of normal tissues, including embryo tissue, producing no significant change. It is also contradicated by the more recent results of Nakagawa and Nakagawa (1956) who found homogenates of neither spleen, liver, nor kidney from normal rats to reduce liver catalase. Of special interest is the work of Sawasaki (1953) who prepared the toxohormone fraction from human placenta according to the original method of Nakahara and Fukuoka and found it to be entirely without effect in 100-mg. doses. It may be recalled that embryonic and placental tissues are among the nonmalignant tissues whose metabolism resembles that of cancer tissue. It is possible that the discrepancy may have arisen from the difference in the source of normal tissue in relation to the test animal (mice) or that what little effect the normal tissue homogenates exerted was not regarded by these latter authors as significant. In any event, it must be admitted that the catalase depression reported by Day and associates is only of a small degree, and it is hardly to be compared with the marked effect of cancer tissue homogenate or of toxohormone concentrates. When Day and collaborators said that “the nature of the depression . . . involve[s] n rather complex physiological process, n dynamic process apparently
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involving a delicate compensatory mechanism under stress,” they probably had the normal mechanism of regulating liver catalase level in mind. Adams (1952, 1953, 1955) carried out extensive experiments concerning the maintenance of liver catalase activity in normal mice, and he arrived a t the conclusion that testicular and adrenal hormones, as well as traces of copper and manganese, are of importance in this mechanism. He also showed that testosterone, which is responsible for the sex difference in catalase activity in normal mice, requires the presence of riboflavin for its action on the enzyme level. Although it seems probable that there exists some sort of hormonal control to regulate the normal level of liver catalase, it does not necessarily follow that toxohormone operates through the mediation of such a mechanism. Nor does Adams’ idea that the function of toxohormone may be “to protect this liver enzyme (catalase) from hormonal influences” seem quite lucid. Whether or not these hormonal influences are capable of exerting any protection against the catalase depressing action of toxohormone is a more pertinent question. Begg (1951b) reported that injections of testosterone do not restore the catalase level in rats bearing large tumors. It would seem probable that in tumor bearing animals the supposed normal regulatory mechanism may no longer be operative, being overwhelmed by the active production of toxohormone.
V. CHEMICAL NATURE OF TOXOHORMONE The principal steps in the purification of toxohormone as originally carried out by us consisted of (1) extraction with water under heat and removing the heat coagulable matter, (2) precipitation with two volumes of absolute alcohol, and (3) removal of ether-soluble substances. The active substance of this fraction is precipitable by half saturation with ammonium sulfate and also by other usual protein precipitants such as trichloroacetic acid, picric acid, cupric sulfate. From these facts, we assumed a t the outset that toxohormone may be protein-like in nature and probably a polypeptide. From the dialysis and ultracentrifugal experiments, Greenfield and Meister (1951) suggested that the active agent might itself be of, or be associated with particles with, a molecular weight of a t least 40,000. The active fraction, when digested with 6 N HC1, was found to yield by paper chromatography the following amino acids : alanine, glycine, serine, proline, aspartic acid, arginine, valine, threonine, phenylalanine, hydroxyproline, a-aminobutyric acid, tryptophan, lysine, leucine, isoleucine, and glutamic acid. Okushima (1952a) hydrolyzed with 10% HC1 the original alcohol precipitate from human cancer tissue and established, also by paper chromatography, similar amino acid composition. He did not find isoleucine, hydroxyproline, a-aminobutyric acid and tryptophan, but noted
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the presence of methionine and a large spot of what appeared to be “underglutamic acid.” There is some indirect evidence which seems to support the view that toxohormone may be of polypeptide nature. Our early experiments showed that the toxohormone content of tumor tissue, as roughly estimated by the relative amounts of tissue homogenate necessary to produce liver catalase depression in normal mice, can be perceptibly increased by injecting a whole protein hydrolyzate or a mixture of pure amino acids into the tumor bearing animals (Fukuoka and Nakahara, 1953). The amino acids necessary for insuring this increased toxohormone content of the tumor were found to be alanine, proline, aspartic acid, arginine, phenylalanine, lysine, leucine, and glutamic acid. We interpreted the findings as indicating that these eight amino acids may be the major components of toxohormone polypeptide and that the increased toxohormone content may be due to the supply of an extra quantity of the building material. It is possible that in order to reveal the effect of the extra supply of amino acids, the basal diet, on which the tumor bearing animals are maintained, should be of protein subdeficient type, as was used in our experiments. If the dietary protein furnishes the optimum amounts of all the necessary amino acids, the superposition of additional amino acids may find no expression in the amount of toxohormone synthesized. The role of amino acids in the synthesis of toxohormone was demonstrated more clearly in our subsequent in vitro experiments (Nakahara and Fukuoka, 195413). Under the influence of the recent discovery of the ATPdependent biosynthesis of the tripeptide glutathione by an enzyme system from liver tissue, we speculated that biosynthesis of toxohormone may be possible in a somewhat similar manner. Upon experimentation, it was readily found that a substance highly active a s toxohormone can be producecl in vitro by incubating a t 37°C. for 24 hours fresh tumor tissue slices in a solution of amino acid mixtures in the presence of ATP. The active material thus obtained was heat stable, water soluble, and alcohol precipitable, exactly as was the toxohormone fraction directly separated from tumor tissue. The alcohol precipitate was active for mice in about one-fifth the amount of the material similarly prepared from the same tumor tissue ( N F strain of mouse sarcoma) the slices of which were used in the biosynthesis experiments. An interesting fact emerging from these experiments was that only three specific amino acids need be included in the reaction mixture, and these three turned out to be arginine, phenylalanine, and leucine. It is important, however, to take into account the fact that small amounts of many amino acids are carried into the reaction mixture b y the tumor slices and hence the special role of these three amino acids cannot be strictly upheld. It is also to be admitted that it is merely an assumption that the,
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active substance produced is a condensation product of the amino acids, and, therefore, is actually a polypeptide. The real value of these biosynthesis experiments may consist, after all, in the control experiments which demonstrated the failure of normal tissue slices to produce toxohormone under the conditions in which tumor tissue slices so readily elaborate the same substances. As may be inferred from the method used, the toxohormone concentrate originally obtained b y us contained much nucleic acid, often amounting to 30% of the weight, Nakagawa et al. (1955), in their attempt to purify toxohormone from Brown-Pearce rabbit carcinoma, followed and confirmed Nakahara and Fukuoka up to the copper sulfate precipitate; they then separated this precipitate into protein and nucleic acid fractions by means of hot tricholoroacetic acid solution and found the latter to be more active than the former. Ent!irely analogous results were also obtained using human lung and stomach cancer tissues. Nakagawa and associates claimed that their nucleic acid fraction was active for mice in doses as low as 0.1-0.2 mg. The fraction gave a negative biuret reaction and a strongly positive Molisch reaction, but an examination by means of the chloroform gel method revealed the presence of a small quantity of protein or peptide. It may be mentioned here in passing that, according to Fuchigami el al. (1956) who investigated in our laboratory the chemical properties of the active material isolated from the urine of cancer patients b y the original method of Nakagawa et al. (the benzoic acid adsorption and alcohol precipitation), neither RNA nor DNA fraction of this material shows the ultraviolet absorption characteristic of nucleic acid. They identified the following amino acids as components of the polypeptides of the material: aspartic acid, glutamic acid, serine, threonine, proline, alanine, valine, leucine, phenylalanine, lysine, cystine, and glycine. Nucleic acid is not the active constituent of toxohormone. As a matter of fact, tumor nucleic acid isolated b y means of Clark-Schryver’s method was found to be devoid of toxohormone activity (Endo, 1954). Moreover, it was possible to obtain a n active fraction free from nucleic acid contamination. From the fact that nucleic acid is attached to many toxohormone preparations, Ono et al. (1955), in our laboratory, assumed that the toxohormone may be a basic polypeptide. They have isolated a basic polypeptide from tumor tissues in the form of a white amorphous powder which was active as toxohormone in doses as low as 10 mg. and which showed no definite absorption peak a t around 280 mp. I n separating this nucleic acid-free toxohormone fraction, they followed the method for the preparation of corticotropin, involving the processes of defatting with acetone, extracting with methanolacetic acid mixture, and precipitating with ether. It is interesting to observe that the nucleic acid-free toxohormone is not precipitable from its solution by the addition of two volumes of alcohol
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a t pH 4.5. The fact that Nakahara and Fukuoka originally achieved the isolation of toxohormone by means of alcohol and acid precipitations may be attributable t o the nucleic acid moiety of their fractions. Greenfield and Meister (1951), incidental to confirming the isolation of toxohormone, reported an unexpected fact that digestion of the active alcohol precipitate with 6 N HCl at 100°C. for 1 2 4 8 hours did not affect the activity and that the active substance in the digest was dialyzable. A complete hydrolysis should have reduced the protein to amino acids, but, as Greenfield and Meister themselves showed, a mixture of all the amino acids detectable in the hydrolyzate of the toxohormone concentrate failed to depress liver catalase activity. Two alternative possibilities, therefore, come t o mind in the interpretation of this experimental result, namely, either that the hydrolysis was incomplete and enough active peptide remained intact, or that toxohormone is a substance not attacked by the protein hydrolyzing process. It is important to point out here that, according to our observations, freshly prepared extract of tumor tissue contains a small amount of dialyzable substance which depresses liver catalase activity when injected into normal mice (Nakahara and Fukuoka, 1954a). By separating the dialyzate into alcohol precipitate and filtrate, it was found that the filtrate, instead of the precipitate, was active. Yields varied, but on an average the total dialyzate amounted to about 10 mg. per gram of fresh tumor tissue, and the ratio of alcohol precipitate to filtrate was something like 1:30. The filtrate was brownish yellow, sticky, hygroscopic matter, and active for mice in 50-mg. doses. Toxohormone as originally isolated by us, or by Greenfield and Meister, is nondialyzable, but it is interesting to note that this nondialyzable toxohormone fraction, when digested with papain or pepsin, yields a dialyzable substance which is potent i n vivo, exactly as the active substance in the dialyzate of tumor tissues (Nakahara and Fukuoka, 1954a). Digests were heated a t lOO"C., coagulum formed was centrifuged off, and the clear solution was dialyzed against 200 cc, of distilled water for 5 days a t the temperature of 2-3°C. Dialyzates were then precipitated by alcohol, yielding about 40 mg. of precipitate and 200 mg. of filtrate per 1 gm. of the starting material before digestion. These facts definitely suggest that toxohormone occurs in two forms, one nondialyzable and the other dialyzable, and that the latter may be derived from the former b y the enzymatic splitting process. Taking advantage of the knowledge that toxohormone in the form of alcohol precipitate can be largely converted into dialyzable form without damage to its activity, Ono et al. (1956a) subjected their nucleic acid-free toxohormone fraction to enzymatic hydrolysis for 4 days. They used 1% pepsin and, adjusting the reaction of the digest to the slightly alkaline side,
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added one-third the volume of saturated solution of sodium picrate under stirring. About 30 minutes later, they noticed a gradual formation of precipitate which, upon microscopic examination, was found to consist of fine needlelike crystals. After recrystallization, the yield of the picrate was about 5 mg. from 1g. of the original material before digestion. The identical crystals were obtained upon repeated trials. I n all cases the crystalline picrate proved to be active in 0.2-mg. doses for mice. I n a spot test, this active picrate gave a blue color with ninhydrin after one week, contrary to the rapid reaction of free amino acids. It also gave one spot in paper chromatograms, with Rf of 0.2 in an acetic-butanol system and of 0.016 in a lutidine system. As determined b y paper chromatography, the amino acid composition was found to be: aspartic acid, glutamic acid, serine, threonine, proline, hydroxyproline, alanine, valine, leucine, isoleucine, phenylalanine, lysine, glysine, and arginine. Cystine, methionine, tryptophan, and tryosine were not detected. Assuming one molecule each of threonine and valine to be present and other amino acids in integral ratio, the minimum unit of constituent amino acids was calculat,ed t o be 30 moles, the molecular weight being close to 4000. The picrate crystal decomposed a t 300°C; its melting point was undeterminable. Whether or not this crystalline picrate represents that of pure toxohormone is a moot point. Peptides of many sorts must be assumed to be present in the pepsin digested material, and the probability of some of them forming a mixed picrate crystal with toxohormone cannot be regarded as small. Further studies alone can settle this point. Our present concept of the nature of toxohormone is that this unique toxin in its elementary form may be a relatively small molecule and that it may occur in cancer tissue for the most part either as aggregates of such elementary forms or in close association with some other substance, constituting the toxohormone of the usual or nondialyzable form. In the latter case, the associated substance might be nucleic acid, and the enzymatic action, which yields the dialyzable form, may simply be to dissociate the two. The dialyzable form may thus be thought to correspond to the nucleic acid-free toxohormone, neither of which is precipitated by alcohol. However, the toxohormone in the urine of cancer patients is precipitated by alcohol in spite of the absence of nucleic acid in the same fraction. Does this mean that the nondialyzable toxohormone consists of aggregates of smaller molecule forms? There is much to be done in elucidating the precise chemical nature of toxohormone.
VI. MODEOF ACTIONOF TOXOHORMONE Many substances are known to inhibit catalase activity in vitro, and animals’ tissues, normal as well as cancerous, are now known to contain such inhibitors. Hargreaves and Deutsch’s (1952) “kochsaft factor” is an
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inhibitor of this sort, representing nonspecific tissue constituents capable of reducing Fe"' of catalase to Fe" (Endo et al., 1955; Ceriotti and Spendrio, 1955) without catalase depressing action in vivo. Toxohormone differs from these substances. It does not inhibit catalase :tctivity in vitro as early demonstrated by Greenstein (1942, 1943) and by Nakahara and Fukuoka (1949). Price and Greenfield (1954) more recently reiterated that the tumor fractions which show a marked effect on liver catalase of normal animals in vivo do not lower either the catalase activity or Soret band absorption in vitro. Conclusive evidence that the decreased catalase activity of liver of tumor bearing animals is attributable to the actually smaller amount of catalase contained in the liver was obtained by Price and Greenfield (1954). By subjecting normal rat liver and the liver of tumor bearing rat to the identical treatments of absorption and elution (using calcium phosphatecellulose columns) and studying spectrophotometrically the chromatographic peaks so obtained, these authors showed that there may actually be five times as much catalase in normal liver as in the liver of the tumor bearing animal. They also isolated crystalline catalase from these two kinds of liver and noted that there is no qualitative difference whatever between the two. Prima jacie, the presence of an actually smaller amount of catalase in the liver of tumor bearing animals as compared to that of normal animals can be accounted for either by the slower rate of the synthesis of catalase or by the shorter life-span of the enzyme in these animals. Here, previous kinetic studies of Greenstein (1943) may be regarded as adequate in ruling out the second possibility. He demonstrated, among other things, that the rate of spontaneous inactivation in vitro of liver catalase is identical between tumor bearing and normal animals. There seems to be, then, no alternative but to conclude that the action of toxohormone may be to suppress the process of catalase synthesis. Hirsch and Pfutzer (1955), seeing that the dialyzate of the cell-free fluid of ascites tumor (though activc in vitro) is entirely devoid of action in vivo, made an interesting suggestion that a high molecular substance of protein nature may be acting as carrier for the catalase-inhibiting substance. They tried to explain the lack of in V ~ V Oaction of the dialyeate by assuming that the inhibitor, without this protein carrier, may pass through the liver too rapidly to attain the concentration necessary to effect the lowering of liver catalase activity. This, however, is based on the gratuitous hypothesis that the in vivo and 1'11 vitro active substances are identical. We now know that nonspecific tissue constituents of many sorts are involved in the in vitro inhibition of wttiliise, while toxohormone is active only rn uivo. How does toxohormone suppresh the synthesih o f liver catnlasc'? Having
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found that administration, either per 0s or parenterally, of ferric chloride or iron-rich materials (dried liver or blood) appreciably prevented the decrease of liver catalase activity of tumor bearing animals, we tried to visualize the mechanism of the toxohormone effect on liver catalase synthesis in terms of decrease of available iron (Fukuoka and Nakahara, 1951). The effect of injecting an isolated toxohormone fraction was also found to be almost completely nullified when test mice have been fed for one week on a diet containing 5 mg. of ferric chloride per 300 g. This latter finding was duplicated by Kosuge et al. (1951) using the toxohormone fraction from a cancer patient’s urine. Von Euler (1952), however, reported his failure to confirm fully these same results; he observed only slightly higher liver catalase activity in iron-treated, rather than untreated, tumor bearing animals. The discrepancy here may have stemmed from a difference in the type of tumors used, since the countereffect of additional iron may vary according to the amount of available toxohormone. Our experiments were carried out with the N F mouse sarcoma which is of relatively low toxohormone activity. However this may be, it must be pointed out that so far it has been impossible to demonstrate any special capacity of toxohormone to bind iron i n vitro. Resegotti and von Euler (1954) later reported that the decrease of liver catalase activity in ascites tumor bearing rats can be prevented and the activity held near normal by the injection of tartrate of trimethylcolchicine acid methylester; injections of pure colchicine showed no such effect. Since both substances blocked the growth of tumor to about the same extent, the different effects on the liver catalase cannot be ascribed to the different growth rates of tumors. Moreover, cases were found in which liver catalase activity was normal in the tartrate treated rats with profuse growth of ascites tumor. The i n evitro effects on catalase of incubating the liver homogenate from tumor bearing and normal rats with the solution of the tartrate were identical. Resegotti and von Euler suggested, purely hypothetically, that tartrate of colchicine acid methylester acts as “anti-toxohormone.” The elucidation of the mechanism of this “anti-toxohormone” effect will be of great importance. On the basis of the hypothesis of Fukuoka and Nakahara (1951) that toxohormone may suppress the synthesis of the liver catalase by impairing the utilization of iron, Ono et al. (1956b) carried out an investigation on the porphyrin level of the liver, Harderian glands, and urine of tumor bearing rats and on the effect of toxohormone on the porphyrin metabolism in normal rats. They demonstrated that t,here is a considerable increase of protoporphyrin contents in the liver and of coproporphyrin excretion in urine of tumor bearing animals. Injections of toxohormone fraction also showed nn unmistakable tendency for these prophyrins to increase in
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normal rats. According to their view, these results indicate an underutilizntion of porphyrin caused by thc impairment of iron metabolism or of the synthesis of protcin moiety of hemoproteins. KO :Idtlitional or nonphysiological porphyrin, not found in normal mts, was detected in the liver or urine of tumor bearing or toxohormone injected rats. I n order to understand the mode of action of toxohormone, it may be of help to look for some known chemical substance which can reduce liver catalase activity in viuo. One such substance seems to be allylisopropylacetylcarbamide (“sedormid”). Schmid et al. (1955) found that oral administration to rats of this substance in 80-mg. daily doses brings about a marked fall of liver catalase activity with a corresponding marked rise in hepatic porphyrin concentration. They concluded that sedormid blocks the formation of catalase in the liver and that this metabolic block may explain the markedly increased concentration of porphyrin in the liver. Heim et al. (1955) called attention to 3-amino-l,2,4-triasole with which they have been able to produce in normal animals a marked decrease of liver catalase activity. Sugimura (1956) found, however, that 3-amino-1,2,4triasole inhibits catalase activity also in vitro when in the presence of cofactors contained in liver extract. He found, moreover, that the i n vivo depression of the liver catalase following the injection of this substance is not accompanied by notable increase of porphyrin in the liver. VII. TOXOHORMONE IN GENERALTUMOR-HOST HELATIONS There is evidence for assuming that the marked decrease of liver catalase activity in cancer bearing hosts may be only a salient expression of a deep-seated and more fundamental disturbance which toxohormone brings about. It was suggested earlier that toxohormone may adversely affect, not only the synthesis of catalase, but also of other iron-containing proteins. In neoplastic disease there is a group of findings (such as anemia, lowered hemoglobin, and decrease of plasma iron), in man as well as in animals, all pointing to the disturbance of iron metabolism. This interference with iron utilization may have a t its root ttn injury to the synthetic mechanism of specific ferro-protein ferritin, and the decrease of plasma iron which precedes that of hemoglobin may also be attributable to a similar disturbance in the metabolism of t,issue iron. The relative decrease of liver ferritin seems to be demonstrable in cancer patients. The porphyrin content of the blood of tumor bearing animals is appreciably increased even before the decrease of hemoglobin becomes apparent (Sugimura el al., 1956). It may be reasonable to assume that the marked lowering of the synthesis of liver catalase cannot occur without concurrently involving changes in the general iron metabolism and particularly in the hepatic iron reserve.
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Of interest in connection with this problem is the recent findings of Iijima et nl. (1956) that toxohormone injections produce in the liver of normal mice changes in non-hemin iron frzictions similar to those occurring in the liver of gastric cancer patients. These authors prepared toxohormone concentrate from human gastric cancer tissue according to the original method of Nakahara and Fukuoka, and observed that 60 mg. was sufficient to induce, 24 hours later, (a) a decrease in the ferritin fraction to 30% from the normal level of 50%, (b) an increase in the nucleic acid iron fraction to 27% from the normal ratio of about lo%, and (c) a n increase in the hemosiderin fract,ion to about 55% from the norm of 38%. The changes they found in gastric cancer patients were quite comparable to those in toxohormone-injected mice. It would appear that toxohormone, which suppresses the hepatic catalase synthesis, brings about a t the same time an imbalance of various nonhemin iron fractions of the liver. These findings, if confirmed, may effectively connect the decrease of the liver catalase to anemia in neoplastic disease, bringing both of these important phenomena under the influence of toxohormone. It is freely admitted that obscurity prevails as to tlhe exact mechanism involved, yet there seems to be sufficient evidence already to lead us to believe that toxohormone may affect the metabolism of iron-containing proteins in general. Since these proteins include many substances of much biological importance, it seems not unreasonable to suspect that the essential cause of the so-called cancer cachexia may ultimately be found to be related to the toxohormone activity. No hasty conclusion should be drawn in dealing with such a complex phenomenon as cachexia in cancer. The subtraction of body substances by growing tumor, the effect of secondary hemorrhage or of incidental infection, the possibility of hormonal disturbances, all these may have parts to play in producing the cachectic condition as one sees it in the terminal stage of cancer. With due consideration for all these complicating factors, however, the fact remains that there is a demonstrable toxohormone-associated disturbance of iron metabolism in cancerous organisms constituting sufficient ground for suspicion that toxohormone may be fundamentally responsible for the final shaping of the cachectic condition in cancer. It may not be out of place to discuss here the marked thymus involution which, as is well known, constantly occurs in tumor bearing animals. Because thymus involution is sometimes accompanied by adrenal enlargement, attempts were made in the past to consider these phenomena in cancer in the light of the adaptation syndrome of the so-called stress reaction (Savard, 1948; Begg, 1951a). However, while involution of the thymus is a marked and constant phenomenon in tumorous animals, aclrend enlargement is often so slight as to be practically negligible. The
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enlargement of the adrenals is said to be accompanied by a marked reduction in their ascorbic acid and lipid contents and is interpreted as indicating a reduced cortical activity. The decrease of the lipid content, however, does not seem to be of great magnitude since we found (unpublished observations) that no difference in the organ’s specific gravity can be detected between normal and tumor bearing mice. An important fact here is that thymus involution can be induced in normal animals by toxohormone. By injecting into normal mice a toxohormone fraction (a copper sulfate precipitate prepared from N F mouse sarcoma tissue) in doses of 10-20-mg., Fukuoka and Nakahara (1952) found that, a t 24 hours to 3 clays after the injection, the average thymus weight was reduced to about 29 mg. per 100 g. of body weight, from the average normal of 113 mg. in males and 147 mg. in females. This reduction in thymus weight generally corresponded to that found in mice bearing tumors of 3-6 g. Injections of similarly prepared fraction from normal tissues showed no such effect. It is interesting to note that in these experiments, while marked thymus involution was constantly induced, there was no evidence of concomitant adrenal enlargement. We advocated as likely that thymus involution may be connec+ecl with the disturbance of nitrogen metabolism and that it may be looked upon as an early sign of the wasting of body protein in cancerous animals. It is well known that various conditions such as malnutrition, infection, intoxication are closely associated with thymus atrophy, and it would appear that toxohormone, through its adverse effect on protein synthesis, may bring about a similar thymus change. This point of view leads us again to the idea that toxohormone may play a significant part in producing the so-called cancer cachexia. Although both the liver-catalase-decreasing and the thymus weight reducing actions are carried by many tumor fractions so far tested, there is a possibility that the active substance concerned may not be identical (Ono et al., 1955). Further studies may make it necessary to accept a secondary cancer toxohormone of thymotropic type.
VIII. CONCLUDING REMARKS The biological significance of toxohormone has been considered largely from the point of view of its systemic effects on tumor bearing animals. This is natural, since the whole toxohormone problem originally arose as an attempt to discover the chemical basis of the tumor-host relations. There is, however, another aspect to the study of toxohormone which may well prove to be of fundamental importance but which so far has not received the attention it deserves. The ability of toxohormone to inhibit the synthesis of liver catalase
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should immediately raise the question of the consequence to the physiology of the cancer cells themselves which produce this inhibitor. We have known for years that cancer cells are notably deficient in their catalase activity. May it not be that the catalase deficiency and toxohormone production in cancer cells are causally related? Catalase, as is well known, is closely associated with aerobic cell respiration, and the almost complete absence of catalase in cancer cells may be explained on the basis of its being useless in the metabolism of these cells, Toxohormone action may provide the mechanism which brings about the deletion of catalase in cancer cells. What seems most intriguing here is the perfect harmony and adaptive interplay of the production of toxohormone, on one hand, and the development of a metabolic pattern requiring no catalase, on the other, and it. takes no great flight of imagination to conceive of these two processes taking place as necessary parts of a more complex whole, which, in its totality, characterizes the cancer cell physiology. It is from this point of view that toxohormone may assume possibly a profound significance. I n the words of Greenstein (1954), (I . . . the neoplastic transformation ends in a cell with only the two characteristics [growth and the production of a circulating toxin] to identify it. Growth is a name given to an over-all biological phenomenon, but cancer toxin is a chemical approachable by conventional methods of analysis. At the least, this latter characteristic offers an experimental handle whereby further knowledge of an unusual metabolic feature of the cancer cell may be grasped.” It may be pertinent to point out again that toxohormone is characteristic of all malignant tumors in that these tumors produce the substance in sufficient amount t o bring about certain definite systemic changes in animals bearing them. None of the normal cells does this which means that in normal cells the synthesis of toxohormone, if any indeed occurs, is of negligible proportions. There is, then, a striking difference between malignant and normal cells in respect to the synthetic mechanism. How does a cancer cell synthesize a substance with such peculiar activity? Nakahara and Fukuoka’s (195413) work on biosynthesis in vitro, already referred to, showed that toxohormone synthesis may depend upon the enzyme system, absent or not operative in normal cells. This study, however, has not even scratched the surface of the problem, since the nature of the enzyme system concerned remains entirely unknown. When the difference between the enzyme systems of cancer cells and normal cells (capable and incapable, respectively, of synthesizing toxohormone) is disclosed we will have gone far in the biochemical understanding of the cellular process of malignancy.
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13 EFERENCEK .idams, D. H. 1950a. Hormonal control of liver catalasc. Nature 166, 952. Adams, D.H. l950b. The mechanism of thc liver catalase depressing action of tuinors in mice. Brit. J . Cancer 4, 183. Adams, D. H. 1951a. Further observations on the liver catalase depressing action of tumours. Bril. J. Cancer 6 , 115. Adams, D. H. 1951b. Mouse liver catalase. The antagonistic effect of tumor tissue upon the hormonal control mechanism. Bril. J . Cancer 6 , 409. Adams, D. H. 1952. Hormonal factors influencing liver catalasc activity in mice. Biochem. J . 60, 486. Adams, D. H. 1953. Effect on mouse-liver catalase activity and blood-haemoglobin level of a milk diet deficient in iron, coppcr and manganesc. Biochem. J . 64, 328. Adams, D. H. 1955. Liver catalase in the riboflavin-deficient mouse. Riochem. J. 60, 568. Begg, R. W. 1951a. Systemic effects of tumors in mice. Cancer Research 11, 341. Begg, R. W. 1951b. Steroid hormones and tumor-host relations. Cancer Research 11, 406. Blumenthal, F., and Brahn, B. 1910. Die Katalasewrikung in normaler und in carcinomatoser Leber. Z . Krebsforsch. 87, 436. Brahn, B. 1914. Fermentstudien bei dcr Krebskrankheit. Z . Krebsforsch. 16, 112. Brahn, B. 1916. Weitere Untersuchungen uber Fermente in der Leber von Krebskranken. Sitzber. kgl. preuss. Akad. Wiss. 478. Ceriotti, G., and Spend rio, L. 1955. On the i n vilro anticatalase activity of tumor extracts. Biochiin el Biophys. A d a 18, 303. Day, E. D., Gabrielson, F. C., and Lipkind, J. B. 1954. Depressions in the activity of liver catalase in mice injected with homogcnates of normal mouse spleen. J. Natl. Cancer Znst. 16, 239. Endo, H. 1954. Studies on nucleic acid in the toxohormonc concentrate. Gann 46, 124 (Japanese). Endo, H., Sugimura, T., Ono, T., and Konno, K. 1955. Catalase depressing tissue factors: toxohormone and kochsaft factor. Gann 46, 51. Fuchigami, A., Umeda, M., and Ono, T. 1956. Effect of urine extract of cancer patients on liver catalase in mice. Gann 47, 295 (Japanese). Fukuoka, F., and Nakahara, W. 1951. Mode of action of toxohormone. A third study on toxohormone, etc. Gann 42, 55. Fukuoka, F., and Nakahara, W. 1952. Toxohormone and thymus involution in tumor bearing animals. .4 fourth study on toxohormone, etc. Gann 43, 55. Fukuoka, F., and Nakahara, W. 1953. Amino acids and toxohormonc synthesis. A fifth study on toxohormone, etc. Gann 44, 1. Greenfield, R. E., and Meister, A. 1951. Effect of injections of tumor fractions on liver catalase activity of mice. J. Natl. Cancer Inst. 6, 997. Greenstein, J. P. 1942. Titration of the liver catalase activity of normal and of tumorbearing rats and mice. J. Natl. Cancer Znst. 2 , 525. Greenstein, J. P. 1943. Further studies of the liver catalase activity of tumor-bearing animals. J. Natl. Cancer Inst. 3, 397. Greenstein, J. P. 1954. “Biochemistry of Cancer,” 2nd ed. Academic Press, New York. Greenstein, J. P. 1955. The in n h effect ~ on liver catalase by a tumor. J. Natl. Cancer Inst. 16, 1603.
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Greenstein, J. P., and Andervont, €1. B. 1942. Thc livcr catalase activity of tumorbearing mice and the effect of spontaneous regrossion and removal of certain tumors. J . Null. Cancer 1ns1. 2 , 345. Grcenstein, J. P., and Andervont, 1%.B. 1943. Notc on the liver catalasc activity of pregnant mice and of mice bcnring growing embryonic implants. J . Natl. Cancer Znsl. 4, 283. Greenstein, J. P., Jenrette, W. V., and White, J. 1941. The liver catalase activity of tumor-bearing rats and the effect of extirpation of the tumors. J . Natl. Cancer Inst. 2 , 283. Greenstein, J. P., Andervont, H. B., and Thompson, J. W. 1942. Kidney and blood catalase activity of tumor-bearing anl"ma1s. J . Null. Cancer Inst. 2, 589. Hargreaves, A. B., and Deutsch, H. F. 1952. The in vitro inhibition of catalase by a tumor factor. Cancer Research 12, 720. Heim, W. G., Appleman, D., and Pyfrom, H. T. 1955. Production of catalase changes in animals with 3-amino-1,2,4-triazole. Science 122, 693. Hirsch, H.H., and Pfutzer, W. 1953. Uber den Katalasehemmstoff in Ascitestumoren. 2.Krebsforsch. 60, 611. Hirsch, H. H., and Pfutzer, W. 1955. ttber die Ultrafiltrierbarkeit des Katalasehemmfaktors aus dem Ehrlichschen Mauseascitestumors. 2.Krebsforsch. 60, 609. Iijima, N., Matsuura, K., and Fujita, Y. 1956. Iron metabolism in gastric cancer. Japan J . Cancer Clin. 2 , 113 (Japanese). Iwatsuru, R., Kato, I., and Tamaki, H. 1954. Effect of K.I.K. factor on various liver enzymes of animals. Gann 46, 643. Kawamorita, Y., Kosuge, T., Kikawa, T., Nakaide, Y., and Shiroshita, K. 1951. Catalase depressing factor from Brown-Pearce carcinoma and Kato sarcoma of rabbits. Acta Med. Hokkaidon. 26, 71 (Japanese). Kawamorita, Y., Suzuki, S., and Kasai, M. 1951. Catalase depressing factor in gastric juice of gastric cancer patients. Acla Med. Hokkaidon. 26, 110 (Japanese). Kosuge, T., Shiroshita, K., Nakaide, Y., Yoshida, H., and Goda, I. 1951. Effect of Ferric chloride on the activity of catalase depressing factor isolated from cancer urine. Acta Med. Hokkaidon. 26, 67 (Japanese). Kuzin, A. M., Sharoukhova, K. S., and Chudinova, I. A. 1955. The effect of tumor extracts on the catalase and the coenzyme of the liver of normal mice. Biokhimiya 20, 126. (In Russian). Lucke, B., Berwick, M., and Zeckwer, I. T. 1952. Liver catalase activity in parabiotic rats with one partner tumor bearing. J . Natl. Cancer Znst. 13, 681. Miyajima, S. 1955. Studies on toxohormone prepared from human cancer ascites. Gann 46, 111 (Japanese). Nakagawa, S. 1952. Liver catalase reducing substance in the urine of cancer patients. Proc. Japan Acad. 28, 305. Nakagawa, S., and Nakagawa, S. 1956. Intracellular distribution of liver catalase reducing substance in malignant tumor tissues. Proc. Japan Acad. 32, 298. Nakagawa, S., Kosuge, T., and Tokunaka, H. 1955. Purification of the liver catalasereducing substance occurring in cancer tissues, with special reference to its activity. Gann 46, 585. Nakahara, W., and Fukuoka, F. 1948. A toxic cancer tissue constituent as evidenced by its effect on liver catalase activity. Japan. Med. J . 1, 271. Nakahara, W.,and Fukuoka, F. 1949. Toxohormone: a characteristic toxic substance produced by cancer tissue. Gann 40, 45. Nakahara, W., and Fukuoka, F. 1950. Purification of toxohormonc. A second study on toxohormone, etc. Gann 41, 47.
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Nakahara, W., and Fukuoka, I?. 1954a. Dialyzable form of toxohormone. A sixth study of tnxnhornione, etc. G m n 46, 67. Nakahara, W., and Fukuoka, 1'. l954b. I
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Cancer Research Department, Royal Beatsan Memorial Hospital, Glasgaw, Scotland
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1. Introduction . . . . . ........................... 11. Chemically Induced Sarcoma.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Chance Infection by Viruses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Host Factors Influencing Transmissibility. . . . . . . . . . . . . 1. Seasonal Influence.. . . . . . . . . . . . . 2. Genetic Susceptibility. . . . . . . . . . . . . . . . . . . ................ V. Tests for Virus.. . . . . . . . . . . . . . . . . . . VI. Evaluation of Negative Experiments .................... VII. Histogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Influence of Solvents on Carcinogenesis with 1 :2:5:6-Dibenzanthracene.. IX. Methylcholanthren ...................... X. Immunological Evi ...................... XI. Attempts to Infect
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1. Macroscopic and Microscopic Evidence. . . . . . . . . . . . . . . .
XIII. Fractionation of Tumor Homogenates, . . . . . . . . . XIV. Selective Action of Radiations. . . . . . . . . . . . . . . . . 1. Ultraviolet Rays.. ......................
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XV. Epithelial Tumors in Fowls. . . . . . . . . . . . . . . . .
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XVII. Species and Tissue Susceptibility. References. . . . . . . . . . . . .
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I. INTRODUCTION Spontaneous tumors in fowls cover the same range of histological types as is encountered in mammals and there is nothing in their microscopic appearance to suggest that they differ fundamentally from the tumors of other species. Nevertheless, the best known avian t,umor is the Rous sarcoma No. 1 whose transmissibility by cell-free filt,ratea was first described by Peyton Rous (1911). Subsequently, a number of spontaneous fowl sarcomata were found to be transmissible by cell-free preparations and in each case the histogenesis of the original tumor was faithfully reproduced. Attempts to propagate mammalian sarcomata by cell-free filtrates 179
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invariably failed and this difference between spontaneous avian and mammalian sarcomata has remained one of the most remarkable facts of cancer research. 11. CHEMICALLY INDUCED SARCOMA The introduction of experimental carcinogenesis by means of coal tar made possible the comparative study of tumors in different species induced by a common exogenous factor. Murphy and Landsteiner (1925) injected ten fowls with chick embryo pulp and tar, and in two of these birds tumors developed, one of which was a sarcoma. This tumor (chicken tumor No. 9) was successfully transplanted through 11 serial passages by Sturm and Murphy (1928) and behaved like mammalian transplantable sarcoma, growing progressively and metastasizing freely in new hosts. Nevertheless all attempts to transmit chicken tumor No. 9 by cell-free extracts failed. Similar independent experiments by Choldin, Leitch, and Russell were reviewed by the present author (Peacock, 1933) who described two series of sarcomata induced by intramuscular injection of tar or of 1:2: 5 :6-dibenzanthracene dissolved in lard. Subsequently, the results of transmission of these and other induced fowl sarcomata were published in a series of papers (Peacock, 1935a,b; Peacock, 1946; Peacock and Peacock, 1953). By no means were all of these chemically induced tumors transmissible, but those that were successfully transplanted were named after the Glasgow Royal Cancer Hospital, GRCH 1, 2, etc., in chronological order. These tumors were similar in macroscopic and microscopic characteristics and were easily distinguished from the Rous sarcoma No. 1. Two other available “filterable” tumors, Fujinami myxosarcoma and McIntosh sarcoma No. 5, resembled the Rous No. 1 type much more closely than the GRCH series. 111. CHANCEINFECTION BY VIRUSES The conditions under which the GRCH series were induced were considered to be free from the risk of chance contamination with known fowl tumor viruses. The birds were kept in a farm laboratory where no previous experiments had been carried out, and all the apparatus and instruments used were new. The obvious precautions of heat sterilization immediately before and after use were observed and all injections were checked by two people. Subsequent work has shown that such precautions are advisable when tumors are to be tested for possible virus content. The following example of chance contamination illustrates the point. A series of animals including domestic fowls were subjected to a modified cholecystostomy operation with a view to sampling bile before and after intravenous injection of 3 :4-benzpyrene. One of the fowls subsequently developed a tumor around the cholecystostomy opening in the
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abdominal wall, and it w:is thought, that this rniglit be an inducetl tumor caused by contamhiation of the tissues with the carcinogenic hydrocarbon or its metabolites. However, the characteristics of the tumor on both naked eye and microscopic examination were those of the Fujinami myxosarcoma which was being propagated in the same animal house at the Glasgow Royal Cancer Hospital. Inoculation of fowls and ducks with extracts of this tumor gave rise to typical Fujinami sarcoma in both species, and the tumor was thus proved to be due t o chance infection of the cholecystostomy wound by the Fujinami virus, though how such contamination occurred was never established. Had this tumor been of any other origin it would have been difficult or impossible t o establish the significance of the virus in relation to the experimental procedures. The well established association between leukemia and sarcoma in fowls (reviewed by Oberling and Guerin, 1954) gives further reason for caution not only in planning the experimental induction of tumors by chemical or physical carcinogens, but also in the selection of healthy stock free from leukemia. The fowls used in our earliest attempts to induce sarcoma by chemical carcinogens were of unknown pedigree, but for the past twenty years we have used only two breeds, namely Brown Leghorns obtained from the Institute of Animal Genetics, Edinburgh, and White Leghorns obtained from the Northern Poultry Station, Reaseheath, Cheshire. All these birds were of known pedigree and both stocks are practically free from spontaneous leukosis. Our primary objective was to induce a variety of sarcomata and to test each for possible virus content by the injection of cell-free extracts into new hosts. It gradually became evident that the type of sarcoma that can easily be induced in fowls is a fleshy, whorled, spindle cell tumor, probably derived from voluntary muscle though never showing the transverse striation characteristic of mammalian rhabdomyosarcoma. Sixty-three tumors of this type were induced by tar or by carcinogenic. polycyclic hydrocarbons in various solvents, but only 17 of them were successfully transplanted and thus became available for etiological studies. The apparently capricious transplantability was found to be related to two controlling factors, (1) seasonal variations in susceptibility, (2) genetic. resistance or susceptibility.
11’. HOST FACTORS INFLIJENCING TRANSMISSIBILITY 1. Seasonal InJuence
The period of relative resistance to growth of transplantable sarcomata was found t o be between about June and November and corresponds with the period of molt during which there is a diminution in proliferative activ-
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ity most noticeable in the shedding of feathers and in the absence of egg laying. This is accompanied by marked involution of the ovary and oviduct; during the same period, established grafted sarcomata often undergo complete or partial retrogression followed in some cases by recurrence when the period of proliferative activity returns in the early winter. No such seasonal variation was observed by Kreyberg and Nielsen (1936) in the case of transplanted mouse sarcoma, and it is obviously related to the physiology of the fowl. Murphy and Sturm (1941a) concurrently carried out similar experimental studies (Table I) in carcinogenesis in fowls as a continuation of their earlier work mentioned above and showed that the seasonal effect applied also to the induction of sarcoma after a single injection of 1:2 :5 :6-dibenzanthracene dissolved in bensol. TABLE I Seasonal Variation in Induction Time" Injection Date October December January February-March a
Range (weeks) 6-12 4.5-8 3-6 3-5
Average Induction Time (weeks) 9 5.6 4.7 4.06
From Murphy and Sturm. 1841%
It is interesting that Michalowsky (1928, 1929) was able to induce teratoma in the testes of cocks by intratesticular injection of 5% zinc chloride, but only in the early spring. Bagg (1936) repeated and confirmed Michalowsky's experiments and extended them by injecting gonadotropic hormone into the testicles of adult cocks followed by 5% zinc chloride solution in midJune, and thus was able to induce teratoma two months later in the season than Michalowsky. Anissimova (1939) also induced rapidly growing teratomata at the site of injection of 501, zinc chloride, and a t the suggestion of Professor Michalowsky attempted to transplant these tumors with some success. Some of the tumors grew in new hosts but some retrogressed. The primary tumors were induced by injection of zinc chloride in March, but the tumors from a bird killed on July 14, 1937 grew in fowls (sex not specified) inoculated subcutaneously and intraperitoneally. From a second tumor bearing bird killed on June 26, 1937 pieces of tumor were inoculated subcutaneously, intraperitoneally, intramuscularly, and intratesticularly into two fowls. These birds were killed 3 months later, presumably about September. In one of the birds, the transplants had retrogressed, but in the other, the grafts persisted and showed the presence of a variety of epithelial and mesodermal tissues and some infiltrative growth
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in the muscles of the new host by large cells which were not identified. But, the tendency for most of the teratoma tissue was to undergo further differentiation, and cartilage and bone formation and cysts lined by columnar epithelium were seen. Falin and Gromzewa (1939) obtained teratomata by intratesticular injection of 10% zinc sulfate instead of the chloride and again noted the seasonal effect by injecting birds on March 17, 21, 28, and April 17, 1938. Three out of twenty-nine cocks injected in March gave tumors but none of seven cocks injected in April. Thus the zinc ion seems to be tumorigenic in these experiments, and the results could not be attributed to nonspecific traumatic or irritative effects, as shown by the negative results of intratesticular injection of gonadotropic hormones alone in the experiments of Bagg (1936). This type of experiment seems to offer further possibilities in the direction of attempts to induce new varieties of tumor, e.g., combining zinc ions with local injection of carcinogens into the resulting teratoma. 2. Genetic Susceptibility
Susceptibility to grafted sarcoma was noted by Rous (1910). The original tumor, in a Barred Plymouth Rock hen, from which chicken tumor No. 1, generally called Rous sarcoma, was derived was a t first successfully grafted only into blood relatives, but later into unrelated stock. However, close relationship between the original tumor bearing bird and those used successfully for grafting was not found to be necessary for some other tumors. Indeed the blood relatives of the Rous sarcoma No. 1 bird seem to have belonged to a naturally susceptible strain in which, e.g., tumor No. 18, which arose in a Brown Leghorn, grew better than in blood relatives (Row and Lange, 1913). The same kind of susceptibility to grafted tumors was encountered by us in the case of the GRCH series (Greenwood and Peacock, 1945). Two tumor strains were tested. One of these, GRCH 13, was derived from a sarcoma induced by intramuscular injection of coal tar in a Barred Plymouth Rock hen of unknown pedigree. This tumor grew well in Brown Leghorn chicks and was maintained in birds of this strain through 23 serial passages, but failed to grow in related Barred Plymouth Rock chicks. The other tumor was sarcoma GRCH 15, which originated in a pedigreed Brown Leghorn Cock No. K293, of the Edinburgh strain. This tumor was grafted into Brown Leghorn birds of known, but varied, relationship to the original tumor bearing bird, and again it became evident that what determined progressive growth was not so much the degree of blood relationship as some unspecified susceptibility of a hereditary character. On reviewing the results of several years of blind grafting into birds
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that were phenotypically alike, it was found that some genotypes were highly susceptible and others resistant to the GRCH 15 cells. The nature and origin of this phenomenon was not determined, but it would account for the apparently capricious behavior of tumors grafted into heterozygous stock. V. TESTSFOR VIRUS Consideration of the variable factors already discussed made it clear that tests for filter-passing agents in new tumors of unknown etiology would be difficult to assess unless they gave positive results. In tests on Brown Leghorns, the inoculations were carried out without reference to pedigree so that no bias would be exercised for or against a hypothetical virus. I n retrospect, however, it was found that a fair proportion of susceptible birds had received cell-free inoculations, and these as well as the resistant birds gave uniformly negative results. OF NEGATIVE EXPERIMENTS VI. EVALUATION
The problem of evaluating negative results is one of the most difficult. in cancer research, but one that must be faced realistically. By simple dilution of an active virus preparation, one can always obtain negative results with inoculations that still theoretically contain virus. It can, therefore, be argued that any negative results after inoculation of cellfree extracts are examples of insufficient dosage. To offset this type of argument, we used whole tumor homogenates after first grinding them with sand or high-speed mincing to disintegrate most of the tumor cells. This type of homogenate is usually the first stage in the preparation of filtrates, and in the case of virus induced tumors, yields a high proportion of positive results. I n the case of the GRCH series of chemically induced sarcoma, however, such simple treatment very rarely gave positive results (Peacock, 1946; Peacock and Peacock, 1953). The value of line-bred birds for comparative studies is obvious, but such stock was not readily available until recently. I n 1948 we obtained from Reaseheath some White Leghorn birds of the 9th generation of brother-tosister mating. From these birds we have carried on brother-to-sister mating and a t the time of writing have 16th generation (F16) birds for assay of tumor extracts. While even these birds are not as nearly homozygous as many strains of line-bred mice in current use, they nevertheless give very consistent results with the virus-induced tumors Rous No. 1 and Mill Hill 2, and yield practically 100% positive results with cell grafts of tumors induced in birds of the same st,rLLin.The result,s of tests with such tumors are, therefore, easier to assess than earlier results with unrelated or less closely related birds.
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YII. HISTOGENESIS Most of the tumors in the GRCH series arose ak or near the site of injection of carcinogens in the pectoral muscles or in the fascia1 plane between the superficial and deep muscle layers. The cytological characters and staining affinities suggest that they are derived from muscle rather than from undifferentiated connective tissues. I n this respect, they differ from the available transmissible avian tumors of spontaneous origin, most of which seem to be of fibroblastic or endothelial origin. I n the absence of histologically indistinguishable tumors of spontaneous and of induced origin, further comparison in regard to virus etiology cannot be pursued. It seemed important, therefore, to try to induce tumors of the Rous No. 1 type, but so far no such tumor has been induced in any of our experiments, although GRCH 3 and 13 occasionally approximated to th e histological appearance of Fujinami sarcoma. It is possible that tumor No. 9 of McIntosh and Selbie (1939) is an example of successful induction of this kind of tumor. It arose a t the site of injection of tar dissolved in lard as a fibrosarcoma which was later transplanted successfully, and after four passages it was transmitted on several occasions by cell-free filtrates. The tumor originally diagnosed as a fibrosarcoma “at the second passage became less differentiated, and consisted of loosely packed, long spindle cells, with considerable myxomatous degeneration.” The present author critically reviewed the 10 tumors described by McIntosh (1933) and by McIntosh and Selbie (1939) and considered that only tumor No. 9 was acceptable as a chemically induced tumor that was later transmissible by cell-free extracts. The other tumors were complicated by spontaneous leukemia, or were histologically different from the tumors a t the site of injection of the carcinogen or from the grafts from such tumors. Murphy and Sturm (1941b) were even more critical of the evidence in favor of virus etiology in the McIntosh series of chemically induced sarcoma in the fowl.
VIII. INFLUENCE OF SOLVENTS ON CARCINOGENESIS WITH 1 :2 :5 :6-DIBENZANTHRACENE Murphy and Sturm (1941a) compared the carcinogenicity of 1 :2 :5 :6dibenzanthracene dissolved in (1) benzol, (2) lard, (3) chicken fat and found striking differences in t,he localization of the resulting tumors. (1) When beneol solution was injected, tumors arose at the site of injection and (‘crystals of the injected dibenzanthracene were found generally in the center of the tumors in amounts suggesting that almost the total amount had remained local. I n fact from one tumor we were able to recover over 70 per cent of the amount injected.” Each bird in the
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benzol series received a single injection of 0.8 cc. of a 3% suspension equivalent to 24 mg. of dibenzanthracene. Thus the effective quantity of dibenzanthracene in this experiment would be about 8 mg. or less. The induction time was relatively short, and 48 of the 53 birds injected developed progressive tumors after an average time of 555 weeks and autografts yielded tumors in 22 out of 23 tested. (2) When lard was used as solvent, 4 doses of a 0.4% suspension were injected separated by intervals of 3, 2, and 2 months, giving a dose of about 12 mg. at each site and a total of about 48 mg. In Ohis experiment among 21 birds injected into the pectoral muscles, 5 developed tumors at the site of injection which were recognized clinically between 18 and 56 weeks after the first injection, with an average induction time estimated at 36.4 weeks. All the tumors grew progressively and metastasized. Eight birds developed lesions other than at the site of injection, and these included 2 cases of myelogenous leukemia, 1 of lymphatic leukemia and adenocarcinoma of uncertain origin, 1 carcinoma of the ovary, and 1 fibroadenoma of the ovary. (3) Ten birds were injected with 24 mg. dibenzanthracene dissolved in chicken fat in the breast muscles on each side (total 48 mg.). One bird developed a tumor a t the site of injection after 18 weeks. There were metastases in the liver which also showed marked fatty degeneration. Four other birds died between 3 and 7 weeks after injection with evidence of extensive liver damage but without local tumor at site of injection. The importance of the solvent in determining the result when equal doses of dibenzanthracene were used is obvious and is related apparently to the rate of absorption and site of metabolism of the carcinogen. In our experiments, no dibenzanthracene was found a t the site of injection in the dibenzanthracene-lard series 3 months after injection, and in subsequent experiments Chalmers (1934) found that dibenzanthracene dissolved in chicken fat or egg yolk fat was rapidly removed from the site of injection and after 1 week was not detectable. It may be that the use of lard as a solvent for the tar in McIntosh’s experiments in some way determined a more general action of the carcinogens leading to remote carcinogenesis involving blood-forming organs and the liver and ovary as in the experiments of Murphy and Sturm (1941a). In the present, author’s experiments, a few remote tumors were encountered in birds that had received local injections of tar or of dibenzanthracene dissolved in lard (Peacock, 1946), but they were not attributed to the preceding treatment. Unless very large numbers of control birds are kept, the etiology of such remote tumors cannot be assessed with any certainty. In the case of pure-line birds, however, the probability of remote tumor incidence can be assessed and the influence of various solvents and vehicles for carcinogenesis might best be evaluated by experiments on such birds.
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IX. METHYLCHOLANT-HI~ENE-INDUCED SARCOMA Oberling and Guerin (1950) studied among other induced tumors, one that developed 32 months after injection of an oily solution of methylcholanthrene. The tumor grew slowly after transplantation, but attempts to transmit it by cell-free filtrates failed at the second and third passage. At the fourth passage, attempts at transmission with glycerinated tumor also failed, but a month later in t,he fifth passage, tumor kept in 50% glycerin and refrigerated for 1-4 weeks gave positive results. Filtrates were also positive a t the fifth and sixth passages but failed at the seventh passage. An important point in regard to this tumor is that no filterable tumor was being maintained in Oberling's laboratory at the time. Later Vigier and Guerin (1952) attempted to induce tumors in chick embryo tissue mixed with methylcholaiithrene and Scarlet Red and inoculated into adult birds, following the technique used successfully in the case of mice by Rous and Smith (1945). Their results were disappointing in so far as the induction of new types of tumor were concerned, but they obtained three sarcomata a t the site of injection which resembled those obtained by injection of carcinogens into adult fowls, and these behaved like the majority of such induced sarcomata in serial passage, being transmissible by cells but not by cell-free filtrates. Our own experiments on similar lines yielded precisely similar results (Peacock and Peacock, 1953), and we were unable to obtain evidence that the embryo tissues contributed to those tumors that grew progressively, although identified epithelial remnants were present early in the course of the experiment and even presented a histologically carcinomatous appearance. It is a t least certain that the majority of chemically-induced sarcomata studied so far yield no positive results when cell-free extracts are injected into susceptible birds. X. IMMUNOLOGICAL EVIDENCE Andrewes (1936) found neutralizing antibodies for Ilous No. 1 sarcoma in the sera of pheasants previously inoculated with a noninfective, tarinduced fowl sarcoma. Since normal fowl embryo inoculation failed to stimulate similar antibodies for Rous No. 1 sarcoma, Andrewes concluded that the nonfilterable tar tumor contained a factor antigenically related to the virus of Rous sarcoma No. 1. However, he could not demonstrate the hypothetical virus in the tar sarcoma by test inoculation of cell-free extracts. Andrewes successfully inoculated cells of the fowl tar sarcoma into pheasants and considered that this was evidence of infection of the pheasant cells by a virus in the fowl sarcoma. However, as fowls and pheasants can interbreed it does not seem impossible that fowl cells should grow in pheasant hosts.
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Andrewes (1931, 1933) found that the viruses of spontaneous fowl sarcomata were antigenically related, and Foulds and Dmochowski (1939) demonstrated both neutralizing and complement fixing antibodies for Rous sarcoma virus in the sera of a rabbit that had been injected with cellfree filtrates of a nonfilterable dibenzanthracene-induced fowl sarcoma, RFD 2. Dmochowski and Knox (1939) also demonstrated complement fixing antibodies for Fujinami and Rous sarcomata and Mill Hill 2 endothelioma in rabbit serum after injection of R F D 2 extracts filtered through gradocol . considered that the membranes with average pore diameter 0 . 8 ~ They antigen was probably extrinsic to the fowl because (a) normal fowl tissues do not excite the antibody, (b) absorption of the antiserum with normal fowl cells does not affect the reaction, (c) filtrates of Fujinami sarcoma grown in ducks or fowls react in the same way. An alternative explanation might be that several types of tumor cells are antigenically alike, but are different from normal cells. It does not seem to be necessary to postulate that antigenic differences between tumor cells and normal cells are due only to the presence of viruses in the former. The unsuccessful inoculation of virus tumors often indicates resistance or immunity to the virus and in the case of Rous No. 1 sarcoma, resistant birds also fail to grow tumors after direct cell grafts from actively growing tumor. No such spontaneous or acquired resistance to Rous No. 1 virus could be demonstrated after retrogression of GRCH 17 sarcoma (Peacock and Peacock, 1953). On the whole, immunological evidence in fowls is difficult to interpret, and this may be due to the unpredictable presence of saprophytic or nonpathogenic viruses. The extensive work of Burmester and Gentry (1954) on avian visceral lymphomatosis illustrates well the complexity of the problem due to the widespread distribution of carriers of lymphomatosis virus among poultry flocks. Duran-Reynals et a2. (1953) showed that chicks hatched and reared in isolation from lymphomatosis carriers had no antibodies for Rous No. 1 sarcoma in their sera and remained free from lymphomatosis, whereas genetically related chicks reared in contaminated surroundings and subsequently showing a high incidence of lymphomatosis did have antibodies for Rous No. 1 sarcoma in their sera. XI. ATTEMPTSTO INFECT CHEMICALLY INDUCED TUMORS Mellanby (1934) induced sarcoma in fowls a t the site of injection of 1 :2 :5 :6-dibenzanthracene or of tar. These tumors were essentially similar to the GRCH sarcomata and were, like them, transplantable but not transmissible by cell-free filtrates. Mellanby (1938) inoculated fowls with both the tar- and the dibenzanthracene-induced sarcomata and with Rous sarcoma No. 1, and all three tumors grew concurrently. They remained distinct, however, and there was no evidence that the induced sarcoma cells
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became modified by the Rous virus since grafts from the induced sarcoma reproduced the same type of growth; but filtrates prepared from either tumor in such birds yielded only Rous sarcoma or negative results. Carr (1945) showed that the virus content of transplanted, chemically induced sarcoma in birds also bearing Rous sarcoma No. 1 was not greater than that of their normal tissues.
XII. ESSENTIAL DIFFERENCES BETWEEN CERTAINFOWL TUMORS 1. Macroscopic and Microscopic Evidence
The available evidence suggests, to the author, that the well known and much studied virus sarcomata of fowls are essentially different from most of the chemically induced sarcomata so far studied. Not only are there obvious differences in the histogenesis and growth characteristics of the two classes of tumor as judged clinically and by naked eye and microscopic studies of the tumors, but the ease of demonstration of the specific viruses in the one group and the persistent failure to demonstrate infectivity in the other with a few exceptions is one of the most consistent results over the past twenty-five years and is the experience of all who have made comparative studies of such tumors. 2. Electron Microscopic Evidence The advent of electron microscopy raised hopes of demonstrating elementary virus bodies in infective extracts of tumor such as Rous sarcoma No. 1. Unfortunately, the methods of preparation of infective filtrates necessarily provides a multitude of small particles of cellular origin, and similarly prepared filtrates of normal tissues or of chemically induced tumors yield apparently identical pictures (Howatson, 1953), despite biological evidence of difference in regard to infectivity. Sections of Iious No. 1 sarcoma or fresh tumor cells spread thinly by various techniques occasionally yield electron micrographs showing small intracytoplasmic uniform dense particles of the theoretical size of the specific virus (Claude et al., 1947), but the rarity of such appearances is surprising in view of the high infectivity of many such tumors. Epstein (1955) has described particles about 0.1p in diameter in cytoplasmic vacuoles of the cells of Rous No. 1 sarcoma grown as a n ascites tumor. By mounting the individually separate cells on Formvar films, he was able to obtain thin enough preparations for electron microscopy and found the characteristic particles in from 1 in 50 to 1 in 2000 cells in preparations from different tumors, although no particles were seen in some tumors; there was little variation in samples from any one tumor. Epstein (1956) claims that there is a close correlation between the infectivity of Rous ascites tumors and the percentage of cells with vacuoles containing characteristic particles of 70 mp diameter.
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Rouiller et al. (1956) working in Professor Oberling’s laboratory have recently published very clear electron micrographs showing discrete oval bodies about 110 mp diameter, with a denser center, in thin sections of Mill Hill 2 endothelioma (generally referred to by French writers as the Murray-Begg endothelioma). These small bodies occurred extracellularly and were present. in 33 tumors examined. In our laboratory, similar particles to those described by other workers have occasionally been seen in thin sections (Laird, 1956), but never in such large numbers as illustrated by Epstein (1956) and by Rouiller et al. (1956). All who have made such studies agree that virus-like bodies are not always demonstrable, and the significance of such bodies when present is uncertain. Carr (1953) has attempted to compute the probability of finding recognizable virus particles in sections of 0.1 p thickness suitable for electron microscopy and concludes that they might be found only in 8% of sections under the best conditions. He also reviews the evidence about the cycle of infectivity of the known sarcoma viruses, and suggests that failure to demonstrate infectivity, especially in slowly growing tumors in birds more than 100 days old, does not prove that viruses are not involved in their etiology. On the other hand, there is no positive evidence for the presence of virus in such experiments. Bernhard and Oberling (1953) have suggested a theoretical explanation on the lines of cyclic development of virus with a particulate phase and a diffuse phase, the latter being the commoner. On the whole, the results of electron microscopy, although technically excellent, leave the problem of the relationship of viruses to malignant growth unsolved. XIII. FRACTIONATION OF TUMOR HOMOGENATES Purely mechanical separation of virus from tumor homogenates is probably never complete owing to the presence of innumerable normal cellular constituents of protein nature of about the same physical characteristics as the virus. Fractionation by means of selective procedures, such as digestion with enzymes, offers further prospect of purification; much work has been done on these lines, and with some success, in the case of the Rous No. 1 sarcoma in particular. This work has been reviewed by Harris (1953) and need only be mentioned here because similar techniques have been employed, with negative results, in the case of chemically induced sarcomata.
XIV. SELECTIVE ACTIONOF RADIATIONS i. Ultraviolet Rays Rous (1913) showed that ultraviolet rays can be used to destroy the cells of his sarcoma No. 1 without destroying its infectivity. Baker and Pea-
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cock (1926) made use of the sterilizing action of ultraviolet rays to destroy bacteria in dilut,e filtrates of Rous sarcoma without destroying the infective virus. 2. Ionizing Rays
X-rays or radium have been used to destroy tumor cells without loss of infectivity in the case of sarcomata 11 and 13 (Murphy and Sturm, 1941a), Rous No. 1, and Fujinami sarcomata (Peacock, 1946). Murphy and Sturm (1941b) showed that in vitro doses of 20,000 r did not destroy the infectivity of spontaneous sarcomata 11 and 13 of Furth and Stubbs, but rendered nontransmissible the cells of strain 16 induced originally by methylcholanthrene and of strain 870 induced by dibenzanthracene. Comparative studies of cell-free filtrates of these tumors confirmed the presence of infectivity in strains 11 and 13 and noninfectivity of the chemically induced strains. I n our experience, doses of the order of 23,000 r are required to stop all cell growth or migration in tissue cultures of fowl sarcomata (Peacock, 1946), and such doses cannot be employed clinically. Doses of about 6000 r administered to small, grafted Rous or Fujinami sarcomata cause them to retrogress but do not destroy the virus. Such treatment is followed by recurrences in the inflammatory proliferating cells at the periphery of the irradiated area. This type of localization of virus is essentially similar to that described by Kous et al. (1912) a t the site of injections of diatomaceous earth, and by Pentimalli (1916) a t the site of thermocautery in Rous sarcoma bearing birds. Similar localization sometimes occurs in the ovary following hemorrhage a t ovulation and in sites of minor infection and irritation such as occur in the wing web around metal number tags. No such localization has been recorded in birds bearing chemically induced tumors or transplants from such tumors (Peacock and Peacock, 1953).
xv.EPITHELIhL
T U M O R S IS FOWLS
Carcinoma of tsheovary is one of the commonest spontaneous tumors in fowls, and other epithelial tumors are also recorded in the literature (Felclman, 1932). Nevertheless few carcinomata have been induced experimentally until quite recently. I n a very interesting experiment, Duran-Reynals (1952) painted the skin of young chicks repeatedly with a solution of methylcholanthrene and obt,ained skin tumors which were histologically classified as squamous carcinoma but which showed the characteristic inclusions of fowl pox. The tumors grew as carcinomata in grafted fowls, but cell-free filtrates induced fowl pox, not carcinoma. We repeated this painting experiment on our White Leghorn chicks, which are free from fowl pox, but obtained no skin
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tumors (Peacock and Peacock, 1953). However, Rigdon and Brashear (1954) have described small skin tumors a t the site of repeated painting with methylcholanthrene which, though histologically resembling squamous carcinoma, failed to grow progressively. At present, no transplantable fowl carcinomata are known to be available for study, though a few have been transmitted for one or more serial passages by cell grafting. Foulds (1937) published a well documented record of a transplantable carcinoma apparently originating in the oviduct of a fowl that had been injected intravenously two and one-half years previously with a suspension of Mill Hill 2 endothelioma. Three other birds similarly injected failed to develop Mill Hill 2 or other tumor. The tumor was transplanted through 12 generations and revealed remarkable variations in histological appearance including a sarcomatous strain and tumors resembling human mixed salivary gland tumor. Bielschowsky and Green (1945) described an adenocarcinoma of the kidney after 87 weeks of experiment in one of two cocks given 5.5 g. 2-acetylaminofluorene orally over a period of 46 weeks. The other bird, similarly treated, was killed a t the same time and showed evidence of leucosis in the spleen and liver. We confirmed the induction of adenocarcinoma of the kidney in White Leghorn fowls treated by repeated injection into the lumen of the crop of a 1.5oJ, aqueous suspension of 2-acetylaminofluorene (Peacock and Peacock, 1954). In the same experiment, we observed three cases of primary carcinoma of the bronchus, one associated with separate primary carcinoma of kidney, another with hepatoma, and in another bird squamous carcinoma of the palate and squamous papilloma of the esophagus (Peacock and Peacock, 1954). We have not encountered spontaneous carcinoma of the lung or bronchus in fowls, and, according to Cowen (1950), the only case reported was that of Apperly (1935). We had previously described two cases of squamous carcinoma of the crop at the site of repeated injection of 1% solution of 2-acetylaminofluorene in arachis oil (Peacock and Peacock, 1949). A further example of squamous carcinoma a t the site of injection of 2-acetylaminofluorene in the crop was reported, and carcinomata were induced also by repeated injection into the crop of 5% methylcholanthrene dissolved in arachis oil. An anaplastic carcinoma occurred in the crop a t the site of injection, in a second bird, adenocarcinoma of the proventriculus, and in a third, malignant hepatoma associated with adenoma of the kidney (Peacock and Peacock, 1954). Campbell (1955) fed eight Brown Leghorn hens aged 6 months with gelatin capsules containing 25 mg. 2-acetylaminofluorene dissolved in 0.6 ml. sesame oil daily except Sundays for 3 months. Three out of five survivors for four years developed epithelial tumors, including hepatoma,
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carcinoma of oviduct and ovary. There were no primary tumors of the lung nor of the crop as in our experiments, and this may be due to the different method of administration. Campbell (1956) has described hepatoma associated with varying degrees of liver damage, regeneration, and cirrhosis in fowls fed with alkaloids of Senecio jacoboea. This result is similar to that obtained by Schoental et al. (1954) in rats. Thus in fowls, as in mammals, a variety of chemically induced epithelial tumors has been reported, but unfortunately none of them has been successfully transmitted in series. Some of our birds with internal tumors were found dead after several hours, and the prospects of successful transplantation were poor. But two birds were killed when moribund and immediate attempts t o transmit the adenocarcinoma of the kidney from one of these failed in seven birds (Peacock and Peacock, 1956). Grafts of a squamous carcinoma of the crop also failed to grow in four birds.
XVI. LYMPHOSARCOMA (GRCH 22) I X D U C E D BY 2-ACETYLAMINOFLUORENE White Leghorn fowl No. 3189 which had received repeated injections into the crop of a 1% solution of 2-acetylaminofluorene in tricaprylin or in arachis oil was killed six and one-half years after the first injection because it was losing weight and had ulceration of the vent. Multiple tumors were present and, as some of these were located in the kidney and macroscopically resembled the adenocarcinomata already observed, they were inoculated with the provisional diagnosis of kidney tumors. However, histological examination of the primary tumors and of the successful grafts showed that the tumor was composed of undifferentiated round cells resembling lymphocytes and it was classified as a lymphosarcoma. The tumor grew rapidly following intraperitoneal or intramuscular injection of homogenates and a t the first passage a preliminary filtrate through No. 1 Whatman filter paper also yielded a positive result. This has not been successfully repeated and may have been due to technical fault or t o the passage through the paper of (a) cells or (b) infective agent. No subsequent evidence of infectivity has yet been obtained in a further 9-serial passage. The use of protamine solution, advocated by Moloney (1956) as a selective precipitant for Rous sarcoma virus was employed in three experiments with GRCH 22 lymphosarcoma but without positive result. I n view of the histological similarity to lymphomatosis, tfests were made by incubating fertile eggs laid by birds that had been inoculated with (a) cells, or (b) filtrates of GRCH 22 tumor. On the 15th-17th day of incubation, the livers of the embryos were removed, homogenized, and inoculated intraperitoneally into eight White Leghorn chicks, 3 weeks old. No evidence of
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transmission of GRCH 22 has been obtained in this experiment after 6 months following injection, but as spontaneous lymphomatosis is slow to develop, only further observation will show whether this tumor is infective or not. XVII. SPECIES AND TISSUE SUSCEPTIBILITY The skin of the fowl seems to be remarkably resistant to the action of carcinogenic polycyclic hydrocarbons. Our observations of squamous carcinoma of the crop at the site of injection of 2-acetylaminofluorene came as a surprise, since this carcinogen does not commonly cause this type of tumor in mammals. I n view of the ease with which sarcoma can be induced in fowls by the carcinogenic polycyclic hydrocarbons, this seems to indicate a tissue resistance rather than a species resistance. The absence of,sebaceous follicles in fowls was thought to be a possible factor in this resistance, but even implantation of pellets of carcinogens into the lumen of the preening gland, which is morphologically a collection of sebaceous glands, failed t.0 induce any neoplastic reaction (Peacock and Peacock, 1949, 1954). It is possible that the squamous epithelium of the skin and of mucous membranes of fowls respond to different types of carcinogen. Carr (1956) has recently described multiple renal tumors in chicks under 20 days old inoculated intramuscularly with erythroleukemia virus ES4. The tumors occur about 20-30 days later. He considers these renal tumors to be adenocarcinomata, but the illustrations could be described as adenomatous or hyperplastic growths without obviously invasive characters. Transplantation of these renal tumors was unsuccessful but the accompanying leucosis was transmissible and yielded both sarcoma and erythroleukemia. This is in keeping with the well known potentiality of leucosis viruses to induce both sarcoma and leukemia. Direct injection of ES4 virus preparations into the kidney induced sarcomata, leukemia, and renal tumors in birds up to 25 days old a t the time of inoculation, and the earliest kidney tumors were found 17 days later, but were never obtained without leucosis. I n view of the short duration of the experiment (24 days), the absence of invasive growth or metastasis, and the failure of grafts to reproduce the renal tumors, it might be advisable to suspend judgment on the classification of these renal lesions as adenocarcinomata. However, they are an interesting example of the well known cytotropic versatility of the fowl leucosis viruses. Kidney tumors resembling adenocarcinomata were described by Foulds (1937) in birds bearing Mill Hill 2, and the present author has frequently encountered this type of growth which at first sight looks like adenocarcinoma.
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XVIII. DISCUSSION The balance of evidence supports the view that chemically induced tumors in fowls resemble induced tumors in mammals in regard to histogenesis, variety, and transplantability. Only sarcomata have been serially transmitted. I n most cases, the transplantable tumors grow slowly but progressively and are late to metastasize. Spontaneous retrogression after grafting may occur and probably depends on hereditary and hormonal characters of the host. Few claims to cell-free transmission have been made and some of these may have been due to faulty techniques, but some may be accepted as above criticism on the available evidence. The interpretation of such results is a problem in itself and will depend on personal points of view. It may be useful to the reader to follow the present author’s line of reasoning, whether in agreement with it or not. Sporadic instances of spontaneous sarcoma in the domestic fowl have been studied in many parts of the world. Many of these tumors could not be transmitted by any of the methods that were used, but some were grafted by cell inoculation to new hosts. I n subsequent passages, some, if not all, of these sarcomata of spontaneous origin were transmitted by cellfree extracts. No epizootic outbreaks of sarcoma have occurred even in laboratories where these tumors have been studied. Occasionally, apparently normal birds have neutralizing antibodies for sarcoma viruses in their serum. The evidence, therefore, is that potential sarcoma virus is widespread among poultry throughout the world, but that it is not necessarily pathogenic. The techniques of poultry breeding have established flocks of widely differing characteristics in regard to many diseases and probably in regard to potent,ial tumor viruses. For example, the Edinburgh flock of Brown Leghorns and the White Leghorn flock used in our experiments are apparently free from spontaneous leucosis and fowl paralysis and do not carry fowl pox. I n birds of unknown pedigree, there may be random distribution of potential sarcoma or leucosis virus. The conditions that initiated the spontaneous virus sarcoma are unknown. However, under experimental conditions, virus-induced sarcoma can be localized at the site of reactive proliferation following mechanical trauma, injection of foreign bodies (Rous et al., 1912), cautery (Pentimalli, 1916), X-ray or radium burns (Peacock, 1946), local infection (Peacock and Peacock, 1953), or injection of methylcholanthrene (Carr, 1942). Cell-free extracts of such localized tumors reproduce the characteristic tumor associated with the particular virus. The conditions under which such experimental localization occurs may preclude the possibility of accidental inoculation of virus, e.g., when ther-
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mocautery is used, but the birds itre known to harbor the virus already. Had any of the birds that bore the original sarcoma from which virusinduced sarcomata were derived been the subject of injection of a carcinogen, it seems likely that they would have yielded infective tumors transmissible by cell-free extracts. The occasional reports of cell-free transmission of chemically induced sarcoma may, therefore, be explained by the presence in the experimental bird of latent or potential virus before the experimental procedure. McIntosh and Selbie’s sarcoma No. 9 and Oberling and Guerin’s methylcholanthrene sarcoma could be explained in this way. Nevertheless, the great majority of chemically induced fowl sarcomata cannot be transmitted by cell-free extracts, and there is no logical reason t o postulate a virus etiology for them against the evidence. If we depended on histological diagnosis for other diseases as we do for cancer, we might well postulate a common cause for many acute bacterial diseases. How could we distinguish between the causes of acute inflammation, for example? Assuming that chemically induced tumors in birds are due to different etiological factors from those of the virus group, they seem to be strictly analogous to chemically induced tumors in mammals, including man. As such they are valuable in throwing light on the etiology and mechanism of carcinogenesis. Yet it may be wise to consider whether chemically induced tumors are any more representative of cancer as it occurs in nature than are virus induced tumors. Apart from industrial and occupational cancers in man, little is known about the etiology of spontaneous cancer. Excessive exposure of the skin to the sun’s rays or artificially to ultraviolet rays is known to cause epidermoid carcinoma in man and in rodents. Cancer of the penis is causally related t o chronic balanitis and can be prevented by early circumcision, but the causal factor has not been identified. Recently Pratt-Thomas et al. (1956) obtained squamous carcinoma of the cervico-vaginal epithelium of female dba-C-strain mice after biweekly intravaginal applications of human smegma for 14 months or more. Chemical analysis revealed 3% cholesterol, but none of the well known carcinogens. Although these experiments were very well designed and controlled, the authors are not yet able to say what factor is responsible for t,he carcinogenic effect. It is reasonably certain that most of the synthetic chemical carcinogens that have been studied in the past twenty-five years or so are not commonly available in nature and are not likely, therefore, to be the cause of spontaneous tumors. An exception must be made in the case of 3 :4-benzpyrene and other carcinogens present in coal tar, because these substances are widely distributed in all urban communities and presumably have been for many hundreds of years.
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As we postulate in the case of viruses that they are not responsible for all cancers, so we must assume that no single chemical or group of chemical carcinogens is the cause of all cancers even of a particular histogenesis, for example squamous carcinoma of the skin. Thus it is reasonable to conclude that cancer is a special type of cell reaction that may be excited by a great variety of factors having in common perhaps nothing but their carcinogenic property.
HEFERENCES Andrewes, C. H. 1931. J. Pathol. Bacteriol. 34, 91-107. Andrewes, C. H. 1933. J . Pathol. Bacteriol. 37, 27-44. Andrewes, C. H. 1936. J . Pathol. Bacteriol. 43, 23-33. Anissimova, V. 1939. Am. J . Cancer 36, 229-232. Apperly, F. L. 1935. Am. J. Cancer 23, 556-557. Bagg, H. J. 1936. Am. J. Cancer 26, 69-84. Baker, S. L., and Peacock, P. R. 1926. Brit. J . Exptl. I’uthol. 7, 310. Bernhard, W., and Oberling, C. 1953. Bull. assoc. franc. Ltude cancer 40, 178-185. Bielschowsky, F., and Green, H. N. 1945. Nature 166, 780. Burmester, B. R., and Gentry, R. F. 1954. Cancer Research 14, 34-42. Campbell, J. G. 1955. Brit. J . Cancer 9, 163-169. Campbell, J. G. 1956. Proc. Roy. SOC.Edinburgh B66, 111-130. Carr, J. G. 1942. Brit. J . Exptl. Pathol. 23, 221-228. Carr, J. G. 1945. Brit. J. Exptl. Pathol. 27, 1-3. Carr, J. G. 1953. Bull. assoc. franc. ktude cancer 40, 407-412. Carr, J. G. 1956. Brit. J. Cancer 10, 379-383. Chalmers, J. G. 1934. Biochem. J . 28, 1214-1218. Claude, A., Porter, K. R., and Pickels, F. G. 1947. Cancer Research 7, 421-430. Cowen, P. N. 1950. Brit. J. Cancer 4, 245-253. Dmochowski, L.,and Knox, R. 1939. Brit. J . Exptl. Pathol. 20, 466472. Duran-Reynals, F. 1952. Ann. N . Y . Acad. Sci. 64, 977-991. Duran-Reynals, F., Burmester, B. R., Cottrall, G. E., and Bryan, E. 1953. Cancer Research 13, 408-414. Epstein, M. A. 1955. Nature 176, 784-785. Epstein, M. A. 1956. Brit. J . Cancer 10, 33-48. Falin, L. I., and Gromaewa, K. E. 1939. Am. J . Cancer 36, 233-236. Feldman, W.H. 1932. “NeopIasms of Domestic Animals.” Saunders, Philadelphia Foulds, L. 1937. Am. J. Cancer 31, 404-413. Foulds, L.,and Dmochowski, L. 1939. Bril. J . Exptl. Pathol. 20, 458-465. Greenwood, A. W., and Peacock, P. R. 1945. Brit. J. Exptl. Pathol. 26, 357-361. Harris, R. J. C. 1953. Advances in Cancer Research 1, 233-271. Howatson, A. F. 1953. Brit. J. Cancer 7, 393-400. Kreyberg, L., and Nielsen, S. S. 1936. Am. J. Cancer 26, 533-540. Laird, H. M. 1956. Personal communication. McIntosh, J. 1933. Brit. J. Exptl. Pathol. 14, 422434. McIntosh, J., and Selbie, F. R. 1939. Brit. J . Exptl. Pathol. 20, 49-63. Mellanby, E. 1934, 11th Ann. Repf. Brit. Empire Cancer Campaign p. 82. Mellanby, E. 1938. J. Pathol. Bacteriol. 46, 447-460. Michalowsky, L. 1928. Virchow’s. Arch. pathol. Anat. u. Physiol. 267, 27. Michalowsky, L. 1929. Virchow’s. Arch. pathol. Anat. u. Physiol. 274, 319. Moloney, J. 1956. J. Natl. Cancer Inst. 16, 877-888.
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Murphy, J. B., and Landsteiner, K. 1925. J . Exptl. Med. 41, 807-816. Murphy, J. B., and Sturm, E. 1941a. Cancer Research 1, 477483. Murphy, J. B., and Sturm, E. 1941b. Cancer Research 1, 609-613. Oberling, C., and Guerin, M. 1950. Bull. assoc. franc. dtude cancer 97, 5-14. Oberling, C., and Guerin, M. 1954. Advances in Cancer Research, 2, 353-423. Peacock, A,, and Peacock, P. R. 1949. Brit. J . Cancer 3, 289-295. Peacock, A., and Peacock, P. R. 1956. Brit. J . Cancer 10, 110-113. Peacock, P. R. 1933. J . Palhol. Bacteriol. 96, 141-152. Peacock, P. R. 1935a. Am. J . Cancer 26, 37-48. Peacock, P. R. 1935b. Am. J . Cancer 26, 49-65. Peacock, P. R. 1946. Cancer Research 6, 311-328. Peacock, P. R., and Peacock, A. 1953. Brit. J . Cancer 7, 12e130. Peacock, P. R., and Peacock, A. 1954. Brit. J . Cancer 8, 147-153. Pentimalli, F. 1916. Sperimentale 70, ( 3 4 ) . Pratt-Thomas, H. R., Heins, H. C., Latham, E., Dennis, E. J., and McIver, F. A. 1956. Cancer 9, 671-680. Rigdon, R. H., and Brashear, D. 1945. Cancer Research 14, 629-631. Rouiller, C., Haguenau, F., Golde, A., and Lacour, L. 1956. Bull. assoc. franc. 6tude cancer 43, 10-22. ROW,P. 1910. J . Exptl. Med. 12, 696. ROUS,P. 1911. J . Exptl. Med. 13, 397-411. ROLE.,P. 1913. J . Exptl. Med. 18, 416. ROUS,P., and Lange, L. B. 1913. J . Exptl. Med. 18, 651. ROUS,P., and Smith, W. E. 1945. J . Exptl. Med. 81, 597-646. ROW,P., Murphy, J. B., and Tytler, W. H. 1912. J . Am. Med. Assoc. 68, 1751. Schoental, R., Head, M. A., and Peacock, P. R. 1954. Brit. J . Cancer 8, 458-465. Sturm, E., and Murphy, J. B. 1928. J . Exptl. Med. 47, 493-502. Vigier, P., and Guerin, M. 1952. Bull. assoc. franc. dtude cancer 99, 175-183.
ANEMIA IN CANCER Vincent E. Price and Robert E. Greenfield Laboratory of Biochemistry, National Cancer Institute, National Instituter of Health, Public Health Service, U. S. Department of Health, Education, and Welfare, Bethesda, Maryland
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I. Introduction. . . . . . . . . , , , . . . . , , . . . 11. Incidence of Anemia.. . . . . . . . . . . . . 1. General Statement.. . . . . . . . . . . .
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2. Methods . . . . . . . . . . . . . . . . . . . . . ........................... A. Determination of Hemoglobin Concentration, Ilcmatocrit, and Red Cell Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Determination of Red Cell Volume, Plasma Volume, and Blood Volume. C. Relation of Blood Volume Data to Various Body Measurements.. . . . . . 3. Findings in Cancer Patients., . , . , . . , . . . . . . . . . , . . , , , , . , . , . . , , , . . . . . . . . A. Hemoglobin Concentration and Hematocrit .............. .............. B. Blood Volume Changes. . . . . . . . . . . . . . , , . . . .................. 4. Summary . . . . . . . . . . . . . . . , , . . . . , . . . . . . . . . , . . 111. Role of Decreased Erythrocyte Formation., , . , . . . . , , . . . . . . . . . . . . . . . . . . . . . .............................. 1. General Considerations, . . . . . , . . . . .............................. 2. Ferrokinetic Studies in Cancer Pa 3. Factors Contributing to the Inhibition of Erythropoiesis. . . . . . . . . . . . . . . . . A. Iron Deficiency.. . , , . , . , . , . . . . . . . . . , , , . . , . . . . . . . . . . . . .
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ion of the Bone Marrow.. . . . . . . . . . . . . . . . . . . . . . . . . 223 D. Infection . . . . . . . . . . . . ., . . . . . . . . . . . . . , , , . , . . , . . _ ,. . . , , . , , , , , . , , , . 225 .......................... 226 4. Discussion ............... , , . , . . . . . . . , , . on . . . . . . . . . . . . . . . . . . . . . . . . . IV. Role of Increased Erythr 1. General Considerations ...................... . . , , . . . . . _ . 226 struction . . . . . . . . . . . 2. Criteria of Increased E A. Red Cell Survival Studies.. . , . . . . . . , , , , . . . . . . . . . . . . B. Excretion of Bile Pigments.. . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 C. Increased Erythrocyte Synthesis, , . . . . . . , . . . . , . . . . . . . . . . . . . . . . . . 235 3. Evidence for Increased Blood Destruction in Cancer Patients.. . . . . . . . . . . 236 A. Studies on Leukemias and Lymphomas.. . . . . . . . . . . . . . . . . . . 236 B. Studies on Carcinomas and Other Neoplasms. , . . . . . . . . . . . . . . . . . . . . . 247 4. Nature of the Destructive Process.. . . . . , . , . . . . . . . . . . , . . . . . . . . . . . . . . . , 255 A. Pathways of Red Cell Destruction., . , . . . . . . . . . , . . . . . . . . . . . . . . . . . . 255 B. Mechanisms Involving Defective Erythrocytes. . . . . . . . . . . . . . . . . . . . . . 257 C. Mechanisms Involving Vascular Defects, . . , . . . . . . . . . . . . . . . . . . . . . . . . 268 5. Discussion ................................................. 278 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... 284 ,
,
,
I. INTRODUCTION The etiology of anemia in cancer patients has been a challenging problem to investigators for many years. Many of the theories as to the cause 199
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of the anemia arose from early studies on leukemia. At the turn of the century, i t was generally accepted that the anemia resulted from invasion and crowding out of the bone marrow by neoplastic tissue, R theory sponsored by the eminent German hematologist, Pappenheim (1906). Hirschfeld (1906), in a detailed study of a leukemic patient with severe anemia, observed hemorrhage in the bone marrow and also noted a remarkable abundance of connective tissue, in some places filling whole areas of the marrow. For this reason he felt that most cases of anemia in leukemic patients could be attributed to the action of a “leukemische Noxe,” i.e., a poison from the leukemic cells, on the bone marrow. He considered that this provided a more adequate explanation for the anemia than that of direct marrow invasion by neoplastic tissue. Because of the excessive amount of hemosiderin in the liver of this patient, he suggested that a hemolytic process might also be a contributing factor. I n 1933, Jaff6 questioned the role played by bone marrow deficiencies since his detailed studies of the bone marrow of 21 patients who had died from leukemia revealed normal or increased erythroid activity in most cases. He observed hemosiderin deposits in the liver cells, Kupfer cells, abdominal lymph nodes, bone marrow, and occasionally in the renal epithelium, which led him to propose that the anemia in both acute and chronic myelogenous leukemia developed as a result of excessive destruction of red cells, and that in the acute leukemias the hemorrhagic diathesis accelerated the rapid decrease in the number of erythrocytes. Although these observations were also made by von Kress (1934), the theory was not widely accepted because of the failure by others to find sufficient amounts of hemosiderin in the liver to account for the anemia (Whipple and Robscheit-Robbins, 1933; Collins and Rose, 1948). Collins and Rose agreed with Forkner (1938, p. 54) that defective blood formation, blood loss, and increased blood destruction may each play a part in the mechanism of anemia in leukemia, but they were of the opinion that the last two processes were only occasional and contributory factors and that disturbance of normal erythropoiesis was not only a commoii accompaniment but an essential pathological feature of the disease. They felt that in myelogenous leukemia the total amount of erythropoietic marrow was usually increased and surmised that the primary factor in the anemia was one of defective maturation. The rapid introduction of radioisotope techniques within the past decade is providing new tools which promise to aid in a more quantitative evaluation of these various theories. The new information derived from such approaches has led, in many instances, t o definite conclusions and increased understanding, in others, to contradiction and variable data. Since the confusion can be traced in many instances to the use of techniques
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which retain, iis yet, intrinsic defects, an attempt will be made in this review t o evaluate these techniques in some detail and to indicate their limitations and potentialities in the study of the anemia associated with cancer.
11. INCIDENCE OF ANEMIA 1. General Statement
I n the past, the term “anemia” in most instances has referred to a low hemoglobin concentration or hematocrit value. On this basis, a high proportion of patients with advanced cancer had a n anemia. If, however, anemia is defined as a deficiency in the total hemoglobin or red cell mass of the body, then a low hemoglobin or hematocrit would be a valid criterion of anemia only in those cases in which there is a relatively stable blood volume. Otherwise, a low hemoglobin concentration may indicate either a deficiency of hemoglobin, or simply a dilution of the normal amount of hemoglobin in a higher volume of blood; conversely, a high hemoglobin concentration may indicate either an increase in the total hemoglobin mass or a state of hemoconcentration. Studies carried out on patients with cancer in an effort to determine the changes in bIood volume have presented conflicting conclusions. Clark et al. (1947b,c), Morton et al. (1952), and Durbn (1955) presented evidence that the blood volume of patients with cancer was depressed. Bateman (1951) and Linden (1955) indicated that there was no change in blood volume, and Kelly el al. (1952) indicated that there was evidence of hypervolemia in over half of the cancer patients they studied. Investigators whose studies revealed changes in blood volume have raised doubt as to the reliability of lowered hemoglobin concentration and hematocrit values as criteria of anemia, and they have suggested that total hemoglobin mass would be a more adequate comparative value. On the other hand, the variability in the blood volumes reported for patients with cancer and the conflicting conclusions reached by various authors raise the question as to the reliability of the methods used to measure, calculate, and present data on blood volume. Since the question of the incidence of anemia in cancer is dependent on the reliability of the methods used, some of the problems which arise in these procedures will be discussed in the next section. 2. Methods
A. Detei~t~incctionof Hertroglobitr Concentmtion, Hematocrit, and Red Cell Count. :L. Hemoglobin conceiitrtrtion. Comparative evaluation of hemoglobin concentrittion between patients is difficult. The rather marked spread of normal henioglobin values from 11.5-18 g./100 ml. blood (Sturgis,
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VINCENT E. PRICE: AND ROBERT
E.
GREENFIELD
1955, p.4) makes the interpretation of the hemoglobin level of a single patient subject t o rather large error. For example, if the value of 11.5 g.% is used as the lower limit of normal, a patient with 16 g. of hemoglobin could have a 28% reduction of hemoglobin before he is classified as anemic, and a patient whose normal level of hemoglobin is 12 g. has to have only a 4y0 depression to fall in the same category. For comparison of general phenomena in different disease states, this problem can be minimized by comparing results obtained on large groups of patients. The normal range can also be effectively reduced by calculating the data from patients according t o sex. The male has a range of hemoglobin from 14-18 g./100 ml. of blood with an average of approximately 16 g.%. The female has a range of 11.5-16 with an average of 14 g.%. At the present time, hemoglobin values from one laboratory cannot be compared to that of another without recalculation as the standards used vary widely. For example, the average normal hemoglobin standards reported from the various laboratories referred to later in this section vary from 12.8 to 16 g. The need for a stable calibration standard such as that proposed (Cannan, 1955) is obvious. b. Hematocrit. An excellent discussion of hematocrit techniques was given by Mollison (1956) and will not be repeated here. Wintrobe (1946, p.72) reported the hematocrit of normal males t o be 47.0 f 7.0 and that of females to be 42.0 f 5.0. There is considerable variation among different investigators as to the factor to be used in the correction of the hematocrit value for plasma trapped in the centrifuged red cells (0&8.5% of the observed value). Chaplin and Mollison (1952) suggested that the per cent correction should vary with the length of the red cell column since the amount of plasma trapped in the lower layers of the packed red cells was found to be smaller than that in the upper layers. Most of the studies on the blood volume of patients with cancer have reported hematocrit values without correction for entrapped plasma. c. Erythrocyte count. Few investigators employ the red cell count as a routine procedure. The amount of time, training, and care needed to prevent large errors are usually out of proportion to the added information obtained (Magath et al., 1936). Red cell counts are now more or less restricted to situations in which the unique information obtained from such a count is needed. B. Determination of Red Cell Volume, Plasma Volume, and Blood Volume. a. General considerations. I n order to measure the total hemoglobin mass, it is necessary to know both the volume occupied by the red cells and the hemoglobin concentration per ml. of packed cells. The red cell volume can be determined most directly by measuring the dilution of tagged red cells. The isotopes chromiumdl and phosphorus-32 can be incorporated into red cells in vitro, thereby tagging the individuals’ own cells. At present
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Cr61 is the isotope of choice for the determination of blood volunie due to its efficiency in labeling the cells, its slow elution, :ind its easily measured gamma emission (Gray and Sterling, 1950). Irou-59 :tiid iron-55 must be administered to a donor to obtain lal>eleclblood for retransfusioii into the individual whose blood volume is to be determined. In clinical studies, these requirements present problems of dosage and technique. In studies using highly inbred laboratory animals, however, the utilization of FeK9is often the method of choice. Many of the earlier studies on the red cell volume of patients with cancer were carried out prior to the extensive use of radioisotopes as a red cell label. The red cell volume was in most instances calculated from the total plasma volume and the hematocrit by the formula: red cell volume = plasma volume X (100/100 - hematocrit) - plasma volume. The plasma volume was most often calculated from the dilution of the dye T 1824 as described by Gregerson (1944). This method has been vigorously criticized and defended over the past decade. As many of the hypotheses and concepts associated with blood volume have developed during the controversy over this technique, a discussion of this procedure and a brief review of its history are necessary for proper evaluation of the available data using this technique in cancer patients. The dye method requires little equipment and as far as the techniques are concerned can be carried out in most hospitals. It is not, however, a procedure which can be easily developed into a routine type of assay, as the measurement on each individual patient requires special consideration if reliable results are to be obtained. As an example, hemolysis or lipemia may lead to large errors which are not readily avoided by reading the plasma dye against a plasma blank of the same patient. An excellent discussion of techniques and precautions necessary to carry out this procedure is given by Mollison (1956). Beyond these difficulties in technique, which can be minimized with experience and skill, are those problems concerned with the proper interpretation of the data obtained. The two main questions which arise are: (1) Does the dilution of T 1824, or any other labeled plasma method, measure only the volume of the plasma within the blood vessels, and if so (2) can the red cell volume or blood volume be measured from the plasma volume with the aid of the hematocrit. Although neither of these questions can be answered completely a t this time, a great deal of information has been accumulated which makes it possible to a t least partially answer both of them. b. Binding of T 18% dye. Many investigations have been carried out on the fate of the dye in the 10 minutes between the injection of dye and the withdrawal of the dye-plasma sample. I t has been shown (Rawson, 1942; Gregerson and Ramson, 1942; Barnes el nl., 194%) that the intra-
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VINCENT E. PRICE A N D ROBERT E. GREENFIELD
venous injection of the dye is followcd by a rapid combination with plasma albumin. Certain investigators (Storaasli et al., 1950; Aust et al., 1951 ; Krieger et nl., 1048) reported plasma volumes measured with T 1824 dye to be greater than those determined with radioactive iodiriated plasma protein or human serum albumin. They suggested that a certain amount of dye escapes from the vascular system in the first few minutes following injection, in the period before the dye becomes firmly bound to the albumin. In view of later findings, the loss of appreciable unbound dye appears unlikely. Crispell et al. (1950), Schultz et al. (1953), and Inkley et al. (1955) on determining the plasma volume of patients simultaneously with T 1824 and Ilsl-labeled albumin found no significant difference in the results obtained by the two methods. Freinkel et al. (1953) injected simultaneously T 1824 and iodinated albumin and measured the concentration of each a t 2-second intervals through the first cycle of circulation and into the second cycle. The curves obtained by the two methods were almost superimposable, the dilutions were consequently very similar, and no evidence for a disproportionate loss of T 1824 was seen. There appeared to be an immediate and stable union between the dye and protein with no evidence for loss of unbound dye. c. Relationship between red cell and plasma volume. The question as to whether the blood volume and the red cell mass can be determined from the plasma volume and the hematocrit has been the subject of numerous investigations over the past four decades. Smith et al. (1921) using the carbon monoxide and viviperfusion method of Welcher to determine the red cell volume of dogs demonstrated that the total mass of red cells determined by these procedures was 20-30% less than the values obtained by calculations based on the plasma volume and venous hematocrit. They postulated that this discrepancy was caused by the false assumption that the venous hematocrit was representative of the ratio of red cell to plasma throughout the body, and they recommended that total blood volume be calculated by adding the plasma volume, obtained by a protein dilution procedure, to the red cell volume. They pointed out that the percentage of the total blood volume occupied by red cells could be calculated by dividing the total red cell volume by the blood volume calculated by the above procedure. This value has come to be known as the “body hematocrit.” Smith et al. suggested that a relatively low red cell to plasma ratio in the capillaries might be the principle reason that the venous hematocrit was not representative of the cell to plasma ratio of the whole body. They suggested that laws governing the flow of simple liquids might apply to blood flow in the capillaries, i.e., that the center of the stream moves a t a rate faster than the average velocity of the stream. On the basis of previous
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microscopic findings indicating that the flow of blood in small vessels was not uniform in all parts of the cross section, they suggested that the peripheral “still space” might represent a body of plasma which was relatively stagnant when compared to the more rapidly flowing “axial stream” of blood in minute vessels. Fahraeus and Lindquist (1931) provided corroborative data to support this hypothesis by showing that blood when passed through progressively smaller glass capillaries of 500p or less had a decreasing proportion of red cells to plasma. Krogh (1929, p.5), Jager (1935), and Vejlens (1948) describe a marginal plasma layer with the thickness approximating the diameter of a red cell, surrounding the axial stream of blood in minute vessels. Although this theory has obtained wide acceptance aiid has become one of the theories by which the concept of “body hematocrit” has been explained, it has been difficult to obtain direct experimental evidence as to its quantitative effect upon the blood volume (Gibson et aZ., 1946b; Barnes et al., 1948a). Peters (1948) and Prentice et al. (1951) oppose the theory of “body hematocrit” aiid contend that the dye space is not synonymous with the plasma volume but includes a part of the volume of the lymphatic system and biliary system. They argue that the presence of dye in the thoracic duct within 5 minutes of its injection prevents any method based on dye concentration from accurately measuring the circulating plasma in spite of any refinement of colorimetric techniques or mathematical manipulations. The excellent review by Gregerson (1951) discusses this question more thoroughly than can be undertaken here. The lack of evidence for an early disproportionate loss of labeled albumin in the rather extensive studies of Moir et al. (1956) on the early disappearance curves makes it probable that the loss of the albumin complex is directly dependent on concentration and is thereby subject to correction by extrapolation of the concentration of labeled albumin to zero time. Although the reason for the discrepancy between the total red cell mass determined by the use of labeled red cells and that determined from plasma volume and hematocrit has remained in question, the presence of the discrepancy has become firmly established by many investigators (Hahn et al., 1941, 1942b; Reeves and Veall, 1949; Nachman et al., 1950; Berson and Yalow, 1952; and others). Gibson et al. (1946a) concluded that the ratio of body hematocrit to venous hematocrit was a constant in both dogs and man with a value of about 0.91 (cf. Hicks et al., 1956). Their conclusion was based on observations made on human subjects whose venous hematocrit ranged from 38.548.1% and on dogs whose hematocrits ranged from 31-62%. Mollison et aZ. (1950) extended these conclusions by showing that the ratio of body hematocrit to venous hematocrit in newborn infants remained relatively constant over a venous hematocrit range of 17.9-66.2
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although their value of 0.87 is slightly lower than that usually obtained in adults. Chaplin et al. (1953), reporting on anemic and polycythemic patients, showed a mean value of 0.910 with a standard deviation of 0.026 for 28 measurements over the hematocrit range from 9-82Oj,. These authors also review some of the earlier comparative investigations and show that the body-venous hematocrit ratio is remarkably constant. They conclude that the small variability that still exists can be explained by the different conventions of the various laboratories which carry out investigations in this field. They suggest that an investigator who does not elect to determine a value for the ratio of body to venous hematocrit under his own conditions of measurement is justified in selecting a value between 0.90 and 0.92. They note that the constancy of the body-venous hematocrit ratio strongly suggests that there is no systematic overestimation of plasma volume, for if there were, relatively higher ratios would be obtained in polycythemic subjects and lower ratios in anemic subjects. Although the dilution of labeled cells is undoubtedly the most direct method of measuring the red cell volume, it may be concluded that the difference between the labeled red cell and plasma techniques has proven to be so nearly constant over such a wide range of conditions that comparison of the two methods using the ratio of body to venous hematocrit seems to be justified in most instances. Steinfeld (1956) showed that a ratio of 0.91 was also valid for the cancer patients he studied by simultaneously measuring the blood volume using CP-labeled erythrocytes and labeled albumin. He showed that the published data on the blood volume of cancer patients could be brought into closer agreement by the use of this correction factor. C. Relation of Blood Volume Data to Various Body Measurements. It has been shown that blood volume per kg. varies quite considerably with body type (Gregerson and Nickerson, 1950). I n a group of 9 healthy students, selected because of their endomorphic body type, the mean plasma volume was 67.8 ml./kg. of body weight with the extreme values ranging from 62.8 to 78.9 ml./kg. Another group of students, selected for their obvious ectomorphic body type, had a mean plasma volume of 101.0 ml./kg. of body weight with a range from 92.4 to 108.8. N. I. Berlin et al. (1951a) suggested that the difference in the red cell volume of the males (29.9 ml./kg.) and the females (27.0 ml./kg.) in the individuals they studied might be explained by the increased amount of fat in the female (cf. Hicks et aE., 1956), and they predicted that the red cell volumes per kg. of “fat free” body weight would give better agreement. They suggested that the blood volume of female patients in debilitating disease states might more accurately be compared to that of normal males because of the loss of body f a t in these conditions. Bush et al. (1955) showed that in swine the blood
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volume per kg. decreases with growth and ascribed this change to the excessive deposition of fat in the adult pig. Anderson et al. (1957), on studying the relationship between gain in blood volume and body weight on simple overeating, reported that there was some iiicrease in total blood volume during the first weeks of overeating with no further change thereafter. The 8 patients who gained weight most rapidly had an average increase of 25% in body weight and an average increase of only 4.5% in blood volume. The average specific blood volume of these individuals decreased from 41.3 to 34.5 ml./kg. during the 20 weeks of observation. Linden (1955) compared plasma volume with the thickness of the subcutaneous fat layer as a percentage of the body circumference. He also estimated the lean body mass from the difference between thoracic and abdominal measurements by using the empirical relationship between these measurements and total specific gravity described by Behnke et al. (1942) and Welham and Behnke (1942) and found a better correlation than when body weight was used. He reports a mean plasma volume per kg. lean body mass of 48.56 f 0.53 ml., o, f 2.99, as compared to a plasma volume per kg. body weight of 35.8 f 1.15 ml., o, = f 6 . 5 3 , in a series composed of 15 male patients with minor physical defects or illnesses and 17 rather obese women, the majority of whom had cholelithiasis. When the patients were arranged according to the amount of subcutaneous fat, nearly all of the males had less fat than the females. The plasma volume in ml./kg. correlated inversely with the fat content, ranging from 45.6 ml./kg. for the thinnest male to 23.6 ml./kg. for the fattest female. The extreme range of the plasma volume per kg. lean body mass was 53.744.2 ml. A second series of patients studied by Linden was composed of rather thin males suffering in most cases from duodenal or gastric ulcers without bleeding. In this series the plasma volume in ml./kg. body weight ranged from 48.5 ml./kg. for the thinnest individual to 37.1 ml./kg. for the fattest individual. The extreme range in plasma volume per kg. lean body mass was 53.045.5 ml. Linden extended his studies to cancer patients and his interesting results will be presented in the next section. A less empirical method of relating blood volume to lean body Inass is to calculate the proportion of lean body mass and adipose tissue in the body directly from total body density. I n order to make such calculations, assumptions are made that the water content of the lean body mass is constant, that all tissues except bone and fat have a similar density, and that bone minerals represent a certain per cent of the lean body mass. If these assumptions are true, any change in the body density must be due to fat. The basic studies of Itathbun and Pace (1945), Pace and Itathbun (1945), and Morales et al. (1945) on guinea pigs, and the studies of Behnke et al. (1953) and others on man have shown these estimates to be reason-
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able in the normal individual. Tissues other than fat and bone have been found to have similar densities. The lean body mass of an adult man was found to contaiii about 72% water, 19% “protein,” 7% mineral, and 2% essential lipid niaterial (Behnke, et nl. 1953). Using such body density techniques, Huff and Feller (1956) and Allen et al. (1956) have made extensive studies on the changes of blood volume with variations in the fat content of normal individuals. From these studies Huff and Feller estimated that adipose tissue contained about 70% as much blood as the lean body mass, whereas Allen et al. estimated 15-20% for this value. In view of the difference in the methods used by these two authors and considering the sources of error which are still present in these techniques, it is probably wisest to conclude only that these calculations indicate that the blood volume of adipose tissue is considerably lower than that of the lean body mass without attempting to quantitate the difference a t this time. Unfortunately the assumptions implicit in the calculation of body fat from body density are not valid in the patient with water imbalance. Furthermore, the ratio of tissue to bone cannot be expected to remain constant in an emaciated individual in negative nitrogen balance. Metastases may also cause actual lysis of bone. The eventual solution of this problem must depend on the development of techniques for the independent measurement of bone mass, tissue, fat, and extracellular fluid in the individual patient. Until such estimates can be made, the comparison of blood volume between individual patients will be subject to rather large variation. The authors are essentially in agreement with Inkley et al. (1955) when he states, “By any current technique in use, plasma volume has definite limitations, and reliance on clinical signs and symptoms as well as other laboratory procedures should not be abandoned in favor of a plasma volume determination, which may vary considerably in any individual or within groups of individuals.” 3. Findings i n Cancer Patients
A. Hemoglobin Concentration and Hematocrit Values. Low hemoglobin concentration and hematocrit values are common findings in patients with malignancy. Shen and Homburger (1951) reported that 60% of 193 patients with advanced carcinoma had a hemoglobin concentration of less than 80% of the normal value of 15.6 g./100 ml. (g.%). Of their 116 cases with low hemoglobin, 52 cases (44.8%) had a hemoglobin concentration between 7040% of normal, 42 cases (36.2%) were 55-69% of normal, and 22 (19%) were below 55% of normal. Morton et al. (1952) reporting on 25 patients with far-advanced cancer of the colon showed that 52% had hemoglobins below 80% of the normal value of 16 g.%. Samuels and
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Bierman (1956) reported that in a group of 158 patients with various malignant neoplasms, a hemoglobin concentration below 80% of the normal value of 12.9 g.% was found in the following frequency: carcinoma, 55%; lymphoma, 77%; chronic lymphocytic leukemia, 75y0’,; chronic granulocytic leukemia, 95%; acute leukemia, 97%. Collins and Rose (1948), in their studies on the anemia of leukemia, reported that significant anemia, with hemoglobins of less than 12 g.%, which is 82% of their standard value of 14.5 g.70, was present in every case of acute leukemia, in 75% of their cases of chronic lymphatic leukemia, and in 65% of their cases of chronic myelogenous leukemia. Severe anemia, below 7.25 g.%, was found in acute leukemia in 64y0 of the patients, in chronic lymphatic leukemia in 50%, and in chronic myelogenous leukemia in 30% of the patients. These figures can only give a general estimate as to the prevalence of the decreased hemoglobin concentration in patients with various types of neoplasms. Details as to the stage of the disease and the general condition of the individual patient greatly influence the percentage of patients with low hemoglobin concentration in any given group. Sturgis (1955, p. 42) states, “From my experience it seems reasonable to estimate that anemia develops in about 750/, of all patients with cancer a t some time during the course of the disease.” In studies on tumor-bearing animals, decreased hemoglobin concentration, red cell count, and hematocrit values have been correlated with the growth of both induced and transplanted tumors in the rat (Dunning and Reich, 1943), the mouse (Taylor and Pollack, 1942), and the embryonic chick (Taylor et al., 1943). Taylor (1945, p.99) was able to show that only a portion of the decrease in hemoglobin concentration seen in DBA mice with a transplanted carcinoma could be explained by the increased blood volume found in these animals (see Fig. 1). B. Blood Volume Changes. Early investigators who studied blood volume in the cancer patient were int,erested in quantitating the amount of blood necessary to adequately prepare a patient for surgery (Clark et al., 1947a). The goal was to restore the patient as nearly as possible to his former state of health. From this viewpoint, the blood volume of the patient prior t o his illness appeared to be the ideal standard for a basis of comparison. This was calculated by multiplying the patient’s previous weight in kg. by a standard value for blood volume of 85 ml./kg. of body weight. The standard blood volume, obtained by the plasma dilution technique, was reported by Gregerson (1944) and confirmed by Stewart and Warren (1945), Clark et al. (1947b), and Kelly et al. (1952). When the blood volumes of a series of cancer patients (Clark et al., 1947c), determined by measuring the dilution of T 1824 dye, were compared to the estimated
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blood volume of the patient prior to his illness, a moderate decrease was found in nearly every case. The decreased blood volume represented primarily a decrease in the total red cell mass. The total red cell volume of their patients with carcinoma of the stomach, colon, and pancreas averaged 50, 51, and 59% respectively, of the standard red cell volume. The total plasma volume varied to a much less extent being 93, 74, and SO%, respectively, of the standard value for plasma volume. Clark et al. (1947a) introduced the concept of “chronic shock” to explain the findings of diminished blood volume and total plasma proteins in patients with cancer and other debilitating diseases and they recommended that the blood volume be restored by transfusion preoperatively. The extensive studies of D u r h (1955) are presented in a similar fashion and confirm the earlier findings a I20 110
100 90 80
70 60 50 40
30 20 1
o
0
1
2
3
4
5
6
7
8
9
TUMOR WEIGHT IN GRAMS
FIQ.1. Indicates the increase in blood volrimc and thc decrease in hemoglobin with the growth of a tumor (from Taylor, 1945).
of Clark et al. In fact, most of the data now available on the blood volume of the cancer patient would indicate a deficit in blood volume if the total blood volume is compared to that of the patient prior to his illness. The question arises as t o whether a decrease of the total blood volume of a chronically ill patient results from the pathological processes suggested by the term “chronic shock” or is simply a compensatory adaptation of the body to a decreased requirement for red cell mass because of weight loss and inactivity. Expressing the blood volume in terms of body weight also leads to misconceptions in patients with weight loss since adipose tissue which contains less blood than lean tissue tends t o be lost more raipdly than other tissues. Consideration must also be given to the possibility that the blood volume contained within the central vessels may not contract proportionally with the loss of body weight. Therefore, in the absence of any specific effect of the neoplastic process, the total blood volume of the patient with cancer
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would tend to decrease because of weight loss, and the specific blood volume (ml./kg.) would tend to increase. When the data of Clark et al. (1947~)was recalculated on the basis of ml. of blood per kg. of actual body weight, the specific blood volumes on the patients with carcinoma of the pancreas, lung, and esophagus were not elevated but were within the normal range. The patients with carcinoma of the coloii had a significant reduction in specific blood volume, having 64 ml. of blood per kg. against a control standard of 85 ml./kg. It must be concluded in view of the expected rise of the blood volume, when expressed as ml./kg. of body weight, that at least in carcinoma of the colon there was a reduction in blood volume beyond that expected from weight loss, and probably in the others also. This reduction of specific blood volume in patients with carcinoma of the colon was confirmed by Morton et al. (1952). In this study, the specific blood volume (ml./kg.) of 25 patients with carcinoma of the colon, determined by the dilution of T 1824, was compared to a standard of 85 ml./kg. which was originally obtained from normal individuals averaging 15-20% fat.' Since these tumor patients had an average loss of 13% in body weight, their blood should more correctly be compared to a standard obtained from lean individuals who have a higher blood volume per kg. of body weight.2 I n spite of this tendency to minimize the difference and in spite of the fact that blood volume was related to the patient's observed weight rather than his previous normal weight, there was an 18% depression in specific blood volume, a 30% depression in specific red cell volume, and only an 8% depression in specific plasma volume. It would appear that in these cases there was a rather remarkable reduction in the blood volume which is attributed largely to a loss in red cell mass. Bateman (1951) reported on the blood volume of 33 cases of faradvanced carcinoma, measured by the dilution of T 1824. The total blood volume of tumor patients was compared to the blood volume of normal individuals of the same height, obtained from the relationship between height and blood volume reported by Gibson and Evans (1937). The total blood volume of the patient when compared to such a standard appeared to bear no direct relationship to the disease and ranged from 73-149% of the standard.
* The question has been raised (N. I. Berlin el al., 1955) as to whether the observed values for blood volume, plasma volume, red cell volume, circulating hemoglobin, and circulating protein in Table I1 of Morton el al. (1952) were calculated on the basis of observed or standard weight. The author has informed us that the observed weight was used. The standard weight was used only to calculate the per cent depression of the observed weight. 2 Linden (1955) reported a mean blood volume of 92.5 ml./kg. for a series of 35 lean males with gastric or duodenal ulcers, calculated from the dilution of T 1824 dye.
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The great variability in the values obtained by Bateman can be partially explained by her selection of a standard based solely on height without any consideration of weight. The total blood volume of individuals of a given height varies greatly according to body frame, muscularity, and obesity. I n order t o illustrate the variability introduced by body build, the per cent deviation of the total blood volume of individual patients with mammary tumors from that calculated from height, as reported by Bateman, are plotted in Fig. 2. The per cent deviation of the patient’s body weight from a standard weight, calculated from an empirical relationship between weight and height (Lorentz, 1936), was also plotted. It can b e seen that the total blood volume varied from that calculated from height as the body weight varied from the standard body weight based on height. It can be seen from Fig. 2 that if one corrected the blood volumes for the variations related to deviations in body weight, they would fall within the normal range. When the 33 cases reported by Bateman were also recalculated by the method of Morton et al. (1952) by relating their specific blood volume t o the arbitrary value of 85 ml./kg., a decreased blood volume was found in 24 patients although only 9 were below 80% of the standard and only 2 were below 70% of the standard. Kelly et al. (1952) reported on the blood volume, body water, and circulation time of 33 patients with far-advanced neoplastic disease. The normal value for specific blood volume, calculated by the dilution of T 1824, ranged from 72-99 ml./kg. with a mean of 85 8.9 ml./kg. of body weight. I n over half of the cancer patients, an increased blood volume was found. The suggestion was made that the anemia seen in far-advanced cancer patients may be partially explained by the dilution of red cells. In Fig. 3, the specific blood volume data (Kelly et al., 1952) on patients with carcinoma of the breast, testes, cervix, lung, lips, nasopharynx, as well as melanoma, multiple myeloma, mycosis fungoides, lymphosarcoma, and Hodgkin’s disease, were corrected for body hematocrit and replotted in terms of ml./kg. body weight against the patient’s observed body weight as a per cent of a standard body weight calculated by the arbitrary relationship of Lorentz (1936) between weight and height. It can be seen that there is a rather good correlation, considering that there has been no separation of patients as to sex and considering the wide variety of tumors involved. Those patients whose body weight fell below the standard weight tend t o have a higher amount of blood per kg. of body weight than those whose body weight exceeded the standard weight. Such a relationship suggests that part of the apparent increase or decrease in specific blood volume of these patients also can be accounted for by the inverse relationship between specific blood volume and deviation from normal body weight. This relationship between body weight and specific blood volume did not
+
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ANEMIA IN CANCER
hold for patients with leukemia in that a rather marked hypervolemia was associated with normal or elevated body weights. Linden (1955) has recently attempted to relate the blood volume of the
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PATIENTS
FIG.2. 0 Per cent deviation of the patient’s total blood volume from that calculated from height (from Bateman, 1951). 0 Per cent deviation of the patient’s actual body weight from a standard weight calculated from height by the following formula: (Ht., cm. - 100) - (Ht,., cm. - 150)0.25 = kg. body weight (Lorentr, 1936).
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BODY W E I G H T - % OF STANDARD
FIQ.3. Indicates the relationship between specific blood volume of patients with cancer and thc observed body weight expressed as a per cent of a standard body weight calculated from an empirical relationship between weight and height (Lorentz, 1936). Replotted as described in the text from Kelly el al. (1952).
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VINCENT E. PRICE AND ROBERT E. GREENFIELD
cancer patient directly to lean body mass. The lean body mass was calculated indirectly, as described above, through the relationship between specific gravity, subcutaneous fat, and body circumference. The blood volume was calculated from the dilution of T 1824 using the hematocrit. Twenty-seven of the 47 cases studied had cancer of the stomach. Eleven of these cases had stenosis, emesis, or edema. The material studied also included 5 patients with cancer of the rectum, 3 with carcinoma of the pancreas, 2 with carcinoma of the lung, 2 with carcinoma of the cecum, and single patients with hepatic carcinoma, carcinoma of the larynx, carcinoma of the lip, carcinoma of the colon, carcinoma of the prostate, carcinoma of the intestine, and a carcinoma from the mediastinum. The blood volume of these cases averaged 91.7 f 1.45 ml./kg. of lean body mass and was found to be equal to the standard normal values expressed in terms of lean body mass. The red cell deficit was found to be fully compensated by the increase in plasma volume except in 4 cases with stenosis of the intestine and emesis. In these cases the disorder of the fluid balance was thought to have been responsible for the plasma volume deficit. Linden concluded that the organism, in spite of protein depletion and weight loss, is capable of maintaining the blood volume at a level corresponding to the lean body mass and compensates for red cell deficit by increasing the plasma volume. All but 16 out of the 47 cases Linden studied had a body weight between 80-100% of normal. The blood volume of these patients (83.7 f 1.55 ml./kg. of body weight) compared closely to that calculated for a series of patients with gastric and duodenal ulcers (84.0 ml./kg. of body weight) and varied considerably from the specific blood volume calculated for a series of fat patients (66.2 ml./kg. of body weight). The subcutaneous fat layer expressed as a fraction of the body circumference was found to compare closely between the cancer patients and those patients with ulcers while the series consisting predominantly of female patients with cholelithiasis had a much thicker layer of subcutaneous fat and therefore a higher ratio of subcutaneous fat to body circumference. As the tumor patients of this series indicated no change in blood volume, expressed in terms of lean body mass, i t was of interest to see if plotting the specific blood volume against the patient’s deviation from standard body weight (Lorentz, 1936) would support a similar conclusion. It can be seen (Fig. 4) that the correlation was fairly good. When the blood volumes are corrected for the variation related to deviations in body weight, most of the blood volumes fall within the normal range. Therefore, the graph correction of changes in blood volume due to variation in body weight agrees with Linden’s own findings, based on an empirical calculation of body fat, that in this series of patients there was no significant
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change of blood volume which cannot be explained on the basis of the proportion of adipose tissue in the various patients. N. I. Berlin et al. (1955) measured the blood volume of 66 patients with far-advanced carcinoma using P32-labeled red blood cells. The use of fatfree body weight as an index of comparison was discussed, but postponed on the basis that further development of technique was necessary before such a method was practical. Although the authors were well aware of the dangers of erroneously interpreting data based on body weight in the presence of rapid weight loss, it was decided that actual body weight was the best way available to present the blood volume data. On this basis they concluded that 21 patients, or 31.8% of the patients studied, were anemic as they had a red cell volume below 24 ml./kg. of actual body weight,
90
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BODY WEIGHT - % OF STANDARD
FIG.4. Indicates the relationship between specific blood volume of males with carcinoma of the stomach and observed body weight expressed as a per cent of a standard body weight based on height (Lorentz, 1936). Data corrected for body hematocrit. Calculated from data of Linden (1955).
which was the lower limit of the normal range. As pointed out by the authors, it should be emphasized that this value is a minimal figure, as in most instances the patients had lost rather considerable body weight and consequently the patient’s red cell volume should more correctly be compared to a value approaching that for lean body mass. In order t o show the relationship of specific blood volume to the loss of body weight, the specific blood volume of those patients reported by N. I. Berlin to have carcinoma of the stomach or esophagus were corrected for body hematocrit and plotted (Fig. 5) against the body weight, expressed as a per cent of the empirical standard of Lorentz (1936). The correlation suggests that part of the variability of the specific blood volume is related to loss of “adipose tissue.” The fact that the line of regression crosses the 100% mark below the normal average specific blood volume suggests that there may be a moderate reduction of specific blood volume in these patients.
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VINCENT E. PRICE A N D ItOBERT E. GREENFIELD
Reilly el al. (1956) measured blood volume by the dilution of Cidllabeled red cells. Although the average specific blood volume of the patients studied was not significantly different from that of the normal controls, the specific blood volumes of patients with leukemia, carcinoma of the stomach and esophagus, and carcinoma of the colon were significantly elevated. Red cell mass and plasma volumes were calculated from the hematocrit. The average red cell mass of all cancer patients, 25.9 ml./kg., was significantly lower than that of the normal controls, 29.6 ml./kg. The average plasma volume, 41.6 ml./kg., was significantly increased from that of the normal control, 35.9 ml./kg. The hematocrit of patients with cancer in all categories averaged lower than normal.
50
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I 60 70 80 90 100 110 B O D Y W E I G H T % OF S T A N D A R D
@I 120
-
Fro. 5. Indicates the relationship between specific blood volume of patients with carcinoma of the stomach or esophagus and the observed body weight expressed as a per cent of a standard weight based on height (Lorenta, 1936). Replotted as described in the text from the data of N. I. Berlin el al. (1955).
4. Summary The use of hemoglobin concentration as a criterion of anemia has been criticized previously on the basis that a concentration value cannot distinguish between an actual change in total hemoglobin mass and an apparent change resulting from alterations in blood volume. It has been suggested that total hemoglobin mass, by reflecting changes of blood volume, more directly measures the oxygen carrying capacity of the vascular system. Since the total hemoglobin mass will vary markedly with the size and weight of the individual, a suitable basis for comparison must be selected. In this section, an attempt was made to show that much of the reported change in specific blood volume of patients with cancer could be related to
AXEMI.4 I N CASCER
217
deviations of the observed body weight from :L standard weight formula of Lorenta (1936) and was strikingly similar to the changes of specific blood volume resulting from dietary gain or loss of weight. The variations in specific blood volume, which remained after correction was made for those changes related to deviations of body weight, varied with the type of tumor but, in general, were only slightly lower than normal. It would therefore seem that both hemoglobin concentration and hematocrit values would be reasonable criteria of anemia until the fundamental relationships needed to compare the total hemoglobin mass from one patient to another are more exactly delineated. It has long been known that a great majority of cancer patients have low hemoglobin and hematocrit values which vary greatly from patient to patient but as a rule become progressively lower as the disease progresses. Until more satisfactory criteria for anemia are available the best conclusion that can be made a t this time is that most cancer patients develop an anemia a t some stage during their disease.
111. ROLE OF DECREASED ERYTHROCYTE FORMATION 1. General Considerations
Until recently, investigations on the role of decreased erythrocyte synthesis in the development of anemia in the cancer host have been hampered by the lack of methods for estimating the rate of red cell synthesis. Even at autopsy i t is impossible to estimate the total amount of erythropoietic tissue in the cancer patient as only limited areas of marrow can be viewed if undue mutilation is to be prevented, and extramedullary erythropoiesis increases this difficulty. Microscopic examination can be very misleading unless numerous samples from various areas are studied since the remaining bone marrow often hypertrophies in the presence of bone marrow destruction. Until the extensive use of isotopes and tracer techniques became established, the number of reticulocytes in the peripheral blood served as the best criterion of the rate of synthesis. In more recent years, the level of the hemoglobin maintained in the presence of known rates of red cell destruction has provided some estimate of the overall capacity for erythrocyte synthesis in the host. A major advance in studies on blood formation was made in 1950 by Huff et al. who reported a technique for estimating the rate of erythrocyte synthesis by use of radioiron. In this procedure the turnover of the plasma iron was calculated by multiplying the total plasma iron (plasma iron concentration x plasma volume) by the rate a t which iron disappears from the circulating plasma following the injection of plasma labeled with tracer amounts of radioiron. An estimate of red cell iron turnover was approximated by multiplying the plasma turnover by that proportion of
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VINCENT E. PRICE AND ROBERT E. GREENFIELD
the injected radioactive iroii found in the red cells after a period of equilibration. Many attempts have been made to find simpler relationships between the metabolism of iron and erythropoiesis. Finch et al. (1949a) found in patients with cancer a decreased rate of radioiron incorporation into red cells. Although the rate of incorporation was found t o be related to erythropoiesis, it was also influenced by the size of the iron pools of the body and the amount of unlabeled iron originating from red cell destruction. The fact that reticulocytes will take up iron directly in the circulating blood, by-passing the bone marrow completely, indicates the difficulties encountered in studying the kinetics of the curves describing radioiron utilization. Attempts have also been made to employ the radioiron clearance rate alone to characterize erythropoiesis without considering the plasma iron levels (Wasserman et al., 1952). Although the clearance time lengthened in aplastic anemia and shortened with increased erythropoiesis, it was so dependent on the plasma iron level that neither the clearance rate nor the plasma iron level had much meaning alone. Bothwell et al. (1957) attempted to show the relationship between plasma turnover and erythropoiesis by comparing the plasma turnover with the reticulocyte count. Their findings suggested that the turnover of iron in other than blood-forming tissue may amount to as much as 50% of the normal turnover and may double in conditions leading to high serum iron. In patients with severe Cooley’s anemia, the plasma iron turnover was increased without a corresponding rise in the reticulocyte count suggesting that the hyperactivity of the marrow was largely ineffective insofar as the delivery of viable erythrocytes to the circulation was concerned. Although the plasma iron turnover was confirmed to be a sensitive index of marrow activity, it was of limited value in estimating effective erythropoiesis unless combined with a measurement of the total iron incorporated into red cells. By using the equations of Huff et al. (1950), a reasonable estimation of the rate of red cell synthesis at the time of measurement can be obtained. Care must be exercised in extrapolating these rates over periods of time as the dynamic equilibrium between destruction and formation makes the turnover rate sensitive to change. Bothwell et al. (1957) showed marked depression of plasma iron turnover within 24 hours in rabbits following irradiation, in dogs following blood transfusion, and in patients during nitrogen mustard therapy. The absence of a related body measurement makes the comparison of turnover rates between individuals difficult. If the turnover values are expressed as total mg. of iron removed from the plasma or taken up by the red cells in 24 hours by the individuals, the turnover values will reflect variations in their size. On the other hand, rates expressed in terms of
ANEMIA IN CANCER
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milligrams of iron per kilogram reflect those variations of blood volume which arise from body build, water balance, or during the wasting processes of chronic disease. A further source of error arises from interpretation of the curves describing the disappearance of plasma iron. Measurements from these curves are taken within the first few hours following the injection of labeled plasma, a t a time when proteins such as albumin are known to be equilibrating with extracellular spaces. The inability to describe certain curves in which iron is slowly removed from the plasma as a single phenomenon (Wasserman et al., 1952) arid the unreasonably high values obtained in polycythemia Vera (Sharney et al., 1954) suggest that a similar equilibration may occur between the intravascular and extravascular ironbinding protein. There is also evidence suggesting that in the normal individual as much as 15% of the iron initially incorporated may be returned to the plasma by the breakdown of a population of short-lived cells (London et al., 1950a,b; Neuberger and Niven, 1951; Schapira et al., 1955). Any calculations made from the initial slope in the presence of such processes would tend t o overestimate the amount of iron turned over through the plasma. Recently several investigators have proposed more complex models of iron kinetics in order to minimize these defects (Sharney et al., 1954; Pollycove and Mortimer, 1956; Huff and Judd, 1956). In spite of these reservations, the techniques described by Huff et al. (1950) remain the best general method now available for estimating the rate of erythropoiesis. 2. Ferrokinetic Studies i n Cancer Patients
Huff et al. (1950) studied the plasma and red cell iron turnover of patients with leukemia. The plasma turnover for 7 patients with myelogenous leukemia averaged 0.84 mg./kg./day as compared to 0.35 mg./kg./day for the controls. The average red cell turnover for these patients was 0.42 mg./kg./day, compared to 0.26 mg./kg./day for the controls. Five of the patients had both plasma and red cell iron turnover values above the normal range, 1 being elevated by 4-fold. The remaining 2 patients had normal values for plasma iron turnover, but one had a decreased red cell iron turnover. The average plasma iron turnover of 19 patients with lymphatic leukemia was 0.50 mg./kg./day and the average red cell iron turnover was 0.38 mg./kg./day. Thirteen patients had a plasma iron turnover above the normal range, 4 had normal values, and 2 were depressed. Thirteen had elevated red cell iron turnover values, 1 was in the normal range, 4 were depressed, and in 1 instance the value was not reported. h similar range of plasma iron turnover values was reported for myeloid metaplasia, myelogenous leukemia, and lymphatic leukemia by Bothwell et al. (1957).
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A very extensive study on the iron metabolism of patients with advanced carcinoma was carried out by Hyman and Harvey (1955). Although they report that red cell production was elevated in a majority of the patients studied, examination of their data indicates that their conclusions are based on the elevated values which they obtain for the “fraction of plasma iron removed per hour.” Since the plasma iron was reduced in these patients, an increased fraction of plasma iron would have to be removed per hour to keep the net amount of iron utilized even a t normal levels. By similar logic, it is evident that a larger fraction of the red cell iron will be renewed in an anemic patient than in a normal patient with an equivalent rate of red cell formation. For this reason, it is also incorrect to use the fraction of red cell iron removed to evaluate the rate of erythropoiesis. In addition, certain mathematical inaccuracies have entered into the calculations of some of the turnover values reported (Table I of Hyman and Harvey, 1955). Recalculation of the data shows that in their series of 45 patients with far-advanced carcinoma 51% have plasma iron turnover values in the normal range of 20-37 mg./day, 33% were below this range and 16% were above. The red cell iron turnover of only 36% of the patients were in the normal range of 14-38 mg./day, whereas 62% were below the normal range, and 2y0 were above. Miller el al. (1956) carried out similar studies on patients with faradvanced carcinoma using the techniques of Huff et al. (1950). In order to avoid the inaccuracies of relating such studies to body weight, turnover values were expressed as mg. iron/hr./kg. of red cell mass. In 21 of 37 studies, the plasma iron turnover was within the normal range of 0.5691.149 mg. iron/hr./liter R.B.C. I n 14 studies, the plasma iron turnover was elevated above the normal range and in only 2 of 37 studies was it decreased. In 20 of 36 studies, the red cell iron turnover was within the normal range of 0.569 to 1.033 mg. iron/hr./liter R.B.C.; in 11 studies it was increased, and in 5 it was decreased. It should be noted that normal turnover values expressed as mg./hr./liter R.B.C. do not indicate that sufficient iron is being utilized to maintain a normal hemoglobin level, but rather indicate that it is sufficient to maintain the observed hemoglobin level. Therefore, patients with severe anemia and with a reduced total hemoglobin mass may have a turnover of iron/liter R.B.C. within normal levels and still have insufficient iron being incorporated into hemoglobin to maintain the total red cell mass within the normal range. It would appear that in the absence of marked changes in blood volume a better comparison would be obtained by expressing the turnover values as mg./hr./liter of blood. Recalculation of the data into these terms reduces the number and the extent to which turnover values are elevated and increases the number and degree of those reduced. It does
ANEMIA IN CANCER
22 1
not, however, significantly change the number falling in the normal range, and it does not change the general conclusion that a high proportion of the patients studied had normal or slightly increased turnover values, which usually failed to compensate sufficiently to prevent the appearance of anemia. Perhaps the most striking finding of these studies on the rate of erythropoiesis in anemic cancer patients is the number which show essentially normal turnover values. In view of the marked destructive process present in many of these patients, it would be expected that the rate of synthesis would be increased. In the next section some of the factors which may decrease the rate of synthesis will be discussed. 3. Factors Contributing to the Inhibition of Erythropoiesis
A. I r o n Deficiency. In certain instances, iron deficiency anemia develops from chronic blood loss of ulcerative neoplastic lesions. Lesions in the proximal colon are especially prone to give rise to anemia by this process. Clark et al. (1945), reporting on 21 cases of carcinoma of the proximal colon, found that those patients with severe anemia had low grade, large, ulcerating, penetrating, nonobstructing carcinomas. They report a relationship between the duration of symptoms and the degree of hemoglobin depression but no relationship between the degree of anemia and the extent of local growth, regional metastases, or involvement of the liver. Thirteen of these patients had a hemoglobin of less than 10 g./100 ml. of blood. Nineteen of the patients exhibited slight to marked hypochromasia and nine showed varying degrees of microcytosis. The range of serum iron was from 22 t o 76 pg/lOO ml., while values obtained on normal controls varied from 99 to 158 pgyo. The concentration of serum iron was reported as being definitely lower in those cases which had a moderate to severe anemia than it was in those with a low normal or slightly decreased hemoglobin concentration. The one patient observed long enough before operation to permit the employment of adequate iron therapy showed an increase in hemoglobin from 6.6 to 11.4 g.yo after receiving 0.73 g. of ferrous sulfate daily for 7 weeks. The reticulocytes increased from 1.5 to 3.50/0. It was pointed out that the absorption of iron may also be defective, as achlorhydria, indigestion, and diarrhea are all symptoms of lesions of the right half of the colon. Lesions on the left side of the colon are usually discovered before the anemia becomes severe. The red blood is expelled unmixed with the feces leading to early recognition, and the solid nature of the stool leads to early obstruction. I n those cases in which anemia does develop it is similar to that observed in the right half of the colon. I t has been pointed out by Ley (1956) in his excellent review on the
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VINCENT E. PRICE A N D ROBERT E. GREENFIELD
classification of the anemias of cancer patients that iron deficiency may develop following definitive operative procedures. He also emphasizes the prevalence in women of “simple” or “idiopathic” iron deficiency anemia, which may on occasion be attributed to the neoplasm when in actuality it may have developed quite independently. In cancer patients, less than half have a bleeding process sufficiently severe to account for their anemia. Shen and Homburger (1951) found that in only 33 of 113 patients could the anemia be completely explained on the basis of hemorrhage and in only 48 patients could it be partially explained on this basis (cf. Bateman, 1951). They found no cases of nutritional [idiopathic] iron deficiency anemia in their series of patients. B. Blz Deficiency. It is generally recognized that gastric cancer occurs with a higher frequency in patients with pernicious anemia (Kaplan and Rigler, 1945; Mosbech and Viedbach, 1950; Norcross et al., 1952). Although the reported incidence varies widely, the careful survey of Zamecheck et al. (1955), under conditions in which the patients could be accurately diagnosed and followed throughout most of their clinical course, revealed that approximately 10% of these patients developed cancer before they died. The development of megaloblastic anemia in the above cases has been attributed to the loss of normal gastric secretions due to mucosal atrophy with resultant depression of vitamin BIZabsorption. Heinle et al. (1952) demonstrated that vitamin B12 labeled with Cow could be recovered in high yield (72-96%) in the feces of patients with pernicious anemia, whether in remission or relapse, when administered in daily doses of 5 pg. If, however, the labeled Blz was accompanied with normal gastric juice or some other source of intrinsic factor, less of the vitamin Blz could be recovered in the feces (3ooj,). Because of the high incidence of megaloblastic anemia following totJal gastrectomy (Paulson and Harvey, 1954), a procedure nearly always performed because of gastric carcinoma, similar studies to those of Heinle et al. have been carried out in these patients (Swendseid et al., 1953; Ley and Sharpe, 1954). A failure to absorb vitamin B12 was found which could be corrected by administering normal gastric juice with the vitamin. Comparable studies on patients with cancer would be desirable, but, in view of the small amount of gastric secretion needed t o absorb BIZ, it is unlikely that there is sufficient destruction of the normal epithelium which secretes intrinsic factor to lead to the development of a secondary megaloblastic anemia. Of the total number of carcinomas of the stomach, only a small fraction arise in patients with pernicious anemia. Oppenheim et al. (1945), reporting on anemia in gastric cancer, found only one case of hyperchromic
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macrocytic anemia out of 122 patients with gastric cancer. Although, as shown in Table I, 24 out of the 78 patients with anemia had a macrocytic normochromic anemia, they did not give a reticulocyte response to the intramuscular injection of liver extract. The authors felt that the macrocytosis was on a different basis than Addisonion pernicious anemia and was perhaps related to an associated liver insufficiency. In a similar fashion, oral ferrous sulfate administered to patients with microcytic normochromic anemia had little effect on the cell count or hemoglobin concentration although it did correct the microcytosis. It can be seen from Table I that most of the patients had a normochromic anemia of unexplained origin. TABLE I The Nature of the Anemia in Patients with Gastric Cancer“ Hemoglobin content of erythrocytes Hyperchromic Normochromic Hypochromic 0
Pat,icnts with Macrocytosis
Normocytosis
Microcytosis
1 24 0
0 35 0
0 14 4
Oppenheim et ~ 1 (1945). .
C. Fibrosis and Invasion of the Bone Narrow. Tumor encroachment and the accompanying fibrosis of the hone marrow is rarely a primary factor in the etiology of the anemia of cancer patients. For example, Sheri and Homburger (1951) in their study of patients with carcinoma could find no correlation between the extent of metastases and the degree of anemia. The one group of patients with cancer in which the replacement of bone marrow may be of significance is characterized by having immature cells of the erythroid and myeloid series in the circulating blood. This hematological picture is indistinguishable from that found in patients with bone marrow replacement due to other causes and has been called “leukoerythroblastosis.” This characteristic blood picture has been most commonly seen in association with carcinoma of the breast (West el al. 1955) and the prostate (Rundles and Jonsson, 1949). Both of these tumor types are recognized t o have bone metastases in a high proportion of cases. Direct evidence as to the state of the hone marrow in these patients is difficult to obtain. West et al. (1955) reported that only 5 out of 15 bone marrow aspirations could be counted. Three of those counted had a normal distribution of cells, and 2 had an increase in erythroid elements. It was possible to identify tumor cells in the aspirations of 8 patients in this study. The frequent failure to obtain satisfactory samples was attributed to fibrosis of the marrow. Rundles and Jonsson (1949) resorted to the use of the
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trephine when they encountered difficulty with marrow aspiration. By selecting areas with tumor nodules, roentgen abnormalities, and areas of bone pain, they were able to find tumor cells in 17 of 30 patients with leukoerythroblastosis from carcinoma of the prostate. I n those areas not invaded by tumor cells, the bone marrow was reported as normal. Much consideration has been given to the question as to whether, even in these patients with leukoerythroblastosis, the anemia results from an absolute decrease in the rate of erythropoiesis or from the inability of the hematopoietic tissue to adequately compensate for an accompanying destructive process. Vaughan (1936), after careful clinical studies and autopsy examinations on patients with leukoerythroblastosis, secondary to carcinoma, concluded that many of her patients had increased amounts of erythroid marrow and also an increase in the proportion of erythroid cells in the marrow. She could find no relationship between the degree of metastatic spread to the bones and the presence or absence of leukoerythroblastosis, suggesting that the anemia was not dependent on mechanical blockage of the bone marrow. Although Rundles and Jonsson (1949) in studies on cancer of the prostate reported a correlation between the severity of the anemia and the degree of metastatic spread to bone, West et al. (1955), studying breast cancer, could find no such relationship. The findings by Ley (1956) that the red cell survival time of both autogenous and homologous red cells was decreased in patients with leukoerythroblastosis secondary t o cancer of the breast, certainly indicates that a process of destruction is involved in a t least some of these patients. He quite correctly pointed out, however, that both destruction and synthesis of red cells must be altered since the shortened survival curve of these patients seldom indicates a rate of destruction of red cells which exceeds the capacity of the known compensatory mechanisms of normal marrow. The hematological picture of leukoerythroblastosis can often be confused with myelogenous leukemia, for in both conditions there may be a stimulation of the bone marrow with release of nucleated red cells (Collins and Rose, 1948). Erythrocyte disturbances of the pernicious anemia pattern often can precede the onset of myelogenous leukemia. The elevation of the white count may distinguish between the two pictures since usually the white count in leukoerythroblastosis is normal or low when corrected for nucleated red cells. The anemia associated with chronic lymphatic leukemia presents a different picture. Although infiltration of tumor cells into the bone marrow is more marked and the anemia more severe, nucleated red cells appear in the peripheral blood with much less frequency. A hematological picture similar to that of leukoerythroblastosis was found in animals bearing far-advanced transplanted tumors (Sterling et al.,
ANEMIA I N CAA'CER
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1958). It was found that animals bearing rat hepatoma 3683 and lymphosarcoma R 2788 had large numbers of immature and nucleated red cells in the circulating blood, which had been formed in response to a severe process of red cell destruction in the tumor-bearing animal (see Section IV,4). In some of the rats bearing these tumors, reticulocytes and immature cells accounted for over 90% of the red cells. Tracer studies with Fe69using the techniques of Huff et al. (1950) revealed a red cell iron turnover from 2 to 6 times greater than that of the normal controls. The hematological picture in these animals, although similar to that associated with myelophthisis, was not associated with any evidence of physical interference with the bone marrow. Although the marrow was extruded from the femurs intact and fixed as a tissue, there were no tumor cells found, and the bone marrow was hyperplastic with a predominance of immature cells of the erythroid series. Although the lymphosarcoma showed local lymph node invasion and was once successfully transplanted by the transfusion of blood from the host into normal animals, there is no evidence that the hepatoma metastasizes from the site of subcutaneous injection before death of the host from anemia and cachexia. The possibility that this anemia arose secondary t o Bartonella infection arising in the tumor-bearing animal was carefully considered and excluded. The occurrence of this hematogical picture in the rat associated with a syndrome more clearly characterized by red cell destruction than by a reduced rate of red cell synthesis suggests that the appearance of young cells in t,he circulating blood may merely indicate an overstimulation of the bone marrow. On the other hand, Wintrobe (1956, p.592) has suggested that in instances in which a production deficit in the bone marrow has forced the spleen and liver to take over the formation of erythrocytes that immature cells may find their way into the circulating blood due to a lack in these organs of the regulatory mechanisms found in the bone marrow. Such a hypothesis would explain the immature cells in the blood stream of these rats as arising from the extramedullary hematopoietic centers which develop as the body compensates for the overwhelming destructive process. It seems probable that in patients with reduced amounts of bone marrow one could expect that immature cells might appear in the circulation earlier regardless of whether they arise from the bone marrow or from extramedullary foci. D. Injection. It has been shown by Wintrobe's group (Bush et al., 1956) that the iron turnover values of patients with infection are in the normal range and do not increase to compensate for the shortened survival of the erythrocyte in these patients. Because of the similarity of the development of anemia in the presence of infection and in cancer, most investigators have eliminated patients with obvious infection from the cancer
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VINCENT E. PRICE AND ROBERT E. GREENFIELD
patients studied. To the extent infection coexists with cancer, it undoubtedly contributes to the development of anemia. Although the relative importance of infection in the anemia of cancer patients cannot be measured at this time, there is little evidence to suggest that it is a major cause of the anemia in most patients.
4. Discussion The evidence indicates that a majority of patients with cancer, although possessing the ability to synthesize cells a t a normal rate, do not compensate for the increased destruction of red cells by a greatly stimulated red cell production. Only in the exceptional case does the turnover rate double that of the normal individual. In some of these patients, this lack of a markedly stimulated synthesis may possibly be explained by metastases into the bone marrow and by fibrosis. However it is difficult to explain this limited capacity of synthesis entirely on the basis of insufficiency in the amount of hematopoietic tissue. It can be hoped that we will have a clearer understanding of this problem when additional information on the metabolism of iron and of such new hematopoietic factors as erythropoietin becomes available in the cancer patient. IV. ROLEOF INCREASED EHYTHROCYTE DESTRUCTION 1. General Considerations
In 1938 Schiodt, in a theoretical treatise, presented two possible mechanisms for the normal pathway of erythrocyte destruction: (1) The theory of longevity: that red cells live for a certain finite length of time and then are removed and destroyed as the result of some process of aging or senescence. (2) The theory of random destruction: that a certain fraction of red cells are removed each day by some random process extrinsic to the red cell which is nonselective with regard to the age of the erythrocyte. Subsequent studies have more than adequately proven the theory of longevity and have shown that the normal life span of the human erythrocyte is 110-120 days (Hawkins and Whipple, 1938; Mollison and Young, 1942; Shemin and Rittenberg, 1946; Callender et al., 1945, 1947). Abnormal destruction of erythrocytes may occur in two ways: (1) By finite shortening of the life span due to the formation of defective erythrocytes which are destroyed a t an earlier age. This mechanism implies that all of the red cells of a given age live for a certain definite but shortened span of life and then are destroyed. This process is also called accelerated senescence (Sheets et al., 1951). (2) By random destruction due to factors which affect all of the cells so that both old and young cells are destroyed
A N E M I A IN CANCER
227
without regard to their age. Where random destruction is present the cells have no definite life span, and their length of life can be described only in terms of a n average period of survival or 50% survival time. I n carrying out red cell survival studies in disease states, it is valuable to distinguish where possible between finite-shortening and random destruction since, in general, finite-shortening is evidence for an intracorpuscular etiology for the destructive process, whereas random destruction usually points to an extracorpuscular cause. In a number of disease states, cross-transfusion between the patient and a normal individual has helped to determine whether the destructive factor is intracorpuscular or extracorpuscular in nature (Lloyd, 1941 ; Dacie and Mollison, 1943). If matched red cells from a normal donor are transfused into a patient with increased erythrocyte destruction and disappear more rapidly than normal, it is assumed that the lesion is extracorpuscular in nature since the normal cells which have been transfused presumably have no intracorpuscular defect. If, on the other hand, the normal cells are destroyed a t a normal rate, it is assumed that the patient has no extracorpuscular defect, and the lesion must be in the patient’s own cells. Conversely, when matched red cells from the patient are injected into a normal individual and disappear more rapidly than normal, it is assumed that the lesion is intracorpuscular since the normal recipient presumably has no extracorpuscular defect. If, on the other hand, the patient’s cells disappear a t a normal rate in a normal recipient, it is assumed that no intrinsic defect is present in the patients cells, and therefore the lesion in the patient must be extracorpuscular in nature. Examples of intracorpuscular lesions which lead to destruction of defective cells are seen in pernicious anemia, paroxysmal nocturnal hemoglobinuria, and familial hemolytic anemia. Examples of extracorpuscular lesions are seen in acquired hemolytic anemia and hemorrhage. Intra- and extracorpuscular lesions are not mutually exclusive, however, for in blackwater fever both processes are seen: Cells taken from a patient during an acute attack are destroyed rapidly when transfused into a normal recipient, and, on the other hand, cells from a normal donor are destroyed rapidly in the patient during an acute attack. Presumably an extracorpuscular factor is creating random damage to both the red cells of the patient and of the normal donor during the acute attack so that the patient’s damaged cells, when transfused into a normal recipient, are rapidly destroyed and the cells of the normal donor, when transfused into the patient, are first damaged and then destroyed. It should be emphasized that intracorpuscular defects do not always lead to finite shortening. In sickle-cell anemia, for example, cells of all ages may be damaged a t random under condit,ions of low oxygen tension. As a
228
VINCENT E. PRICE AND ROBERT E . GREENFIELD
general rule extracorpuscular lesions do lead to random loss. It is not inconceivable, however, that under certain conditions they might lead to destruction of only the older cells causing finite shortening of the life span. Since the life-span methods are laborious and take a long period of time to carry out, clinicians must place considerable reliance on the older methods of determining the presence of increased erythrocyte destruction. These methods also will be discussed briefly in the following section. 2. Criteria of Increased Erythrocyte Destruction
A. Red Cell Survival Studies. Except for overt hemorrhage, which will be discussed in a later section, the most concrete evidence for increased destruction of erythrocytes in cancer patients has come from measurements of the survival of tagged red cells in the patient’s circulating blood. A number of excellent reviews (Ashby, 1948; R. Berlin, 1951; Dornhorst, 1951; Eadie and Brown, 1953; Strumia, 1956; Mollison, 1956) have covered in detail both the technical and theoretical considerations regarding these methods. Unfortunately, each of the available techniques has important limitations which need to be considered in interpreting the data derived from them. The techniques for measuring red cell survival may be divided into those in which cells of all ages are randomly labeled and those in which cells of only a limited age group are labeled. In some techniques donor cells are used, and in others the patient’s own cells are labeled. There has been considerable discussion and little agreement among various investigators as to which of the life-span techniques are acceptable, and which are not. Upon examination of the various techniques it will be seen that they complement each other, and in the absence of a single perfect technique it is fortunate that a number of different ones are available. Each technique furnishes some information that others do not provide. The one needed for a given study will depend in large measure on the type of information required. a. Life-span methods in which cells of all ages are labeled. The principle methods in this group are the Ashby and the radiochromate techniques. The Ashby technique (Ashby, 1919; Young et aZ., 1947; DeGowin et al., 1950) involves transfusion into the patient of red cells from a suitable donor of a different serologic type, but still compatible with the patient’s blood. Either the ABO or M N blood group systems (Wiener, 1934) may be used. At intervals after the transfusion, a sample of blood is withdrawn from the patient, and the patient’s cells are removed by their agglutination with the appropriate blood-typing antiserum. The nonagglutinated donor cells remaining in the blood sample are then counted. Since the donor cells are of a random age distribution with approximately the same number of cells
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A N E M I A I N CANCER
of each age being present, a certain, nearly constant, number of donor cells are destroyed each day by senescence. In the ideal case, a linear fall in the concentration of circulating donor cells is observed throughout the normal life span of about 120 days. It is important that very potent and highly specific antisera be used. Eadie and I. W. Brown, Jr. (1953) emphasize the importance of making preliminary curves with known mixtures of the cells over the range of concentrations expected in the experiment. Sheets et al. (1951) have presented an excellent report on the interpretation of Ashby curves. The technique has the disadvantage of being very laborious. Recently the Ashby technique has been modified (Hurley and Weisman, 1954; Eadie and Brown, 1955) by use of group-specific hemolytic antisera to remove the recipient's cells by differential hemolysis (Todd and White, 1911). This technique is much more accurate in that no agglutinated clumps of recipient cells are present to obscure the field and to trap donor cells in the counting procedure. An example of a survival curve of normal donor cells in a normal recipient using this modified technique is illustrated in Fig. 6. I00
eo
%
60
40
20
' 0
20
40
60
80
100
120
140
DAYS
FIG.6. Survival of crythrocytes transfused from normal donors into normal rccipiand thc radiocnts, followed simultaneously by a modified Ashbg technique, 0-0, (from Eadie and I. M'. Brown, Jr., 1955). chromium technique, 0-0
The use of sodium chromate containing the isotope Cr5' to randomly label red cells of all ages was first carried out by S. J. Gray and Sterling (1950). It has been shown that nearly all of the radiochromate is bound to the globin moiety of hemoglobin (S. J. Gray and Sterling, 1950; Necheles et al., 1953). Hemoglobin is oxidized by the chromate ion to form methemoglobin (Hughes Jones and Mollison, 1956) so that it is important to avoid
230
VINCENT
E. PRICE AND ROBERT E. GREENFIELD
the use of high concentrations of chromate. The usually accepted limit is 20pg. of chromium per ml. of blood. The radiochromate should be added slowly with adequate mixing to avoid local concentrations of radioisotope since it is important that cells be labeled as uniformly as possible. If these precautions are taken, it appears that the red cells are not significantly injured by the labeling process (Hughes Jones and Mollison, 1956). Increasing number of investigators have come to feel that washing the labeled cells does more harm than good since the small amount of CrS1which does not bind to red cells is rapidly excreted in the urine. Ebaugh et al. (1953) labeled erythrocytes with Cfll and then lysed them before their injection. No transfer of the radiochromium to intact red cells of the circulating blood was found, and presumably after the breakdown of erythrocytes there is no problem of transfer of released radiochromium to other red cells. This is confirmed by the experimental curves which do approach zero. The chromium technique has been widely used since i t has a great advantage in that the patient's own cells can be removed, tagged with Cfll, and then reinjected. Since cells of all ages are labeled, theoretically the concentration of tagged cells in normal people should fall in a linear fashion as in the Ashby technique. However, when the chromium technique is carried out on donor cells simultaneously with the Ashby technique, by either differential agglutination (Ebaugh et al., 1953; Read et al., 1954) or hemolysis (Eadie and Brown, 1955), the radiochromium leaves the circulating blood more rapidly than do the donor cells, as measured by the Ashby technique as illustrated in Fig. 6. Ebaugh el al. (1953) have suggested that this discrepancy may be due to elution of chromium from the labeled erythrocytes and have estimated a half-time of 77 days for the elution process. Read et al. (1954) ; Eadie and Brown (1955), and Hughes Jones and Mollison (1956) have estimated the half-time for the elution process to be 79, 77, and 64 days, respectively. Necheles et al. (1953) have found that during in vitro dialysis of chromium-labeled human erythrocytes against isotonic saline, chromium was eluted a t the rate of about 1% per day, which corresponds to a half-time of 75-80 days, and is in close agreement with the in vivo findings mentioned above. In contrast to the findings of the above workers, Hyman et al. (1956) failed to find a discrepancy between the radiochromium and Ashby techniques. Examples of their curves are shown in Fig. 7. It will be observed that in this study the Ashby curves were not linear and the donor cells disappear more rapidly in the early portion of the curve than in the later portion. One wonders whether the technique of chromium labeling has not damaged a considerable fraction of the cells, causing them to be destroyed more rapidly. It would be valuable to label and transfuse a portion of the normal donor cells by the chromium technique and, a t the same time, to
23 1
ANEMIA I N CANCER
inject the bulk of the donor cells directly without chromium treatment to see if under these circumstances the discrepancy reported by the other workers might appear. In view of the inconsistency of these findings with those of other laboratories and its importance to the interpretation of the large amount of data which Hyman's group has obtained on patients with carcinoma, it is hoped that additional studies on the survival of red cells in normal volunteers using the two techniques will soon be published. Until additional data is obtained, the nature and extent of this discrepancy between the Ashby and chromium curves cannot be defined. If, by chance, further experience should show that Hyman et al. are right and that there is no significant discrepancy between the two techniques, then a number of profound problems with regard to the curvilinear nature of the survival curves arise which will require serious study. 7-
1
\\\
01
0
' 10
I
20
I
'
30
40
' 50
a
," 60
1
70
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90
100
110
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DAYS
FIG. 7. Survival of erythrocytes of normal donors upon transfusion into normal recipients, using the radiochromium and Ashby terhniques simultaneously. It will be seen that in this study the Ashby curves were not linear and that no significant discrepancy between the two techniques was observed (from Hyrnan el al., 1956).
I t might be noted that if one examines the curves reported in the literature one is impressed by the fact that a t,ruly linear curve by either technique is uncommon. Some factors which may contribute to the nonlinearity of the survival curves by these techniques are summarized here: (1) An inconstant rate of red cell synthesis in the donor. A linear curve is predicated on the assumption that red cell synthesis in the donor has been at a constant unvarying rate. If this is not so, there will be more cells of some ages than there will be of others. (2) An inhomogeneous population of cells with different life spans (Sheets et al., 1951). Such a population might arise
232
V I N C E S T E. PRICE .\ND ROBERT E. G R E E N F I ELD
as the result of genetic factors or from the mishandling of cells, as in damage during prolonged storage (Ebaugh et nl., 1953). (3) Random losses of short duration, as in menstruation (Callender et al., 1947; R. Berlin, 1951). (4) Uptake of more chromium by older cells, as suggested by Ebaugh et al. (1953). (5) Elution of chromium, as suggested by Ebaugh el al. (1953). (6) Damage to cells by high concentrations of chromium, occurring from the use of C P preparations of low activity or from inadequate stirring during the labeling procedure (Ebaugh et al., 1953). b. Life-span methods in which cells of a limited age group are labeled. Cells of a limited age group may be labeled by the in vivo incorporation of an isotope into the hemoglobin of red cells during their synthesis over a short period of time. In some cases, the cells are labeled and their disappearance followed in the same individual; in other cases, the erythrocytes are labeled in one individual and then transfused into a compatible recipient to follow their disappearance. The former technique, by avoiding the transfusion of cells, has no problem arising from damage of cells during transfusion and has the advantage of studying the individual’s own cells in their normal environment. The fact that labeling occurs over a number of days is a serious defect which lessens the sensitivity of the method. Conversely, transfusing the labeled cells into a second individual not only subjects the cells to possible damage during transfusion but also introduces the cells into a foreign environment. It has, however, the real advantage that the cells may be removed after a very short period of time after injection of the isotope so that those cells which are labeled are all of nearly the same age. Both techniques have a common disadvantage in that when the erythrocytes die some of the isotope which is released is reused for the synthesis of new red cells. The most useful isotopes for this type of study have been Fe55, Fe59,NI5 in glycine, and C41in glycine or lysine. Radioactive iron was first used for tagging erythrocytes by Cruz et al. (1941). They used erythrocytes labeled with radioiron for blood volume studies but ruled out its use for measuring the life span of red cells because the iron from senescent red cells was so rapidly reutilized for resynthesis of new red cells that very little fall in the radioiron concentration in the blood occurred a t the end of the life span (Hahn et al., 1942). They also showed that upon destroying red cells with acetylphenylhydrazine there was rapid reutilization of the FeS9for the formation of new red cells (Cruz et al., 1942). Several years later, Finch et al. (1949) showed that by administration of large amounts of nonradioactive iron intravenously, the reutilization of radioiron could be reduced in the dog from 75 to 16%. By intravenous administration of saccharated iron oxide to minimize reutilization, I. W. Brown, Jr. and Eadie (1953) have carried out survival studies in several
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.4XEMIA I S CASCEIt
species and have discussed the mathematical analysis of survival curves (cf. Amatuzio and Evans, 1953) to correct for reutilization, as well as for a process of random destruction of unknown etiology, which they also found in their animals. Although radioiron may be of considerable value for certain studies on the life span of erythrocytes in animals, it is not of general use clinically because of the problem of reutilization and because the radioactivity is not eliminated from the body until physical decay occurs. I n the biosynthesis of heme, the 4 nitrogen atoms (Shemin and Rittenberg, 1945, 1946) and 8 of the 34 carbon atoms arise from glycine. The carbon atoms all arise from the a-carbon atom of glycine (Muir and Neuberger, 1950; Radin et al., 1950; Wittenburg and Shemin, 1950). The carboxyl carbon of glycine is not used for porphyrin synthesis but is incorporated into globin, the protein moiety of hemoglobin (Grinstein et al., 1948). This is the basis for the use of Nib- and 2-C1*-glycinein life-span studies. Shemin and Rittenberg (1946) gave N16-glycine orally for 3 days and isolated hemin from samples of blood taken a t intervals thereafter. A curve obtained by this technique from a later paper of London et al. (1950a) is shown in Fig. 8. The life span in this normal male subject was W V
x 05 W
sz
0.4
t-
z O3 W
y
02
W
H 0
2
01 0
11 20
t
I
I
40
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80
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1
I
100 120 140
TIME
"
I
I
L
160 100 200 2 2 0 2 4 0
IN DAYS
FIG.8. Erythrocyte survival studies in a normal male following injection of NL6-glycine. 0-0 represents the concentration of "6 in hemin isolated from hemoglobin. - represents the conccntration of "6 in stercobilin isolated from the feces (from Imidon el nl., 1950a).
calculated to be 120 days. Three characteristics of the curve should be pointed out: (1) The uptake of glycine into the heinin required about 24 days to reach the maximum level. (2) The specific activity of the hemin does not fall off sharply a t the end of the life span, only 50% of the cells dying within the period of 120 f 14 days. (3) The final curve does not fall to zero, indicating that there is some reutilization. Lysine labeled with CI4a.t the t-carbon atom has been used in life span
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VINCENT E. PRICE AND ROBERT E. GREENFIELD
studies by Bale et al. (1949). The lysine is primarily incorporated into the protein moiety, and the curves obtained are very similar to those with glycine. The estimated life span in the dog was 115 days. B. Excretion of Bile Pigments. Sribhishaj et al. (1931) showed t,hat when 1 g. of hemoglobin is given intravenously to dogs approximately 40 mg. of bile pigment was excreted through a bile fistula. This fact was used to make one of the first reasonably accurate measurements of the life span of the red cell (Hawkins and Whipple, 1938). Dogs were made anemic by acute hemorrhage or with acetylphenylhydrazine and allowed to recover from their anemia. After 120 days, the excretion of bile pigment from the fistula reached a peak as a result of the destruction of a large number of senescent red cells which had been rapidly synthesized in response to the sudden anemia. James et al. (1953) fed N16-glycine to a normal type 0 man and after maximum incorporation of N16 into the hemoglobin had occurred, the labeled erythrocytes were transfused into a type A recipient. Njtrogen-15 appeared in the stercobilin of the feces on about the 70th day, increased to a maximum on the 12211d day and then receded. Calculated from the stercobilin data, the life span of the infused cells in the recipient was 115 days, whereas in the donor of the labeled cells, it was 119 days. The appearance of N16 in the stercobilin correlated with the disappearance of NlS in hemin isolated from the erythrocytes. From the hemin data, the life span was 113 days; from the Ashby technique, the life span was 110 days. This shows a striking agreement between the various techniques used. Despite the correlation between hemoglobin destruction and bile pigment excretion which has been demonstrated above, more recent studies have shown that these relationships are at best qualitative rather than quantitative for two reasons: (1) London et al. (1950a) have shown that in the 8 days immediately following administration of labeled glycine, N16 appears in a very sharp peak in the stercobilin of the feces. The N16 then falls to a low plateau which starts to rise on about the 70th day as shown in Fig. 8. The second peak represents breakdown of red cells. The first peak, however, occurs before any destruction of mature circulating erythrocytes is known to occur. It represents a t least 11% of bile pigments being excreted and may rise to 30 to 40% in pernicious anemia (London and West, 1950), and congenital porphyria (London et al., 1950b). Similar findings were reported by C. H. Gray et al. (1950). This early peak is as yet unexplained but may represent bile pigments from unutilized hemoglobin precursors, or from immature defective red cells, or from other heme proteins such as catalase, myoglobin, and the cytochromes. N. I. Berlin et al. (1951b), in life-span studies on patients with polycythemia Vera with glycine-2-CI4,have reported incorporation of isotope into Ehort-lived
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.4NEMI.4 IZI CANCER
cells. This may well represeiit one source of the early peak of bile pigments (cf. Evans, 1954). (2) Although most of the heme pigment is degraded to a group of tetrapyrroles which react with Ehrlich’s aldehyde reagent, and as such are labeled as urinary and fecal “urobilinogen,” some are degraded further to dipyrroles, such as the bilifuscins, and to monopyrroles, which do not give a red color with Ehrlich’s aldehyde reagent (Seidel et al., 1947, 1948). Therefore excretion of bile pigments is only a semiquantitative measure of the rate of erythrocyte destruction. Frequently urobilinogen excretion is related to the total circulating hemoglobin and presented as the “hemolytic index” (Miller et al., 1942) : hemolytic index
=
urobilinogen (mg./day) x 100 total circulating hemoglobin (g.)
The hemolytic index of normal individuals ranges from 8 to 20. C . Increased Erythrocyte Synthesis. The body responds to red cell destruction by mobilizing its forces as rapidly as possible to replace the blood loss. A good example of this is shown by the experiments of Harne e2 al. (1945), who subjected monkeys to hemorrhage and followed their reticulocyte counts over a period of months. Following the hemorrhage, the expected marked rise in reticulocyte count occurred, as shown in Fig. 9, followed by a return to more normal levels, with smaller fluctuations RETICULOCYTES
m 30
-
(
-
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-
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FIG.9. Illustrating the reticulocyte response in monkeys following removal of blood amounting to 1% of the body weight (from 0. G. Harnr el al., 1945).
approximately 8 days apart. This caoiitinued until the period of 94 to 117 days when a second marked rise was observed. This second peak resulted from the response of hematopoietic centers to the sudden loss by senescence of the large population of cells which had been formed in response to the sudden hemorrhage 100 days previously. Actually these experiments of Harne et al. represented one of the first accurate measurements of the life span of the erythrocyte. This classic experiment serves a t this point to d e m o n s h t e that, a t repeated intervals of 100-120 days following any acute process of blood destruction, there will be an elevation in the reticulocyte count and corresponding increases in the rate of iron utilization for
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VINCENT E. PRICE A N D ROBERT E. GREENFIELD
erythropoiesis, each rise being somewhat lower than the previous one until a relatively constant rate of erythropoiesis is restored. More recently the rate of clearance of tracer amounts of radioiron from the plasma has been used as a measure of the rate of iron turnover and is frequently markedly elevated in the presence of compensatory erythropoiesis following an abnormal rate of blood destruction. Since this has been discussed in detail in a previous section, there is no need to consider it further a t this point. 3. Evidence for Increased Blood Destruction in Cancer Patients
Evidence for increased blood destruction in patients with leukemia and solid tumors has been accumulated slowly over a period of half a century. Hirschfeld in 1906 observed a marked increase in hemosiderin in a leukemic patient with severe anemia and considered the possibility of an increased rate of blood destruction. Paschkis (1927) observed that the very dark stools in myeloid leukemia were suggestive of increased red cell destruction. Jaff6 (1933, 1935) noted an increased hemosiderosis, suggestive of increased blood destruction, in the presence of elevated erythropoiesis. Von Kress (1934) and Klima (1935) also observed marked hemosiderosis in leukemic patients. Von Kress went so far as to consider increased blood destruction in leukemia to be a proven fact. Barker (1938) and Watson (1938) reported increased excretion of bile pigments in some, but not all, cases of leukemia. Many others reported the existence of hemolytic anemia in cancer (cf. R. Berlin, 1951; Dacie, 1954) and pertinent references to them will be made in a later section. Suffice it to say, the first successful attempts to quantitate erythrocyte destruction in cancer patients came with the advent of red cell survival studies, and so this section will be concerned primarily with these studies. A. Studies on Leukemias and Lymphomas. To our knowledge the first published report on red cell survival studies in cancer patients was made by G. M. Brown (1950). Using the Ashby technique, he found that 7 patients with malignant diseases of the hematopoietic system had the following 50% survival times for transfused normal cells as compared to the expected value of 60 days: acute leukemia, 23 days; 2 patients with chronic lymphatic leukemia, 40 and 18 days; chronic myelogenous leukemia, 8 days; plasma cell myeloma, 28 days; and Hodgkin’s disease, 22 and 63 days in 2 patients (cf. G. M. Brown et al., 1951). In 1951, R. Berlin published an important monograph describing detailed studies on red cell survival in 15 patients with chronic myelogenous leukemia and 9 with chronic lymphatic leukemia. The goal of R. Berlin’s study was ‘(to obtain experimental evidence to support the opinion that in most forms of splenomegaly a latent hemolytic qndrome may be the
ANEMIA I N CANCER
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cause of the moderate to severe normochronic anemia usually present in these conditions.” It had been noted that in cases of leukemia with splenomegaly there is urobilinuria, reticulocytosis, and an active bone marrow pattern. R. Berlin wanted to learn to what extent removal of the enlarged spleen caused an improvement of the “hemolytic condition” in the patient. Red cell survival studies, using the Ashby technique, offered a sensitive method for measuring any degree of improvement.
FIG.10. Red cell survival studies by the Ashby technique on patients with chronic myelogenous leukemia (from R. Berlin, 1951).
Five typical Ashby curves from I<. Berlin’s patients with chronic myelogenous leukemia are presented in Fig. 10, w e , and show markedly varying patterns of erythrocyte destruction. Figure 10a, for example, shows n rapid initial fall followed by an almost normal rate of red cell destruction. Figure 106 shows a precipitous destructive process ending in death of the patient. Figure 1OC shows a moderate intial fall followed by a slower but variable rate of destruction. Figure 10D shows an essentially normal curve, even though the patient had marked splenic enlargement. Figure 1OE shows a very rapid but irregular process of destruction. The last patient, whose Ashby curve is shown in Fig. 10E, was subjected to spleriectomy
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V I N C E N T E. PRICE AS11 ROBERT E. GREENFIELD
which resulted in marked clinical improvement, normal blood counts, and a gain of 12 kg. in weight. An Ashby curve following surgery (Fig. 10F) showed a normal survival time of about 60 days. The spleen which was removed weighed 800 g. and had patches of hemosiderosis in the pulp cells and in the blood vessel walls, especially below the splenic capsule. The Ashby curves on patients with chronic lymphatic leukemia showed a similar wide range of erythrocyte destruction. The hemoglobin and erythrocyte counts were moderately or markedly reduced in nearly all of the 24 patients. Ashby studies showed 50% survival times to be lowered from a normal value of 60 days to less than 40 days in 17 of the patients, thus showing the presence of marked erythrocyte destruction. The serum bilirubin was slightly elevated in only 3 patients, but such elevation would generally not be expected except in the presence of damage t o the liver. The reticulocyte counts were elevated in the myeloid series, but less often so in the lymphatic series. The hypotonic fragility was generally normal, being significantly increased in only 3 patients. Of 12 of R. Berlin's patients who died or were subjected to surgery, 5 had pronounced hemosiderosis, especially in the liver and spleen. R. Berlin felt, however, that the hemosiderosis was related to the numerous transfusions these patients had received. The fact that frequent transfusions were needed, however, indicates the probable existence of a marked destructive process in these patients. R. Berlin also felt there was a correlation between the size of the spleen and the rate of red cell destruction. This would appear to be only a tendency since a number of patients with only moderate splenic enlargement had marked reduction in the 50% survival time (cf. Aas, 1952; Verloop, 1955). I n 1951, a number of other investigators came out with preliminary studies on the survival of red cells in patients with leukemia. Ross et al. (1951) published an abstract reporting that Ashby studies carried out on 10 patients with leukemia and lymphoma showed a rate of erythrocyte disappearance 2 or 3 times more rapid than normal. Red cell formation was also generally increased rather than depressed. Only 1 patient had the usual evidence of hemolytic disease, such as reticulocytosis, hyperbilirubinemia, and a positive Coombs test. N. I. Berlin et al. (1951b) published a brief report in which using gly~ine-2-C'~ i t was shown that 1 patient with chronic lymphatic leukemia had a normal life span of 110 days and 2 patients with chronic myelogenous leukemia had shortened life spans of 71 and 76 days respectively. These patients have been included in the more complete report published later (N. I. Berlin et nl., 1954). In 1952, Bottner and Schlegel reported Ashby studies on a series of 28 patients with malignant diseases of the hematopoietic system. Their results
are summarized in Table 11. The survival of transfused cells ranged from 20 days to a normal value of 110-120 days. The erythrocyte count was below 4 niillioii in 24 patients, and the hemoglobins were below SOYo of the standard value in 22 patients. In view of the fact that 18 patients had hemoglobins of less than SOY0, it is remarkable that only 1, Ha., had a significant elevation of the reticulocyte count. In the last column of Table I1 are shown symbols indicating the relative importance of decreased erythropoiesis, E, and increased destruction, D, in the etiology of the anemia. Parentheses denote that the enclosed symbol is of some, but lesser, importance than the unenclosed symbol. In most cases both decreased erythropoiesis and increased destruction played a role in the anemia. In some, however, only one factor appeared dominant. Thus in patient Fr. in both studies ( a ) and ( b ) , the reduction in hemoglobin to 60 and .!joy0 could readily be accounted for by the shortened survival times of 81 and ca. 60 days, respectively and indicated that destruction was the primary cause of the anemia. On the other hand, patient Wei. with a hemoglobin of 4oY0 had a survival time of 102 days, and patient Kl. with a hemoglobin of 60% had a completely normal survival of 120 days, so that in these cases a decreased erythropoiesis appeared to be the predominant cause of the anemia. Except for these latter 2 cases, however, Bottner and Schlegel noted a general relationship between reduction of the hemoglobin levels and shortening of the red cell survival time. In 3 of Bottner and Schlegel’s cases, survival studies were repeated on the same patient with relatively little change in the life span. On the other hand, in patient Gr. 2 successive studies showed a life span of 95 days, but on the 3rd study the survival fell to 63 days. Patient Ba,. with chronic myelogenous leukemia, had a markedly short,ened survival of 50 days which fell to about 20 days just before death. Aas (1952) performed Ashby studies on 16 patients with assorted neoplastic diseases of the hematopoietic system. The 50% survival time ranged from a normal value of 60 days down to only 3 days. Fecal urobilinogens were not performed, but the patients showed little other evidence of increased blood destruction. The reticulocyte counts were seldom elevated, and the Coombs test mas negative in all cases. No correlation was observed between the size of the spleen and the 50Y0 survival time. Two patients with multiple myeloma were studied by Sheets et ul. (1954). I n one patient, donor cells disappeared with a survival time of about 80 days. The patient’s own cells fell from 4.4 to 2.2 million per CU. mm. Death occurred on the 78th day with extensive infiltration of myeloma cells in the skeletal system. There was hyperplasia of erythroid elements. The liver and spleen were normal. In the second patient 2 successive studies were done with survival times of about 80 and 40 days, respectively,
TABLE I1 Evidence for Erythrocyte Destruction" No.
Initials
Sex
Age
Diagnosis
Id. Ma. Pi. Pi. We. We. KL Fi. Bi. Ha. wo. KO. Ba. Ba. Bie. Hei. Ot. Pe. HX. Eb. He. Kr. Kr. Ka. Me. Gr.
F F F F M M M M
62 64 55 56 56 56 36 51 42 46 38 48 43 43 26 43 41 57 20 16 31 25 25 21 24 55
Chr. Lymphatic Leuk. Chr. Lymphatic Leuk. Chr. Lymphatic Leuk. Chr. Lymphatic Leuk. Chr. Lymphatic Leuk. Chr. Lymphatic Leuk. Chr. Myelogenous Leuk. Chr. Myelogenous Leuk. Chr. Myelogenous Leuk. Chr. Myelogenous Leuk. Chr. Myelogenous Leuk. Chr. Myelogcnous Leuk. Chr. Myelogenous Leuk. Chr. Myelogcnous Leuk. Acute Leukemia Acute Leukemia Acute Leukemia Acute Leukemia Acute Leukemia Acute Leukemia Lymphogranulomatosis Lymphogranulomatosis Lymphogranulomatosis Lymphogranulomatosis Lymphogranulomatosis Lymphogranulomatosis
h1 F F F M M
F
M F hl
M F ILI F F hl M F
Lifebspan (days)
Reticulocytcs
(7%)
(x1W
(%I
75
55
so
40 70
3.1 2.0 3.5 4.6 4.0 3.8 3.5 3.2 3.6 2.8 3.9 2.8 2.6 1.6 1.5 2.6 2.7 2.6 1.6
1.1 0.8 2.2 0.6 0.5 0.3 1.8 1.9 1.3 4.9 1.5 0.6 0.6
96 98 105 110 120 103
90 85 65
85 50 ca. 20 20 ca.
H ~ Y R.B.C.
60 76 86 89 92
ca. 70 65 68 80 60 95
80 85 80 60
70 85 50 70 65 35 25 25 50 50 55 45 20
70 30 38 70 50 45
3.5 1.8 1.9 3.9 3.0 2.2
0.3 0.2 0.3 0.2 0.7 0.7 ~
0.8 2.5 0.2 0.3 1.1 1.9
Etiologyd
GI.. Gr.
F F
Fr. Fr. Ke. MO. 0. Wei. Wei.
F
se.
M
F F
M ill
nr ill
55 55 10 22 22 46 58 48 52 52
Ly mphogranulomatosis Lymphogranulomatosis Ly mphosarcomatosis Lymphosarromatosis Lymphosarcomatosis Ly mphosarcomatosis Lymphosarcomatosis Lymphosarcomatosis Plasmacytoma Plasmacytoma
96 63 45 81 ca. 60 92 95 98 102
65
45 30
25
60 50 80 65 85
40 70
2.7 2.5 1.5 2.1 2.0 4.7 3.3 3.9 2.2 3.6
1 .o 2.6 0.7 2.2 2.1 1.1 1 .o 0.9 0.6 0.5 3
Bottner and Schlegel (1952). b Norm31 120 days. ' Per cent of normal. d Relative importance of decreased erythropoiesis. E, and incressed destruction, D. in the etiology of the anemia. Parentheses denote lefis importanre of the e n e l d symbol.
H
3
242
VINCENT E. PRICE A N D ROBERT E. GREENFIELD
for the donor cells. During her 6 months of study, the red count fell from 2.7 million to less than 1 million per cu. mm. before death. At necropsy the bone marrow was infiltrated with myeloma cells. The spleen weighed 650 g. and had increased amounts of hemosiderin in the phagocytes. Hemosiderin was also found in hepatic cells. These 2 patients, therefore, had both increased red cell destruction and invasion of bone marrow which interfered with maximal compensatory erythropoiesis. 8.00
z -
m
3 (3
6.00
w I
k
*5
4.00
L
Ga 2 k
2.00
0 W
n.
v)
0
20
40
60
DAYS
FIQ.11. Red cell survival in 8 patient with chronic lymphatic leukemia whose cells have been labeled with glycine-2-CI4. The upper box shows that the erythrocytes disappear with the logarithmic slope characteristic of random destruction (from N. I. Berlin et al., 1954).
Read et al. (1954) described a patient with severe hypoplasia of the bone marrow. Survival studies using the chromium technique showed a half-life of 21.5 days, which was moderately reduced. Six months later the patient was diagnosed as having lymphatic leukemia with increased red cell destruction. In this case the finding of increased blood destruction actually preceded diagnosis of the underlying disease which apparently caused it. I n order to study the life span of the patient’s own red cells, N. I. Berlin et al. (1954) labeled the erythrocytes of 8 leukemic subjects with glycine-2-C14. Three of the patients had chronic lymphatic leukemia. Of these, 2 had essentially normal mean life spans of 113 and 102 days respec-
.\SEMI.\
I S CANCER
243
tively. The 3rd patient had a markedly shortened life span curve with a mean cell life of about 18 days. When the points on the descending portion of the curve, shown in Fig. 11, were plotted on semilogarithmic paper, they fell on a straight line, indicating random destruction of the labeled cells. The specific activity in this patient rose to 4 times that of the other patients, indicating the greater utilization of glycine in this patient for synthesis of hemoglobin to replace that lost in the severe destructive pro(:ess. This patient had a marked reticulocytosis, ranging from 19 to 26y0. The serum bilirubin was normal, but the fecal urobilinogen was slightly elevated. Of the 5 patients with chronic myelogenous leukemia, 1 had itu : t h o s t normal mean life span of 100 days, and 4 had shortened mean life spans of 70 t o 83 days. N. I. Berlin et al. interpreted the latter 4 curves as being representative of “finite shortening,” which might be caused by the presence of a defect intrinsic to the erythrocyte. A life-span curve of one of thesc patients is shown in Fig. 12A. It will be seen that the curve resembles in shape the normal curve of London et al. (Fig. 8) using N14-glycine except that the midpoint of isotope disappearance occurs a t about 90 days after injection of the labeled glycine instead of 133 days as in the curve of London et nl. Since the disappearance of labeled cells occurs rather sharply and approximates the S-shaped death curve one would expect where cells are dying of senescence after a certain definite life span, this curve may well represent finite shortening. It is possible, however, that the shortening of the curve could also be explained by the appearance of a random destructive process of increasing intensity a t 60-70 days. In the other 3 cases of chronic myelogenous leukemia, it is much more difficult to accept the concept of finite shortening. An example of these curves is shown in Fig. 12B. It will be seen that the specific activity rises rapidly for 8-10 days after injection of the labeled glycine and then more slowly t o a maximum a t 20-30 days. Instead of maintaining the specific activity at nearly the maximum level for a considerable period of time, as in Fig. 12A, it will be seen that the curve starts back down and after about 50 days falls at a fairly linear rate to about 130 days, when it levels off in a curve asymptotic to the abscissa. This means that some of the red cells lived 50 days or less and others as long as 120 days. The top of the curve may be even more complex than it appears in that some of the labeled cells may have been destroyed very early while the specific activity of the hemoglobin was still rising, and, conversely, some isotope may have been incorporated into new cells even after the specific activity had begun to fall. If we neglect this complication, although it may be incorrect to do so, then we might conclude that most of the cells lived 40 days before dying. If one uses the term “finite shortening” in the sense that the cells have lived a t
244
VINCENT E. PRICE AND ROBERT E. GREENFIELD
least a certain minimum length of time before dying then this and the other 2 cases similar to it might fall into this group. This would seem to be an improper use of the term “finite shortening,” however. This is perhaps most simply illustrated by estimating from the life-span curves the number of days required for the specific activity to fall from 75 to 25y0 of the maxi-
FIG.12. Red cell survival in patients with chronic myelogenous leukemia whose cells have been labeled with gly~ine-2-U~ (from N. I. Berlin el ul., 1954).
mum value. In the first case, shown in Fig. 12A, this required 17 days, whereas in 2 of the other curves, the time required was approximately 48 and 53 days. In the 4th case, too little of the curve was presented (N. I. Berlin et al., 1954) to make this calculation although by inspection it could be seen that the time for the middle 50y0 of the cells to die would be quite long. One does not have, therefore, in the latter 3 cases a sufficient number
ANEMIA IS CANCER
245
of cells dying in a certain limited period of time to justify use of the term “finite shortening.” If, instead of postulating defects intrinsic to the red cell as being responsible for the types of curves seen in Fig. 12, we turn to a process external to the red cell, a much simpler mechanism can be found to explain the variation among the life-span curves described by N. I. Berlin et al. in their patients with chronic myelogerious leukemia. It seems very probable that a random destructive process external to the red cell which developed or increased in intensity at different points in the life-span curves could produce the observed variations. I t would be valuable if these curves could be subjected to a rigid mathematical analysis similar to that carried out by I. W. Brown, Jr., and Eadie (1953, see Section IV,2). I n their analysis these workers attempted to correct for variations in the time of synthesis of the labeled cells, for reutilization of the isotope in the resynthesis of new cells, and for varying amounts of random destruction which they found to occur in the animals they studied. Verloop (1955) examined 15 patients with chronic lymphatic leukemia, 7 with chronic myelogenous leukemia, 4 with chronic erythroleukemia and 11 with Hodgkin’s disease. He found the fecal urobilin to be elevated in nearly half of the cases of chronic lymphatic leukemia, occasionally in chronic myelogenous leukemia, in all the cases of erythroleukemia, and in many of the cases of Hodgkin’s disease. The reticulocyte count was of less value as an indicator of red cell destruction, frequently being normal or only slightly higher in the presence of an elevated fecal urobilinogen. In one striking case of erythroleukemia, the reticulocyte count was normal a t 0.8% whereas the fecal urobilinogen was being excreted a t 2050 mg./day as compared to normal values ranging from 25-180 mg./day. Verloop observed that the number of patients with a hemolytic anemia in whom abnormal autoantibodies were found was strikingly low in that only 2 of the patients with chronic lymphatic leukemia and 1 with Hodgkin’s had a positive direct Coombs test. These 3 patients also had an increased osmotic fragility and spherocytes in the peripheral blood. An Ashby study on the patient with Hodgkin’s revealed extremely rapid destruction with a maximum red cell survival of only 4 days. This patient required daily transfusions of 800-1200 ml. to maintain the hemoglobin a t normal values. Boiron et al. (1955) studied the survival of erythrocytes in patients with acute leukemia. Long studies were difficult because of the rapid progression of the disease and the need for repeated transfusions. Glycine2-Cl4 was administered to 4 patients. Of these, 2 patients had little or no incorporation, indicating an almost complete cessation of erythropoiesis. In a third the glycine-2-C14 was incorporated rapidly during the first 3
240
VINCENT 6. PRICE . i N D ROBERT E. QREENFIELl)
days, reached a maximum 011 al~out,the 8th day, ant1 then fell rapidly, in a logarithmic fashion, with it half-life of 18 days, corresponding to a mean cell life of 26 days. The fourth patient died before the study could be completed, but the life span was estimated to be greater than 30 days. Nine patients were studied by the Ashby technique. Three died early in the study and the other 6 had 50% survival times of 60,40, 5, 5, 30 and 25 days, as compared to a normal of 60 days. One patient who had a survival time of 40 days during an acute stage of the disease subsequently went through a period of remission during which a second Ashby study was performed. The 50% survival during the period of remission fell to 20 days, showing in this case a lack of correlation between red cell survival and the severity of the disease. This is in contrast to the findings of Bottner and Schlegel (1952) and Renfer (1955). Miller et al. (1956) found a similar lack of correlation in some of the patients they studied. I n an interesting method of approach, Strumia et al. (1955) reported the case of a 67-year old male with monocytic leukemia in which it was shown that the red cell mass fell to about 5301, in 7 days. However, the concentration of radiochromium per ml. of packed cells fell to only 87% of the initial value. This meant that the red cells were being replaced at the rate of 13Q/, in 7 days, or approximately 2% per day. While this is twice the normal rate, it was not the 4-6-fold elevation in erythropoiesis to which a normal bone marrow can expand when called upon. This confirms the findings obtained in ferrokinetic studies (see Section 111) that patients with advanced cancer usually cannot compensate for the increased red cell destruction by a marked elevation in the rate of erythropoiesis. I n most leukemic patients, there were few signs of overt hemolysis. I n certain patients with unusually severe anemia, however, clinical signs of hemolysis appeared. Sheets et al. (1951), in an excellent study on the kinetics of erythrocyte destruction, used as a n illustration 1 patient with chronic lymphatic leukemia with enlarged spleen and lymph nodes, and a picture of extremely rapid erythrocyte destruction. Ten transfusions of 500 ml. each did not significantly raise the red count. At the time of the Ashby study the hemoglobin was 3.7 g.%, the red count was 960,000 per cu. mm., of which 439;b were reticulocytes. A marked increase in osmotic fragility was found; the red cells being completely hemolyzed in 0.50% NaC1. The Ashby study showed that the red cells disappeared precipitously in logarithmic fashion with one-half of the remaining cells being lost each day. I n 1953 Weinstein and LeRoy, using the CF-technique, studied 3 patients with chronic myeloid leukemia and obtained half-lives of 38, 23, and 9 days, respectively. Normal values ranged from 25 to 39 days with an average of 32 days. The third patient mentioned had an enlarged spleen,
A S K M I A I S C.\SCER
247
a 4+ direct Coombs test, a normochromic anemia with a hemoglobin of 8.5 g.%, and a very high reticulocyte count of 30%. Wasserman et al. (1955) reported the case of a 61-year old male with chronic lymphocytic leukemia, a hemoglobin of 6.0 g.%, a red count of 2 million per cu. mm., a reticulocytosis of 9.5%, marked spherocytosis, and a markedly positive Coombs test. The marrow had a normoerythroblastosis of 4oYo7,.The fecal urobilinogen was markedly elevated a t 760 mg./day. An Ashby study revealed rapid random destruction of the red cells with a 50% survival time of only 2.5 days. Following x-irradiation a t a dose of 1000 r to the spleen the spherocytosis disappeared, the anemia improved, and the reticulocyte count fell toward normal. Marked general improvement, shrinkage of the spleen, and a fall in serum bilirubin and fecal urobilinogen was obtained on three occasions by a course of intravenous therapy with ACTH. The case was diagnosed as an autoimmune spherocytic hemolytic anemia occurring 1 year after the onset of chronic lymphyocytic leukemia. B. Studies on Carcinomas and Other Neoplasms. With the exception of G. M. Brown (1950), all of the earlier red cell survival studies were limited to patients with leukemias and lymphomas. A number of more recent investigations have extended these studies to patients with carcinomas and a few other neoplasms. G. M. Brown (1950) in his first report described the Ashby curves of 5 patients with carcinoma of the lung, parotid, prostate, breast, and uterus. He found their 50% survival values to be 39, 33, 53, 33, and 16 days, respectively, as compared t,o a normal value of 60 days. In 1954, Hyman presented the first of several comprehensive studies which demonstrated that a shortened red cell survival was commonly found in patients with a wide variety of neoplasms. This study examined evidence for erythrocyte destruction in 30 patients with carcinoma of the breast, prostate, lung, tongue, esophagus, stomach, and kidney, and in addition 1 patient with malignant melanoma, 2 with Hodgkin’s disease, and one with multiple myeloma. Of the 34 patients, 13 had suitable blood types for Ashby studies. The data on these 13 patients are summarized in Table 111. Of the 13 patients, 2 had essentially normal life spans with a 50% survival of 50 days or over, 5 had moderate shortening to 3&50 days, and 6 had marked shortening ranging from 8 to 28 days. In the patient with malignant melanoma a 50% survival time of 26 days was reduced to ahout 8 days just before death. Of the 31 patients i l l the group with metastic carcinoma, 4 had hemoglobins greater than 12 g.%, 8 were classified as having mild anemia with hemoglobins in the range of 10-12 g.%, 14 had inoderatc nnemius with 8-10 g.yOand 5 had severe anemias with hemoglobins of less than 8 g.%. Bone nit~rrowaspirations on 23 patients showed 70%, to h a w
248
VINCENT E. PRICE AND ROBERT E. GREENFIELD
infiltration of the marrow with tumor cells. I n spite of this infiltration, 3 had an increased number of erythroid cells, 18 were normal, and only 2 had a decrease in erythroid elements. Of 29 patients examined for overt evidence of hemolysis only 2 were considered to have significant signs of hemolysis, a conclusion based largely on elevations of the reticulocyte count to 8.3 and 12.6% respectively. It should be noted, however, that 1 patient with carcinoma of unstated origin had a positive direct and indirect Coombs test, an elevated urinary urobilinogen, and a reticulocyte count of 3.4%, which is slightly increased. Hyman et al. emphasized that although their red cell survival studies showed markedly increased rates of red cell destruction other signs of destruction were relatively minor. The findings of their study represent the type of evidence for erythrocyte destruction seen in a large group of cancer patients. I n 1954 Sheets et al. reported Ashby studies on 4 patients with faradvanced cancer. One patient with far-advanced cancer of the breast, with widespread bony metastases, had life spans of 30 and 48 days in 2 successive studies. Her red cell count remained level a t about 4 million cells, and it was estimated that to maintain this level the rate of release of new erythrocytes was 4-6 times normal. This patient, therefore, had rapid erythrocyte destruction with relatively little interference with erythropoiesis. A 2nd patient with adenocarcinoma of the rectum, with metastases to bone, had a rapid loss of the donor cells. Her own red cell count fell rapidly during the study from 2.8 down to 1.2 million cells per CU. mm., at which time the patient died. The Ashby study was not completed, but the donor’s cells had been lost from the circulation a t the rate of about 50% in 20 days. At necropsy much of the bone marrow was replaced with carcinoma cells, but there were many islands of erythroid and myeloid activity. The spleen was markedly enlarged, weighting 650 g. and contained areas of necrosis as well as multiple centers of hematopoiesis. Hemosiderin was present in both the spleen and liver. This case, therefore, had rapid red cell destruction and invasion of the bone marrow, which may have interfered with compensatory erythropoiesis. In 1954 and 1955, Rerifer published two reports relating the anemia of cancer patients to the rate of increased blood destruction and to the spread of the neoplasm. Their investigations were based on Ashby studies in 19 patients with a wide variety of malignant tumors. The patients fell into three groups depending on the length of their survival curves. Group I contained 4 patients with bronchogenic cancer, mammary cancer, Ewing’s sarcoma, and lymphatic leukemia, respectively. These patients had faradvanced tumors and a markedly shortened red cell survival time, with an average of 44 days. The average patient survival was 4.5 months. Group I1 contained 7 patients: a cancer of the tongue, a mammary cancer,
TABLE I11 Evidence for Erythrocyte Destructions
KO.Initials
J.H. J.H. E.B.
RIP.
P.G. H.J. T.Bo1. P.D. R.C. P.S. T.Bar. M.M. M.M. R.L. Y.Y. a
Urobilinogen Diagnosis
Cancer of kidney Cancer of kidney Cancer of breast Cancer of breast Cancer of breast Cancer of breast Cancer of breast Cancer of breast Cancer of esophagus Cancer of tongue Mult. myeloma Malignant melanoma Malignant melanoma Hodgkin’s disease Hodgkin’s disease
50%b Survival
Iteticulocytesc
(days)
( 76)
38 15 19 46 54 32 ca. 31 30 28 46 ca. 50 26 ca. 8 15 12
Fecald
1.8 -
8.3 2.9 3.2
Urinarye
Evidence of
Osmotic!
J1ech.g
(yesaline)
(yo) 1.1
1-160 1-640 1-160
0.45-0.30 0.45-0.35 0.45-0.30 0.45-0.30
2.2 6.1 1.3
-
-
-~
(E.V.ye) (dilution) 230 140 71 107
Fragility
1-320 -
-
1.5 -
-
-
-
-
2.2
105 -
Neg.
0.40-0.25
2.0
-
-
-
-
12.6 1.7
263 38
-
0.45-0.30 0.40.30
5.9 1.6
-
1-80
Slight Slight Moderate Slight Slight Inadequate None Inadequate Inadequate None Inadequate None None Marked None
;P 2,
M
5
2 o
;P
3
Compiled from Hyman (1954).
* Normal 60 days, Ashby technique. Upper limit 3%. Upper limit 200. 4 Upper limit 1-160. f Normal onset of hemolysis below 0..50Yosaline. 0 Upper limit 5%. c
d
to A
W
250
VINCENT E. PRICE AND IWBEItT E. GREENFIELD
a cancer of unknown origin, a round-cell sarcoma, a lymphosarcoma, a reticulosarcoma and a treated x-ray-sensitive tumor of unstated origin. These patients had generalized or widely-metastasized tumors. They had an average erythrocyte life span of 85 days and an average patient survival of 8 months. Group I11 had 9 patients: a multiple myeloma, a chronic myelogenous leukemia, a bronchogenic cancer, a tumor of the brain stem, an astrocytoblastoma, a chronic lymphatic leukemia, a lymphoid-cell reticulosarcoma, a mammary cancer under treatment with Perandren, and a treated metastatic x-ray-sensitive tumor of unknown histology. These patients had nearly normal life spans with an average of 110 days. The tumors in this group were early and had not rapidly progressed during the period of study. Only 1 patient of the latter group died during a period of 12 months.
O
/ //
/o;
4b
6b
,b Days
Ibo
IlO
IbO
'
FIQ. 13. Showing the general relationship between degree of anemia and red cell survival time in a number of cancer patients (from Renfer, 1955).
When the red cell survival time of the 19 patients was plotted against the erythrocyte count a good correlation was found between them, as shown in Fig. 13. Most of the points hover close to a straight line although several do not. Patients a, b, and c fall above the line. They had unusually high erythropoiesis as shown by an increased number of erythroblasts and reticulocytes in the blood stream. Patients n, m,and s, who fall well below the line, had diffuse permeation of the bone marrow by tumor cells with decreased erythropoiesis, which presumably accounted for their low erythrocyte count. I n a stimulating discussion, Renfer pointed out that the correlation between red cell survival and erythrocyte count in this fashion made available a method of locating disturbances in the equilibrium of
ANEMIA IN CANCEIt
25 1
red cell formatioil and destruction. He emphasized also the inconstancy of the “hemolytic intensity” during the period of study, and he related the clinical spread of malignancy to the increased rate of red cell destruction, as did Bottner and Schlegel (1952). Renfer felt that the close relationship between the degree of the anemia, the spread of the tumor, and the patient’s red cell survival time pointed to the dominating cause of the “tumoranemia” as being the result of increased erythrocyte destruction. Hyman and Harvey (1955) in a second report on the pathogenesis of anemia in patients with carckoma extended the previous study (Hyman, 1954) to 45 patients with widely disseminated carcinoma who had not received radiotherapy, tumor chemotherapy, or blood transfusions. Anemia was present in 90% of the patients a t the outset of the investigation and eventually in all cases. Ashby studies were carried out on 17 patients with carcinoma. One patient had a normal Ashby curve, 3 had slight increases in the rate of destruction, and 13 had moderate to marked increases in red cell destruction, with 50% survival times of 35 days or less. In two instances, a second study done a t a later time interval showed an increase in the rate of destruction as the disease progressed. This report essentially reaffirmed the findings of their earlier study. Most people have reported marked shortening of the red cell survival time in a large proportion of patients with cancer. Johnston (1956) after carrying out red cell survival studies in 10 patients with carcinoma, using both the Ashby and radiochromate techniques, found only 3 patients with a shortened survival time, whereas 7 were normal. Detailed results are not given in the report, but the hemoglobins ranged from 6.5 to 11 g.%, all 10 patients had a reticulocytosis, and in no case was there evidence of blood loss by hemorrhage. Of the 3 patients with shortened red cell survival, 1 was only slightly reduced to 100 days, and 2 had considerable shortening. Of these 2 patients, 1 had an elevated serum bilirubin and a positive Coombs test, which suggested in this instance the possibility of overt hemolysis. The second patient had cancer of the breast with widespread metastases to the skin and a hemoglobin of 6.8 g.%. In a n initial study, using both the Ashby technique and CrS1,a normal curve was found for the first 26 days after which increased destruction began to appear. The CrS1was all gone by the 86th day, and a second study showed very rapid destruction with 30% of the labeled cells disappearing in 6 days. The onset of hemolysis in this patient was said to have coincided with a pronounced deterioration of her clinical condition. Ross and Emerson followed their initial abstract (Ross et al., 1951) by detailed studies on the etiology of anemia in cancer patients (Chodos et al., 1956; Miller et al., 1956). Their study included 30 patients with carcinoma, 1 with lymphosarcoma, and 1 with fibromyxosarcoma. Four patients with localized lesions had no anemia. Of those with metastases, 12 had no anemia,
252
VINCENT E. PRICE A N D ROBERT E. GREENFIELD
and 6 had a mild anemia with hemoglobins of 10.5-12 g.%, 14 had a moderate anemia of 7.0-10.4 g.%, and 2 had a severe anemia of less than 7 g.%. Patients with overt blood loss, overt hemolytic anemia, infection, or primary liver or renal disease were excluded from the group. The reticulocyte count was slightly elevated (3-5y0) in 4 patients. The Coombs test was negative, and the serum bilirubin was within normal limits in all patients. The hemolytic index was elevated to a value ranging from 29 to 38 in 6 patients, as compared to a normal value of 8-20, indicating an increased excretion of bile pigments. Ashby studies on 10 patients showed that the red cells were destroyed a t a normal rate in 5 patients, and a t a n accelerated rate with survivals ranging from 25 to 80 days in the other 5 patients. Two of the latter patients showed rapid destruction early in the study, followed by a normal rate for the remainder of the curve. One patient with a resected cancer of the sigmoid with metastases had a normal red cell survival even during the last month before death. Another patient with resected cancer of the breast with numerous metastases to bone had normal curves on two occasions 11 months apart despite progression of the tumor. The hemoglobin level also remained normal. These cases serve to illustrate the highly variable pattern of red cell destruction seen in various cases of malignancy. Hyman et al., in 1956, published a report in which they attempted to evaluate the important question as to whether or not the erythrocytes of cancer patients are of an abnormal type with a shorter life span than normal red cells. Four types of experiments were carried out: (1) transfusion of whole blood from cancer patients into volunteer recipients: (2) transfusion of red cells from patients into volunteers; (3) transfusion from volunteers into volunteers, as a control study, and (4) transfusion of whole blood from volunteers into patients. In examining the data, it should be kept in mind that only patients having hemoglobin of greater than 11 g.% were used, which would exclude most of the patients with severe or even marked erythrocyte destruction. The Ashby technique was used for some studies and radiochromate for others. I n a number of studies, both techniques were used on the same individual. It will be remembered from the section on methods (Section IV,2) that a unique finding of Hyman el al. in this study was that there was no discrepancy between the curves obtained with the Cr6' and Ashby techniques when the donor blood was labeled with radiochromate. This was discussed in Section IV-2. Suffice it to say that a wide range of normal control values were found ranging from 23 to 52 days for 5Oy0 survival times and from 57 to somewhat over 100 days for 10% survival times, i.e., the time at which only 10% of the labeled cells remain in the circulating Hyman el al. (1956) believe that it is misleading to use the 50% survival figure and that survival curves should be interpreted only if they arc carried nearly to completion. This is certainly true if one is trying to obtain an accurate evaluation of the kinetics of
dNEMI.5 I N CANCER
253
In their first series of experiments, whole blood from 15 cancer patients was transfused into normal volunteers. The erythrocytes of 1 patient had a shortened 50% survival time of 19 days and a 10% survival time of about 47 days. The remainder fell within a range of 23 to 42 days for the 50y0 survival times and 61 to 100 days for the 10% survival times. These were comparable to the range found in the group of normal volunteers. This means that 1 of the 15 patients appeared to have slight shortening of the red cell life as compared to the normal controls. Six of the 15 patients were studied by both the Ashby and C P techniques and, as in the normal controls, no significant discrepancy was found between the curves obtained by the two methods. In the second series of experiments, the red cells were separated from the plasma before their injection. The erythrocytes of 3 of the 15 patients had 50% survival times shortened to 15, 20, and 22 days, and 10% survival times shortened to about 35, 28, and 27 days. The remainder were essentially within the limits of the normal controls, so that the survival curves of 3 of the 15 were significantly shortened by this technique. Although the above experiments were a step in the direction of determining whether the erythrocytes of cancer patients were defective or not, they have two limitations. The first is the marked variation in the survival of the erythrocytes of normal donors upon transfusion into apparently normal recipients. The shorter control values may conceivably reflect the presence of a random destructive process of unknown cause in apparently normal humans, as suggested by Mollison (1956) and as described by I. W. Brown, Jr. and Eadie (1953) in several species of animals. The second limitation is the restriction of the study to patients with hemoglobins of 11 g.% and above. In a search for defective erythrocytes, it would be preferable to study the red cells of patients who have a marked anemia so as t o be sure that the erythrocytes are sufficiently defective that one would expect them to be removed upon transfusion into a normal recipient if such a mechanism was the cause of the erythrocyte destruction. Because of these two limitations, it is difficult to state whether the finding of increased destruction of the erythrocytes of 4 of 30 cancer patients, upon transfusion into apparently normal recipients, is significant or not. I n the final series of experiments, whole blood from normal volunteers the survival curves. However, when one is examining survival curves having variable kinetics, the entire shape of the curve has significance. A number of workers use simply the end point a t which the tagged cells are no longer found in the circulating blood. This will give a figure representing the maximum life of the labeled cells, but it gives no information on the cells which have been destroyed earlier. As the end point is difiicult to determine, some investigators have used the point at which 10% of the labeled cells still survive. I n our discussion of this paper, we find that it is valuable to use both the 5oTcand 10% survival values.
254
VINCEKT E. PRICE ARTD ROBERT E. GREENFIELD
was injected into cancer paticnts. In 4 of the 17 studios, thcre was shortening of the 50% survival to 13-17 days and in 6 there was shortening of the 10% survival time to 19-45 days. Upon examiiiing the curves, one is impressed by the variable shapes of the different curves. For example, 1 of the patients has a precipitous early fall after which the curve descends at a normal slope, as if a markedly increased rate of destruction had occurred early in the study. In 2 others the initial slopes are almost normal, for a chromium curve, but after 20-30 days the rate of destruction sharply increases, suggesting the sudden appearance of a destructive process. These findings are similar to those of Bottner and Schlegel (1952), Renfer (1955), and Miller et al., (1956). In this group, therefore, 7 of the 17 studies showed abnormal rates of erythrocyte destruction. This is a fairly large proportion if one considers that patients with moderate to severe anemia were excluded.
FIQ.14. Serial red cell survival studies, using the Ashby technique, on IL patient)wiih metastatic carcinoma of the breast (from Sohier d al., 1957).
Recently Sohier et al. (1957) have reported a detailed investigation in which serial red cell survival studies were performed on 4 patients with metastatic carcinoma of the breast and on one patient with malignant melanoma. Although the initial life span studies were nearly normal, signs of increased destruction appeared in later studies which became very marked in the month before death. One of these serial studies is illustrated in Fig. 14. The initial life span of 120 days fell to 105 days and then to 60 days just before death. It might be observed that although in the initial Ashby study an overall life span of 120 days was observed there was a period in July when 30% of the labeled cells disappeared within 2 weeks time. This was accompanied by n fall in the hemoglobin concentration of
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about 17% and followed by an elevation of the reticulocyte count to about 8%. It would seem that this sequence of events might represent the transient appearance of the destructive process which became so pronounced toward the end of the study. At death the patient had a hemoglobin of about 7 g.% and a markedly elevated reticulocyte count of over 50%. It would seem that this excellent and detailed study should provide a pattern for future investigations in that by continuously following the rate of disappearance of tagged cells during serial studies it is possible to more accurately locate and measure fluctuations in the rate of the destructive process and to correlate these fluctuations with the clinical status of the patient. I n the investigations reported above, red cell survival studies have beeii carried out on approximately 220 patients with leukemia and lymphomas and on 77 patients with carcinomas and other solid tumors. In both groups about half the patients studied had normal curves or only moderate shortening with red cell survival times of 70 days or more, and half had marked to severe shortening to survivals times of less than 70 days. These figures, of course, do not reflect the true indicence of processes of abnormal erythrocyte destruction in cancer patients since in the various studies they have not necessarily been selected from a representative group of patients. It is also likely that some of the patients with little or no abnormal erythrocyte destruction a t the time of study may have such a process a t some later stage of their disease. The data do show, however, that processes of abnormal erythrocyte destruction are prominent in many cancer patients and, where present, undoubtedly play an important causative role in the anemia of the patient. These investigations also repeatedly emphasize the fact that the most satisfactory criterion of increased red cell destruction in cancer patients is obtained by a direct measurement of the survival of tagged erythrocytes, particularly by serial studies using the Ashby or radiochromium techniques which permit continuous evaluation of the destructive process in the patient. Of the other criteria, the excretion of fecal urobilinogen and the reticulocyte count are still of considerable value, whereas the urine urobilinogen, serum bilirubin, osmotic fragility, and Coombs test are generally normal even in the presence of a marked destructive process. The latter tests may on occasion, however, provide other information of value regarding the status of the cancer patient. A.
Nature of the Destructive Process
A. Pathways of Red Cell Destruction. There are four main pathways by which red cells may be destroyed within the body in pathological conditions:
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a. Lysis. They may be lysed within the blood stream. The hemoglobin released binds to the newly defined “haptoglobin” in the plasma (Jayle and Boussier, 1955), which leaves the blood stream slowly and is presumably removed by the reticulo-endothelial system in the liver. Hemoglobin in excess of the haptoglobin-binding capacity of the plasma is excreted by the kidneys, causing hemoglobinuria (Laurel1 and Nyman, 1957). b. Phagocytosis. They may be phagocytized directly from the circulating blood by components of the reticulo-endothelial system in the spleen, liver, bone marrow, and lymph nodes. c. Hemorrhage. They may escape from the blood stream through breaks in the vascular wall and be either lysed, phagocytized, or lost from the body by external hemorrhage. d. Thrombosis. When a defect occurs in the vascular wall, a thrombus is formed. Entrapped red cells are lysed or phagocytized within the thrombus. Although the destruction here occurs within the vascular tree, it is not within the circulating blood. Regardless of the pathway involved most or all of the hemoglobin not lost through the urine appears eventually in cells of the reticulo-endothelial system, where it is degraded. The ring of the porphyrin moiety is opened and converted to bilirubin. The bilirubin passes to the liver where the glucuronide is formed (Schmid, 1956) and excreted through the bile into the intestines, where it is converted by bacteria into a complex group of compounds labeled “urobilinogen.” These four pathways of red cell destruction fall into two general groups when classified as to the nature of their initial lesion. The first two pathways, lysis and direct phagocytosis, are characterized by the fact that the destructive process is initiated by the presence of malformed or injured circulating erythrocytes. If the defect is severe, the red cells are lysed directly, and if it is less severe, the damaged cells are picked up from the circulating blood and phagocytized by components of the reticulo-endothelial system. The second two pathways, hemorrhage and thrombosis, are characterized by the fact that the process begins as the result of injury to the endothelial lining of the blood vessels which results in the loss of essentially normal cells from the circulating blood, either by hemorrhage from the blood vessel or by being entrapped in thrombi within the vessels. In the first group, abnormal cells are removed from the blood stream by normal processes. I n the second group, normal cells are removed by abnormal processes. In considering the mechanisms of red cell destruction, it will be convenient to consider first those mechanisms involving primary damage to the red cell, realizing that the actual mechanism of destruction may be
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either by lysis or by phagocytosis, and secondly, to consider those mechanisms in which the primary lesion is in the blood vessels, causing hemorrhage and thrombosis. B. Mechanisms Involving Defective Erythrocytes. a. Evidence for a n intrinsic defect in the erythrocytes of cancer patients. Intrinsic defects of erythrocytes may cause either finite” or ‘(random” shortening of their survival curves : (1) Finite shortening results from accelerated senescence of the erythrocytes. N. I. Berlin et al. (1954) reported that when erythrocytes of patients with chronic myelogenous leukemia were labeled with C14-glycine they appeared to have a finite and shortened life span. Examination of their curves, which have been discussed in Section IV,3, reveal only one with the S-shaped curve which would be expected if finite shortening were present, and even this curve (Fig. 12A) could be obtained by the appearance of a random destructive process of increasing intensity a t 60-70 days. N. I. Berlin et al. (1954, 1957) point out that although their own studies would suggest that the defect is intrinsic, the Ashby studies of R. Berlin (1951, see Section IV,3) would implicate an extrinsic factor as causing accelerated destruction of the red cells since with the Ashby technique the cells being observed are those of a normal donor rather than the patient’s own cells. A similar argument would hold for the Ashby studies of G. M. Brown (1950) and Ross et al. (1954) on patients with chronic myelogenous leukemia in which an accelerated rate of destruction was reported (see Section IV,3). It is obviously impossible to select between defects of intrinsic or extrinsic origin by use of the patient’s cells alone. A demonstration of the presence or absence of an intrinsic defect in the erythrocytes of a given patient could he obtained by the use of cross-transfusion experiments similar to those reported by James et al. (1953, see Section IV,2). I n such an experiment, the patient’s own cells could be labeled with C14-glycine and the cells of a normal donor with N15-glycine. When incorporation of the NI5-glycine approached maximal levels, cells of the normal donor would be transfused to the patient. If the N15 cells had a normal life span whereas the C14 cells of the patient still showed finite shortening, it would be presumptive evidence of an intrinsic defect in the patient’s cells. If, on the other hand, the N15 donor cells disappeared in a curve similar to that followed by the C14 cells of the patient, it would be quite convincing evidence that an extrinsic factor was affecting both groups of cells in a similar manner. It may be concluded that there is little evidence as yet for the occurrence of accelerated senescence in the erythrocytes of cancer patients. (2) Random shortening would be found where newly-formed cells were so defective that they were removed by the reticulo-endothelial system
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without regard to their age. Neuberger and Niven (1951) and N. I. Berlin and Lot2 (1951) in studies with isotopically-labeled glycine have reported random shortening of the life span of newly formed cells in rabbits and rats, respectively, following acute hemorrhage. It is possible that a similar phenomenon might be found in cancer patients during periods of rapid and severe erythrocyte destruction. Unfortunately, the random shortening reported in the above studies could be caused either by formation of severely defective erythrocytes or by vascular injuries secondary to the shock of acute hemorrhage. It would have been worthwhile to carry out cross-transfusion experiments in these instances to determine whether the newly-formed cells were actually defective or not. b. Evidence for injury by extrinsic factors. A considerable literature has developed in the search for toxic substances or immune bodies which might inflict injury on the circulating erythrocytes of cancer patients and lead to their destruction. Research in this area has progressed along two lines: (1) The examination of cancer tissues for the existence of a hemolytic toxin, and (2) the examination of red cells from cancer patients for evidence of injury which might implicate the hemolytic activity of such a toxin or of some immune phenomenon. ( 1 ) Evidence for tumor toxins and hemolysins. One of the main pathways pursued by early investigators on anemia in cancer was the possibility that tumors produced toxins which had a destructive action against red cells. Much of this history has been reviewed by Weil (1907). At the turn of the century, Metchnikoff had been studying the hemolytic action of extracts of various normal tissues. In describing the approach of these early investigators t o the problem, Weil reports, “It had always been common knowledge that a characteristic of malignant tumors was to produce cachexia and secondary anemia in their victims. Marchand (1902) interpreted the necroses, thromboses, hemorrhages, and round cell infiltrations as evidences of a toxic influence. He went even further; he suggested that the disturbance of tissue equilibrium, to which Weigert had attributed the rapid advance of tumor cells, was due to the destructive influence of this toxic substance on the surrounding tissues. It has frequently been suggested that the anemia of tumors is probably due t o the action of some toxin elaborated by the tumor and diffused into the circulating blood.” Panzacchi (1902) was the first to demonstrate the lysis of erythrocytes by tumor extracts. This was followed by reports of Micheli and Donati (1903) and Kullmann (1904). Kullmann prepared extracts of tumors of the breast and uterus, and on testing them against washed red cells of man, rabbit, sheep, and dog obtained hemolysis in every case, indicating tJhat the toxin of trumors was a general rather than specific hemolysin. In his own paper, Weil (1907) demonstrated that extracts of normal orgatis (liver and kidney) varied considerably in their hemolytic potency and that the hemolytic
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activity of extracts from organs thoroughly freed from blood was very low, and in some cases entirely absent. Although red cell extracts did not hemolyze erythrocytes of themselves, they did increase the hemolytic activity of bloodless organs. When red cells were treated with red cell extracts, they became sensitized. If the sensitized cells were then washed and added to organ extracts, they would hemolyze. In studies on tumors, Weil found that necrotic tumors were much more hemolytic than nonnecrotic ones. I n the necrotic tumors, the hemolytic principle was entirely different in that the addition of red cell extracts to the necrotic tumor extracts did not increase their hemolytic activity, and the hemolytic principle of the necrotic extracts was dialyzable whereas that of nonnecrotic tumors was not. He concluded that the anemias of malignant tumors are probably due in part to the hemolytic and toxic action of such products of necrosis. Little of further significance happened in this field for 40 years. In 1947, Gross reported experiments suggesting “a direct destructive action of a filterable substance contained in the tumor cells, on the erythrocytes of the host.” His detailed studies were summarized in a later paper (Gross, 1948). Briefly, his experimental technique was this : Working under aseptic conditions, tumors were removed and ground with 5-10 volumes of isotonic saline. The suspensions were centrifuged a t 5,000 r.p.m. for 10 minutes and then either recentrifuged twice or passed through a No. 3 Seitz filter. 0.5 ml. of this extract was then added to 1.0 ml. of a 1% R.B.C. suspension and incubated a t 37°C. for 246-3 hours, a t which time any intact cells were centrifuged down and the degree of hemolysis observed as negative, doubtful, or up to 3+ for complete hemolysis. A 10% extract of mouse mammary carcinoma on the average gave 2+ hemolysis against mouse erythrocytes, whereas a 20% extract gave negative results against human, chicken, guinea pig, and rabbit erythrocytes, and a one 1+ result against rat erythrocytes. On the other hand, a 20% extract of a mammary cancer obtained from rats gave 3+ hemolysis with mouse erythrocytes and only doubtful t o 1 hemolysis with rat erythrocytes. It appears therefore that the effect was not species-specific, but that mouse erythrocytes were very sensitive to hemolysis in this test. Extracts of normal tissues, such as liver, spleen, kidney, muscle, and ovaries gave no hemolysis. Only extracts of placenta or of mammary glands of pregnant or nursing females gave positive results. Extracts of mouse sarcoma 180 and of the lymph glands and spleen of leukemic mice also gave positive results. Extracts of normal liver inhibited the hemolytic action of tumor extracts. The extracts were stable to heating a t 61°C. for 30 minutes, but8 were completely destroyed a t 68°C. Incubating the extract a t 37” for 3 hours destroyed most of the activity. In a later report, Gross (1949) extended these interesting studies to
+
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human cancer. In his initial experiments using assay periods of 236-3 hours, only negative results were obtained. He did get weakly positive results, usually I f , with a variety of human carcinomas upon extending the incubation period to 48 hours, whereas control tubes gave negative results, as did extracts of such normal organs as liver, muscle, thyroid, breast, and placenta. I n addition, certain extracts gave an agglutination phenomenon which was destroyed on heating the extracts to 56°C. for 30 minutes. The test erythrocytes used were of the same blood type as the patient from whom the tumors were removed. Gross makes no mentioii of comparative results with extracts of good viable tumor as compared to those of necrotic or hemorrhagic areas. At about the same time, Salaman (1948) observed that saline extracts of a transplantable mouse sarcoma agglutinated mouse and rabbit erythrocytes. Very little hemolysis was seen except after long periods a t room temperature or if the experiments were carried out at 37°C. The hemagglutinating activity of the tumor extracts was very unstable, falling to onehalf in one hour but could be regenerated and protected by BAL (British anti-lewisite) and sodium thioglycollate. Extracts of mouse liver, spleen, kidney, heart, lung, brain, lymph nodes, and thyroid gave no agglutination but those of the reproductive system, such as testis, ovary, uterus, and placenta did. Rabbit erythrocytes were more readily agglutinated by the mouse tissue extracts than were mouse erythrocytes. The presence of complement had no detectable effect on the hemagglutinating activity. Adelsberger (1951a,b) studied the hemolytic behavior of the erythrocytes of normal and tumor-bearing animals. She found, unexpectedly, that on prolonged incubation in saline, the erythrocytes of normal C3H mice were hemolyzed to a much greater extent than those of mice bearing the C3H mammary tumor. She incubated the cells in an antitumor rabbit serum and in normal rabbit serum and again found greater hemolysis when erythrocytes of normal C3H mice were used then with those of tumorbearing mice. However, results with the antitumor serum did not differ significantly from those with normal control serum. When tests were carried out with tumor suspensions, much more rapid hemolysis was seen, the hemolysis occurring in 3-24 hours instead of the 1-4 days required with saline or serum. In 16 tests, the red cells of tumorbearing mice were hemolyzed to the same degree as those of normal controls, in 7 there was less hemolysis, and in 14 there was no hemolysis at all. The hemolytic activity of the tumor suspensions fell rapidly on standing at room temperature or a t 37°C for a few hours. In a later paper (Adelsberger and Zimmerman, 1954), essentially similar results were found with a variety of transplantable brain tumors from C3H mice. The increased hemolysis of normal erythrocytes that Adelsberger found with tumor suspensions is similar to the finding of Gross with tumor
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extracts. The additional finding by Adelsberger of increased resistance of the red cell of tumor-bearing animals to hemolysis is of considerable interest. She felt that the red cells of tumor-bearing mice might have taken up an antibody which inhibited hemolysis. Another possibility, however, is that the population of red cells in the tumor-bearing animals may have been younger and more resistant to hemolysis. Ponder (1948b) reported the appearance of red cells with an increased resistance to hypotonic saline and to lytic substances, such as saponin and digitonin, following massive hemorrhage, and also in a patient with severe anemia secondary to metastatic gastric carcinoma. Chalfin (1956) also reports an increased osmotic resistance in the young cells formed in response to repeated hemorrhage. Sterling et al. (1958)) in studies on the fragility of red cells from rats bearing transplanted tumors of various types, found a high proportion of young cells with increased resistance to hypotonic saline. In a careful and detailed study, Ponder and Nesmith (1952) investigated the question of the identity or nonidentity of hemolytic substances found in tumors and normal tissues. They found that the differences in both hemolysins and agglutinins could be attributed simply to concentration differences of the substances in the tumors and various tissues and not to qualitative differences in the substances. Tumor homogenates usually contained greater concentrations of the hemolysin than homogenates of normal tissues, and fluid from necrotic centers of the tumors had maximal activity. They found that the lytic material of fresh extracts of botjh tumors and normal tissues was unstable, falling to one-quarter of their initial value after standing for 2 hours a t 25"C., but that on prolonged incubation more lytic material was released, presumably by enzymatic activity. This would account for Gross' finding of hemolytic material in human tumors after long incubation. Possibly the initially active material disappeared during the long period of time usually required to obtain human autopsy material. Ponder and Nesmith found that lytic substances extractable with organic solvents were similar to those present in saline extracts and that the lytic material had two alcohol-soluble fractions, one of which was soluble in ether, the other being insoluble. Both fractions upon electrophoresis a t pH 5.6 separated into two components, each of which had hemolytic activity. Although mouse red cells were lysed more rapidly by mouse tissuc extracts than the red cells of other species, Ponder and Nesmith observed that this should not be interpreted as showing that the lysins are speciesspecific. Instead the differences in rate of hemolysis appeared to result from differences in resistance to hemolysis of cells of the various species, so that in his system they could be arranged in a resistance series: mouse < man < rabbit < sheep; with mouse cells being the most susceptible to hemolysis. From the data of Weil, Gross, Adelsberger, and Ponder, one may con-
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clude that lyt,ic substances may be extracted from neoplastic tissues, and in lesser amounts from normal tissues. One is still faced with the problem as to whether these tissue lysins have any causal relationship to the anemia of cancer patients. Several facets of this problem merit further consideration : ( a ) The fact that lytic substances can be extracted from neoplastic tissues does not mean that they are released from tumors in vivo and play an active role in erythrocyte destruction (cf. Ponder, 1951 ; Pirofsky, 1957). Such extraction procedures invariably involve destruction of the cellular architecture, releasing substances which under in vivo conditions may never enter the circulating blood. (b) If lytic substances do enter the blood stream from tumors are they excreted by viable tumor cells or are they products of autolysis from iiecrotic areas’? Bruckmann and Westheimer (1945) considered the tissuc lysins to be products of autolysis. We have no satisfactory information on the rate a t which substances from dying and necrotic areas of tumor may enter the circulating blood. The rate would, of course, be dependent upon whether or not there was a circulation in the capillaries of the dying or necrotic area. Since necrosis generally results from a defective circulation in a given area of the tumor, it seems probable that most of the capillaries would be thrombosed, thus blocking escape of substances by this route. In this case, material would have to escape over considerable distances by diffusion, so that the rate would bear an inverse relationship to molecular size. At the periphery of the necrotic area, the rate of diffusion would be slowed by the presence of cell malls and connective tissue barriers which have been only partially destroyed by the autolytic process. If the lytic substances were of fairly large molecular weight it seems that the rate of their escape into the blood stream would be rather slow. This same general conclusion was reached by Mider et al. (1948) for the nitrogenous components of tumors. (c) At best the lytic substances which have been extracted from tumors are rather weak in activity, as emphasized by Ponder (1951). Usually several hours to several days are required to bring about hemolysis. Bruckmann and Westheimer (1945) have shown that shaking reduces the amount of hemolysis by 3-4-fold. What then is the effect of continual mixing found in most parts of the circulation? In view of their weak activity one must also ask if the lytic substances are removed before they can be effective. If so, areas of relative stasis, such as may be found within the sinusoidal vessels of many tumors, may be important factors in the lytic process. (d) There is considerable evidence that the alcohol-ether soluble fraction of Ponder contains fatty acids, and so they are classified as being “soaplike substances,” in contrast to the alcohol-soluble, ether-insoluble
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fraction which are given the term “lysolecithinlike substances” (Ponder, 1951). There is considerable evidence that the fragility of dog’s red cells is increased after a meal of fat (Longini and Johnson, 1943; Johnson et aZ., 1944) and also that a high-fat diet is followed by an increased excretion of bilirubin (Loewy et al., 1943). The lipemia of cancer patients and tumorbearing animals has been frequently described, but as yet its etiology is unknown. I n view of the above findings, one must determine whether or not lipemia is a cause of increased red cell destruction in cancer patients. A new development in this field has come from Pirofsky, who in 1956 reported the presence of an agglutinin and hemolysin in extracts of leukemic cells which were active against trypsinized erythrocytes. Similar results were obtained with leukemic cells of the lymphocytic, granulocytic, and monocytic series. In recent detailed studies, he has sought to determine whether these agglutinins and hemolysins were similar to or different from p-glucuronidase, lytic materials from tumor tissues, anti-H agglutinin, or coating antibodies eluted from the erythrocytes of patients with acquired hemolytic anemia. They were found to be different in each case. Erythrocytes treated with the leukemic extracts were found to give the dusky green color generally associated with choleglobin formation (cf. Ponder, 1951). An inhibitor was found in both normal and leukemic serum which completely prevented hemolysis and markedly diminished agglutination and choleglobin formation by the extracts; therefore, the activity of these systems in vivo remains in question., Possible ways that these substances from leukemic cells might bring about erythrocyte destruction in vivo were discussed, including the possibility that by increasing choleglobin formation they might cause premature senescence of the erythrocyte. It was pointed out, however, that the significance of the agglutinin and hemolysin in vivo still has t o be demonstrated. Rosenthal et al. (1955) report that in their patients with chronic lymphatic leukemia regression in the size of the spleen and lymph nodes, which they assume to result from the destruction of young lymphocytes, lymphoblasts, and plasma cells, was accompanied by a reduction in hemagglutinin titer. If the hemagglutinin described by Pirofsky was released as a result of destruction of leukemic cells one might expect to see an elevation of hemagglutinin titer during periods of lymphoid regression, rather than a diminution. It may be concluded that evidence that substances from neoplastic tissues play a significant role in vivo in the destruction of circulating erythrocytes is still circumstantial. Much more work is required to firmly establish a place for them in the etiology of anemia in cancer. (2) Evidence j o r extrinsic dejects of the red cells of cancer patients. This section is concerned with defects of circulating erythrocytes which arise
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from external causes, such as immune phenomena and toxic substances. Red cells which are injured by extrinsic factors may agglutinate with other cells and be removed by the reticulo-endothelial system, or they may swell, due to permeability changes in the red cell wall, and finally lyse. Ponder (1948a, p. 30) lists the stepwise changes which the red cell goes through during the action of a typical lysin: biconcave disc + crenated disc + crenated sphere + smooth sphere + prolytic sphere + ghost. These changes are reversible until an actual increase in volume takes place during formation of the prolytic sphere. The smooth sphere has a normal red cell volume, but since it is rounded it has a smaller diameter than the normal disc, These abnormally and so is frequently described as a “micro~pherocyte.”~ shaped cells may be removed by the reticulo-endothelial cells in the spleen or they may proceed to lysis directly in the blood stream, producing hemoglobinemia. They are also more fragile in hypotonic saline solutions. Therefore, clinically, spherocytosis and an increased hypotonic fragility are generally considered to be signs pointing to extrinsic injury of the red cells. A third finding which is considered to be pathognomonic of red cell damage is a positive Coombs test. This is a test for the detection of weak and “incomplete” antibodies which was developed in its present form by Coombs et al. in 1945 but was actually first applied by Moreschi in 1908. It was used by Boorman et al. (1946) to distinguish certain types of acquired hemolytic jaundice from congenital hemolytic jaundice (cf. Loutit and Mollison, 1946). Because of its importance, before going further it may be well to include definitions taken from Mollison (1956, pp. 175,299), of several of the terms used in this field and also to describe the Coombs test. Hemolysins: Antibodies capable of lysing red cells in uitro. Complete antibodies: A serum which is capable of causing agglutination of cells suspended in saline is said to contain complete antibodies. For example, the serum of group 0 persons contains anti-A and anti-B saline agglutinins. Incomplete antibodies: If a serum fails to agglutinate a saline suspension of red cells but will agglutinate cells suspended in a colloid medium, such as plasma, serum, or albumin, it is said to contain incomplete antibodies; such antibodies will sensitize cells to an antihuman globulin (Coombs) serum. Most anti-Rh sera are of this kind. The Coombs (Antiglobulin) Test: When cells suspended in saline are exposed to the action of an incomplete antibody they are not agglutinated, but they do adsorb antibody on their surface. Antibodies are globulins. In the direct Coombs test the globulin, or antibody, adsorbed to the cell surface is detected by exposing the red cells to an antiglobulin serum which
‘
These spherocytes are to be distinguished from the malformed and thickened, but still discoidal, spherocytes seen in hereditary spherocytosis (Ponder, 1948a; Dacie, 1954).
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causes a rapid agglutination of the cells. The antiglobulin serum is prepared by injecting human serum or gamma globulin intraperitoneally into a rabbit twice a t 1-month intervals and harvesting the serum 10 days after the last injection. I n the indirect Coombs test, red cells containing a known antigen are used to examine unknown sera for the presence of incomplete antibody, or conversely, sera containing known incomplete antibodies are used to determine the presence of the corresponding antigen on the surface of the red cells. I n either case, antiglobulin serum must be added, in a second step, to bring about agglutination of the red cells. Examination of the literature on anemia in cancer indicates that spherocytosis, increased hypotonic fragility, or a positive Coombs test occur rather infrequently in patients with carcinoma. Shen and Homburger (1951) for instance found 3 cases with one or more of these findings in 116 patients with carcinoma. Chodos et al. (1956) found a negative Coombs test in 24 patients with carcinoma. Exceptions to this have been the ovarian tumors, in which a hemolytic process with spherocytosis and increased fragility is rather frequently found (West-Watson and Young, 1938; Singer and Dameshek, 1941; Dacie, 1954). Patients with these findings are generally classified as having an acquired hemolytic anemia (Dacie, 1954). The signs pointing to extrinsic lesions of the erythrocytes are much more frequently found in neoplasms of the hematopoietic system, particularly in the lymphoid series (Rosenthal et al., 1955). The frequency varies greatly from study to study. Haden (1939) concluded that a “hemolytic type” of anemia in leukemia was rare. R. Berlin (1951) reports a n increased hypotonic fragility in 2 of 15 patients with chronic myeloid leukemia and in 1 of 9 patients with chronic lymphatic leukemia. Coombs tests performed on 14 patients were negative. He does not mention the occurrence of spherocytosis. Rosenthal et al. (1955) studied 20 patients with chronic lymphatic leukemia, 3 with lymphosarcoma, and 1 with giant follicular lymphoblastoma, all of whom were classified as having autoimmune hemolytic anemia. He found positive Coombs tests in all 20 patients on whom it was performed. Serum agglutinins were found in 11 of 19 patients, but hemolysins were negative in the serum of 12 patients examined. Spherocytosis was found in all 24 patients a t some stage of the disease. Fragility studies were not reported. Seaman et al. (1957) report the presence of overt hemolytic phenomena in 52 out of 212 cases of chronic lymphocytic leukemia. Therefore although extrinsic lesions of the erythrocytes cannot, at the present time, be implicated in more than a small number of cancer patients, they are a factor to be concerned with in the destructive processes to be found in some patients, particularly those with tumors of the ovary and
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of the hematopoietic system. Our interest centers around three problems: (1) How are the red cells injured? (2) What, if any, is the role of the spleen in the etiology of these extrinsic lesions of the erythrocytes? (3) How can the magnitude of this route of red cell destruction be measured in a given patient? None can be answered satisfactorily a t this time. As to the first problem, Rosenthal et al. (1955) felt that the primary cause of the red cell injury is autoimmune in nature. They suggested that antigens may be formed in malignant cells of the lymphocytic series which have configurations in common with some antigen in the erythrocytes, and that antibodies formed in response to the antigen of the malignant lymphocytes might act on the closely related antigen of the erythrocyte. Although an immune technique is used to determine the presence of a globulin coating on the red cell, Wagley et al. (1948) point out that this does not necessarily imply that the primary lesion of red cells was caused by an immune mechanism (cf. Ponder and Ponder, 1954). A positive Coombs test is found in erythrocytes injured by phenylhydrazine (Roth and Frumin, 1957). The latter authors, in studies on acquired hemolytic anemia, found that the severity of the disease was directly related to concentration of serum agglutinins and inversely to the degree of red cell coating. Incubation experiments showed a protective effect of the coating which was manifested by an increased viability of red cells under adverse metabolic conditions. They conclude that “the question as to the nature of the factors encountered in acquired hemolytic anemia will have to remain open, regardless of the increasing number of reports dealing with blood group specific (or cross-reacting) ‘auto’ antibodies found in autoimmune hemolytic disease.” The second problem about which a considerable interest has been focused is the question of whether the spleen plays a significant role in causing extrinsic lesions of the erythrocytes. Evidence for such a role comes largely from the beneficial effect of splenectomy in some patients with leukemia, and from a comparison of the clinical syndrome found in these patients with that found in other types of acquired hemolytic anemia in which splenectomy is frequently of therapeutic value. It will be remembered from Section IV,3 that R. Berlin (1951) carried out his red cell survival studies on leukemic patients to evaluate the effect of splenectomy on the rate of erythrocyte destruction. Six splenectomies on patients with chronic myeloid leukemia resulted in a return of the Ashby curves to normal in two cases, one of which is illustrated in Fig. 10s. In two of the cases, the results were only fair; in another two, little or no benefit was observed. The varied benefits of splenectomy are rtlso emphasized by Stats et aE. (1947) and by Dnmeshek and Welch (1954). The latter authors present a comprehensive summary of the concept of hyperplenism and the various theories as to its etiology (cf. Doan, 1949; R. Berlin, 1951; Aas, 1952).
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Since in neoplasms of the hematopoietic system the spleen is one of the organs most heavily invaded by neoplastic cells, it seems to the reviewers that extrinsic damage to the erythrocytes may result from a disruption of normal anatomical structures of the spleeii, and therefore, in a very real sense, be a n example of a condition in which lesioris of the blood vessels may’actually lead to injury of circulating erythrocytes. Stats et ul. (1947) describe an “arteriul” hyperemia of the spleen characterized by a n “active hyperemia, overfilling of the pulp with blood, or collapsed venous sinusoids in a lake of blood.” With such a disturbed pattern of circulation, it is probable that progress of the erythrocytes through some areas of the circulation will be greatly impeded; in other areas it may be prevented entirely. In those areas in which the circulation is impeded, the red cells will be in a condition of stasis and be subjected for longer periods of time to concentrations of enzymes and lytic substances which normally act to destroy abnormal erythrocytes held back by the spleen (see Ponder, 1948a, pp. 323,337). Much more study is needed to adequately evaluate the role of the spleen in the etiology of extrinsic lesions of the red cells in the leukemias and lymphomas. The third problem is that of determining the magnitude of red cell destruction occurring in a given patient as a result of extrinsic damage to circulating erythrocytes. A positive direct Coombs test does not necessarily mean that the affected cells will have a shorter life span. Mollison (1956, p. 165) points out that, “Red cells ‘coated’ with antibody are not necessarily destroyed a t an increased rate.” Loutit and Mollison (1946) transfused Coombs positive cells from 4 patients with acquired hemolytic anemia into normal recipients and found that the cells disappeared from the circulation at a normal rate. Similar questions arise as to the significance of spherocytosis. Ponder (1948a, p. 36) point out that spherocytosis is reversible until a n actual increase in red cell volume occurs. Many of the clinical reports of spherocytosis in leukemic patients are of microspherocytosis, which is characteristic of the first and presumably reversible stage. Does such a reversal actually take place in the spherocytes of patients with leukemia’? If so, are a significant number of spherocytes removed by the spleen and reticuloendothelial system before reversal back to the normal discoidal shape can take place? Obviously the simple report of the occurrence of spherocytosis is only suggestive that destruction may be impending. It does not give us quantitative information as to the number of cells destroyed. Changes in osmotic fragility are usually reported in terms of the range of saline concentrations over which lysis takes place. Although they indicate the presence of abnormally fragile or resistant cells, such values do not adequately reflect the number of cells which are abnormal, or whether they actually have a shortened period of survival within the circulation.
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VINCENT E. PRICE AND ROBERT E. GREENFIELD
It would seem therefore that a positive Coombs test, spherocytosis, and an increased osmotic fragility are simply indicators that abnormal destruction may be taking place, but they give no indication of the magnitude of the process involved. The number of cells destroyed may range from few to very large numbers depending on the rate a t which the injured cells are removed from the circulation by the reticulo-endothelial syitem. It is hoped that radioisotope techniques, such as those of Jandl et al. (1956), may eventually provide us with adequate tools to answer this problem in the near future. If one reexamines the evidence for intrinsic or extrinsic defects of circulating erythrocytes as being the cause of erythrocyte destruction in cancer patients, it will be seen that the evidence is largely suggestive and circumstantial, and at best limited to a rather small proportion of cancer patients. Little quantitative evidence is available to demonstrate that defects of circulating red cells play a major and significant role in the etiology of the anemia of most cancer patients. C. Mechanisms Involving Vascular Defects. Vascular defects may cause erythrocyte destruction externally by hemorrhage, or internally by extravasation of blood into the tissues, and by the trapping of red cells within the vascular tree during thrombus formation. a. External loss. Overt hemorrhage has long been accepted as a major factor in the anemia of cancer patients, whenever it has been observed (cf. Shen and Hornburger, 1951). Most studies on the “hemolytic anemia” of cancer patients carefully exclude those with overt hemorrhage. Little consideration has been given to the fact that these patients undoubtedly have processes of internal destruction going on at the same time. Owen et al. (1954, 1955) have reported a method for detection of gastrointestinal hemorrhage following the appearance of radiochromium in the feces from erythrocytes labeled with Cr61. Potentially, by such a technique one could measure the amount of radiochromium lost from the bloodstream, and by determining its recovery in the feces one could estimate the amount of erythrocyte destruction which could be accounted for by gastrointestinal hemorrhage. From the difference between this value and the amount lost from the circulating blood, one could obtain an estimate of the amount of red cell destruction which could not be accounted for by the gastrointestinal hemorrhage. b. Internal destruction: Extravasation of blood into tissues and trapping of red cells within thrombosed areas have long been accepted as complications of tumor growth, but comparatively little attention has been given to them as significant factors in the anemia of cancer patients. A few recent exceptions should be mentioned. Castle (1950, p. 379) in discussing anemias due to hemorrhage states that: “When the hemorrhage is internal, espe-
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269
cially when diffusely into tissues rather than into a body cavity, the effect on hemoglobin regeneration is not simply that of external blood loss, but may resemble that of increased blood destruction.” Eadie and I. W. Brown, Jr. (1953) in a general discussion of destruction due to extracorpuscular factors mentioned one factor as being hemorrhage into tumor areas in the course of malignancy. West et al. (1955) in their description of a patient considered to have a “hemolytic type of myelophthisic anemia, secondary to metastatic cancer of the breast” (see Section IV,3) reported that after treatment with cortisone, “There was marked improvement in general, particularly in regard to her hemorrhagic diathesis and in her ability to maintain red blood cell values a t a steady level without further transfusion.” Ponder and Ponder (1954) in studies on the anemia of tumor-bearing C3Hmice posed the questions: ((Isthe occurrence of the anemia associated with the appearance of the tumor, which may be white, black because of hemorrhage into its substance, or a mixture of the two, and is there evidence that the anemia is a hemolytic anemia rather than one due to hemorrhage, very considerable quantities of blood being found in the large black tumors?” He goes on to describe the mammary tumors: “The appearance of the tumors, whether white or black (because of extravasated and hemolyzed blood) is not related to their sizes; small black tumors and large white tumors are found with equal frequency. Sections of the white tumors show that they are composed of adenocarcinoma tissue, with a tendency to necrosis a t the center of the tumor; sections of the black tumors show that the black color is due to hemorrhage into necrotic regions, together with a hemolysis of the extravasated red cells to form pools of laked blood. This laked material is strongly hemolytic (Ponder and Nesmith, 1952) and since hemolytic materials can be isolated even from white tumors, it is probable that the sequence of events in the formation of a black tumor is necrosis, extravasation of blood, and laking of the red cells by the lysins which are present locally.” “In about 30% of the tumor-bearing mice the skin and subcutaneous tissues have a yellow appearance which is seen as the tumors are dissected out. This yellow appearance, which is presumably related to red cell breakdown, is as commonly seen when the tumor is white as when it is black. I t is therefore likely to be the result of a generalized blood destruction (a hemolytic anemia), for in a t least half of the cases it cannot be explained as a result of the absorption of pigments from the extravasated blood in the tumors.” In the spleens of their tumor-bearing mice, Ponder and Ponder found extensive extramedullary hematopoiesis and also hemosiderosis of the pulp. The hemosiderin granules were localized almost exclusively in macrophages. Thus, Ponder and Ponder gave an excellent description of hemorrhage
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VINCEKT E. PIlICE ,4ND ROBERT E. GREENFIELD
into necrotic areas of a tumor with hemolysis of extravasated red cells. They were not able to evaluate quantitatively the importance of the hemorrhagic process in the tumor on the anemia of the host. For this reason they turned again to search for defects in the erythrocytes as being the cause of the anemia (Ponder and Ponder, 1954). The interest of the reviewers in the problem of anemia in cancer was aroused when, in the course of radioisotope experiments on rats bearing the Dunning lymphosarcoma, it was found that the tumors contained a large amount of iron which could not be accounted for by the iron of the circulating blood (Price and Greenfield, 1955). The correction for the iron of the circulating blood was made because the great difference in vascularity of various tumors could of itself account for marked differences in their iron content. The correction was made by injecting cells labeled with C P into the blood stream shortly before the animal was sacrificed. By determining (1) the ratio of iron to CrS1in the circulating blood, and (2) the amount of CrS1in the various tissues, it was possible to determine the amount of iron in each tissue which was left in the tissue from the circulating blood. By subtracting this value from the total iron of the tissue i t was possible to determine the amount of noncirculating iron. It will be seen in Table IV that the tumors have accumulated a very large amount of TABLE IV Distribut,ionof Iron in Normal and Tumor-Bearing Animals Animal
Normal. Tumor-bearing6 a
Liver
Tnmor
494 454
724
-
Blood
2570 1930
Average body weight 243 g. Average body weight 232 g.: average tumor weight 29.0 g.
noncirculating iron, even greater in total quantity than the iron of the liver, which is the major iron storage organ of the body. It will also be seen that the accumulation of iron in the tumor is largely balanced by a decreased amount of hemoglobin iron in the circulating blood. The question immediately arose as to the relationship of the iron in the tumor to the anemia of the host. I n order to explore this problem, red cells of normal animals which had been labeled with radioiron were injected into rats bearing a variety of tumors. The concentration of labeled cells in the circulating blood was followed through samples withdrawn from the tail. Three typical experiments are illustrated in Fig. 15. Each curve is the average of 2 or 3 animals. With
27 1
ANEMIA IN CANCER
lymphosarcoma R2788, the labeled cells were injected 16 days after tumor implantation, whereas with the Yoshida ascites tumor and the iutraperitoneal Morris hepatoma No. 3683 the labeled cells were injected only 1 day following tumor implantation. During the early stages of tumor growth, little change in the concentration of labeled cells was observed. As the tumor grew larger, however, a pronounced progressive fall in the concentration of labeled cells occurred. DAYS 16
18
19
FOLLOWING TUMOR IMPLANTATION 20
21
22
YOSHIDA
A S C I T E S TUMOR
@
3 0
\
0
\
\
\ ' A
2 HEPATOMA
3683,
I P
\
\
' . ,
FIG. 15. Survival of erythrocytes tagged with Fe'g after transfusion into normal rats, 0 , and tumor-bearing rats, A. Each symbol represents the average value for a group of 3 animals.
At the end of the experiment, cells labeled with radiochromium were injected in order to obtain the filial blood volume by use of a differential counting technique for FeK9and Cr5', and also to serve as a label of the circulating blood remaining in each organ after sacrifice. The distribution of radioiron in the blood, tumor, liver, and spleen of animals bearing these three tumors is shown in Table V. It will be seen that a fairly large fraction of the radioiron which has disappeared from the blood stream was recovered in the tumor (Greenfield and Price, 1956).
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VINCENT E. PRICE A N D ROBERT E. GREENFIELD
TABLE V Distribution of Fe60 Countsa in Tumor-Bearing Rats Following Injection of Labeled Red Cells Animal Tumor
Mam X
AC
&o
X Ac
M6?0
Heptoma 3683, I.P.
Lymphosarcoma R 2788, S.C.
Yoshida ascites
N
T
N
T
N
T
Blood Tumor Liver Spleen
90. 2 1.6 -
67.0 17.3 4.5 -
84.6 1.2 6.3
58.4 21.4 5.4 4.4
89.7 4.8 -
50.3 23.8 14.3 -
Recovery
91. 8
88.8
92.1
89.6
94.5
88.4
-
In terma of par cant of injected radioactivity
The liver had only a moderate elevation in radioactivity. The spleen of the animal bearing the Morris 3683 hepatoma had a lower radioiron content than did that of the normal control. Similar studies were carried out on the Morris 2226 Harderian gland carcinoma, the Morris 4956 fibrosarcoma, the Heston 8971 mouse fibrosarcoma, the Essner 134 ascites tumor, and on spontaneous mammary tumors of CJI mice obtained from Dr. Andervont. Among the various tumors there was a great variation in the time at which the labeled red cells began to disappear from the circulation and also in the rate a t which they disappeared. In animals where the loss of red cells was later and small in magnitude, the tumors grew much larger, cachexia appeared late, and the animals lived for a longer period of time. Even in these cases, a major share of the radioiron missing from the blood stream was recovered in the noncirculating radioiron of the tumor (Greenfield and Price, 1958). The above studies were carried out on pools of several animals to lessen the problem of biological variation. Red cell survival studies on individual animals, shown in Fig. 16, demonstrate a marked variation in the response of the individual animals as to the time of onset and the magnitude of the destructive process. In 2 of the tumor-bearing animals, an initial period of destruction is observed, followed by a period of normal survival, and then a second period of increased destruction. This irregularity is similar to the findings in cancer patients reported by Bottner and Schlegel (1952), Renfer (1955), and Ross and Miller (1956) (see Section IV,3). The above experiments showed that the anemia of tumor-bearing animals could a t least in part be accounted for by the loss of hemoglobin iron into the area of the tumor. Three possibilities presented themselves: First,
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ANEMI.2 IN CANCER
that the trimor destroyed its own vascular system causing hemorrhages ant1 thromboses of t,he vessels of t,hr tumor; secoiidly, that the tumor somehow caused hemolysis tlo o(:cur within the circulating blood, releasing hemoglobiii which might then be rcnioved from the blood stream by the rapidly growing tumor to be utilized as a source of nitrogen, and thirdly, the least likely possibility, that the tumor damaged erythrocytes leading to their phagocytosis by the spleen and reticulo-endothelial system and that for some reason a large fraction of the released radioiron was taken up from the plasma by the tumor.
I [
I
0
I
5
I
10
I
15
I 20
25
I
DAYS
FIG. 16. Variation of time and rate of destruction of Fe69-labeled erythrocytes in individual rats bearing the R 2788 lymphosarcoma. 0 normal rats, 0 tumor-bearing rats.
It seemed possible that a distinction between these possibilities might be made by tagging the globin moiety of hemoglobin with radiochromium since the chromium might be deposited at the site of red cell destruction. Therefore red cells labeled with Cr6' were injected into animals bearing the Dunning lymphosarcoma, and the animals were followed until a considerable fraction of the labeled cells had disappeared from the circulating blood. Erythrocytes labeled with Fe69were then injected to label the circulating blood, shortly before the animals were sacrificed. The distribution of CrS1in the blood, tumor liver, and spleen are shown in the first column of figures of Table VI. It will be seen that a large fraction of the radiochromium which had disappeared from the blood stream had been deposited in the tumor. Only a small amount was in the liver and spleen (Greenfield and Price, 1956). This experiment demonstrated that the chromium of labeled red cells was deposited in the tumor and gave presumptive evidence that the globiri
274
VINC!ENT E. PRICE AND ROBERT E. GREENFIELD
TABLE VI Fate of Cr61 in Tumor-Bearing Rats4
Tissue
Intact erythrocytes
Lysed erythrocytes
Blood Tumor Spleen Liver Urine Other tissues
67 26 2 2
2 4 6
Total recovery
97
4
29
37 19 97
In terms of per cent of injected rndioactivity.
moiety of hemoglobin was entering the tumor along with the iron moiety. This made even more unlikely the possibility that damaged erythrocytes had been phagocytized by the spleen and reticuloendothelial system and that the iron released had been taken up by the tumor from the plasma. It did not distinguish between the two possibilities as to whether the labeled hemoglobin entered the tumor in intact red cells or whether it was cleared by the tumor from the blood stream after being released from red cells by a hemolytic process. In order to determine which of these two processes was occurring, chromate-labeled red cells were hemolyzed and tracer amounts were injected into the blood stream over a period of several hours. Several injections were made over a period of days in order to simulate as closely as possible the prolonged passage of hemoglobin into the tumor. The chromium-labeled hemoglobin disappeared from the blood stream rapidly, with a half-time of about 2 hours. Eighteen hours after the last injection, the animal was sacrificed and the tissues, blood, urine, and feces were examined for radiochromium. The level of radiochromium in the circulating blood was so low that use of a second isotope t o tag the circulating blood was unnecessary. The data obtained are shown in the last column of Table VI. It will be seen that by far the largest amounts of isotope were in the liver and in the urine and very little was in the tumor. This shows that when chromate-labeled hemoglobin is slowly injected, the CrS1does not go to the tumor but, instead, is deposited in the liver or excreted into the urine. This is in marked contrast to the fate of hemoglobin when injected in intact cells, where most of the radiochromium which had disappeared from the blood stream was recovered in the tumor, and only a small amount was in the liver and spleen (Greenfield and Price, 1956, 1958). In order to directly compare the fate of radioiron and radiochromiumlabeled erythrocytes, cells tagged separately by the two techniques were
275
ANEMIA IN CANCEIt
mixed and injected into normal rats. Dunning lymphosarcoma was then implanted subcutaneously into half of the animals. I n Fig. 17 will be seen the concentrations of hemoglobin, FeK9,and (21.6’ in the circulation of the normal and tumor-bearing animals. I n this experiment the hemoglobin level is a measure of the host’s own cells; the radioiron is a measure of young newly-formed cells, and the radiochromium is a measure of transfused cells of all ages. It will be seen that in the tumor-bearing rats the three curves fall away simultaneously from the control curves on about the 8th day following tumor implantation. The curves appear to describe the same process although the hemoglobin curve does level off on about the 13th day because of increased blood formation.
loo
50
t
t
1 0
5
10
15
DAYS
PIC. 17. Survival of Fe3g and CW-tagged erythrocytes transfused simultaneously into rats bearing the R 2788 Iymphosarcoma. Solid symbols represcnt normal animals; open symbols represent tumor-bearing animals.
A second goal of this experiment was to compare the fate of the radioiron- and radiochromium-labeled cells within the tumor. On the 17th day, the animals were sacrificed and the tumors removed and sectioned grossly into areas of white viable tumor, areas of hemorrhage, and areas of necrosis. The results of this study are shown in Table VII. It will be seen that the concentrations of both FeS9and CrS1are lowest in areas of viable, nonhemorrhagic tumor, highest in areas that are markedly hemorrhagic, and intermediate in areas of moderate hemorrhage and necrosis. As the FeK9and
276
VINCENT E. PRICE A N D ROBERT E. GREENFIELD
TABLE VII Distribution of lW and Cr61 from Tagged Red Cells Transfwed Simultaneously into Animals Bearing Lymphosarcoma R 2788
~~~
Viable, nonhemorrhagic Moderately hcmorrhagic Markedly hemorrhagic Nccrotic
~
18 63 113 74
3.6 22 31 24
33 92 159 147
20
69 125 82
Cr51have approximately the same distribution patterns, it provides further evidence that the iron- and radiochromium-tagged protein moiety of hemoglobin were deposited together in the tumor by the same process. From the concentration of radioiron in the various areas of the tumor and from the average ratio of radioiron to total iron in the blood during the period of study, a calculation can be made as to the amount of iron which had entered the tumor as red cells. The calculated values, in the last column of Table VII, indicate that iron from red cells accounts for a high proportion of the total iron in the tumor. Studies on the pathology of these tumors have been carried out in collaboration with Dr. Ross McCardle. The Dunning lymphosarcoma showed areas of hemorrhage restricted for the most part to the boundary between the capsule and the tumor and to the connective tissue trabeculae between the tumor nodules. On microscopic examination, numerous macrophages loaded with hemosiderin were seen in the areas surrounding the hemorrhage and moving away from the hemorrhagic area. On microincineration of adjacent sections, the iron of the macrophages appeared as small orange red masses of ferric oxide in the capsule and trabeculae. Almost no iron was in the actual tumor nodules or even in the areas of hemorrhage. This was not unexpected since in the areas of hemorrhage the hemoglobin had a low iron content of only 0.33'%, whereas in the macrophages hemosiderin had a very high iron content of 20-30%. Because of this, large amounts of iron could be accumulated in macrophages in and around the tumor in the presence of relatively small amounts of visible hemorrhage. I n the process of removing the tumor from the animal, many iron-laden macrophages had been left behind in the yellowish mucoid material which lined the connective tissue bed surrounding the tumor (cf. Ponder and Ponder, 1954). Undoubtedly in the earlier experiments on the fate of erythrocytes labeled with radioiron, much of the isotopic iron which had been
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277
deposited in the areas of the tumor was i l l these pcripheral, iron-ledcn macrophages which were slowly moving out, from the area of the tumor. If this radioiron could have been included with that of the tumor, the yield of radioiron which had been deposited in the area of the tumor would have been even higher. Although in lymphosarcoma R 2788 the vascular lesions were primarily hemorrhagic in nature, other tumors had different patterns of vascular damage. For example, the spontaneous C3H mammary tumor had many large sinusoids, many of which were thrombosed, leading to necrosis in the area. I n this situation, relatively little blood escaped by hemorrhage, and the fall in concentration of tagged cells within the circulating blood was not so much from their loss from the vascular tree as it was from a marked expansion of the blood volume within the growing tumor, which served to dilute out the coilcentration of tagged erythrocytes. In these animals, anemia, cachexia, and death occurred relatively late. Because of the vascularity of these tumors the amount of lioncirculating iron remaining in the hemoglobin of the thrombosed vessels was quite large in advanced stages of tumor growth. I n other tumors such as the Morris 2226 Harderian gland carcinoma and the 3683 hepatoma, the more familiar pattern of central thrombosis and necrosis seen in many transplanted tumors was found. The necrotic material frequently contains considerable amounts of extravasated blood in varying states of degradation, for although the capillary and venous beds of a tumor are often damaged, the arterioles generally remain quite free from injury (cf. Willis, 1952) and may permit extravasation of considerable amounts of blood into the necrotic area. Although the rate of such processes may be slow, over a period of time the amount of hemoglobin iron deposited in a tumor by this route may be quite considerable. In these tumors, ulceration of the skin leading to infection and loss of blood by external hemorrhage are also undoubtedly importaiit factors in the aneniia and cachexia which ultimately lead to the death of the animal. In the Morris 4956 fibrosarcoma, the tumor consists largely of connective tissue elements and extracellular fluid so that only a few very small vessels are seen. Here, although central necrosis may occur, almost no iron is lost by hemorrhage or thrombosis, and the tumor becomes massive, frequently ranging from one-third to two-thirds of the total body weight. Symptoms of cachexia appear very late. Here again ulceration of the skin leading to infection and loss of blood by external hemorrhage ultimately causes anemia, cachexia, and death in the very late stages of tumor growth. Ultmann et al. (1956) have reported a similar lack of abnormal erythrocyte destruction in rabbits bearing the Brown-Pearce carcinoma. I t is apparent that the pattern of vascular involvement varies greatly
278
VINCEXT E. PRICE A N D ROBERT E. GREENFIELD
from tumor to tumor. In lymphosarcoma R 2788 where hemorrhage of blood into tissues surrounding the tumor nodules is an important phenomenon, relatively little necrosis is seen. Therefore macrophages move into the edge of the area of hemorrhage, rapidly destroy the extravasated blood, store the iron, and presumably degrade the protein moiety to amino acids which are then returned to the body pools via adjacent functioning capillaries. Because of this process, relatively little hemorrhage is seen although a large amount of iron has been deposited in the area of the tumor. On the other hand, in those tumors where thrombosis and necrosis predominate, the “hemorrhagic appearance” varies greatly depending on the vascularity of the tumor and on the amount of blood which is extravasated into the necrotic area. Although many of these tumors may appear more “hemorrhagic,” the amount of red cells destroyed may be no more or even less than in a non-necrotic hemorrhagic tumor, such as lymphosarcoma R 2788, since hemoglobin is much less rapidly removed from the necrotic area. 5. Discussion
Is there evidence that vascular lesions such as those described in rats play an important role in the erythrocyte destruction found in cancer patients? Unfortunately, here too, direct quantitative evidence in the human patient is lacking. Furthermore, it appears that such evidence will be very difficult to obtain. Since man is about 200 times as large as a rat, the tumor must become much larger before there will be sufficient red cell destruction to produce significant changes in the circulating blood of the cancer patient. As the tumor grows larger there is more time for metastases to develop, thus making quantitative recovery of the tumor difficult. Metastases to vital organs may also greatly complicate the clinical condition of the patient. The longer period of tumor growth will also greatly lengthen the time available for the movement of iron-laden macrophages from the area of the tumor. The fact that man is the host adds still further to the complexity of the problem in the human patient. The optimal conditions for carrying out a quantitative study are obtained with a localized circumscribed tumor, and with man, of course, this is just the type of tumor which demands treatment without delay. The final problem in a truly quantitative study is recovery of the isotope in the tissues after a suitable period of time in order to terminate the experiment. With man this becomes a very difficult problem, which was of course very simple in the rat. I n view of the difficulty of obtaining adequate quantitative data in the human, it seems that initial efforts will have to be directed toward qualitative and semiquantitative techniques if one is to see if findings in the
A N E M I A IN C A S C E R
279
human are a t least consistelit with those processes found in animals. From the experiments in the tumor-bearing rat, and from the studies of Jandl et al. (1956), it is possible to outline an approach which may help clarify the problem in the human. One might, for example, take a patient with cancer and a marked anemia, but no signs of overt hemorrhage or infection, and label his erythrocytes with radiochromate. If the level of radiochromate falls quite rapidly, it will indicate a rapid rate of erythrocyte destruction and show that this patient is suitable for further study. If circulating erythrocytes are being destroyed by lysis, one would expect most of the chromium to be deposited in the liver or excreted in the urine. If defective circulating erythrocytes are being removed by phagocytosis, one would expect a large fraction of the radiochromium to be deposited in the spleen, as demonstrated by Jandl et al. (1956). On the other hand, if the erythrocytes are being destroyed as the result of a vascular defect, one would expect the radiochromium to be deposited in the area of the tumor. To carry out these studies it would, of course, be valuable to label the circulating blood with a second isotope before removing the tissues to be examined. On the other hand if the erythrocyte destruction is very rapid, one could simply wait until most of the labeled cells had left the blood stream and then search for the location of the radiochromium. A second type of experiment which could readily be extended to cancer patients would be initiated by labeling the patient’s blood with radiochromium shortly before surgery or death and determining the ratio of radiochromium t o hemoglobin in the circulating blood. From this ratio, and from the concentration of radiochromium in various areas of the tumor, one could calculate a value which would represent the amount of hemoglobin iron remaining in the area which had been in the blood stream at the time the circulation ceased. By subtracting this value from the total iron content of the tissue, one could obtain a measure of noncirculating iron which was in the area of the tumor a t the time of injection of the labeled cells. With either of the above approaches, certain problems will be encountered. If the tissues are obtained a t surgery the timing will be easier, but getting adequate specimens of normal tissue will be much more difficult. At autopsy, obtaining tissues will be easier, but the timing may be more difficult. The chromate-labeled cells must be injected soon enough before death to get adequate equilibration iu d l parts of the circulation but if death should then be prolonged by more than a fern days, errors may arise from: (1) ii change of the ratio of radiochroiniuni to hemoglobin iron in the circulating blood; (2) deposition of chrornate-ltibeled cells within the tumor, and (3) movement of some of the noncirculating iron away from the area of the tumor. The extent of these errors will vary greatly from case to case.
280
VINCENT E. PRICE AND ROBERT E. GREENFIELD
If the delay is not too great, the exact figures may be altered, but the general picture of iron deposition within the tumor could still be observed. The data obtained by either of the above techniques should be correlated with the pathology of the tumor, and here microincineration would be a valuable adjunct. I t is particularly important that in selecting tumor areas for study that the pieces of tissue be removed from areas of hemorrhage and of necrosis as well as from areas of good viable tumor. Since the primary goal of the hospital pathologist is to obtain an accurate diagnosis of the type of tumor involved, the natural tendency is to select areas of white, firm, viable tumor upon which an accurate diagnosis can be made rather than to select less satisfactory areas from the diagnostic standpoint. Such a study will therefore require close cooperation with the pathologist. It should be emphasized that techniques such as the above are only semiquantitative and would only indicate whether the processes found in the cancer patient are similar to those in tumor-bearing animals. A truly quantitative study would require measurement of the amount of tagged erythrocytes which has left the circulating blood and recovery of the missing radioisotope from all areas of the body. This would be very difficult in most cases of advanced cancer. In the absence of available quantitative data in human cancer patients, we can only examine those points which may support or oppose a vascular mechanism of erythrocyte destruction. What do we know about the destructive process: (1) In most patients the process is characterized by an abnormally rapid disappearance of tagged erythrocytes from the circulating blood, accompanied frequently by an elevated fecal urobilinogen and a reticulocytosis. These are signs of a destructive process, but they do not give evidence as to the mechanism involved. (2) Defective erythrocytes and evidences of an “overt hemolytic phenomenon” are found in only a few of the patients involved. This tends to oppose but does not rule out a mechanism involving defective or injured erythrocytes. (3) External hemorrhage or hemorrhage into the gastrointestinal tract is frequently found in patients with anemia. This emphasizes the frequency of vascular injury in cancer patients. (4) Internal hemorrhage and thrombosis are well known in the pathology of tumors. This shows that vascular lesions do occur, but it does not give evidence as to the magnitude of erythrocyte destruction which may result from such lesions. (5) It is known that tumors metastasize frequently by the invasion of veins. Willis (1952) emphasizes that such invasion is not peculiar to sarcomas but is very commori in carcinomas also. At the site of initial invasioii of a blood vessel and a t each of the sites of metastatic implantation, one would expect hemorrhage to occur. Bottrier and Schlegel (1952), Renfer (1955), and IZoss atid Miller (1956) observed t i general correlation between the onset of the anemia and the spread of the
ANEMIA IN C A S C E R
28 1
malignant process. This might be expected if vascular lesions arising in the course of the metastatic. spread of thc iieoplasm play an important role in erythrocyte destruction. Although the vascular lesions caused by metastases may be an important factor in the anemia of cancer patients, the growth of the metastatic implants in vital centers such as the lungs, liver, adrenals, and central nervous system may in many cases cause clinical complications which would overshadow the importance of the anemia in bringing on cachexia and death of the host. (6) Several investigators have remarked about the irregular character of the erythrocyte destruction observed in their life-span studies (Bottner and Schlegel, 1952; Renfer, 1955; Miller et al., 1956). This was also observed in our studies with individual animals. It seems probable that greater irregularity would be seen in small animals, such as rats, than in man, because in rats a single lesion might produce a rather profound loss of blood, whereas in man the lesion would either have to be much larger or there would have to be many more lesions. With more lesions, one would expect a less irregular pattern of red cell destruction since it is probable that they would not occur a t the same time. Therefore, when a n irregular pattern is seen in humans it would be indicative of a rather large hemorrhagic lesion a t some site around the tumor and in tumors which are more widely metastasized, and have many foci, one would expect a more constant rate of erythrocyte destruction. An example of the latter would be seen in the hemorrhagic diathesis observed in some patients with leukemia. In any case one would expect the anemia to become more severe as the tumor grows larger and inflicts more serious damage on the vascular system. Thus in early stages of tumor growth, little or no anemia would be observed since the host would be able to compensate for the rather minor losses of blood which were occurring a t intervals. Only with advanced growth and spread of the tumor would one expect the rate of erythrocyte destruction to exceed the capacity for red cell formation. It can be seen from the above that there is an abundance of knowledge about vascular lesions resulting from neoplastic growth. There is as yet, however, no quantitative data in the human patient as to the amount of erythrocyte destruction which may be caused by these lesions. Studies in the rat show that even small hemorrhagic lesions may cause rather large amounts of erythrocyte destruction. The problem is to determine the importance of similar phenomena in the cancer patient. Although a vascular mechanism of erythrocyte destruction could explain many of the findings in the cancer patient, there are two findings which cannot be adequately explained from our existing knowledge of the processes involved.
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(1) The first problem is the lack of markedly stimulated erythropoiesis in most cancer patients. While erythropoiesis is occasionally stimulated or depressed, the level on the whole is within normal limits. Several possibilities must be considered in this regard. The first possibility is that destruction of the bone marrow reduces the capacity for an elevated erythropoiesis in many cancer patients. In other words, while myelophthisis does not completely eliminate the bone marrow, it may seriously compress its available space so that even with extramedullary centers functioning, an elevated rate of erythropoiesis cannot be maintained. Although myelophthisis may be a factor in the lack of compensatory erythropoiesis, it must be considered to be only a contributing factor rather than a primary factor in the anemia of most cancer patients since most of the patients are able to incorporate iron into erythrocytes a t normal rates, and only a few have markedly decreased rates of incorporation. One complicating factor merits discussion here because of its possible relationship to the vascular lesions of tumors. It will be recalled from the introduction that Hirschfeld (1906) in an autopsy study of a patient with leukemia observed marked hemorrhage in the bone marrow and an abundance of connective tissue which filled whole areas of the marrow. He attributed this to a leukemic toxin and felt that it might be the main cause of myelophthistic anemia. In 1935, Jaff6 in postmortem studies on patients with acute leukemia gave descriptions such as the following, in a Negro boy of 4 yr., “The bone marrow (femur, sternum) was extremely congested with blood. There were only a few fat cells and the majority of the sinusoids and capillaries were collapsed, the blood cells being located outside the preformed blood spaces.’’ In a similar examination in a Negro boy of 11 yr., Jaff6 states: “The bone marrow was extremely hemorrhagic, looking like a lake of blood crossed by an occasional compressed capillary.” I n view of these descriptions one wonders if frequently hemorrhage into the bone marrow followed by its organization with fibrous tissue may not destroy more of the marrow in many cases than the tumor itself. West et al. (1955) did marrow aspirations on 14 patients with breast cancer and found suitable marrow for differential cell counting in only 5 cases in spite of repeated attempts in each unsuccessful case. If areas of the marrow were filled with fibrous tissue, one would expect difficulty in obtaining suitable specimens of marrow, and the so-called “dry tap” becomes significant. On the other hand, other hematologists feel that there is insufficient myelofibrosis in cancer patients to account for lack of compensatory erythropoiesis. They suggest a mechanism involving some sort of marrow inhibition, such as that described by Dameshek in the syndrome of “hypersplenism,” with which the anemia of leukemia has frequently been
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classified. It is unfortunate that more adequate quantitative techniques for measuring the effective volume of hematopoietic tissue within the body are not available. Until some such solution is a t our disposal, the significance of these destructive processes in the marrow on the erythropoietic potential of the cancer patient cannot be adequately evaluated. A second possible reason for the lack of compensatory erythropoiesis may be that the continual deposition of hemoglobin iron in the area of the tumor may create a state of iron deficiency within the cancer patient. The low serum iron found in most cancer patients suggests that the supply of available iron may be under some stress (cf. Finch et al., 1950). On the other hand, Shen and Homburger (1951) found the hemoglobin content of erythrocytes to be low in only 13 out of 65 cancer patients who had an anemia but no external blood loss. It is possible that in the condition of chronic blood destruction found in most cancer patients enough iron is moving back from old areas of hemorrhage that with the iron being absorbed from the gastrointestinal tract, the iron being made available from wastage of muscle and other tissues, and the iron stores of the liver, the body is able to nearly compensate for the iron being lost into new areas of hemorrhage and thrombosis. This suggests that erythrocyte destruction may have to be very acute and severe before a hypochromic anemia will be observed, and it indicates that iron deficiency of itself may not be a n adequate explanation for the lack of elevated erythropoiesis in most cancer patients. It is possible, however, that the utilization of iron for hemoglobin synthesis may be a t the expense of other tissues, and in this way lead to the wastage of muscle and to cachexia in the cancer patient. Whipple (1948, pp. 14-15) has shown that €he utilization of iron for the synthesis of hemoglobin has first priority and that in iron deficiency the hemoglobin level will be maintained at the expense of other body tissues. I n our tumor-bearing animals, anemia appeared to be a major factor in the onset of weight loss and cachexia, whereas deposition of nitrogen in the tumor was a relatively unimportant factor. For instance, animals bearing the Morris 4956 fibrosarcoma, with no signs of blood destruction, could grow large tumors of 30% of the total body weight with relatively little weight loss and cachexia until finally ulceration of the skin led to external blood loss and infection, which then precipitated a rather rapid downhill course. A third possibility for the lack of compensatory erythropoiesis may be that under prolonged and chronic conditions of elevated blood destruction, the body may not be able to maintain a continually elevated level of erythrocyte production. An evaluation of this possibility must await future investigation. (2) The second problem in cancer patients which is difficult to explain
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by a vascular mechanism of erythrocyte destruction is the finding of spherocytosis, an increased red cell fragility, or a positive Coombs test, which indicate the presence of injured circulating erythrocytes. As stated above, these findings are frequently observed in chronic lymphatic leukemia and in patients with ovarian tumors, but they are uncommon or rare in other cancer patients. If subsequent studies on human patients support a vascular mechanism of erythrocyte destruction, these findings will require explanation. One cannot help but wonder if substances released during the clotting mechanism may not have an injurious effect on red cells although there is no satisfactory evidence for this at the present time. It is apparent from the above discussion that we still do not have an adequate understanding of the sequence of events which lead to anemia in the cancer patient. It is hoped that a reorientation of our approach to give more consideration t o the significance of vascular mechanisms of erythrocyte destruction may lead to more rapid progress in this field. ACKNOWLEDGMENTS We would like to express our appreciation to Dr. Jesse P. Greenstein for his encouragement, cooperation, and criticisms in the preparation of this manuscript.
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Verloop, M. C. 1955. Acta Med. Scand. 161, 367-380. von Kress, H. 1934. Deut. Arch. klin. Med. 176, 359-390. Wagley, P. F., Shen, S. C., Gardner, F. H., and Castle, W. B. 1948. J . Lab. Clin. Med. 38, 1197-1203. Waaserman, L. R., Rashkoff, I. A., Leavitt, D., Mayer, J., and Port, S. 1952.J . Clin. Invest. 81, 32-39. Wasserman, L. R., Stats, D., Schwarta, L., and Fudenberg, H. 1955. Am. J . Med. 18, 961-989. Watson, C. J. 1938. “Downey’s Handbook of Hematology,” Vol. 4,255&2554. Hoeber, New York. Weil, R. 1907. J . Med. Research 16, 287-306. Weinstein, I. M., and LeRoy, G. V. 1953.J . Lab. Clin. Med. 43, 368-376. Welham, W. C., and Behnke, A. R. 1942.J . Am. Med. Assoc. 118, 498-501. West, C. D.,Ley, A. B., and Pearson, 0. H. 1955. Am. J . filed. 18, 923-931. West-Watson, W. N.,and Young, C. J. 1938.Brit. Med. J . 1, 1305-1308. Whipple, G. H. 1948. “Hemoglobin, Plasma Protein, and Cell Protein.” C. C. Thomas, Springfield, Illinois. Whipple, G. H., and Robscheit-Robbins, F. S. 1933. J . Expll. Med. 67, 671-687. Wiener, A. S. 1934. J . Am. Med. Assoc. 103, 1779. Willis, R. A. 1952. “The Spread of Tumors in the Human Body.” Mosby, St. Louis, Missouri. Wintrobe, M. M. 1946. “Clinical Hematology,” 2nd ed. Lea & Febinger, Philadelphia. Wintrobe, M. M. 1956. “Clinical Hematology,” 4th ed. Lea & Febinger, Philadelphia. Wittenberg, J., and Shemin, D. 1950.J . Biol. Chem. 186, 103-116. Young, L. E.,Pflatzer, R. F., and R d e r t y , J. A. 1947. J . Lab. Clin. Med. 83, 489-501. Zamcheck, N., Grable, E., Ley, A., and Norman, L., 1955. New Engl. J . Med. 363, 1 103-1110.
SPECIFIC TUMOR ANTIGENS L. A. Zilber N. F.
Gomoleyo Institute of Epidemiology and Microbiology, Moscow, U.S.S.R.
Page
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . 291 11. Antigens of Filterable Tumors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 111. Antigens of Nonfilterable Tumors. . . . . . ....................... 296 IV. Antigens of Human Tumors.. . . . . . . . . . . . . . . . . . . . . . . . . . . 300 V. Detection of Specific Tumor Antigens with thc Help of Anaphylaxis Following Desensitization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. The Reaction of Passive Anaphylaxis. . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Localization of the Specific Antigen in Tumor Cells . . . . . . . . . 312 VIII. Time of Appearance in the Cell of the Specific Antigen.. . . . . . . . . . . . . . . . . . 314 IX. The Nature of the Specific Tumor Anti ific Tumor Antigen., . , , , , , , , , . . 319 X. Adsorption upon the Red Cells of t XI. Discussion. . . . . . . . . . . . . . . . . . . . . . ............................. 321 XII. Conclusion. . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . ................................... 325
I. INTRODUCTION The tumor cell, just like any normal one, contains various kinds of antigens such as species- and organ-specific antigens, blood group substances and histocompatibility factors, and heterophile antigens of the Forssman type. However, the present publication deals principally with a different question, via. whether the tumors do contain any antigens which are absent in the tissues of the normal organism. The peculiarities of the antigenic structure of tumor and normal cells as revealed by immunological methods will also be discussed. These problems have been studied by numerous investigators, but until recently the data available did not warrant any definite conclusions (cf. Haushka, 1952). In some respects the problem is still disputable. However, substantial evidence has recently been provided to show the antigenic differences between the tumor and normal cell. The causes underlying these differences may be quite different. Filterable tumors contain certain viruses which act as the etiological factor of the t,umors; these viruses are antigens which are absent in normal cells. Tumors may also contain some foreign viruses as well as bacteria without etiological relationship to cancer (Taylor and MacDowell, 1949; Law 291
292
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and Dunn, 1951). In tumors traiisplanted from one aninial to another, there may occur isoantigens in consequence of genetic differences between hosts and grafts. Presumably autoantigens may also arise in the tumors. Viruses and carcinogenic substances may disturb protein synthesis in t h e affected cells so that proteins may be formed with a different structure and composition from normal cells. At the present time, some diseases are known which are caused by autoimmunization (cf. Vorlaender, 1954; Dausset, 1954; Cavelti, 1955). Finally, the metabolic disturbances associated with the carcinogenic process may result in the disappearance of some antigens from normal cells, a peculiar simplification of their antigenic composition. Different combinations of these factors are possible so that analysis of the antigenic distinctions of the tumor cell may frequently involve certain difficulties. I n view of the peculiarities of the antigenic structure of tumors of different origin, it seems advisable to divide the data available on tumor antigens into two principal groups: antigens of filterable tumors and antigens of nonfilterable tumors. A special group will be formed by human tumors. A discussion of the evidence presented below warrants certain suggestions concerning pathogenesis of malignant growth.
11. ANTIGENS OF FILTERABLE TUMORS The principal antigenic distinction of the cells of filterable tumors from normal cells is due to the antigens of the virus contained in the tumor cells. The antigenic properties of the tumor viruses have been the object of rather detailed studies (cf. Oberling and GuBrin, 1954; Harris, 1953; Dmochowski, 1953) so that a discussion of the relevant data does not seem necessary. It will be noted, however, that almost every effort to isolate these viruses from the antigenic components of normal tissues of the host proved futile. Thus, Rous virus obtained by different methods in a highly purified state takes part in the precipitation and complement fixation test with sera of animals immunized with normal chick tissues (Barrett, 1941; Furth and Kabat, 1941; Dmochowski, 1948; Kabat and Furth, 1940), and it is difficult by means of these reactions to distinguish it from the antigens of normal tissues. These data indicated the presence in the Rous virus of two antigens one of which is common to the antigen of the tissue in which the virus propagates (Amies and Carr, 1939). However, rabbit antisera against chick tissue do not neutralize the virus, whereas antitumor sera do neutralize it (Barrett, 1941). Hence, it may be taken that the normal component is not a composite part of the virus but is only associated with it.
SPECIFIC TUMOIl ANTIGENS
293
This assumption is confirmed by a study of the influenza virus. Even the highly purified virus when cultivated in chick embryos or in mouse lungs contains normal chick and mouse antigens respectively (Knight, 1946; Smith et al., 1955). It hardly seems probable that such dissimilar antigens (chick and mouse) are components of the same virus. They apparently become associated with it when cultivated in the respective tissues. A study of other tumor viruses likewise reveals the close association of these viruses with the antigenic components of normal cells. Evidence has recently been produced to substantiate the assumption that the particles of the Ilous virus are agglomerates cemented by a ballast protein of high density (Gessler et al., 1956). The aggregates are destroyed by treatment with a mixture of freon and heptane which releases pure 10-20 mp virus bodies not sedimenting upon prolonged centrifugation a t 144,OOOg. The cementing protein might perhaps belong to the tissue in which the virus is reproducing, and this explains the antigenic community of the virus and tissue from which it has been isolated. It is claimed by some workers (Gorodilova and Shabad, 1946; Bennison, 1948; Imagawa et al., 1948) that Bittner virus can be distinguished from normal tissue of the mammary glands of mice by means of the precipitation reaction and the complement fixation test. No such differences have been revealed in other studies. The sera of rabbits immunized with the biologically active sediments of ultracentrifuged extracts of mammary tumors did not differentiate, in the complement fixation test, the extracts of tumors and normal tissues of genetically identical mice irrespective of the presence or absence of the virus in these extracts (Dmochowski et al., 1952). However, sera of rabbits and rats immunized with Bittner virus neutralize its biological activity while those of animals immunized with normal tissues lack this capacity (Andervont and Bryan, 1944; Green et al., 1946; Bittner and Imagawa, 1955). Bittner virus, probably more than any other, is associated with the antigenic components of normal tissues, and this greatly impedes its serological identification. The virus of the rabbit Shope papilloma is more than others free of normal tissue components so that it can readily be revealed by the Complement fixation test (Kidd, 1938a; Narcissov, 1956). I t will be noted, however, that tumor extracts containing the ROUS,Shope, and Bittner viruses can readily be differentiated by the anaphylaxis reaction following desensitization. This question will be discussed later (Radsikhovskaya, 1950; Dyadkova, 1954; Levina, 1956; Artamonova, 1956). I n spite of the varying degree of association of the tumor viruses with normal cell components, these viruses behave as antigens foreign to the organism in which they induce tumors. It is held by most workers that tumor viruses, like any other viruses, get into the cell from the outside a n d
204
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hence their antigens are of exogenic origin (cf. Zilber, 1946; Oberling and GuBrin, 1954). Mention should be made of one peculiarity of the tumor viruses which is important for the study of these viruses, viz. their capacity to be adsorbed on the red cells. This capacity has long since been mentioned by Pentimalli (1935) with regard to the Rous virus. A more detailed study of this problem has been taken up in this laboratory on Rous and Shope viruses (Zilber et al., 1953; Ludogovskaya and Morgunova, 1956). Rous virus was found to be well adsorbed when the virus-containing extract was brought into contact with the red cells of chicks, rabbits, and guinea pigs. Thoroughly washed red cells, after adsorbtion, induced a typical sarcoma when implanted to chicks. The virus could also be detected in the red cells by means of the passive agglutination reaction: rabbit erythrocytes carrying the adsorbed virus are agglutinated by sera of rabbits immunized with the chick sarcoma (Zilber el al., 1956). The serum of rabbits immunized with normal chick tissue agglutinated the virus-adsorbing red cells, but only in higher concentrations. Formol-treated virus is likewise adsorbed by erythrocytes. Inhibition of passive agglutination yielded clear-cut results in the experiments with Rous virus. Immune antitumor serum, when mixed with the tumor extract, lost its capacity to agglutinate the virus-adsorbing red cells, whereas this capacity is retained when the serum is mixed with the extract from the normal muscle. The results are summarized in Table I. With chick red cells no results have been obtained in similar experiments although they adsorbed Rous virus. In the experiments with Shope virus (Zilber el al., 1953; Artamonova, 1956), a strain adapted to the domestic rabbit was used. The papillomata of domestic rabbits contain the virus when implanted, but its concentration is rather low and the inoculation is successful with the papillomatous tissue dilution 1.100 but rarely with higher dilutions. When extracts from the papillomatous tissue were brought into contact with the red cells of rabbits and of some other animal species (guinea pigs, chicks), the virus was adsorbed by the latter. This could be demonstrated by inoculating the rabbits with thoroughly washed red cells. The papillomatous extracts could be made completely devoid of the virus by repetitive adsorption. Upon immunization of rabbits with the virus adsorbed on erythrocytes, sera have been obtained which showed the complement fixation reaction with the papillomatous extract (Narcissov et al., 1956). According to Bittner (1947), the virus of the mammary cancer of mice can be adsorbed by the red cells of noncancerous mice. This virus is concentrated in the blood cells of cancerous mice in higher concentrations than
296
SPECIFIC TUMOR ANTIGENS
TABLE I Reaction of Passive Hemagglutination and Inhibition of Passive Hemagglutination with Extracts from Chick Sarcomaa
+
Serulnl
Rabbit Erythrocytes
-
After contact with sarcoma extract
1 3 5 85 86 801 807 815 833 834 856 a
_ _
Serum
Extract
Hemagglutinating Titers ~~
:3a .4guinst surcomn 5n 59 61 194 833a 834a 801 807
b
Rabbit Ser um
NN
+
Serum Extract of Normal Muscle
Against chick muscle Nonimmunized rabbits
~~~
320 320 640 320 320 1280 640 20-4Ob
40
0-10D 0 0 0 0 0 0 0 0 0 0-1 01)
0 0 10 0 0 0-10 0-10-20 0 0-40b
16O-32O 320 320 320 320 16Ck320 160-64ob
0-40 0-40*
0 0 0 0 0 0 0 0
0-lob 0 0 0 0 0 0 0
0 0 10
0 0 0
Zilber et al. (1956). The serum titer varied from one experiment to another within the range mentioned.
in the serum (Bittner, 1945). The conclusion is thus indicated that tumor viruses are adsorbed by the red cells. It will be noted that this adsorption is not accompanied by any agglutination. Agglutinins, and eventually hemolysins, frequently occur in extracts of the Shope papilloma and of the Rous sarcoma acting upon rabbit and other red cells in low dilutions. But these are not agglutinins or hemolysins of the virus. Probably, we are dealing here with tissue products or with normal antibodies. The agglutinins can be eliminated by heating to 60"-65" and the hemolysins by adsorption on activated charcoal. The following are the conclusions from the above evidence. 1. Filterable tumors contain specific antigens which are lacking in normal tissues and represent the substance of the virus.
296
1,. A . ZILBER
2. Virus antigens of these tumors may be adsorbed upon erythrocytes of various animals modifying their serological properties. 3. Antigens of tumor viruses have components in common with the tissues in which they reproduce and identification of these viruses by serological methods is sometimes very difficult. The question of the presence in the filterable tumors of other antigens which are absent in normal tissues will be taken up below.
111. ANTIGENSOF NONFILTERABLE TUMORS Two types of such tumors should be distinguished, viz. primary tumors induced by various carcinogenic factors and treatments, and transplanted tumors. A special group is comprised of the so-called spontaneous tumors of animals and humans with a nonidentified etiology. The study of antigens of transplantable tumors of any origin involves some difficulties associated with the genotypic differences between the donor of the tumor and the recipient. Most conclusive are experiments carried out on inbred animals, although in these cases prolonged passage of the tumor might induce some modifications of its antigenic structure. I n most cases the study of the. antigens of animal tumors has been carried out by comparing antibodies of animals immunized with the tumor tissue or with its various components with those of animals immunized with the respective materials of normal tissues. In many experiments a study was also made of the antibodies of cancerous animals. Kidd (193813, 1940, 1946) has the merit of a detailed study of a special antigen in the Brown-Pearce rabbit carcinoma. Saline extracts of this tumor fixed the complement with sera of rabbits of the blue cross strain implanted with this tumor. No reaction was obtained with extracts from tissues of normal kidney, liver, spleen, and red bone marrow, or pus of various origins as well as from tissues of other rabbit tumors. I n the sera of commercial rabbits inoculated with the Brown-Pearce carcinoma, antibodies have been revealed which reacted not only with tumor extracts but with those of normal tissues as well (MacKenzie and Kidd, 1945; Friedewald and Kidd, 1945). It has been concluded that the Brown-Pearce tumor contains an antigen which is different from those of normal tissues. This is probably a ribonucleoprotein (Kidd, 1940; MacKenzie and Kidd, 1945) combined with the microsomes of the tumor cells (Kidd, 1946). Kidd’s experiments were performed on a tumor which had originated in a rabbit of another strain although transplanted to inbred rabbits. Some workers (Jacobs and Houghton, 1941; Ginsburg-Kalinina et al., 1950) failed to reproduce Kidd’s data by grafting the tumor to commercial animals but his data have been confirmed by some others (Ellerbrook et al., 1952; Lippincott et al., 1953).
MPECIFlC T U M O R ASTIGENS
297
Gardashyan (1956) used as antigen in this laboratory the protein fraction precipitated by acetic acid a t pH 4.5 from a slightly alkaline tumor extract. A positive complement fixation reaction was obtained in 23 out of 39 rabbits with the Brown-Pearce tumor. Of 107 sera of rabbits immunized with the tumor tissue, 97 gave a positive reaction. A considerable part of these sera did react, although a t a lesser titer, with the protein fraction from normal organs. However, sera of rabbits immunized with the methanol precipitate from the tumor prepared after Cox et al. (1947) reacted only with the antigen from the tumor tissue but not with that from tissues of normal organs. Thus the Brown-Pearce tumor contains an antigen which is diff ererit from those of normal rabbit tissues. Such a n antigen has also been detected by Kidd (1946) in the transplantable V:! carcinoma which resulted from malignization of the rabbit papilloma (Kidd and ROUS,1940). In Maculla’s experiments (1947, 1948), the nucleoprotein fractions prepared after Mirsky and Pollister (1942) from various transplantable inice tumors proved immunologically different from those of normal mouse organs. This difference was found in a strictly standardized test of complement fixation by sera obtained through immunizatiori of rabbits with various preparations from the tumors. Antigens of cancerous and embryonic tissues were found to be closely related. In 6 tumors, components have been revealed similar to those of embryonic liver although not all of them possessed adult liver components. Antitumor sera invariably reacting with the spleen of a n adult did not react with the embryonic spleen. Antigenic differences of the nucleoproteins of the rat sarcoma from those of the liver, spleen, and other organs have been detected by Manoilov et al. (1953). I n Rovnova’s experiments (1956), sera of rabbits immunized with the transplantable M-1 sarcoma were adsorbed by the spleen tissue stored over a month in formol. Such sera lost the antibodies against the liver and spleen extracts of those rats with whose tumor extracts they gave the complement fixation reaction. According to some observations, alcohol extracts of mouse tumors, but iiot of normal tissues, exhibit a complement fixation reaction with sera of mice in which the tumors have been grown. The reaction proved negative with control sera (Hoyle, 1940). The ether- and alcohol-soluble lipid antigen has also been revealed in other transplaiitable and some spontaneous mouse tumors (Bunting, 1943) as well as in niitochoiidria of the rat lymphosarcoma (Rapport and Graf, 1954, 1955). Aptekman et ul. (1949) succeeded in iniinuiiiziiig the King A rats with lipid extract from ~ a r c o maof the same strain of rats. An antigen of L: lipid character has been isolated by Luiid (1955) from ascitic Ehrlich mouse carcinoma and Ioshida ascitic rat sarcoina. The sera
298
L. A . ZILBER
of rabbits immunized with the antigen from cancerous cells yielded a positive complement fixation reaction with both cancerous and sarcomatous cells. Adsorption of these sera by the red cells and leucocytes did not reduce the titer of the sera. The antiserum to the sarcoma antigen did not react with the cancerous antigen. The lipid nature of the antigen detected in all these studies is by no means certain. According to Miller (1955), alcohol extracts of rat sarcoma and lymphoma contain a protein immunologically different from the protein of alcohol extracts of normal muscles of the same strain of rats. She noted a pronounced difference in the precipitation reactions of sera obtained through immunization with the protein from alcohol extracts of tumors and of normal tissues. The reacting antigen of the alcohol extracts obtained by other investigators is possibly also lipoprotein. Liver cathepsins of healthy rats and those of transplantable hepatomas have been compared by Mayer and Barrett (1943) in the reactions of precipitation, complement fixation, and anaphylaxis. It appears from their data that, although related, the cathepsins are not identical. A number of publications deal with a comparative study of the antigenic structure of leukemic and normal mouse tissues. Dulaney et al. (1949) used the complement fixation test for studying the antigenic composition of the cytoplasmic fractions (mitochondria and submicroscopic particles) of leukemic and normal tissues of inbred strain mice with the aid of sera of rabbits immunized with these fractions. In part of the experiments, the sera were exhausted by leukemic and normal mitochondrial fractions. No antigen has been detected in these experiments which would be appropriate only to the leukemic tissue although a quantitative difference was noted in the reactivity of the preparations from leukemic and normal tissues. Nor were any distinct antigenic differences noted between nuclear antigens of leukemic and normal tissues (Arnesen et al., 1949). However, sera obtained through immunization with leukemic preparations exerted a much stronger cytotoxic effect upon leukemic cells than those obtained by immunization with preparations from normal tissues (Dulaney and Arnesen, 1949). Similar relationships were reported by Nungester and Fisher (1954) for mouse lymphosarcoma. Werder et al. (1950, 1952) communicate about a distinct difference in the neutralization capacity of rabbit sera obtained through immunization with leukemic and normal mouse cells. Moreover, the specific antibodies proved more thermostable thaii the antibodies against normal cells. The precipitation reaction in the gel did not reveal any ailtigenic differences between the lymphosarcoma h u e and the normal lymphoid tissue (Korngold and Pressman, 1954a). Antigens of various animal tumors have been studied in this laboratory
SPECIFIC TT'MOIZ A N T I G E N S
299
by means of several imniunological reactions. The principal objective of these studies was t,o find out whether the tumors contain antigens which are absent in norninl tissues. At first the strictly standardized complement fixation test m m used with differelit transplantable sarcomas originally induced by 9,10-dimethyl-1,2-benzanthracerieand by other carcinogenic substances (Zilber ct al., 1948; Zilber and Narcissov, 1949; Narcissov and Zilber, 1949). As antigen, use was made of the protein fraction precipitated a t pH 4.5 from alkaline tumor extracts and the antigen dose was defined by the amount of protein determined by the micro-Kjeldahl method. Rat sera were studied prior to tumor implantation and a t varying intervals thereafter. Out of 425 sera of rats inoculated with M-1 sarcoma (Shabad's strain), positive results have been obtained 11-14 days after implantation in 21.6 to 28.50/,, after 15-19 days in 34.8 to 42.0%, and after 25 days in 77.3 t o 87.0%. A positive reaction with sera of healthy rats was noted in 1.75y0. Thus with the progress of tumor growth the percentage of positive results increased. Rats in which the tumor was removed surgically, while the reaction was markedly positive, exhibited much less frequent relapses and proved more resistant against second inoculation. Upon resorption of the tumor, the antibodies were found a t a high titer and disappeared soon after the completion of this process when pronounced immunity set in (Narcissov and Ebert, 1956). The above experiments have not been carried out on inbred animals so the results might have been due to isoantigenic differences between the cells of the tumor and of the animals to which it has been transplanted (cf. Gorer, 1937, 1938, 1942, 1956). However, this suggestion is a t variance with the experiments in which rats were immunized with the nucleoproteiri fraction from rat sarcoma. The sera of these rats reacted with the tumor antigen but not with the antigen prepared in a similar way from the liver and lungs of healthy rats. Immunization of the rat with the latter antigen did not result in the formation of antibodies either to the tumor antigen or to those of normal organs (Zilber and Narcissov, 1949). Experiments with primarily induced tumors likewise contradict the assumption that the tumor antigen detected in the transplantable rat sarcoma is an isoantigen. In these experiments (Narcissov and Abelev, 1956), rats were injected subcutaneously with 9,10-dimethyl-l12-benzanthracene or with niethylcholanthrene. Antigens prepared from induced tumors were used for the complement fixation test with sera of the same rats from which the tumors were taken. The fraction obtained upon centrifugation of the clarified tumor homogenate a t 18,000 r.p.ni. during 1 hour was used in these experiments. Along with the supernatant, the sediment containing mitochondria and microsomes and dissolved in a dilute solution of NaOH and then neutralized was used as the antigen. Of 23 rat
300
L. A . ZILRER
sera, 13 gave a distinct positive raction of complement fixation with the mitochondrial-microsome fraction from tumors of the same animals while the protein content of the antigen was 1-2 mg. per milliliter. The supernatant showed a reaction in 7 cases. These experiments have been carried out not with transplantable but with induced tumors grown from the cells of the host himself. Hence they could not contain any isoantigens. The presence of antigens in tumors not transmissible by filtrates has been confirmed by immunological tests on inbred animals. According t o Gross (1943, 1945), Stoerk and Emerson (1949), Aptekman and Lewis (1951), Lewis and Aptekman (1951, 1952), Goldfeder (1945) and Foley (1952a,b), resorption of tumors induced in certain strains of rats and mice and transplanted t o the same strains calls forth immunity to these tumors. The resorption was caused by ligating the tumors, by X-ray irradiation of the grafted tumor fragments, by treatment with A-metapterin, etc. These results have been attributed to mutation of the tumor as caused by repeated transplantations (Haushka, 1952). Foley (1953), however, observed immunity in C3H-He mice following ligation and resorption of sarcoma induced in these mice by methylcholanthrene and transplanted only once or twice. It is hardly possible to conceive of any tumor mutation under such conditions. Similar experiments with spontaneous cancer in inbred mice did not give any positive results (Fardon and Prince, 1953; Foley, 1953).
IV. THEANTIGENSOF HUMAN TUMORS The study of human tumor antigens has been carried out for the most part by comparing the capacity of sera of animals immunized with the tumors or their extracts to react. with extracts or various preparations from tumor and normal tissues. Pioneer work in this field has been done by Hirszfeld et al. (1929), Witebsky (1929, 1930), Witebsky and Morelli (1933), Lehman-Facius (1928), Morelli (1934) a.0. They showed that sera of rabbits immunized with tumor suspensions are active in the complement fixation test with alcohol tumor extracts and do not react with alcohol extracts of normal organs. However, these sera reacted with red cells and when exhausted by them ceased to react with alcohol extracts of cancerous tumors as well. Thus, antibodies of the sera at issue belonged to group-specific, but not cancer-specific, antibodies. Kobayashi (1956a,b) and Kobayashi and Kawasaki (1956) studied alcohol extracts from stomach carcinoma and from the placenta. Some differences have been found in the capacity of sera of animals immunized with these extracts and with fractions obtained therefrom to react in the precipitation test with antigens of the cancerous and normal tissue.
SPECIFIC TrMOll ASTIGENS
301
Witebsky et al. (1956) recently reported a different reaction in the coinplement fixation test of sera obtained through immunization with extracts of cancerous and normal thyroid tissue. These sera did not contain any group antibodies nor had they been freed from them through absorption upon the red cells. The normal thyroid extract reacted with the respective serum a t a much higher dilution than with the thyroid cancer antiserum whereas the tumor extract reacted with the cancer antiserum a t a much higher dilution than with the thyroid antiserum. However, the difference proved only a quantitative one. Any attempts to eliminate from the cancer antiserum the antibodies against normal antigens proved futile. Successful experiments of this kind have been reported by Saakov (1952, 1956). He adsorbed antibodies against normal antigens from cancer antisera by exhausting them in a suspension of dermatol particles previously kept in contact with saline extracts from normal tissues. Such a serum, obtained for example by immunization with a salt extract from pulmonary cancer, could not react any more with normal lung extracts but still reacted with salt extracts from pulmonary cancer and with those of the uterus and stomach cancers although the titer decreased from 1 :320 to 1 :40. Kosyakov et al. (1955) eliminated from the tumor antisera antibodies against normal antigens by adsorbing them upon spleen tissue which had been previously preserved in 5y0 formol for more than a month and then carefully rinsed in tap water. Tumor antisera, reacting in the complement fixation test with tumor extracts as well as with spleen and liver extracts, after adsorption reacted only with tumor extracts. According to V. S. Korosteleva (1956), for exhaustion of the antitumor sera normal tissues may be used preserved in 80% glycerol. The aqueous-salt extracts from formol-treated tumor tissues reacted in the complement fixation test with tumor antisera but not with those against some normal tissues. Korosteleva et al. (1956) obtained from these extracts (by acetone precipitation with subsequent hydrolysis) a protein which reacted in the complement fixation test with the tumor antisera but did not react with the hepatic and splenic antisera. The protein of this substance consisted of 15 amino acids of which 13 have been identified. No antigenic activity has been displayed by the free lipids contained in the salt extract. Manii and Welker (1940, 1943, 1946) immunized rabbits with proteins of human tumors adsorbed on aluminum cream. Toward the 6th month part of the sera of these rabbits were free of precipitins to normal serum protein but still contained those against the cancerous one. These sera proved active in high dilutions in the precipitation tests with autolysates of various tumors but did not react with the autolysates of normal organs. The authors claim that the cancerous protein differs from the normal one. This protein may presumably also be contained in the blood of cancer patients since the cancerous antiserum precipitated sera of these patients
302
L. A . ZILBER
as well. The precipitins against cancerous proteins persisted in rabbits for more than 2 months after the disappearance of the precipitins against serum proteins. Attention is also due to the data indicating the simplification of the antigenic structure of tumors. Seligman et al. (1955) compared the antigenic structure of leucoblasts and of normal human leucocytes with the aid of the precipitation reaction in the gel. They showed that leucoblasts lack one of the antigenic components invariably contained in normal leucocytes but they failed to reveal in the leucoblasts any antigens that would be absent in normal leucocytes. The simplification of the antigenic structure has also been noted in animal tumors. Microsomes and mitochondria of the rat liver cancer induced by Buttergelb lack any capacity to react with organ-specific antihepatic antibodies (Weiler, 1954, 1955, 1956a). At the same time, antigens have been discovered in the hepatoma tissue which are absent in healthy liver. But they are not to be considered as specific tumor antigens since they have been found in healthy organs as well. The organ-specific antigen disappears from the cells undergoing carcinogenesis prior to their transformation into cancerous cells. Similar data have been obtained in the cancer of hamster kidneys induced by stilbestrol (Weiler, 1956b,c). Malignization of the cells may, therefore, be accompanied by a loss of some antigens contained in normal cells. Apparently no definite conclusion is indicated by the above evidence as to the presence of specific antigens in spontaneous and nonfilterable tumors. Rather conclusive positive results have been obtained in some studies although negative results have been obtained in others. The technique adequate for the detection of antigenic peculiarities of some tumors proved inefficient when applied to others. The preparations produced in a similar way from different transplantable tumors and tested in the complement fixation reaction did not yield consistent results. Thus, out of 199 sera of rats with transplantable M-1 sarcoma induced by benzpyrene, positive results have been obtained with the antigen of this tumor 25 days after transplantation in 77.3 to 87.0%. However, similar experiments with the transplantable sarcoma 45 induced by 9,1O-dimethyl-l,2dibenzanthracene yielded positive results only in 25.2% (Narcissov and Ebert, 1956). In contrast to the spindle celled M-1 sarcoma, this one was a fibrosarcoma. No consistent results have been obtained with different tumors in a search for a distinct and constant differentiation of antigens of tumor and normal tissues by means of sera of animals immunized with various components of these tissues. It is only eventually that antisera could be obtained which reacted with extracts from cancerous but not normal tissues. Other ap-
SI'EC'IFIC' TUMOR ANTIQEXS
303
proaches had therefore to be lookrd for in studying the problem of specific ti imor antigens.
v. DETECTIONOF
SPECIFIC TUMOR ANTIGENS WITH THE HELP O F ANAPHYLAXIS FOLLOWING DESENSITIZATION
The difficulties involved in the regular detection of specific tumor antigens might have been due to the low number of these antigens among the main bulk of tumor proteins indistinguishable from the antigens of normal cells. It seemed advisable, therefore, to discover the protein fraction of the tumor cells with which the specific antigen is associated and thus obtain it in a more concentrated form. Use also had to be made of a reaction which would work with a mixture of antigens with a varying ratio of specific and nonspecific components and which was sensitive enough to reveal even insignificant amounts of the specific antigen. After many failures, it has been realized that nucleoprotein fractions of tumors contains a greater amount of one specific component than any other. Accordingly, further study has been carried out with these particular fractions. Our presumption was that nonfilterable tumors may contain masked viruses which should be isolated in the nucleoprotein fraction. Murphy et al. (1928) showed that Rous virus is linked precisely with this fraction. Similar results have been obtained by Radsikhovskaya (1950) in this laboratory: the nucleoprotein fraction of the chick sarcoma proved active in a dose of 0.005 mg. I n our first experiments, the nucleoprotein fractions were obtained with the method used by Mirsky and Pollister (1942), but the overwhelming majority of subsequent experiments have been carried out with the nucleoprotein fractions prepared by Belozersky's method (1942). From a slightly alkaline extract obtained from triturated tumors and clarified by centrifuging, a fraction precipitating a t pH 6.0 has been removed. Weak acetic acid was then added to the supernatant to bring it to pH 4.5. The sediment thus formed was washed with acidified water and dissolved in neutral saline. The preparations obtained in this way presented a mixture of nucleoproteins and some other proteins extracted from the cells and precipitated under the above conditions. The protein content has been determined in every one of the above preparations by means of the micro-Kjeldahl method. Preparations from normal tissues have been obtained in a similar way. To study these preparations, use was made of the anaphylaxis reaction modified as follows.
304
1,. A . ZILBER
The nucleoprotein fraction obtained from the tumor tissue by the above method was administered subcutaneously to guinea pigs, iisually in a dose of 4 to 10 rug. protciii. Thc aninials were desensitized through intravenous adiniiiistration of a preparation from the respective normal tissue 25-30 days later. (The injections were made iiito the femoral vein, and for this purpose the skin above it was cut with scissors at the distal end of the hind limb and the material injected into the exposed vein by means of a syringe.) Desensitization was begun by administering the preparation from a normal tissue in a dose equal, on a protein basis, to one-half or one-fourth of the sensitizing dose. The guinea pigs usually responded to this injection by a moderat,e or weak anaphylactic shock. Greater doses may also be TABLE I1 Anaphylaxis Following with Desensitization
Guinc Pig NN
Preparation
Proteir Dose bg.1
Test for Completeness of Desensitization
Desensitization
Sensitization
Preparation
Protein Dosc Reactior (mg.)
Preparation
Protein Dose Reactior
~
231
232
233
220
Cancer of 8.5 mammary gland Cancer of 8.5 mammary gland Cancer 8.5 mammary gland Nonsensitized x
Normal mammary gland Normal mammary gland Normal mammary gland
4.4
++
3.0
+
3.0
+
X
X
Y
Mouse 6.4 hepatoma Mouse 6.4 hepatoma Mouse 6.4 hepatoma Nonsensitized x
Normal liver
3.0
Normal liver Normal liver
Normal mammary gland Normal mammary gland Normal mammary gland
8.8
-
6.0
+
6.0
+
Y
X
~
111 112 113 114 ~
KEY:x
= Experiment not performed.
- = No anaphylactic phenomena observed. + = Drop of temperature, scratching. + + = Scratching, sneezing, coughing. + + + = Hard picture of nhoek. + + + + = Lethal shork.
Normal liver
6.3
2. 25
+++ ++
Normal liver
6.3
2.25
-
Normal liver
4.5
+ +
X
Y
Y
-
305
SPECIFIC TUMOR ANTIGENS
administered, but these call forth strong reactions after which the animals recover with difficulty. It is advisable, therefore, to use several smaller doses. After complete recovery of the animal from shock and the return of the temperature to normal (usually within 2 hours after the injection), a test for completeness of desensitization was made. The animal was treated again with an equal or larger dose of the same preparation from a normal tissue. If this treatment caused no reaction, the animal was considered to be completely desensitized to this dose. If, however, the injection was followed by anaphylactic phenomena, a third injection of the same or larger dose was made 2 hours later. It is most advisable to carry desensitization to the same level of protein dosage as that used for sensitization.
Second Test for Completeness of Desensitization
Preparation
Challenge Injection
Protein Dose Reactior (mg.1
Preparation
Protein Dose Reaction (mg.)
X
X
Cancer of mammary gland
8.5
++
Normal mammary gland
9.0
-
Cancer of mammary gland
8 .5
+++
Normal mammary gland
9.0
-
Cancer of mammary gland
8.5
++
X
Y
Y
Cancer of mammary gland
8.5
-
X
x
S
Mouse hepatoma
6.4
+
Normal liver
6.3
-
Mouse hepatoma
6.4
Normal liver
6.3
-
Mouse hepatoma
6.4
++ ++
X
X
Mouse hepatoma
6.4
-
X ~
X
Sometimes desensitization against normal tissue antigens was effected after two injections in increasing amounts while in other instances more injections proved necessary. Eventually, with highly reactive guinea pigs,
30G
1,. .4. ZILRER
the procedure continued the following day. The first intravenous dose on the second day was the same as the last dose of the preceding day. In other similar cases, desensitization was commenced with a subcutaneous double sensitizing dose in order to continue desensitization on the next day intravenously in the way described above. When the guinea pig was completely desensitized to the desired dose of the preparation from normal tissue, it was injected with a preparation from a tumor tissue in a dose (on protein basis) not exceeding the last desensitizing one. If the animals reacted to this dose by anaphylaxis, this indicated the presence in the preparation of tumor tissue of an antigen lacking in the normal tissue. This is illustrated by the data of Table 11. Each preparation obtained both from tumor and from normal tissue was tested for sterility, while lack of toxicity was ascertained by administering it intravenously to a nontreated guinea pig in a dose equal to the maximal dose used in the experiment. If the preparation proved toxic (which was the exception), it was discarded just like nonsterile preparations. In each experiment with human tumor, the donors of normal and tumor tissue belonged to the same blood group. Desensitization was always carried out by. using a preparation from normal tissue corresponding in type to the tissue in which the tumor originated, e.g. guinea pigs sensitized to prepara tions from the gastric carcinoma were desensitized by means of normal mucosa of the stomach ; in sensitization to hepatoma, desensitization was carried out by normal liver, etc. This rule was ignored only in the case of sarcomas in which experiments the nucleoprotein fraction from muscle tissue was used for comparison. Tables I11 and IV summarize the data obtained by means of these methods. With the aid of the anaphylaxis reaction, antibodies have been detected against tumors which arose in inbred animals and were transplanted to them. Fink et al. (1953) and Fink et al. (1955) showed that inbred mice BALBc sensitized by preparations of the homologous fibrosarcoma 5-621 respond by shock to the administration of the extract of the lysed tissue of this tumor. Mice immunized through inoculation of the tumor tissue into the tail in some cases proved immune to subsequent transplantation. Antibodies detected by the anaphylaxis reaction could not be revealed by the precipitation and complement fixation test or by other methods. The specificity of tumor antibodies has also been demonstrated by Korngold and Pressman (1954b) :iodine-labeled antibodies against lymphosarcoma of rats have localized mainly in the tumors. Sera of rabbits immunized with normal organs contain no such antibodies. Thus it will appear that tumor tissues are not identical to the respective normal tissues in antigenicity. Accordingly, it might be suggested that they contain an antigen which is absent in normal tissues.
307
SPECIFIC TUMOR ANTIGEKS
TABLE I11 Experiments wit,h Animal ‘rumors Animal
Tumors ~
Rlicc
Reference ~~
-
.
- .. _..
Krocker’s sarcoma Mammary carcinonia induccd by methylcholanthrenc Ehrlich’s canccr Sarcoma 263 induccd by carcinogens i n miw Sarcoma 298 C-57 black and subcultured to Sarcoma 656 mice of same strain Hepatoma induced by orthoazoaminotoluene in inbred mire and subcultured to micc of samc strain Hepatoma induced by orthoa&oarniiiotoluciic i n inbred mice Ascitic Ehrlich’s cancer
i!
Rats
*
Sarcoma 465
transplantable sarcomas originally induced by carcinogens
Spontaneous sarcoma Spontaneous fibroadenoma Spontaneous adenocarcinoma
Rabbits Brown-Pearce carcinoma Carcinoma from Shope papilloma Primary carcinoma induced by 9-10-dimethyl-l,2benzanthracene Chicks
Spontaneous tubular adenoma of kidney Spontaneous cancer of oviduct Spontaneous round-celled sarcoma Spontaneous round-celled sarcoma Spontaneous round-celled sarcoma Sarcoma induced by methylcholanthreoc Lymphomatosis
..
-
Freiman (1950) Levina (1956) Freiman (1950) Freiman (1950) IRvina (1956)
1‘.A. Korosteleva (1956) Levina (1956) Gelstein (1957) 7‘. A. Korosteleva (1956) Levina (1956) Freiman (1950) Freiman (1950) Freiman (1950) Levina (1956) Levina (1956) Levina (1956) Gardashyan (1950) Artamonova (1956) Dyadkova (1953)
Dyadkova Dyadkova Dyadkova Dyadkova Dyadkova Dyadkova Dyadkova
(1956a) (1956a) (1956a) (1956a) (1956a) (1956a) (1956b)
I n another series of experiments, it has been attempted to ascertain whether tumors of different localization have a common or different antigenic structure (Shershulskaya, 1952). Guinea pigs were immunized with the preparation of some tumor, e.g. stomach carcinoma, desensitized by the respective normal tissue (gastric mucosa) and then were tested for susceptibility to the preparations of various other tumors such as uterine carcinoma, mammary carcinoma, etc. The aiiimals reacted to this procedure, thereby indicating that the preparations of different tumors have some antigens in common. It is quite obvious that such common antigens
308
L. A. ZILBEIt
TABLE IV Experiments with Human Tumors" Human Tumors Hepatoma Leucoses Carcinoma of the stomach and its metastases Carcinoma of the esophagus and its metastases Metastases of carcinoma of the pancreas Carcinoma of the cervix of the uterus Carcinoma of the lung Carcinoma of the corpus of the utcrus Sarcoma of the soft tissue Carcinoma of thc mammary gland and it.s metastases Papilloma of the urinary bladder Carcinoma of the urinary bladder Carcinoma of the ovary Cholangioma
Reference Zilher el al. (1949) Zilher and Parnes (1949) Shabad and Medvedev (1950) Parnes (1953) Shershulskaya (1951, 1952) Gorodilova and Shershulskaya Gorodilova and Shershulskaya Gorodilova and Shershulskaya Gorodilova and Shershulskaya Gorodilova and Shershulskaya Gorodilova and Shershulskaya Gorodilova and Shershulskaya Gorodilova (1951, 1952) Dyadkova (1953) Levina (1952) Levina (1952) Shabad and Medvedev (1950) Gorodilova and Shershulskaya Shabad and Medvedev (1950) Shershulskaya (1952)
(1956) (1956) (1956) (1956) (1956) (1956) (1956)
(1956) ,
The results obtained in all these experiments are quite consistent. Guinea pigs sensitized by nucleoprotein preparations from tumor tissue and desensitized by similar preparations from n o d tissue responded by shock to the challenge dose of the Preparations from tumor tissue. It is only rarely, when the material was taken from sick people treated with X-rays, that the specific antigen could not be constantly revealed. By means of the anaphylaxis reaction carried out in citro according to the above scheme, antigens from tumor tissue have been recently revealed in the plasma of cancerous patients (Makari, 1955a.h). @
may be species or group ones or varieties of other antigens of normal tissues. However, the antigens of different tumors are not identical since complete desensitization to antigens of another tumor (in the case a t issue the uterine carcinoma or mammary carcinoma) did not eliminate the susceptibility to the antigen with which they have been sensitized (stomach carcinoma). It may be suggested that the tumor antigen has a complex structure in that one of its components is common to different tumors while the other is peculiar only to those with a definite localization. This problem has also been studied by comparing the antigenic structure of leukotic tissues affected with different forms of leukosis (Zilber and Parnes, 1949). I n these experiments, guinea pigs were sensitized with a preparation of the spleen of a patient who had succumbed to one of the forms of leukemia (e.g. lymphatic), desensitized by a preparation from normal spleen, and then tested with spleen preparations from patients who had succumbed to other forms of leukemia (e.g. hemocytoblastosis or
SPECIFIC TITMOR .ZNTIGI"h'S
309
niyelosis). The anaphylactic reaction was always the case whereas guinea pigs desensitized to another form of leukosis rctained thcir susceptibility to the form of leukosis by which they have bcen sensitized. However, the animals desensitized to the form of leukosis by which they have been sensitized lose the susceptibility to all other forms. Thus leukotic tissues in different forms of human leukemia possess both common and different antigenic components in addition to normal ones. The discussion of the data obtained by means of the anaphylaxis reaction with desensitization raises the question as to the character of the antigenic differences between tumor and normal tissues. Are they due to qualitative changes undergone by antigens in the course of malignization or they are of a quantitative nature? On the supposition that some antigen is contained in the cancerous cell in much greater amounts than in the normal cell, it may be assumed that guinea pigs desensitized to a certain dose of normal proteins containing this antigen will not be desensitized to the same dose of tumor proteins since the antigen content of this dose is greater. To answer this question, experiments have been carried out with leukemic material (Parnes, 1957). Guinea pigs were sensitized by preparations from a normal spleen and completely desensitized to a certain dose of this preparation. Then the same dose of a leukotic spleen preparation was administered. Were the leukotic preparation to contain a much greater amount of some antigen than the preparation from a normal spleen, anaphylaxis would have been observed. This, however, was not the case. Therefore, the above results cannot be attributed to a quantitative difference in the normal antigen content of the tumor and normal cell. I n our first experiments (Zilber et al., 1949; Gardashyan, 1950; Shershulskaya, 1951), the sequence of the procedures was sometimes reversed. That is, guinea pigs were sensitized by preparations from normal tissues and desensitized by tumor preparations. For the challenge dose, use was made of the preparation from the normal tissue. These experiments were carried out with uterine carcinoma, with human ovary carcinonia, with Brown-Pearce carcinoma of rabbits, and with some other tumors. Guinea pigs sensitized by the preparations from normal tissues and desensitized by those from cancerous tissues usually did not react to the administration of the preparation from normal tissue. I n connection with the above evidence communicated by Weiler (1954, 1955), similar experiments have recently been carried out with hepatoma of inbred mice (Gelstein, 1957). The results proved quite different : guinea pigs sensitized by liver preparations and desensitized by certain doses of hepatoma preparations reacted to the administration of the same doses of the hepatic preparation.
310
1,.
A . ZILBER
It is the object of further inquiry to decide whether these results are due to the fact that hepatoma contains but few normal hepatic cells, or are to bc attributed to the changes in the antigenic structure of the hepatomic cells as communicated by Weiler (1954, 1955), or to some other factors. Since antigenic simplification has also been noted in leukemic cells (Seligman et ul., 1955), it seems advisable to subject antigens of different tumors to a large-scale investigation. It is quite probable that in those cases when tumor tissue does not contain any normal cellular elements besides stroma desensitization of guinea pigs sensitized by normal tissue with the aid of the cancerous one will not be complete enough. It will also be noted that tumor tissue should be carefully separated from the normal tissue since otherwise its preparations might contain a certain amount of normal components which may affect the results of the experiments. The next question to be considered in discussing the evidence on anaphylaxis following desensitization is whether antigenic differences are due t o differences in the cellular composition of the tumor and normal tissue. In some respects tumor tissues are similar to embryonic ones. Thus, morphologically, leukotic cells are similar to immature elements of white blood. I n some experiments (see above), antigenic community has also been established between tumor and embryonic tissues. However, no positive results have been obtained with human embryonic spleen in desensitizing guinea pigs sensitized by the antigen from leukotic spleen (Parnes, 1957). In these experiments use was made of the spleen of a 24-28 week old embryo of the same blood group as the leukemic adult spleen. Guinea pigs sensitized by the preparation from leukemic spleen and desensitized by the embryonic preparation (and in part of experiments additionally desensitized by normal adult spleen) remain susceptible to the leukotic antigen. Although in all experiments with human tumors the experimental and control material belonged to persons with identical blood groups, the antigenic composition of similar tissues from different humans was not necessarily identical. This is because not all blood groups known were taken into account and identity of blood groups is not irresistibly linked with a complete antigenic identity of organ and tissue elements. However, consistency of the data obtained in a vast number of experiments with most diverse tumors, those of inbred animals exclusively, precludes any suggestion that these factors might have appreciably influenced the experimental results. The objections which can be raised against some of the above experiments do not, therefore, invalidate the principal conclusion indicated by the experiments, viz. tumors and their respective normal tissues are not identical with regard to antigenic composition of the cells and tumors con-
SPECIFIC TUMOR ANTIGENS
311
tain antigens which are absent in normal tissues. Moreover, malignization may presumably be accompanied by a simplification of the antigenic composition of the cells so that the tumor cell may be devoid of some antigens of the normal cell.
VI. THEREACTIONOF PASSIVE ANAPHYLAXIS To demonstrate the presence in the tumors of specific antigens use was also made of the reaction of passive anaphylaxis. Attempts have long been made to use this reaction for the detection of anaphylaxis antibodies in the sera of patients. Pfeiffer (1910), Kelling (1910) and others treated guinea pigs with serum of cancer patients and 36-48 hours thereafter with extracts of human tumor. The challenge dose was administered via the peritoneum, and the drop of temperature was used as an indication of anaphylaxis. No conclusive results were obtained. I n our experiments, sera to be tested were administered subcutaneously to guinea pigs in doses of 0.5-5.0 ml. After 48 hours, this was followed by intravenous administration of the nucleoprotein fractions of the tumors. The reaction was estimated by means of the scale presented in Table 11. Gardashyan’s (1956) experiments were carried out with sera of 16 rabbits implanted with the Brown-Pearce tumor. I n 12 cases, anaphylaxis was noted in guinea pigs treated with rather high doses of the tumor nucleoprotein, viz. 14-20 mg. Lesser doses did not elicit any reaction. Eventually, a weak anaphylactic reaction was caused in some cases by the administration of the tumor nucleoprotein to guinea pigs treated with serum of healthy rabbits. Positive results have been received by Dyadkova (1953) in similar experiments with sera of rabbits with the Brown-Pearce tumor and a tumor induced by 9,1Odimethyl-2-benzanthracene as well as with sera of patients affected with mammary cancer. I n another series of experiments (Shershulskaya, 1957), sera of rabbits immunized with extracts of human stomach and uterine carcinoma were injected subcutaneously to guinea pigs in doses of 2 ml., and 48 hours thereafter the same animals were treated with nucleoprotein fractions of these tumors. The guinea pigs reacted to this treatment by anaphylaxis provided the dose of these fractions equaled 3-5 mg. A similar reaction was also elicited by the respective preparations from normal tissues. Passively sensitized guinea pigs, when desensitized to normal tissue preparations, responded by anaphylaxis to the administration of tumor preparations. The reaction rate was here reduced so that larger doses were necessary to elicit it. Guinea pigs passively sensitized by sera of rabbits immunized by uterine cancer behaved similarly.
312
L. A. ZILBER
Use was made of this method to study sera of 9 rabbits immunized with stomach carcinoma and sera of 3 rabbits immunized with uterine carcinoma. They have been studied by means of preparations isolated from 11 cases of stomach carcinoma and 3 cases of uterine carcinoma. Results similar to the foregoing ones were obtained. Control experiments have been carried out with sera of rabbits immunized with normal tissues. Guinea pigs sensitized by these sera, when desensitized by preparations from normal tissues, did not exhibit any reaction even to larger doses of cancerous preparations. In all these cases, the tumors and normal tissues were taken from patients of the same blood group. In the overwhelming majority of cases of passive anaphylaxis, the reactions were slightly pronounced. These results are borne out by one experiment of passive anaphylaxis following desensitization and are presented in Table V. Thus passive anaphylaxis may likewise be used for studying tumor antigens. The data obtained also indicate the antigenic nonidentity of tumor and normal tissues. Mention should be made of the fact that according to Dyadkova (1953) guinea pigs desensitized by the serum of a patient with mammary cancer exhibited a clear-cut anaphylaxis reaction to the administration of a preparation from the tumor excised from the same patient. Similar relationships have been noted with regard to the primarily induced rabbit tumor. Here also, guinea pigs sensitized by serum of an affected rabbit reacted to the administration of the preparation of the same tumor. In all these cases the reaction could not certainly be induced by isoantigens.
VII. LOCALIZATION OF THE SPECIFIC ANTIGENIN TUMORCELLS A special study was made concerning the localization of the tumor antigen in the cell (Zilber et al., 1955). Nuclei, mitochondria, and microsomes have been isolated from the homogenate of the rat M-1 sarcoma by means of a centrifuge and a separator and freed from solved proteins. The remaining proteins were fractionated by means of ammonium sulfate (up to 33,66, and 100%) into the first and second globulin and the albumin fraction. The particulate fractions were dissolved in a weak alkali and then neutralized by acetic acid. All the preparations thus obtained were tested in the reaction of complement fixation with sera of rats affected with M-1 sarcoma. The doses of the preparations were estimated on protein basis. With each of them 30-74 sera were tested. The serological activit,y was greatest in the mitochondria1and microsomul protein as well as in the first globulin fraction which reacted with some three-quarters of the sera studied. A much lesser serological activity was exhibited by the second globulin
TABLE V Passive Anaphylaxis Following Desensitizations NN
Pigs
Sensitization Antigen
54 75 76 82 53
h u m of
94 92 86 7 8
46 48 49 50
52 0
Desensitization
Dose (ml.)
Antigen
Protein Dose Reaction (mg.)
2 2 2 2 2
Mucosa of normal stomach
2.4 2.4 2.4 2.4 2.7
Serum of rabbit immunized with carcinoma of uterus
2 2 2 2 2
Normal Normal Normal Normal Normal
1.7 1.7 1.7 1.9 4.8
Serum of rabbit immunized with norma1 gastric mucosa
2 2 2 2 2
Normal stomach Normalstomach Normalstomach Normal stomach Normal stomach
rabbit immunized with carcinoma of stomach
Shershulskaya (1957).
uterus uterus uterus uterus uterus
-
1.5 3.0 5.0 5.0 5.0
+ ++ ++ + +
+ + + + -
+ ++ + + ++
Second Desensitization
Antigen
Challenge Dose
Protein Dose Reactior (mg.1
Antigen
Mucosa of normal stomach
6.8 5.8 5.8 5.8 6.8
-
Normal Normal Normal Normal Normal
2.9 2.9 4.3 7.8 7.8
-
Carcinoma of uterus
5.0 5.0 7.5 7.5 7.5
-
Carcinoma of stomach
uterus uterus uterus uterus uterus
Normal stomach Normal stomach Normalstomach Normalstomach Normal stomach
-
-
Carcinoma of normal stomach
Protein Dose Reaction (mg.)
3.5 3.5 5.2 5.2 5.2 2.4 2.4 4.6
-
+ + + -
-
+ +
4.6 6.4
++
8.0 7.5 7.5 10.0 10.0
-
-
314
L. A . ZILBER
fraction, by albumin and proteins of the nucleus. These reacted with some one-quarter and one-third of the cases. Out of 68 healthy rat sera, only 4 showed a slight positive effect. Electrophoresis of the above fractions (Abelev, 1956) showed that more active fractions are similar in composition, a component with a motility cm2/sec predominating in them (0.17 M veronal buffer, of 4.4-4.6 X pH 8.6). The second globulin and the albumin fraction contained slight admixtures of this component which was absent in the nuclear extracts. This component which is present both in the granules and hyaloplasm of the cells is probably responsible for the serological activity of the above fractions. The sera of rats with transplantable M-1 sarcoma reacted not only with microsomes and mitochondria from the tumors but with the same fractions from the lungs and spleens of healthy rats (15 out of 25 sera gave positive reactions). Similar relationships have been reported by Friedewald and Kidd (1945). Sera of rabbits implanted with the Brown-Pearce tumor likewise reacted in some cases not only with the tumor antigen but also with that from normal tissues. The antibodies eliciting this reaction are called by these authors ‘(induced” antibodies. It follows from the above evidence presented by Narcissov and Abelev (1956) that in many cases mitochondria and microsomes show the reaction of complement fixation with sera of rats from whose tumors they have been isolated, and these were primarily induced, not transplanted, tumors. It may be suggested, therefore, that the tumor antigen linked with mitochondria and microsomes does not belong to isoantigens. VIII. TIMEOF APPEARANCE IN
SPECIFIC ANTIGEN Of great interest are the studies on the time of appearance of the specific antigen in the course of carcinogenesis. It has already been mentioned that by means of anaphylaxis following desensitization, Levina (1956) in this laboratory as well as T. A. Korosteleva (1956) in Shabad’s laboratory have revealed in the hepatoma of mice an antigen which is absent from healthy liver. According t o Korosteleva, this antigen is to be found not only in the transplantable hepatoma but also in that primarily induced by orthoaminoazotoluene (OAAT). She has also studied the antigenic composition of the liver a t early stages of carcinogenesis (1954). In mice fed on OAAT in sunflower oil, one month after the commencement of the diet an antigen was found in the liver which was absent in the liver of mice kept on pure sunflower oil with no carcinogen. No such antigen has been detected in the pulmonary tissue of these mice. THE
CELLOF
THE
SPECIFIC T U M O R .4NTIGENS
315
However, i t proved different from the hepatoma antigen, being a complex of liver proteins with OAAT and possessing the specificity of the latter. Similar compounds have been obtained by Creech (1947, 1949, 1952), Korosteleva (1951), and others. By means of the anaphylaxis reactioii following desensitization, Gelstein (1957) has studied in tjhis laboratory liver antigens of inbred mice painted with OAAT a t various stages of carcinogenesis. The mice were sacrificed 2-8 months after the beginning of painting, as well as after the appearance of hepatoma. It follows from these experiments that during the precancerous stage, morphologically characterized by diffuse hyperplasy of the liver tissue and by hepatic cell adenomata, it is already possible to reveal both the antigen with the specificity of OAAT and the one similar to the hepatoma antigen. Neither of these antigens was adsorbed by the red cells. Thus, the changes in the antigenic structure of the cells can be revealed prior t o malignization. This has also been mentioned in the above experiments by Weiler (1955, 1956a-c).
IX. THENATUREOF THE SPECIFICTUMOR ANTIGEN The specific component of the tumor may be of a different nature and origin. It may constitute the antigen of a virus, either active or masked, or an autoaiitigen formed in the course of carcinogenesis. It might also bc suggested that the specific component is a complex formed by the virus and modified cell proteins. The study of this problem involves great methodical difficulties. Accordingly, the experiments cited below should be regarded only as the first step toward the solution of the problem. It is only natural that this study should start with tumors that undoubtedly contain the specific antigen, viz. filterable tumors. The question to be answered was whether the tumors contain, besides the virus antigen, other antigenic substances lacking in normal tissues. In other words, are the antigenic differences between normal and tumor tissue due only to the presence in the former of the viral antigen or are there some other antigenic differences between them? To answer these questions, experiments were set with Shope papilloma of rabbits in which the antigenic composition of the papillomatous tissue, purged of the virus by repetitive adsorption on red cells, was compared with normal rabbit skin. In most of the experiments, not the red cells but their stronia were used because partial hemolysis eventually noted upon fourfold adsorption infected the supernatant with hemoglobin. These experiments carried out by Artamonova (1956) showed that guinea pigs
316
L. A . ZILBEIt
sensitized by the papillomatous extract and desensitized by the extract of normal skin retain their reactivity to the former of these extracts. On the other hand, guinea pigs sensitized by normal extracts and desensitized by tumor extracts did not react thereafter to the administration of the normal tissue extract. It might, therefore, have been suggested that the virusdeficient papillomatous tissue contains an antigenic component that is absent in the normal tissue. Since papillomatous tissue contains a soluble viral antigen, which is denied by Kidd (1938a) but corroborated by some of the data provided by Narcissov et al. (1955), we have desensitized guinea pigs sensitized by papillomatous extracts not only with the extracts from normal tissue but also by the stroma of red cells which have adsorbed Shope virus. In these experiments, we have also invariably noted antigenic differences between the virusdevoid extracts of papillomatous tissue and those from normal tissues. Thus the filterable tumor may contain not only the viral antigen, but another one which is absent in the respective normal tissues. This conclusion is also indicated by some immunological evidence. Rous (1913) has long since shown on chick sarcoma that immunity to the tumor cell and to the virus is due to different mechanisms. This question has also been studied in this laboratory by Radiskhovskaya (1952). She immunized Leghorn chicks with formol-treated filtrates of minced sarcoma tissue. The immunity was tested 14-22 days after the last administration of the preparation. For this purpose, active Rous virus was administered in the form of the supernatant filtrate obtained after the centrifugation of the triturated sarcoma tissue. In 22 of 57 immunized chicks, the tumors did not develop, and the chicks remained healthy for 2 months whereas other animals contracted sarcoma. Sixteen of these 22 virus-immune chicks were inoculated with a suspension of tumor cells in a dose of 0.2-0.5 ml. of 10% suspension. All these chicks exhibited sarcomata simultaneously with control, healthy chicks inoculated with the same suspension. It will be noted that chicks in which spontaneous resorption of sarcoma took place proved healthy both after the administration of the virus and the suspension of tumor cells. According to Burmester and Belding (1947), in chicks immunity to the cells of the lymphoid tumor is not accompanied by immunity t o the virus of visceral lymphatosis. In the experiments carried out by Postnikova (1956a), the papillomatous tissue of rabbits was autotransplanted to the muscle of the tibia while several days prior to this procedure a suspension of papillomata of another rabbit containing an active Shope virus was rubbed into the rabbit skin. In 12 out of 19 experimental rabbits, autotransplants have developed
SPECIFIC TUMOR ANTIGENS
317
whereas none of them exhibited any papilloma at the site of application of the viral suspeiision which in healthy control rabbits has elicited papillomata. Thus, immunity to the virus is iiot accompanied by immunity to the cells originated by this virus. It follows from the above data that the cells of viral tumors must contain at least two different antigens which are absent in normal cells. One of them is a protein of the virus responsible for the immunity to this virus. The other antigen is presumably an autoantigen and causes immunity to tumor cells. It is hardly plausible that this autoantigen is an isoantigen since it appears in animals treated only with the virus but not with the cells. The immunity to cells accompanying spontaneous resorption of the chick sarcoma induced by Rous virus is apparently due to a similar antigen. Thus the conclusion is indicated that immunity to the initiation of the viral tumor and immunity to its transplantation are caused by different mechanisms. Similar relationships are probably to be admitted for spontaneous and induced tumors. According to MacDowell et al. (1934,1935), inbred mice immunized with small doses of leukemic cells of the same strain of mice and refractory to subsequent transplantation of these cells contracted spontaneous leukemia in the same percentage of cases as nonimmunized mice. Our experiments (Zilber, 1949) have been carried out on rats in which spontaneous resorption of the transplantable sarcoma originally induced by 9,10-Dimethyl-l,2-benzanthracene took place. Repetitive transplantations of this sarcoma proved futile. However, after treatment of 42 rats with the same carcinogenic substance, 41 contracted sarcoma. The last immunity test of these rats to transplantable tumor was carried out a t a time when the tumor induced by carcinogenic substances might otherwise have appeared. Accordingly, the results of these experiments cannot be related to the disappearance of the transplantable immunity. Similar data have been obtained by Molkov (1956).The presence of a very strong immunity to the transplantable tumor is not, therefore, accompanied by immunity to the initiation of a spontaneous or induced tumor. Although the interpretation of these experiments may vary, it seems worth noting that the mechanisms of immunity to the initiation and growth of such tumors are dissimilar just as are the mechanisms of immunity to the tumor cell and to the virus responsible for its appearance. In this connection, it seemed worthwhile to attempt a comparative study of the antigenic structure of chick tumors, both filterable and nonfilterable. I n Andrewes (1936) experiments, nonfilterable chick sarcoma induced by coal tar has been successfully grafted to pheasants and produced antibodies to Rous virus. In the pheasant, the tumor grew from pheasant
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but not chick cells, and hence this tumor contained Rous virus. According to Foulds (1937) as well as Dmochowski and Knox (1939), rabbits immunized with nonfilterable chick tumors induced by dibenzanthracene produce neutralizing antibodies to Rous virus. They also produce antibodies which fix complement with the filtrates of Rous sarcoma and of some other filterable chick and duck tumors. Antigen community of filterable and nonfilterable chick tumors has also been pointed out by some other workers (Gottschalk, 1943, 1948). Although in some of these experiments admixture to the nonfilterable tumor of Rous virus is not excluded yet the presence of common antigens in the filterable and nonfilterable chick tumors appears quite probable. Dyadkova (1954) took advantage of the technique developed by the author to compare the antigens of the filterable and nonfilterable chick sarcoma. In her experiments, guinea pigs sensitized by the extract from Rous sarcoma and desensitized by the extract from normal chick muscle still retained the susceptibility to the Rous sarcoma extract as well as to that from the dimethylbenzanthracene tumor. A similar picture was noted in guinea pigs sensitized by the extract of the dimethylbenzanthraceneinduced sarcoma. While desensitized by normal muscle extract, they still remained susceptible to the antigen of the induced and Rous sarcoma. It will appear from these data that Rous and induced sarcoma each contain antigens that are both absent in healthy muscle and related to one another. In further experiments, guinea pigs sensitized by Rous sarcoma extract and desensitized by the extract from induced tumor remained susceptible to Rous sarcoma extract. But after sensitization by extract from induced tumor and desensitization by Rous sarcoma extract, they did not react any more to the administration of the extract from induced tumors. One may conclude that induced sarcoma contains an antigen that is lacking in the normal muscle and that besides this antigen Rous sarcoma contains another one which is absent not only in the normal muscle but in the dimethylbenzanthracene-induced tumor as well. Therefore, it appears quite probable that, apart from the antigen which represents the substance of the exogenic virus, filterable tumors also contain an antigen of tissular origin formed endogenously. It will be noted that Kidd (1946) has long since communicated about the distinct antigen in the rabbit carcinoma Vz which is absent in normal tissues and is of nonviral nature. Great difficulties have been encountered in the methods of studying specific antigens of nonfilterable tumors as well as of human tumors. Since the specific antigens of filterable tumors have the capacity to be adsorbed on red cells, it seemed quite natural to study next this capacity on the antigen of nonfilterable tumors.
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319
X. ADSORPTION UPON THE RED CELLSOF THE SPECIFIC TUMOR ANTIGEN No positive results have been obtained in an attempt to adsorb the tumor antigen on native red cells from the rat M-1 sarcoma (Ludogovskaya and Morgunova, 1956). Absence or presence of adsorption was indicated by the reaction of anaphylaxis following desensitization by the complement fixation test and by passive hemagglutination. Edlinger and Horel (1954) noted that chick erythrocytes adsorb the antigen from rat hepatoma whereby they become agglutinable by the serum of rats with hepatoma. With due observance of the technique of these workers, Gelstein (1957) in her experiments with the transplantable hepatoma of inbred mice was unable t o record any adsorption of the tumor antigen on native red cells of chick or other animals. Nor did the antigen of primarily induced mouse hepatoma adsorb on the red cells. These still scanty data do not yet warrant the conclusion that antigens of the nonfilterable tumors are adsorbed upon the red cells and further studies on other similar tumors are necessary. The problem has been taken up in this laboratory as to the adsorption upon red cells of antigens of human tumors. For this purpose the anaphylaxis reaction with subsequent desensitization has been employed (Ludogovskaya, 1957 ;Shershulskaya, 1957). Various modifications of the experiments have been carried out. In one experimental series, guinea pigs were sensitized by the red cells of rabbit or of the O-group of human subjects which have been in contact with the tumor t.issue extract. They have been desensitized by similar red cells kept in contact with the respective normal tissue and then their susceptibility tested toward red cells kept in contact with the tumor tissue. In another series, sensitization was effected by the nucleoprotein fraction of the tumor and further procedure was similar to that described above. Finally, the third series differed from the first one in that for the challenge injection use was made, not of red cells kept in contact with the tumor extract, but of the nucleoprotein fraction obtained therein. The results obtained are summarized in Table VI. It will appear from the above data that the antigen from human tumors is adsorbed on the red cells in a considerable percentage of cases (Ludogovskaya, 1957; Sherschulskaya, 1957). By means of the anaphylaxis reaction following desensitization, the antigenic capacity of red cells from humans with cancer was compared with that of healthy red cells of the same blood group (Gorodilova and Shershulskaya, 1956). It appears from these studies that in some cases the red cells of cancerous patients are different from healthy red cells and contain an antigen which is absent in the latter and is similar to the tumor antigen.
w
h3
0
TABLE VI Adsorption of Specific Tumor Antigens
Viral Tumors Shope papilloma
Rous sarcoma
Mammary sarcoma in mice Total b 8
+ = adsorption. -
= no sdsorptim.
Number of Experiments 3
5
2 10
+b
-c
Human Tumors
3 0 Stomach sarcoma Esophageal cancer Ovarian carcinoma Mammary carcinoma Pulmonary carcinoma 5 0 Uterine cervix Thyroid carcinoma Glioma Seminoma Chondriosarcoma Osteog. sarcoma 2 0 Gastric polypus 10 0
Number of Experiments
+
17 6 6 7 2 3
14 3 6 6 1 0
2
2
2
2
2 1 1
2 1 1 1 39
1
50
3 3 0 1 1 3 0 0 0 0 0 0 11
Tumors Untransportable by Filtrates Sarcoma M-1
Number of Experiments 4
+0
4
P Sarcoma 116
1
0
1
Sarcoma 464
1
0
1
Brown-Pearce cancer
2
0
2
1
0
1
?N
I
Gu6rin cancer Mouse hepatoma
2 11
0 2 0 11
FI
SPECIFIC TUMOR ANTIGENS
321
The group similarity of the red cells has been observed in these experiments only within the range of the four principal groups. Eventually, positive results might have been due to some groups not taken into account but this could have hardly been always the case. Therefore, it may be concluded that in vivo the tumor antigen is also adsorbed by the red cells; this antigen can be present in the blood stream as a result of necrotic and other processes taking place in the tumor. It will be of some interest to compare these data with those of Calaresu et al. (1955) who have revealed some antigenic differences between the red cells of leukemic humans and mice and the respective red cells of healthy individuals. Thus, there appears ample evidence to show that human tumors, like the viral ones, contain an antigen which is adsorbed on red cells. It is the purpose of further inquiry, being conducted at the present in this laboratory, to ascertain whether human tumors contain still another antigen which is absent in normal tissues. XI. DISCUSSION The above evidence points to the conclusion that the antigens of tumor cells are different from those of normal cells. Rut is this sufficient ground to assume the existence of special tumor antigens which are absent in normal tissues and which are specific for tumor tissues? Of the above data some can be interpreted in a different way while some possibly requires additional control experiments. Yet many of them conclusively indicate the existence of specific tumor antigens. As to the experiments with animal tumors, of greatest interest are those concerning spontaneous and primarily induced tumors. The only possible objection against the evidence brought forward by Narcissov and Abelev (1956) showing that antigens from induced rat sarcoma reacted with sera of the same rats is that they might have detected not the specific tumor antigen but an antigenic complex formed by the carcinogen with the rat protein. That such a complex possessing antigenic specificity of the carcinogen might form is evidenced from the data of Heidelberger and Weiss (1951), Miller and Miller (1947, 1952), Creech et al. (1947, 1949, 1952) and Korosteleva (1954). However, according to Narcissov and Abelev (1956), sera of rats with primarily induced tumors reacted not only with the antigens from these tumors but also with those from grafted tumors where this complex was absent. It should also be rioted that Graham (1955) has reported a positive complement fixation test with sera of cancerous humans and with extracts from the tumors excised from the same patients. I n 12 out of 48 patients, the reaction titer varied from 1 : 16 to 1 : 128. These results could not certainly be due to the protein complex linked with the carcinogen. Finally,
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L. A. ZILBER
mention should be made of the above data of Gelstein (1957) according to which a special tumor antigen can be distinguished in the primarily induced mouse hepatoma from the protein-carcinogen antigenic complex. Thus, one may refute the assumption that the results of the experiments with primarily induced animal tumors might have been influenced by the carcinogen-protein complex. However, the results of experiments in which the complement fixation test has been used may frequently be interpreted as evidence of quantitative, but not qualitative, antigenic differences between the tumor and normal cells. The same question is raised in discussing the data obtained by means of the anaphylaxis reaction. Experimental evidence and some considerations have been presented above to the effect that in this reaction qualitative differences have been revealed in the antigenic structure of tumor and normal cells. Experimental evidence relevant to the nature of specific antigens does not as yet warrant any definite conclusions although some hypotheses can be suggested which can be experimentally verified. The adsorption on red cells of some tumor viruses as well as of specific antigens of human tumors is pretty well grounded although much further inquiry is still needed. No adsorption could as yet be achieved on native red cells of the specific antigen revealed in the nonfilterable animal tumors. This antigen can possibly be adsorbed by tannin-treated red cells. Attention is due to the similarity revealed between viral and human tumors. In both cases, an antigen has been detected which is adsorbed by native red cells. Besides the viral antigen, another one has been revealed in viral tumors which is lacking in normal tissues. The study which is under way might elucidate the presence of such an antigen in human tumors. It might have been assumed that in the filterable tumors the virus disturbs protein synthesis so that proteins are formed which are distinct from normal ones and act as antigens in the organism in which they are formed. Such a process apparently takes place in tumors induced by diverse carcinogenic factors as well as in human tumors. An important problem is to discover those links of protein synthesis which are disturbed in the course of cardiogenesis. If these disturbances result in the formation of autoantigens, it is legitimate to assume that they concern the synthesis of autoantigenic proteins. As mentioned above, the first globulin fraction and the mitochondria1 and microsome fraction of the nonfilterable sarcoma exhibited maximal specific activity. On the other hand, the fractions of diverse tumors which reacted best in anaphylaxis were composed of a mixture of nucleoproteins with other proteins precipitated at pH 4.5. The question naturally arose as to the nature of the protein with which the reactivity of these fractions is linked.
SPECIFIC TUMOR ANTIGENS
323
The answer to this question, a globulin-like fraction has been isolated by ammonium sulfate precipitation from the nucleoprotein preparations used in the anaphylaxis following desensitization test. It was shown that this fraction accounts for the greater part of tumor specificity (Artamonova and Levina, 1957). Guinea pigs sensitized by the tumor nucleoprotein and desensitized by the normal one show a markedly pronounced reaction to the globulin isolated from this nucleoprotein although the globulin does not completely desensitize them to the nucleoprotein. It has been established in studying the masking of Shope virus (Zilber and Artamonova, 1954) that the extract of the cancerous tumor arising from the papilloma, when thoroughly freed i n vivo and in vitro from the serum, masks the virus after a 30-minute contact. The virus masked in this way loses its vaccination capacity whereas the latter is retained in the virus neutralized by antibodies (Postnikova, 195613). A study of the virus masking protein fractions of the extracts showed that the albumin-like fraction, chromosin and histone, lack this capacity whereas the virus is masked by the globulin-like fraction obtained through precipitation with 50% ammonium sulfate. It may be suggested that carcinogenesis involves, first of all, a disturbance in globulin synthesis. There is nothing surprising in this fact since the administration t o the organism of any antigen causes first a disturbance in globulin synthesis, and there arise new gamma-globulins exhibiting the capacity of antibodies. However, antibodies are globulins synthesized in definite cells of the organism which possess this capacity. I n the course of carcinogenesis, globulin synthesis is disturbed in other cells. Hence the globulin thus formed is not a serum antibody, but presumably may react like the latter with the antigen which has disturbed synthesis ofrming with it complex compounds. Eventually, as in malignization of the Shope papilloma, the pathogenic effect of the virus is neutralized. The formation in the cancer cell of proteins differing from normal ones has also been demonstrated biochemically (Zbarsky, 1948; Rondoni, 1955). These differences are probably due to some changes in protein structure but not composition. The above analogy between antibody formation and the carcinogenic process, mentioned also by Rondoni (1955), may be developed still further. The changes in the globulin synthesis called forth by antigens persist after the elimination of the antigen from the organism. A somewhat similar phenomenon is noted in carcinogenesis. Old chick sarcomata are frequently devoid of the virus which has elicited them, and yet growth of the sarcoma continues. Tumors induced by carcinogenic substances keep growing irrespective of whether the carcinogenic substance has been retained in the organism. Haddow (1944) has long since pointed out th a t the carcinogenic substance is not the actual stimulus for tumor growth a n d that the under-
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lying mechanism should be localized within the cells themselves. This mechanism is possibly linked with the disturbances in protein synthesis which result in the initiation of the tumor cell with an altered antigenic structure. The progressive reproductioii of tumor cells is usually attributed to their independence of the growth controlling systems of the organism. This concept has been experimentally substantiated by Alov (1955). In his experiments thyroidin, which stimulates mitosis of normal cells, and cortisone, possessing an antimitotic action, did not alter the mitotic activity of tumor cells in doses which distinctly affect this process in normal tissues. The growth and cell reproduction factors have arisen in the course of evolution, and it is only natural that they are well adapted to cells of normal antigenic structure. When this structure is altered and proteins are formed in the cell differing in their antigenic properties from normal ones, this correlation is upset and the regulating factors become inadequate for controlling growth and propagation of cells with a modified protein. These are, of course, merely hypothetic considerations, but they indicate the possibility of applying immunological methods to the studies of tumor pathogenesis. XII. CONCLUSION The above evidence suggests that the antigenic structure of tumor cells and of the respective normal cells is not identical. This discrepancy is probably due to various factors. In filterable tumors, the cells contain some viruses whose antigens differ from those of normal cells. At the same time these tumor viruses, as do some infectious viruses, contain antigenic components of the cells in which they reproduce thereby impeding the serological identification of these viruses. Yet the study of Shope papilloma demonstrates that the cells of filterable tumors devoid of the virus antigen are dissimilar in their antigenic structure from normal tissues and contain an antigenic component which is absent in the latter. In distinction to the viral component, this one is not adsorbed by the red cells. It is apparently an autoantigen originating in the course of carcinogenesis. The cells of the nonfilterable tumors are likewise distinguished by their antigenic structure from the respective normal cells and contain an antigenic component which is absent in normal cells. Presumably, this component is not adsorbed by native red cells, but the question requires further study. The antigenic structure of cells of human tumors is likewise distinct from that of the normal cells. The specific antigen revealed in these tumors is adsorbed on the red cells.
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325
I n the course of carcinogenesis there occurs also a simplification of the antigen composition of the cells with the loss of some of their antigens. These data concern only a few tumors so that no general conclusions can as yet be made. Complex changes do occur in the antigenic composition of the cells in the course of malignization. Apparently, they begin a t an early data, a t least prior to the initiation of tumor growth. It seems legitimate to assume that these changes of the antigenic structure release the cell from the control of the growth regulating systems of the organism thereby facilitating rapid reproduction which results in the formation of a tumor.
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MacDowell, E. C., Taylor, M. J., and Potter, J. S. 1934. Proc. Soc. Exptl. Biol. Med. 32, 84-86. MacKenaie, I., and Kidd, J. G. 1945. J . Exptl. Med. 82, 41-63. Maculla, E. S. 1947. Yale J . Biol. Med. 20, 343-368. Maculla, E. S. 1948. Yale J . Biol. Med. 20, 465-472. Mayer, M. E., and Barrett, M. K. 1943. J . Natl. Cancer Inst. 4, 65-73. Makari, I. D. 1955a. Brit. Med. J . 17, (4951), 1291-1295. Makari, I. D. 1955b. Texas State J . of Med. 61, 670-673. Mann, L. S., and Welker, W. H. 1940. Am. J . Cancer 39, 360-364. Mann, L. S., and Welker, W. H. 1943. Cancer Research 3, 193-197. Mann, L. S., and Welker, W. H. 1946. Cancer Research 6, 625-626. Manoilov, S. E., Nevler, A. I., and Presnov, M. A. 1953. Voprosy Onkol. 6, 42-49. (The problems of oncology, Symposium). Miller, E. 1955. Growth 19, 187-206. Miller, E. C., and Miller, J. A. 1947. Cancer Research 7, 468480. Miller, E. C., and Miller, J. A. 1952. Cancer Research 12, 547-556. Mirsky, A. E., and Pollister, A. W. 1942. Proc. Natl. Acad. Sci. 28, 344-352. Molkov, J. N. 1956. Voprosy Patogenesa i Immunol. Opukholei, pp. 256-260. (The problems of pathogenesis and tumor immunology, Symposium.) Morelli, E. 1934. Z . Zmmunitatsforsch. 83, 521-526. Murphy, J. B., Helmer, 0. M., Sturm, E. 1928. Science 68, 18-19. Narcisi3ov, N. V. 1956. Voprosy Patogenesa i Immunol. Opukholei, pp. 233-237. (The problems of pathogenesis and tumor immunology, Symposium.) Narckov, N. V., and Abelev, G. I. 1956. Voprosy Patogenesa i Immunol. Opukholei, pp. 243-247. (The problems of pathogenesis and tumor immunology, Symposium.) Narcissov, N. V., and Ebert, M. K. 1956. Voprosy Pplogenesa i Immunol. Opukholei, pp. 212-219. (The problems of Pathogenesis and tumor immunology, Symposium.) Narcissov, N. V., and Zilber, L. A. 1949. Doklady Akad. Nauk S.S.S.R. 66, 229-232. Narcissov, N. V., Artamonova, V. A., and Solovieva, N. J. 1956. Voprosy Patogenesa i Immunol. Opukholei, pp. 238-242. (The problems of pathogenesis and tumor immunology, Symposium.) Narcissov, N. V., Avenirova, Z. A., Stepantschenok, G. I., and Solovieva, N. J. 1955. Voprosy Onkol. 1, 6, 59-64. (The problems of oncology, Periodical.) Nungester, W. J., and Fisher, H. 1954. Cancer Research 14, 284-288. Oberliig, C.,and Guerin, M. 1954. Advances in Cancer Research 2, 353-423. Parnes, V. A. 1953. Zhur. Mikrobiol. Epidemiol. Zmmunol. 9, 64-65. Parnes, V. A. 1957. Eksperimentalnie materiali PO immunologii leikoeov, Moskva. (Experimental data on immunology of leukemia, Dissertation, Moscow.) Pentimalli, F. 1935. Bull. assoc. franc. Btude Cancer 24, 311-320. Pfeser, H.1910. Z. Zmmunitdtsforsch. 4, 458469. Postnikova, Z. A. 1956a. Voprosy Patogenesa i Zmmunol. Opukholei, pp. 25-28. (The problems of pathogenesis and tumor immunology, Symposium.) Postnikova, Z. A. 1956b. Voprosy cmkol. 2, 1, 84-86. (The problems of oncology, Periodical.) Radeikhovskaya, R. M. 1950. Zhur. Mikrobiol. Epiclemiol. Immunobiol. (lo), 27-32. Radaikhovskaya, R. M. 1952. Zhur. Mikrobiol. Epidemiol. Immunobiol. (l),20-22. Radeikhovskaya, R. M. 1957. V o p ~ o s yOnkol. (The problems of oncology. I n press.) Rapport, M. M., and Graf, L. 1954. Proc. Am. Assoc. Cancer Research 1, 2, 39-39. Rapport, M. M., and Graf, L. 1955. Proc. Am. Assoc. Cancer Research 2, 1, 40-41. Rondoni, P. 1955. Advances in Cancer Research 3, 171-221.
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ROUS,P. 1913. J. Exptl. Med. 18, 416-427. Rovnova, Z. I. 1956. Byull. Elcspll. Biol. dled. 41,5, 54-58. Saakov, A. K. 1952. Byull. Eksptl. Biol. Med. 34, 8, 61-65. Saakov, A. K. 1956. Voprosy Immunol. Normalnikh i Zlokacheslvenikh Tkanei, pp. 54-70. Moskva. (The problems of immunology of normal and malignant tissues, Symposium. Moscow.) Seligman, M., Grabar, P., and Bernard, J. 1955. Sang Le, 1, 52-70. Shabad, L.M., and Medvedev, N. N. 1950. Vestnik Akad. Med. Nauk S.S.S.R. 6, 2 8 4 4 . Shershulskaya, L. V. 1951. Byull. Eksptl. Biol. Med. 31, 6, 455-460. Shershulskaya, L. V. 1952. Zhur. Mikrobiol. Epidemiol. Immunobiol. (l),26-33. Shershulskaya, L.V. 1957. Voprosy Onkol. (The problems of oncology. I n press.) Smith, W., Belyavin, G., and Sheffield, F. W. 1955. Proc. Roy. SOC.(London) B143, 504522. Stoerk, H. S., and Emerson, G. A. 1949. Proc. Soc. Expll. Biol. died. 70, 703-704. Taylor, M. J., and MacDowell, E. C. 1949. Cancer Research 9, 144-149. Vorlaender, K. 0. 1954. Acla Allergol. 7, 224-230. Weiler, E. 1954. Slrahlentherapie 93, 213-222. Weiler, E. 1955. Strahlentherapie, 96, 269-270. Weiler, E. 1956a. Z. Naturforsch. llb, 31-38. Weiler, E. 1956b. Brit. J . Cancer 10, 533-559. Weiler, E.1956c. Brit. J. Cancer 10,560-563. Werder, A. A., Jiirschbaum, A., and Syverton, J. T. 1950. Cancer Research 10,248-248. Werder, A. A., Kirschbaum, A., MacDowell, E. C., and Syverton, J. T. 1952. Cancer Research 12, 886-889. Witebsky, E. 1929. Z. Zmmuniliilsforsch. 62, 35-73. Witebsky, E.1930. Klin. Wochchr. 2, 58-63. Witebsky, E., and Morelli, E. 1933. 2. Zmmunillitsforsch. 78, 179-196. Witebsky, E., Rose, N. R., and Shulman, S. 1956. Cancer Research 16, 831-841. Zbarsky, B. I. 1948. Trudy 4 Sessii Akad. Med. Nauk S.S.S.R., pp. 185-192. (Trans. 4 Session Acad. Med. Sci. U.S.S.R., Symposium.) Zilber, L. A. 1946. Virusnaya teorirt proiskhodgenya opukholei. (Viral theory of tumor origin, Moscow, U. S.S.R.) Zilber, L. A. 1949. Uspekhi Sovremennoi Biol. 28, 185-210. Zilber, L. A,, and Artamonova, V. A. 1954. Doklady Akad. Nauk S.S.S.R. 96, 1057-1060. Zilber, L. A., and Narcissov, N. V. 1949. Doklady Akad. Nauk S.S.S.R. 64, 849-852. Zilber, L. A., and Parnes, V. A. 1949. Doklady Akad. Nauk S.S.S.R. 49,257-260. Zilber, L.A., Birjulina, T. I., and Narcissov, N. V. 1956. I’oprosy Onkol. 2, 6, 646-649. (The problems of oncology, Periodical.) Zilber, L. A., Freiman, V. B., Zbarsky, I. B., and Dehov, S. S. 1949. Doklady Akad. Nauk S.S.S.R. 66, 97-100. Zilber, L. A., Narcissov, N. V., Rivkind, T. L., and Baidakova, Z. L. 1948. Vestnik Akad. Med. Nauk S.S.S.R. 3, 36-39. Zilber, L. A., Narcissov, N. V., and Abelcv, G. I. 1955. Doklady Akad. Nauk S.S.S.R. 100, 331-335. Zilber, L. A., Vadova, A. V., Postnikova, Z. A., Barabadze, E. M., and Artamonova, V. A. 1953. Byull. Eksptl. Biol. Med. 36, 3, 57-59.
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CHEMISTRY. CARClNOGENICITY. AND METABOLlSM OF 2-FLUORENAMINE AND RELATED COMPOUNDS Elizabeth
K . Weisburger and
John
H . Weisburger
Laboratory of Biochemistry. National Cancer Institute. National Institutes of Health. Public Health Service. U S Department of Health. Education. and Welfare. Bethesdo. Maryland
..
Page 333 333 334 334 335 335 336 345 346 346 346 347 347 348 348 350 350 350 350 351 352 359 2. 2-Fluorenamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 3. Related Fluorene Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 . . 362 A . 2-Nitrofluorene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 €3 . Derivatives of 2-Fluorenamine . . . . . . . . . . . . . . 364 C. Derivatives of N-2-Fliiorenylacetamide . . . . . . . . . . . . . . . . . . . . . . . 4. Some Physical and Chemical Properties and Carcinogenicity . . . . . . . . . . . . 365 365 A . Ultraviolet Absorption Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 €3 . Molecular Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 C. Oxidation-Reduction React ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 D. Michael Condensat.inn and Carcinogcnicit.y . . . . . . . . . . . . . . . . . . . . . . 368 5 . Tnfluence of Diet.s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 A . General (-1onsiderat.ions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 13. Stork versus Remisynthetic Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .. . ............................................. .. [IT. Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1. Chemistry of Fluorene . . . . . . . . . . . . .............. .. .... 2 . Fluorenamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . 1-Fluorenamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. 2-Fluorenamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. C. 3-Fluorenamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. D . 4-Fluorenamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 3 . Other Fluorene Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. A . 2,7-Substituted Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . B. 9-Substituted Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. C. Fluorene Quinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 4 . Syntheses of Isotopically Laheled Derivat.ives . . . . . . . .. ........................... .. B. Nitrogen-16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................... .. .. I V . Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. N-2-Fluorenylacetamide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. A. Modes of Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effect of Species and Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. C . Synergistic and Antagonistic Effects . . . . . . . . . . . . . . .
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ELIZABETH K. WEISBURGER AND JOHN H . WEISBURGER
C. Protein Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Amino Acid Sripplcmcnts .. ........................ ................................
370 371 373 374 374 375
F. Vitamins. . . . . . .................................. G. Miscellaneous Dietary Constituents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Influence of Hormonal Factors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Steroid Hormones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Thyroid and Hypophyseal Hormones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 C. Pancreatic Hormones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 7. Interaction of Carcinogen and Tissue Constituents. . . . . . . . . . . . . . . . . . . . 382 A. In Vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Vilro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Possible Modes of Combination.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 D. Relation of Binding to Carcinogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
B. Ascorbic Acid. . .
. . . . . . . . . 392
C. Esterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Amidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Deaminases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
395 396 396
VI. Metabolism 1. N-2-Fluorenylacetamide and 2-Fluorenamine. .. A. Analysis of Metabolites B. Analysis of Metabolites by Isotopic Procedures 406 C. Acylation and Deacylation of 2-Fluorenamine . . . . . . . . . . . . . . . . . . . . . D. Hydroxylation of the Fluorene Nucleus followed by Conjugation with 409 Glucuronic and Sulfuric Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Dietary Modifications and the Metabolism of 2-Fluorenamine and . . . . . . . 414 Related Compounds 3. N-2-Fluorenylbenzamide... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. N-(l-Hydroxy-2-fluorenyl)acetamide and N-(3-Hydroxy-2-fluorenyl)acetamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. N-(7-Hydroxy-2-fluorenyl)acetarnide.. . .
VII. Summary and Conclusions
. . . . . . . . . . . . . 419 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
I. INTRODUCTION While cancer is a disease which has been known for thousands of years, research on the origin and development of this affliction is a comparatively recent endeavor. The discovery in the last few decades of pure chemical carcinogens as various hydrocarbons, aromatic amines, and azo dyes has given investigators a number of tools whereby the progressive changes involved in the induction of cancer can be followed. One type of carcinogenic agent, namely N-2-fluorenylacetamide’ (2-FAA, 2-acetylaminofluorene, 2-acetamidofluorene, AAF) , is characterized by its ability to cause neoplasia in a variety of tissues. This remarkable property, in some respects similar to that encountered with spontaneous tumors, aroused the curiosity of investigators in many laboratories and led subsequently to a considerable literature. Several aspects of the carcinogenic properties of N-2-fluorenylacetamide and related compounds have been reviewed briefly by Bielschowsky (1947b), Miller and Miller (1955), and Morris (1955~)although no comprehensive outline on this subject has appeared. 11. HISTORICAL The United States Patent number 2,197,249 entitled “Insecticide” was granted on April 16, 1940 to Houston V. Claborn and Lloyd E. Smith of the U. S. Department of Agriculture. A portion of the introduction to the patent reads: “An object of the invention is to provide a material suitable for use as an insecticide. Another object of the invention is to provide a material which is relatively non-toxic to man and domestic animaIs when taken by mouth and which can be used in place of lead arsenate and other arsenicals for destroying insects without leaving a harmful residue on fruits and vegetables.” Among the various fluorene derivatives described, one of the claims of the patent related to “an insecticide containing as its essential ingredient 2-aminofluorene.” This claim was substantiated, for the compound a t a coilcentration of 40 p.p.m. of water gave a 99% kill of mosquito larvae, and killed all of the second instar of tobacco hornworm at a concentration of 1 part in 400 of water. Thus, only the criterion cited in the second paragraph above, “. . . material which is relatively nontoxic to man and domestic animals . . . ” had to be satisfied before this material could be released for use as an effective insecticide. 1 The nomenclature system rccomrnended by Chemical Abstracts will be used throughout this review.
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ELIZABETH K. WEISBURGER AND JOHN H . WEISBURGER
This problem was undertaken by Wilson et al. (1941), who reported that N-2-fluorenylacetamide (2-FAA), the acetyl derivative of 2-fluorenamine referred to in the patent, had no demonstrable acute toxicity in rats, mice, and rabbits at a dosage up to 50 mg./kg. by subcutaneous injection, and up to 1 g./kg. by feeding. A temporary refusal of rabbits to eat was the only deleterious symptom observed. Owing to the absence of acute toxic symptoms, Wilson et al. (1941) at first coiicluded that high concentrations could be tolerated by continuous feeding. Thereafter, from a large series of long-term feeding experiments these authors showed that time of death, percentage mortality, and increase in body weight were all functions of the dietary concentration of 2-FAA, demoiistrating a chronic toxicity of this compound. From these chronic toxicity studies, the important discovery was made on autopsy of the rats surviving 100 days or more on diets containing various amounts of 2-FAA. It was noted that not only were the livers enlarged, yellow, and nodular in appearance but numerous tumors affecting a great variety of organs and tissues had developed. These results abruptly terminated the service of 2-FAA as an insecticide and opened a new approach using 2-FAA and related compounds t o study the mystery surrounding the genesis of cancer. While the results so far achieved have not attained this desirable goal, some facets of the problem have become somewhat clearer, and there is hope that eventually the intimate mechanism of action of this carcinogen and, perhaps, of others as well will be understood.
111. CHEMISTRY It will be the objective of this section to provide a background of the chemistry of the fluorenamines, their methods of preparation, and some of their reactions. I n addition, the syntheses of some related compounds which have been employed in the study of the mechanisms of carcinogenesis by 2-FAA are discussed. Some familiarity with the properties of these chemicals should be helpful in accounting for their behavior in experiments dealing with their carcinogenicity or metabolism. 1. Chemistry of Fluorene
Fluorene was first isolated from coal tar, and the structural formula was proved to be as shown in formula (1)-a biphenyl with a methylene bridge resulting in an aromatic hydrocarbon with activated hydrogens on the aliphatic type 9-carbon. The chemistry of fluorene has been reviewed by Everest (1927) and by Rieveschl and Ray (1938). It is now generally agreed on the basis of stereochemical (J. H. Weisburger et al., 1950), spectroscopic studies (Merkel and Wiegand, 1947, 1948), and x-ray analyses (Brown and Bortner, 1954; Burns and Iball, 1954) that fluorene pos-
2-FLUORENAMINE
A N D RELATED COMPOUNDS
335
sesses a planar structure. Moreover, fluorene has a benzenoid rather than a iiaphthalenoid structure in that the double bonds are not fixed (Lothrop, 1939). H H //
(1)
In fluorenc, the 2-position is first attacked by electrophilic substituents since this position is para to the biphenyl linkage. In contrast, a diagram showing the distribution of electronic charges as published by Pullman and Berthier (1948) would lead one to assume that the 1- or the 4-position would be attacked first since these positions carried the highest charges. It seems that recalculation of the charge diagram by the more advanced methods presently available would be desirable to determine whether the 2-position does not actually carry the highest charge in agreement with experimental findings or whether fluorene constitutes an exceptional case. 2-Nitrofluorene is the major mononitration product of fluorene although small amounts of the 4-derivative are also formed (J. H. Weisburger, unpublished; M. J. S. Dewar and D. S. Urch, personal communication; cf. Ogata et al., 1953). Further substitution, depending, of course, on the entering group and the nature of substituent already in the 2-position, occurs in the 7-, 3-, or 1-position and to some extent in the 5-position. There is actually no para-position to the 2-position1 but the 7-position acts as if it were in a n extended para relationship to the 2-position. During oxidation of fluorene, the 9-methylene group is generally attacked first with production of fluorenone, melting a t 83434°C. However, fluorenone derivatives can be synthesized by numerous other means. Ring closure of diphenic acids or 2-biphenylcarboxylic acids can be employed. In turn, diphenic acids call be readily prepared from phenanthrenes. Fluorenones can also be readily made by diazotizing properly substituted aminobenzophenones with elimination of the diazonium group and formation of a biphenyl linkage. In other cases, oxidation of certain hydrocarbons such as fluoranthene or retene yields fluorenone derivatives. It is, therefore, not surprising that in many instances more is known of the chemistry of fluorenones than that of the corresponding fluorene derivatives. 2. Fluorenamines
A. 1-Fluorenumine. 1-Fluorenamine was first prepared by Bergmann and Orchin (1949) from 1-fluorenecarboxylic acid according to the scheme: Methyl 1-fluorenecarboxylate hydrazide of 1-fluorenecarboxylic acid + ---f
336
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
l-fluorenecarbonyl azide ---f l-fluorenylurethan + l-fluorenamine, melting point 124-124.6"C. The urethan was hydrolyzed to the amiiie in a sealed tube. Weisburger and Weisburger (1953) found that l-fluorenecarbonyl azide could be readily prepared directly from l-fluorenecarbonyl chloride and sodium aside. By heating the azide with acetic anhydride, N-l-fluorenyldiacetamide, which was easily hydrolyzed to l-fluorenamine, was obtained in good yields. Sawicki and Chastain (1956))who combined some of the steps to simplify this procedure even further, have also prepared a number of various acyl derivatives of l-fluorenamine. B. ,%'-Fluorenumine. 2-Fluorenamine (2-FA) was prepared by Strasburger (1883) by destructive distillation of 4-aminodiphenic acid. Later, Strasburger (1884) showed that the same product was obtained by reduction of 2-nitrofluorene with zinc and hydrochloric acid. Diels (1901) used zinc dust, calcium chloride, and aqueous ethanol to reduce 2-nitrofluorene to 2-FA, melting point 127.5"C. This is essentially the method recommended by Kuhn in Organic Syntheses (1943). Sampey and Reid (1947) have revised this procedure somewhat by decreasing the amount of zinc dust, increasing the reaction time, and adding a small amount of acetic acid to solubilize the 2-nitrofluorene. In our work we have consistently obtained high yields of 2-FA with the Kuhn procedure, modified by employing only one-third the amount of zinc dust and extending the reaction period 2 hours longer. Recently Litvinenko and Grekov (1957) reported quantitative yields of 2-FA by the use of hydrazine and Raney nickel in methanol. Other reducing agents applied for the preparation of 2-FA from 2-nitrofluorene have been stannous chloride (Austin, 1908), sodium hydrosulfite (Buu-Hoi and Ki-Wei, 1944)) and recently iron powder and hydrochloric acid (Gray et al., 1955; Hayashi et al., 1955; Campbell and Temple, 1957). Usually, however, neither in convenience of preparation nor in the yields obtained, do these methods measure up to the procedure given in Organic Syntheses. By catalytic hydrogenation of 2-nitrofluorene in ethanol a t 100 p s i . and 80"C., Rombach and MacGregor (1954) obtained quantitative yields of 2-FA, while we have had similar results by low-pressure hydrogenation (30-50 p s i . ) with platinum oxide catalyst at room temperature in ethanol. However, Campbell and Temple (1957) found that hydrogenation over Raney nickel in ethyl acetate yielded 2,2'-azoxyfluorene. Acetylation of 2-FA gives N-2-fluorenylacetamide (2-acetylaminofluorene) (2-FAA) which was reported by Strasburger to melt at 188°C. Other investigators have observed melting points varying from 186 to 198OC. for this compound. Upon oxidation of 2-nitrofluorene, the corresponding fluorenone is obtained. Reduction of 2-nitro-9-fluorenone with ammonium sulfide (Diels, 1901) afforded 2-amino-9-fluorenone1deep red, m.p. 163°C. Bennett
2-FLITOREXAMINE
A N D RELATED COMPOUNDS
337
aiid Noyes (1930) o1)taiiicd excelleiit yields of 2-an~inofluorenoneby lowpressure catalytic reduction of 2-nitrofluorcnonr, which we have been able to confirm. A diagram showing the calculated distributioii of electronic charges in 2-fluorenamine indicates that the I-, 3-, 5-, 7-, and 8-positions of 2-FA rarry charges of decreasing magnitude in that order (Pullman and Pullman, 19.55). 0 989 1002
H,
loo4
1030
0 998
(11) Diagram of charges for 2-flriorenaminr (from Pnllman and Pullman, 195.5, p. 189)
These five positions do indeed seem to be activated during the metabolism of 2-FA as will be discussed later. On the other hand, the 3- aiid 7-isomers were obtained in the highest yields during attack of 2-FA by electrophilic reagents, with small amounts of l-substituted derivatives being isolated in some cases. The relative distribution of isomers obtained may, however, depend on the rate of substitution a t a particular position which could involve other factors besides the charge on the carbon atom, such as solubility and steric effects. Although the charge diagram makes it possible to determine which positions will be attacked, it does not necessarily follow that the position with the highest charge will correspond to that of the most abundant isomer formed during a reaction. Additional work on the correlations between theoretical and experimental approaches to the problem may serve a useful purpose. a. Nitro derivatives. Nitration of 2-FAA and hydrolysis of the product was reported by Diels et al. (1902) to yield a mixture of 1-nitro-Pfluorenamine and 7-nitro-2-fluorenamine which were separated on the basis of the insolubility of l-nitro-2-fluorenamine in dilute hydrochloric acid. Eckert and Langecker (1928) showed later that the less basic insoluble compound was really 3-nitro-2-fluoreiiamine. However, Sawicki et al. (1956a) have found recently that nitration of 2-FAA does take place to the extent of less than 1% in the l-position (cf. also Ishikawa and Hayashi, 1957), in addition to nitration mostly in the 3- and 7-positions. Nitration of 2-FAA with fuming nitric acid led to the 3,7-dinitro derivative (Ishikawa and Hayashi, 1956). I n nitrating other derivatives of 2-FA, substitution appears to occur almost exclusively at the 3-position. Nitration of 7-methyl or 7-ethyl-N-2fluorenylacetamide or of N-2-fluorenylmethanesulfonamide (Sawicki,
338
ELIZABETH K. WEISBTJIZCEIl AND JOHN H . WEISBUIlGER
195613) also gave a 3-nitro derivative as did N-2-fluorenyl-p-toluenesulfonamide (Bell and Mulholland, 1949; Ishikawa and Hayashi, 1957), 2-carbethoxyaminofluorenoiie and 9-0x0-2-fluorenylbenzamide (Eckert and Langecker, 1928). However, N-2-fluorenylperfluorobutanamide gave largely the 7-nitro derivative (Sawicki et al., 1956a). Nitration of 9-0x0-2-fluorenylacetamide led to results analogous to those with 2-fluorenylacetamide, but nitration of 2-aminofluorenone in concentrated sulfuric acid afforded 2-amino-7-nitro-9-fluorenone exclusively (Eckert and Langecker, 1928). b. Halogen derivatives. Iodo derivatives. Iodination of 2-FAA with iodine monochloride led to the formation of a substance iodinated both in the 7-position of the fluorene ring and on the amide nitrogen (E. K. Weisburger, 1950). The N-iodo substituent was very unstable and could be removed with sodium bisulfite, leaving 7-iodo-2-fluorenylacetamide. The structure of the latter compound was proved by its preparation from the known 7-nitro-2-iodo fluorene. I n order to introduce iodine in the 3-position of 2-FAA (see formula 111),it was found necessary to mercurate 2-FA with mercuric acetate and then replace the organometallic group with iodine after acetylation of the amine (E. K. Weisburger et al., 1951).
&NHCOCH, \
/I
(111)
Bromo derivatives. Campbell et al. (1940) found that bromination of either 7-bromo-2-fluorenylacetamide or N-2-fluorenyl-p-toluenesulfonamide yielded the corresponding 3,7dibromo derivative. Bell and Mulholland (1949) brominated 2-FAA (in acetic acid, chloroform, or carbon tetrachloride) and found that in all cases, 7-bromo-2-fluorenylacetamide was the main product, while a small amount of 3,7-dibromo-2-fluorenylacetamide was isolated as a secondary product. However, reaction of 2-FA with bromine in acetic acid gave 1,3-dibrom0-2-fluorenamine and a secondary product which was probably 3-bromo-2-fluorenamine. Lately, Campbell
2-E’LI;ORENAMINE
A N D RELATED COMPOUN1)S
330
and Temple (1957) have reported that in order to obtain pure 7-bromo-2fluorenylacetamide, uncontaminated by the 3,7-dibromo compound, it was best to nitrate 2-bromofluorene1 reduce to the amine, and acetylate the resulting 7-bromo-2-fluorenamine. Recently, Fletcher and Pan (1956) found that treatment of 2-amino-9fluorenone with ethyl bromide in dimethyl sulfoxide yielded N-2-ethylamino-3-bromo-9-fluorenone and a small amount of 2-amino-3-bromo-9fluorenone. However, N-alkylation did not occur with t-butyl bromide in dimethyl sulfoxide which gave a 75% yield of 2-amino-3-bromo-9-fluorenone. Under similar conditions, 2-FA was converted to both 1-bromo- and 3-bronio-2-fluorenamine, while an excess of the reagent led to some of the 3-bromo derivative and mainly to 1,3-dibromo-2-fluorenamine. A solution of 48% aqueous hydrobromic acid in dimethyl sulfoxide was also an effective brominating agent, giving an 85% yield of 2-amino-3-bromo-9fluorenone from 2-amino-9-fluorenone (Fletcher et al., 1957). Chloro derivatives. Chlorination of either 2-FAA or 9-0x0-2-fluorenylacetamide gave the 3-chloro derivative (Bell and Gibson, 1955). I n order to obtain 7-chloro-2-fluorenylacetamide, 7-nitro-2-chlorofluorene was reduced t o the amine and acetylated (Schulman, 1949). An improved method of preparing the intermediate, 7-nitro-2-chlorofluorene, was devised by Gutmann and Ray (1951), involving the chlorination of 2-nitrofluorene with iodine as a catalyst. Fluoro derivatives. Nitration of 2-fluorofluorene (Miller et al., 1955) gave a 7-nitro derivative, which was also obtained from 7-nitro-2-fluorenamine by means of the Schiemann reaction. Reduction of 7-nitro-2-fluorofluorene1 followed by acetylation, afforded 7-fluoro-2-fluorenylacetamide, m.p. 197-198OC. As far as is known, no other ring-substituted fluoro derivatives of 2-FAA have been prepared. c. Allcyl derivatives. N-Alkyl derivatives. The N-methyl derivative of 2-FA was made by Bielschowsky and Bielschowsky (1952) by alkylation of the sodium derivative of 2-FAA with methyl iodide, followed by hydrolysis to the amine. Little and Ray (1952), who alkylated the sodium derivative of N-2-fluorenyl-p-toluenesulfonamide with methyl iodide, used 50% hydrochloric acid in a sealed-tube hydrolysis of the alkyl sulfonamide to obtain N-methyl-2-fluorenamine. However, Fletcher et al. (1955) reported that N-methyl-N-2-fluorenyl-p-toluenesulfonamidecould be hydrolyzed quantitatively with concentrated hydrochloric acid in boiling glacial acetic acid, a much simpler procedure. N1N-Dimethyl-2-fluoreiiamine was prepared by alkylatioii of 2-FA with dimethyl sulfate, the highest yield obtainable being 60-70Oj, (Weisburger and Quinlin, 1948; Bell and Mulholland, 1949). Alkylation of 2-FA with methyl iodide has given yields of the dimethyl derivative ranging from
340
ELIZABETH K. WEISBUltCEll S N D JOHN H . WEISBURCER
74% (by refluxing in methanol, Vaiiags and Vanags, 1950) to 96% (by reacting in a sealed tube, Wittig et al., 1950). Dimethylation, by treating with methanol in an autoclave, has also been employed, but the yields obtained are not known (Ziegler and Wenz, 1950). The lower yields obtained with dimethyl sulfate may have been due partly to oxidation of 2-FA in the strong alkaline medium. Carrying out the reaction in ail inert atmosphere may have led to better results. Recently Fletcher et al. (1955) have suggested the use of ethyl or methyl phosphates or dialkyl alkylphosphonates to prepare N-alkyl derivatives of fluorenamines. The combination of convenience and, in some cases, improved yields would make this method valuable in certain instances. d. ?'-Alley1 derivatives. In view of the alterations in the carcinogenic activity of N,Ndimethyl-p-phenylazoaniline that were observed by the addition of alkyl groups in the second ring of the molecule (Miller and Miller, 1953), it would be of some interest to test the corresponding alkyl2-fluorenamines. What seems to be a straightforward synthesis leading t o some 7-alkyl-2-fluorenamines has been reported by Sawicki (1954). Nitration of either 2-ethyl- or 2-methylfluorene, both available from 2-acetylfluorene, gave good yields of the 7-nitro derivatives. Reduction afforded the desired 7-ethyl- or 7-methyl-2-fluorenamine. The latter compound was also prepared by Ogata et al. (1953) by a somewhat more difficult synthesis with poorer yields. Condensation of fluorene with chloroacetic acid led t o a mixture of 2- and 4-fluoreneacetic acids which were separated by fractional crystallization. Nitration of 2-fluoreneacetic acid, reduction, and dry distillation of the potassium salt of the amine gave 7-methyl-a-fluorenamine. From 2-acetyl-7-nitrofluorene, Sawicki and Wade (1954) have made 7-acetyl-2-fluorenylacetamide. The spatial configuration of this compound is somewhat similar to that of certain steroids, which could endow it with interesting physiological properties in addition to the effect of the 2-amino group. Experiments on the carcinogenicity of these 7-alkyl derivatives of 2-fluorenamine will be of interest to determine the effect of a blocked 7-position. Unless the in vivo detoxification of such compounds is expedited by conversion of the alkyl groups to a carboxyl, it might be expected that these compounds would be a t least as carcinogenic as 2-FA. Now that adequate syntheses for these substances are available, it is hoped that the results of the biological tests will be forthcoming. e. N-Acyl derivatives of 2-jluorenamine. In view of the combination of carcinogens to tissue proteins, some of the more interesting amides derived from 2-FA are those in which an amino acid is combined with the aromatic amine. Hirs (1949) reacted 2-FA with succinic anhydride to yield N-2-fluoreiiylsuccinamic acid and with bromoacetic acid to give N-2-fluorenyl-
2-FLUOREXAMINE AND
RELATED COMPOUNDS
34 1
glycine. Rombach and MacGregor (1054) coupled phthalyl-protected amino acids with either 2-FA or N-(7-aniino-2-fluorenyl)acetamide. Removal of the protective group with hydrazine hydrate gave the free amine which could be coupled again. In this fashion were prepared mono- and diglycyl and mono- and diphenylalanyl derivatives of the type shown (formula IV) where 71 was 1 or 2 and R either H or CBH6CH2-.
&
(NIICOCIJR),,NII,
Greenstein et al. (1955) have made the carbobenzoxy amino acid and the hydrobromides of the amino acid derivatives of various carcinogenic amines, including 2-FA. By use of the mixed anhydride procedure, both D- and L-forms of 24 carbobeneoxy amino acids were condensed with 2-FA (V). The carbobenzoxy group was removed with dry hydrogen bromide to give the hydrobromide of the amino acid derivative. These compounds were used for studies on enzymatic hydrolysis by tissue homogenates.
&
NHCORNH,
Peck and Creech (1952) prepared 7-isocyanato-2-fluorenylacetamide from 7-amino-2-fluorenylacetamide. The isocyanate reacted with 6-aminohexanoic acid yielding a reference compound 6-[3-(7-acetamido-2-fluorenyl)ureidolhexanoic acid. Condensation of the isocyanate with crystalline horse serum albumin gave a protein conjugate which contained 32 molecules of the fluorene prosthetic group per molecule of protein, as determined by spectrophotometric methods (Creech and Peck, 1952). With bovine serum albumin, there were 30 molecules of fluorene prosthetic group per molecule of protein. The 2-FAA bovine serum-albumin conjugate was used, along with several other hydrocarbon-protein conjugates, as a test antigen against antiserums from 7,12-dimethyl-5-ureylenebenz[n]anthracene-horse serum albumin (Creech et nl., 1955).
342
ELIZABETH K . \I'EISBUItGEIL AND JOHN €1. \VEISBUR(hEII
Various amino acyl derivatives of 2-fluorenamine have also been made for testing as local anesthetics or antispasmodics (Svedres and Jenkins, 1952; Novelli, 1955; Bhargava and Nair, 1957) and were indeed found to have some pharniacologic value. The fact that these compounds would probably be hydrolyzed to 2-FA upon metabolism would, however, preclude their use in medicine. Other amides which have been synthesized are the diacetyl (Morris et al., 1950a) and the fluorinated acyl derivatives of 2-fluorenamine (Sawicki and Ray, 1953a,b; Sawicki, 1956a; Sawicki and Chastain, 1956). These compounds are of special interest since Morris (1955~)found N-(a-fluorenyl)2,2,2-trifluoroacetamide to be more carcinogenic than 2-FAA. Since the organic chemist can often prepare in a relatively short time a compound which requires a year or more for complete biological testing, there are a number of amides from 2-fluorenamine which have not been tested or on which only preliminary reports have appeared. Several of these which seem to be relatively noncarcinogenic are N-2-fluorenylbenzamide (Bachmann and Barton, 1938; Gutmann and Peters, 1953a), N-2-fluorenylphthalamic acid (Ray et al., 1953; Fletcher et al., 1955), and N-2-fluorenyl-p-toluenesulfonamide (Campbell et al., 1940). Sawicki (1952) has made several alkyl and acyl derivatives of the latter compound and other analogs as N-2-fluorenylmethanesulfonamide and 7-mesyl-2-fluorenamine (Sawicki, 195613). To the organic chemist, it is often a frustrating experience to wait for the biological results, but it is only through the slow process of animal tests that any correlation between the structure of a compound and its carcinogenicity can finally be made. However, such studies encompassing several disciplines can often be expedited by collaboration between mutually complementary teams. One successful arrangement of this type, known to the Reviewers, is that existing for several years between Dr. H. P. Morris, of the National Cancer Institute in Bethesda, and Dr. F. E. Ray, of the University of Florida Cancer Research Laboratory, who jointly decide on the structures of the compounds to be synthesized for animal testing programs or for metabolic studies. Frequent communication of preliminary results to the other members greatly aids in shifting research to the most promising approaches. The beneficial effects of interdisciplinary cooperatioii have recently been emphasized by Lacassagne et al. (1956), whose group has achieved impressive results thereby. f. Hydroxy derivatives. Diels (1901) synthesized 2-amino-9-fluorenol by refluxing 2-nitro-9-fluorenone with zinc dust and calcium chloride in ethanol until the mixture was colorless, both the nitro and the 9-keto group being reduced simultaneously. The Diels procedure was used by Miller et al. (1955) to prepare N-(9-hydroxy-2-fluorenyl)acetamide. The metabolites of 2-FAA thus far identified have been the 1-, 3-, 5-, 7-, and 8-hydroxy derivatives, in addition to 2-FAA and 2-FA. Of the
2-FLUOREXAMINE
AND RELATED COMPOUNDS
343
hydroxylated derivatives, the 7-compound is perhaps synthesized most readily by the organic chemist. Ruiz (1928a) discovered that nitration of 2-p-toluenesulfonylhydroxyfluorene gave a 7-nitro derivative which after reduction and hydrolysis afforded 7-amino-2-fluorenol. As far as can be ascertained, this method remained buried in the literature, so to speak, for it does not appear to have been used by any other investigator. Bielschowsky (1945) prepared 7-hydroxy-2-fluorenylacetamide for a standard in metabolic studies by diazotizing 7-nitro-2-fluorenamine1 heating to form 7-nitro-2-fluorenol, reducing, and acetylating the amino group. Since 7-nitro-2-fluorenamine may be fairly readily prepared by either monoreduction of 2,7dinitrofluorene with hydrogen sulfide or by nitration of 2-FAA, this method appears to be a reasonable one. Variations have been reported by Goulden and Kon (1945), by Gutmann (1952), by J. H. and E. K. Weisburger, who also prepared 7-methoxy-2-fluorenylacetamide (1954), and by Hayashi et al. (1955). A procedure which seems to be much simpler and which really is a modification of the early method of Ruiz (1928a) was recommended recently by Bryant and Sawicki (1956). They found that nitration of 2-acetoxyfluorene produced good yields of the 7-nitro derivative. The precursor of 2-acetoxyfluorene1 2-fluorenol, is obtained readily from 2-FA (Ray and Hull, 1949). By contrast, when 2-fluorenol and 2-methoxyfluorene were nitrated, 3-nitro-2-fluorenol and 3-nitro-2-methoxyfluorene were the chief products (Ray and Hull, 1949; Ruiz, 192813). N-(l-Hydroxy-2-fluorenyl)acetamide was made by R. K. and J. H. Weisburger (1954) by nitrating l-fluorenol, separating the 2-nitro-l-fluorenol from the 4-nitro-l-fluorenol by chromatography, reducing the nitro compound, and acetylating the amino group in aqueous buffer in order to avoid acylating the hydroxy group. An attempt was made to simplify the procedure by coupling l-fluorenol with diazotized sulfanilic acid, reducing the azo compound, and acetylating the aminofluorenol. However, the product which could be isolated was 1-hydroxy-4-fluorenylacetamide and not the desired 2-isomer. A second ortho-hydroxy derivative, 3-hydroxy-2-fluorenylacetamide, was synthesized by nitration of 3-fluorenol, followed by chromatography, reduction, and acetylation (E. K. and J. H. Weisburger, 1954). The structures of these compounds were proved by ultraviolet absorption spectra and conversion to oxazoles. Bryant and Sawicki (1956) lately reported a much simpler. synthesis of 3-hydroxy-2-fluorenylacetamide involving coupling of 3-fluorenol with benzenediazorlium chloride, followed by reduction of the azo compound to 2-amino-3-fluorenol and acetylation of the nmine. It would appear that a more direct method of obtaining 3-hydroxy-2-
344
ELIZABETH I<. IYEISBURGER A N D JOHN H . WVEISBUHGh~R
fluorenylacetamide would be to diazotize 3-amino-2-fluorenylacetamide (which is readily available by nitration of 2-FAA and reduction of the nitro derivative) and replace the diazonium group by the hydroxy group. However, Gutmann and Fenton (1955) found that when sodium nitrite was added to 3-amino-2-fluorenylacetamide under conditions generally used for diazotization, a triazole was formed. Similar results with monoacetyl derivatives of other aromatic ortho-diamines had been reported previously by Bergmann and Bentov (1954). The other hydroxy derivatives of 2-FAA have been synthesized starting from the properly substituted nitro carboxylic acids. Nitration of 4-fluorenecarboxylic acid gave as the main product 7-nitro-4-fluorenecarboxylic acid. Substitution of the carboxyl group by an amino group, diazotization, and replacement by a hydroxy group afforded 7-nitro-4-fluorenol which was reduced and acetylated to yield the desired 5-hydroxy-2-fluorenylacetamide (E. K. and J. H. Weisburger, 1955). In a similar fashion, starting from 1-fluorenecarboxylic acid, N-(8-hydroxy-2-fluorenyl)acetamide was prepared (E. K. and J. H. Weisburger, 1956). Boyland et al. (1953) have used potassium persulfate to oxidize amines of the benzene and naphthalene series to ortho-aminophenols. Gutmann et al. (1956a) have attempted to apply this reaction to fluorene, 2-FAA1 and 2-nitrofluorene. No oxidation products of fluorene and 2-FAA were found. However, 2-nitrofluorene did oxidize at the 9-position to yield 2-nitro-9-fluorenone and 2,2'-dinitr0-A~~~'-bifluorene.Oxidation of 2-FA with permonophosphoric acid yielded small amounts of chromatographically detected, but not isolated phosphoric esters (Boyland and Manson, 1957). g. Condensation reactions. Neish (1948b) has suggested that under physiological conditions, 2-FA may condense with certain body constituents t o produce heterocyclic compounds which may actually be responsible for the carcinogenic properties ascribed to the amine. Although there is no experimental evidence for such condensations during the metabolism of 2-FA, these reactions still remain a possibility. It is therefore of some interest to examine briefly such reactions of 2-FA to determine what possible structures could be found and in which position of the fluorene nucleus the ring would probably form. Both Buu-Ho'i and Ki-Wei (1944) and Neish (1948~)condensed 2-FA with dihydroxymalonic ester to give an isatin. However, Neish assumed that ring closure had occurred in the 1-position while Buu-Hoi' suggested the 3-position. Campbell and Stafford (1952) proved afterwards that, the latter view was correct (VI). 2-FA underwent the Skraup reaction to give an indenoquinoline, the structure of which was not definitely proved (Diels and Staehlin, 1902) with regard to whether ring closure had occurred in the 1- or 3-position of
2-FLUORENAMINE
AND RELATED COMPOUN1)S
345
1%
COOEt COOEt
//
HO COOEt
I.
H
the fluorene molecule. Similarly, Hughes et al. (1938) reacted 2-FA with ethyl acetoacetate, followed by ring closure which they thought involved the 1-position; Neish (1948b) assumed also that pyruvic acid and 2-FA led to a cinchoninic acid in which the 1-position was involved. However, in a recent publication, Campbell and Temple (1957) have adduced evidence by chemical and spectroscopic means that the products obtained by Diels and Staehlin (1902), by Hughes et al. (1938), and by Neish (194813) were the results of ring closure in the 3-position of the fluorene nucleus. The one exception seems to be the report of Bremer and Hamilton (1951) who condensed diethyl ethoxymethyleiiemalonate with 2-FA aiid proved that in this case ring closure actually did occur in the 1-position. It thus seems most likely that any heterocyclic compounds resulting from condensation of 2-FA with body constituents during metabolism would contain rings involving the 2- and 3-positions of the fluorene nucleus. C. S-Fluorenamine. 3-Fluorenamine has been made by reduction of 3-nitrofluorene (Hayashi and Nakayama, 1933), which was in turn obtained by small-scale deamination of 3-nitro-2-fluorenamine (cf. Bardout, 1931). Campbell and Stafford (1952) reported that they were unable to deaminate this compound readily. Therefore they converted the nitro smine into 2-bromo-3-nitrofluorene, which when reduced with stannous chloride gave a poor yield of 3-aminofluorene. In contrast, Eckert and Langecker (1928) reported no special difficulties in the deamination of the corresponding fluorenone, 2-amino-3-nitro-9-fluorenone, although they stated that the yields were sometimes variable. A similar type of behavior has been found by Barker and Barker (1954) who attempted unsuccessfully to deaminate 3,6-dinitro-2,7-fluorenediamine by tetrazotization and reduction with ethanol. The corresponding fluorenone, however, was
346
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
deaminated readily, affording 3,6dinitro-9-fluorenone. The metu-directing 9-keto group of the fluorenones may strengthen the basicity of the amine, thus facilitating diazotization and subsequent removal of the amino group. E. K. Weisburger (1955) prepared 3-fluorenamine in large quantities by Wolff -Kishner reduction of 3-amino-9-fluorenone. The latter compound was obtained from 2-biphenylamine by the slightly modified method of Ray and Barrick (1948). Another approach to the synthesis of this amine was developed recently (Weisburger and Weisburger, 1957). The Grignard reagent from 2-bromo-l,4-dimethylbenzenereacted with cyclohexanone to yield a tertiary alcohol readily dehydrated to 1-(2,5-xylyl)cyclohexene. Sulfur aromatized this compound at the boiling point to 2,5dimethylbiphenyl, which in turn could be oxidized to 2,5-biphenyldicarboxylic acid. Ring closure with polyphosphoric acid (incidentally this agent is often superior to sulfuric acid for similar reactions) supplied 9-oxo-3-fluorenecarboxylic acid which was converted to 3-fluorenecarboxylic acid in good yield. 3-Fluorenamine was derived from this intermediate by a modified Curtius reaction. While this series of transformations appears long, it is amenable to large-scale batches and should thus be useful for the preparation of sizable quantities of material. D. 4-Fluorenamine. 4-Fluorenamine was obtained by J. H. Weisburger et al. (1952) from 2,5-dinitrofluorene by monoreduction with hydrogen sulfide, deamination of the nitro amine, and reduction of the 4-nitrofluorene thus formed. Later they discovered that in the monoreduction both possible isomers, 5-nitro-2-fluorenamine and 7-nitro-4-fluorenamine, were formed (J. H. and E. K. Weisburger, 1956). The crystallization procedure used in the earlier work had fortunately separated the desired isomer, 5-nitro-2-fIuorenamine, as the acid sulfate. The fact that the reduction was nonspecific, coupled with the difficulty of obtaining pure 2,5dinitrofluorene, eliminates this as a practical method of synthesis. Neish (1953) has published a six-step synthesis of 4-fluorenamine starting from phenanthrenequinone, but the yields in several steps of his procedure are also low. Schinz et al. (1955) reported a straightforward four-step synthesis of 4-fluorenamine using a Hofmann hypobromite reaction on 4-fluorenecarboxamide. The yields obtained were good, and the starting material, diphenic acid, was readily available, making their method the one of choice. Sawicki et al. (1956b) have also used the Hofmann reaction to prepare 4-fluorenamine. They adapted the synthesis to a smaller scale to prepare 4-fluorenamine-N15and the corresponding acetyl derivative. 3. Other Fluorene Derivatives
A. 2,7-Substituted Compounds. N,N’-2,7-Bisfluorenyleneacetamide is prepared from 2,7-fluorenediamine which is readily synthesized by reduction of 2,7dinitrofluorene with tin and hydrochloric acid (Schulman, 1949).
2-FLUOREXIMINE AND RELATED COMI'0UNI)S
347
2,7-Fluorenediamine itself has been proposed for use in dyes and as an analytical reagent for various determinations (cf. Rieveschl and Ray, 1938). A fairly recent patent also describes the use of sulfonic acid derivatives of acylated 2,7-fluorenediamine as optical bleaches and brightening agents in textiles, paper, felt, plastics, cellulose products, and varnishes (Eberhart and Rarabutes, 1953). While the sulfonic acid group might render such compounds harmless with regard to a possible carcinogenic effect, this point should be established beyond doubt prior to any commercial application. In any case the unsubstituted diamine is unsuitable for uses involving ingestion by, or contact with the human subject. B. 9-Substituted Compounds. 9,9'-BiJluorene and Ag*9'-BiJl~~rene Derivatives. I n 1948 Pinck proposed that the carcinogenic action of 2-FAA was due to conversion to either the 2,2'diacetamido-9,9'-bifluorene, or the corresponding Ag t9'-bifluorene and that these substances might be the actual carcinogens. In order to test Pinck's hypothesis, E. K. Weisburger el at. (1949) prepared these compounds. No attempt was made to separate any isomers of the bifluorene. However, depending on whether the A9s9'bifluorene was obtained by fusion of the 9,9'-bifluorene or by selective reduction of 2,2'dinitr0-A~~~'-bifluorene, two different forms, melting a t 190°C. and 264"C., respectively, were found. It was shown later by Kuhn et al. (1953) that the two different forms of 2,2'-dia~etarnido-A~~~'-bifluorene which were described by Weisburger and co-workers were essentially the same isomer. Kuhn and associates were able to separate the cis and trans isomers of 2,2'-Ag~9'-bifluorenediamine by means of the d-camphorsulfonic acid salts or by chromatography on alumina. However, they could not prove which of their isomers had either the cis or trans form. One problem was that the acetyl derivative of the slower moving amine moved faster on an alumina column than the acetyl derivative of the faster moving amine. It has been proposed by Bergmann (1948) on the basis of various experimental data that Ag*9'-bifluoreneis a diradical (free radical). I n this event no true cis-trans isomers could be expected to exist in a stable form, perhaps explaining some of the difficulties encountered. It seems that determination of the electron paramagnetic resonance spectrum of A9,g'-bifluoreneshould make i t possible to decide definitely whether this compound does exist as a diradical. An experiment by Hutchison et al. (1952) indicated th a t Ag-gt-bifluoreneshowed no paramagnetic resonance absorption at room temperature. Thus, a diradical form makes a t best a minor contribution to the state of the molecule. C. Fluorene Quinones. In the fluoreiie series, very few yuinones have beeii prepared. Koelsch and Flesch (1955) synthesized 1,4,9-fluorenetrione in a seven-step series of reactions, using ethyl anthranilate as the starting material. Lead tetraacetate was used to oxidize 1,4-dihydroxyfluorenone to the quinone. Nenitzescu and Avram (1956) have recently reported the
348
ELIZABETH K. WEISBUIZOER .4ND J O H N H. WEISBURGER
synthesis of 2,7-fluorenequinone. 2,7-Fluorenediol, on oxidation with silver oxide gave the desired quinone as reddish crystals melting at 165-167°C. The compound did not appear to be very stable since even in vacuo it could be kept only 3 to 4 days. It readily formed a quinhydrone type complex with 2,7-fluorenediol. The preparation of certain fluorene quinone imines and of coupling products of these reactive intermediates was reported recently (Nagasawa and Gutmann, 1958). Oxidation of 1-amino-2-fluorenol by chemical means (lead dioxide, ferricyanide) or enzymes (cytochrome c-cytochrome oxidase) yielded the quinone imine, which added to the starting material giving a product coupled at the 4-position of the fluorene nucleus. This compound was susceptible to further oxidation. The quinone imine could react with protein in the presence of an excess of protein rather than of starting material (cf. Section IV,7,C).
4. Synthesis of Isotopically Labeled Derivatives A. Carbon-1.4. Since only one-third of a dose of 2-FAA could be accounted for by colorimetric methods that depended on the presence of a diazotizable amino group (Morris el al., 1948a; cf. Section VI,l,A), it seemed desirable to follow the pathway of 2-FAA by the use of isotopically labeled material. The pioneering work on the synthesis of C14-labeled2-fluorenylacetamide was done by Ray and Geiser (1949, 1950). It seemed reasonable, as was shown later, that the 9-carbon of 2-FAA could be labeled without having it removed from the molecule during metabolism. For this synthesis the Grignard reagent from 2-iodobiphenyl was carbonated with C1402 (from BaC1403)which was swept completely into the reaction vessel with ordinary C 1 2 0 2 . Hydrolysis of the addition complex yielded 2-biphenylcarboxylic acid-C14. Ring closure with sulfuric acid afforded fluorenone-9-P4 which was reduced by means of the Clemmensen method to fluorene-9-Cl4. Nitration, reduction, and acetylation gave the desired N-2-fluoren-9-C14ylacetamide (2-FAA-9-C14). Heidelberger and Rieke (1951) found that by use of a vacuum line (Calvin et al., 1949) during the carbonation step, which eliminated the need for sweeping over C1402 with Cl2O2, use of 80% sulfuric acid for ring closure, and reduction by a Wolff-Kishner reaction, the overall yield of 2-FAA-9-C14 could be increased t o 50% as compared to the 22% yield of Ray and Geiser. The work of Heidelberger and Rieke was independently upheld by Harris et al. (1955), who also obtained very good yields in the preparation of fluorene-9-C14 when a vacuum line and the Wolff-Kishner reduction method were used. The latter authors showed that the Clemmensen reduction of fluorenone affords, in addition to fluorene, 9-fluorenol
2-FLUORENAMINE
A N D RELSTED COMPOUNDS
340
or 9,9'-bifluorene, depending on the length of time the mixture was heated. Similar results had been reported previously by Hitchie (1946) with various methyl fluorenones. The results of Ritchie and of Harris et al. regarding the Clemmensen reduction of fluorenones have apparently been confirmed by Gutmann et al. (1956~).During their synthesis of 2-FAA-9-C14, which was quite similar to the one reported by Ray and Geiser, they employed the Clemmensen reduction with the result that an appreciable amount of high melting material was formed, making it necessary to purify the fluorene-9-Cl4 by sublimation. The contaminant which melted over 210°C. was probably 9,9'-biff uorene, reported melting point 246OC. (Graebe and Stindt, 1896). In summary, it seems pertinent to outline the best methods for the synthesis of 2-FAA-9-C14. The use of a vacuum line during the carbonation step is important to obtain a quantitative transfer of C1402 from the generating vessel into the Grignard reagent without the need for sweeping out the CI4O2with Cl202 which would decrease the specific activity of the final product. Ring closure of 2-biphenylcarboxylic acid should be accomplished under conditions that will not lead to sulfonation of the fluorenone produced, i.e., use of 80% sulfuric acid or, most suitably, of polyphosphoric acid. Reduction of fluorenone to fluorene should be effected with the WolffKishner reaction using a t least a 6-fold excess of hydrazine hydrate (Weisburger and Grantham, 1956) and no alkali (Harris et al., 1955). During reduction of 2-nitrofluorene to 2-FA, most workers have empIoyed zinc dust and calcium chloride in aqueous ethanol. Since catalytic hydrogenation affords quantitative yields of 2-FA without having some of the product adsorbed on a large amount of zinc dust, as in the chemical method, it seems that catalytic hydrogenation in ethanol would be preferable if the apparatus is available. By the use of these precautions in the synthesis, we have obtained an overall yield of approximately 60%, based on the C14 incorporation, of 2-FAA-9-C14 with a specific activity of almost 7 mc./mM. The starting barium carbonate had an activity of 10.5 mc./mM., and the drop in specific activity resulted from dilution with some pure inactive fluorene added prior to the nitration step. By reworking the mother liquors with the help of carrier compounds, it was possible to recover further amounts of radioactivity, so that over 70% of the total activity was finally accounted for in the products. A synthesis of a related compound, N-methyl-C14-2-fluorenamine, has been developed by Little and Ray (1952). The sodium derivative of N-2-fluorenyl-p-toluenesulfonamide was alkylated with meth~1-C'~ iodide, affording an excellent yield of N-methyl-C14-N-2-fluorenyl-p-toluenesulfonamide. Although Little and Ray used a sealed-tube hydrolysis to obtain the free N-methyl-C14-2-fluorenamine, the procedure of Fletcher
350
ELIZABETH K. WEISBURGER AND JOHN H . WEISBURGER
et al. (1955), which involves quantitative hydrolysis of the sulfonamide.by boiling with concentrated hydrochloric acid in acetic acid, seems to be more practical for this reaction. Other labeled amides derived from 2-FA have been prepared by reacting inactive 2-FA with a labeled acid chloride. Thus Ray and Geiser (1950) synthesized 2-FAA labeled with C14 in the acetyl group, while P-labeled p-toluenesulfonyl chloride with 2-FA gave N-2-fluorenyl-p-toluenesulfonamide-836 (Ray and Argus, 1951). Gutmann and Peters (1953a) and Nagasawa and Gutmann (1957) have also made labeled fluorene amides by reacting 2-FA-9-C14 with various acid chlorides for the purpose of studying the metabolism and deacylation of these compounds. The isomeric C14-labeled N-( 1-, 3-, and 7-hydroxy-2-fluoreny1)acetamides have been obtained biosynthetically for use in metabolism studies (cf. Section VI,4 and 5). B. Nitrogen-16. In order that the nitrogen atom of 2-FAA might be traced during metabolism, Argus and Ray (1951) have synthesized the compound tagged with N16. Since this isotope is supplied as a dilute nitric acid solution, methods had to be found to eliminate the water of solution and use the nitric acid efficiently. These difficulties were overcome by employing acetic anhydride as a solvent and concentrated sulfuric acid as a catalyst during the nitration of fluorene. In this fashion, a 79% yield (based on HNl6O3)could be obtained. Reduction and acetylation afforded 2-fl~0renamine-N~~ and N1K-2-fluorenylacetamide.In addition a method of preparing the noncarcinogenic N-4-fluorenylacetamide labeled with Nl6 has been worked out by Sawicki et al. (195613). 4-Fluorenecarbonyl chloride was treated with N16H4N03in dimethylformamide, the N16H3being liberated from the salt by triethylamine. A modified Hofmann-hypobromite reaction on the resulting 4-fluorenecarboxamide-Nl6 yielded methyl-N 1S-4-fluorenylcarbarnate, which after hydrolysis with alkali and acetylation gave N 16-4fluorenylacetamide. C. Iodine-131. An I 131-labeled derivative, N-(7-iod0~~~-2-fluorenyl)acetamide, was synthesized by Weisburger (1950) by iodinating 2-FAA with i~dine-~~~-monochloride. In view of the fact that t'he parent compound is noncarcinogenic (Morris, 1955c), a study of the metabolism of the labeled compound may shed some light on the reasons for its lack of effect. IV. CARCINOGENESIS 1. N-2-Fluoreny Eacetamide
At the time the first report by Wilson, DeEds, and Cox on 2-FAA appeared in 1941, the successful induction of tumors by pure chemicals was an achievement of relatively recent vintage. Certain polynuclear
2-FLUORENAMINE AND
RELATED COMPOUNDS
351
hydrocarbons capable of eliciting neoplasms, mostly a t the point of application to the skin, had been known for less than 10 years. The induction of liver tumors after ingestion of a number of azo dyes had been discovered but 7 or 8 years previously. Tumors of the bladder had been induced in dogs 3 years earlier by 2-naphthylamine. The newly reported 2-FAA not only induced bladder tumors but tumors of many other organs and tissues. Furthermore, the carcinogenic effect was susceptible to modification by species and strain of animal and by hormones and diets. Comparisons have been made between the chemical structure of related compounds and the resulting biological effect. A detailed summary of the results obtained with 2-FAA in terms of the mode of administration, species, strain, and sex, and the type of tumors produced is included in the comprehensive survey of carcinogenic compounds by Hartwell (1951) and by Shubik and Hartwell (1957). The reader is referred to these reports for details. A. Modes of Administration. The ingestion of 2-FAA mixed in a diet produced a wide variety of tumors a t points distant from the portal of entry (Wilson et al., 1941). Effective oral methods of administration include incorporation of the chemical in the diet (1) by solution in a suitable vehicle such as propylene glycol or neutral fat (Wilson et al., 1947b; Morris et al., 1948a), (2) by trituration in a mortar with a small portion of the diet mixture (Miller et al., 1955), and (3) by solution in a volatile organic solvent such as acetone, which is mixed with a portion of the diet and the solvent evaporated prior to incorporation into the diet mixture (Morris et al., 1950a). Besides incorporation in t,he diet, oral administration is made by direct feeding by stomach tube (Armstrong and Bonser, 1944, 1947; Morris et al., 194813, 1955), by spoon (Bonser and Green, 1950), in gelatin capsules (Campbell, 1955), and as a gum acacia suspension in the drinking water (Doniach, 1950). Repeated application of 2-FAA or 2-FA in acetone or benzene solution to the skin induces distant tumors (Bielschowsky, 1944; Harris, 1947; Morris et al., 1950a). A longer latent period is found with skin painting, but the quantity required to induce tumors appears to be much less (Bielschowsky, 194713; Morris et al., 1950a). Exposing subcutaneous homografts of lung tissue to 2-FAA or FA induced bronchogenic carcinomas with great rapidity (Horning, 1950). Partially effective methods include (1) subcutaneous injection of 2-FAA or 2-FA in a suitable solvent (Wilson et al., 1941, 1947b,c; Kirby, 1948; Lacassagiie et al., 1948; Bolis, 1950; Argus and Ray, 1956), (2) intraperitoneal injection (Bolis, 1950; Argus and Ray, 1956), and (3) subcutaneous or intramuscular implantation of crystalline material (Wilson et al., 1947b,c; Peacock, 1947). Other methods included injection of an oily solution into the pharnyx (Heiman and Meisel, 1946), injection of a tricaprylin solution
352
ELIZABETH K . WEISBURGER AND JOHN H. WEISBURGER
into the testes (Guthrie, 1956), or injection into a cecal pouch (Spjut and Eichwald, 1957). These special methods, however, often gave rise to the types of tumors usually found after oral administration. On the other hand, insertion of powdered 2-FAA or an acetone solution of 2-FAA into the ear duct was ineffective (Wilson et al., 1947b; Berenblum, 1954). It may be concluded that the carcinogenicity of compounds derived from fluorene can be determined most reliably by oral administration. This procedure requires fairly substantial quantities of the chemical tested. Repeated application to the skin can be successful with some of the active or moderately active compounds and smaller amounts of material suffice for a test. With few exceptions, other methods of administration are relatively ineffective. B. E$ect of Species and Strain. Other factors being equal, the ability of 2-FAA to induce tumors in animals decreased in the order rat, mouse, hamster, rabbit, fowl, cat, and dog. Of all the animal species so far tested, only the guinea pig and the cotton rat proved refractory to the carcinogenic effect of 2-FAA. a. Rat. This species appeared to be the most responsive to the carcinogenic effect of 2-FAA and has therefore been used most extensively. The maximal observed dosage permitting survival for a sufficiently long period of time for tumor induction was 0.125% of the diet given to Slonaker strain female rats. Ingestion of a ration containing this level of 2-FAA for 95 days led to multiple tumors in as little as 135 days (Wilson et al., 1941). The minimal effective dosage lay in the region of 0.004% in the diet (Wilson et al., 1947a), or 1 mg. per rat per week per 100 g. body weight, by stomach tube for 18 weeks in the Buffalo strain (Morris et aZ., 1948a, 1950a). At the minimal rate, 20 months or longer were required for tumor induction. Bielschowsky (1947b) reported that 4 mg. of 2-FAA per day per rat for 25 weeks caused neoplasms in 90% of rats in less than 42 weeks, but reduction of this amount to 1 mg. per day greatly delayed the appearance of visible tumors. The minimal exposure time amounted to a t least 25 days of ingestion of a diet containing 0.125% 2-FAA, after which the rats exhibited tumors after an average of 554 days. If carcinogen feeding was continued for 75 days or longer, the autopsies revealed tumors after 227 to 295 days (Wilson et al., 1947b). In general, tumors may be expected after 7 to 9 months in rats fed 0.025-0.06% 2-FAA. Effect of Strain. 2-FAA appears to exert a carcinogenic effect mostly on epithelial tissue although a few sarcomas have been observed. Tumors of the liver, mammary gland, and the ear duct appeared most often in rats treated with 2-FAA although the frequency of such tumors depended on variable factors such as amount of carcinogen given, strain,
353
~-FLUOBENAMINE AND RELATED COMPOUNDS
sex, age, and diet. Other tumors have been observed in the bladder, lung, eyelid (Harderian gland), skin, brain, thyroid, parathyroid, salivary gland, lung, pancreas, gastrointestinal tract, kidney, uterus, renal pelvis and urinary tract, muscle, thymus, spleen, ovaries, adrenal, and pituitary. Leukemias have also been induced. That the site affected depended on the strain of rat was noted first by Bielschowsky (1944). Tumors of the urinary tract were absent in Wistar rats, whereas such tumors were frequent in the Slonaker strain used by Wilson et al. (1941). Bielschowsky (1946) compared rats of a piebald strain to those of the Wistar strain and found a low incidence of mammary tumors in the former and a high incidence in the latter. On the other hand, the piebald strain showed a high proportion of intestinal adenocarcinomas which were infrequent in the Wistar strain. Comprehensive studies on this problem were performed by Dunning et al. (1947) and by Symeonidis (1954). Portions of their results relating to the incidence of mammary and liver tumors in various strains of female rats are shown in Table I. Sprague-Dawley, Osborne-Mendel, Buffalo, and TABLE I Effect of Strain on Liver and Mammary Tumor Induction in Female Ratsa
Strain
No. Of
Rats
Sprague-Dawley (9) Osborne-Mendel (S) Buffalo (S) AxC (S) AxC (D) Marshall M-520 (6) Marshall (D) August (D) Fischer (D) Copenhagen (D)
22 21 14 19
10 21 10 10 10 10
Age at Start (Days) 87 138 122 114 139 123 120 110
loo 144
Average Rats Rats Age with with to Death Mammary Liver (Days) Tumors Tumors 392 520 458
450 380 513 215 304 316 540
14 13 4 0 1 2 0 1 2 0
1 0 2 7 5 5 8
7 8
2
Rats with Other Tumors
7 3 5 2 2 1 0 0 0 5
a Data drawn from Dunning ct al. (1947), labeled (D) and Symeonidis (1954), (S). Number of rats refers to the number started in the experiment by Dunning el al. and to the effective number at autopsy in the
tests by Symeonidis.
Wistar rats were prone to mammary carcinoma, whereas liver tumors (benign or malignant hepatoma and cholangioma) were induced readily in AxC, Marshall, August, and Fischer lines. The urinary tract was a particularly susceptible site in the Copenhagen and Slonaker strains. A number of detailed studies describing the characteristics of one or more tumors have appeared in addition to the report of Cox et al. (1947).
354
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
Liver. 2-FAA neoplasms of the liver appeared to be similar to those induced by the carcinogenic azo dyes (Bielschowsky, 1947b; Cox et al., 1947; Firminger, 1955). Studies on the progressive changes induced by 2-FAA have been reported by Cox et al. (1947), Skoryna and Webster (1951), Laws et al. (1952), Matsuo et al. (1953), Matsuo (1954), FritzNiggli (1955, 1956), and Farber (1956). Male rats were considerably more susceptible to liver tumor induction than female rats, especially after partial bone marrow ablation (Stasney et al., 1950b). Metastases, frequently to the lung and lymphatic system, have been noted and transplantable hepatomas have been described (Dunning et al., 1950; cf. Dunham and Stewart, 1953). Recently, Maisin et al. (1957) have presented some of their observations on the histological alterations in the livers of carcinogen fed rats. In addition, Stewart and Snell(l957) have contributed a masterly, illustrated article on the histopathology of liver tumors. Mammary gland. When carcinogen feeding was initiated with mature female rats, tumors appeared after 555 months as free movable subcutaneous nodules (Ross et al., 1953). The neoplasia encompassed ducts, acini, and stroma which were always present although one of these might be dominant. A few of the growths were fibroadenomas. The majority of the mammary tumors developed in female rats although benign growths (Cox et al., 1947; Bielschowsky, 1946) as well as adenocarcinomas (Morris et al., 1950a) have been obtained in males. Engel and Copeland (1948, 1951, 1952a, 1952b) have reported a high incidence of mammary tumors in the AES (Alabama Experiment Station) strain of rats. The prevalence of mammary tumors observed by these authors could possibly be due to the fact that immature weanling rats were given the carcinogen-containing diet. Wilson et al. (1947b) had noted that only 1 of 14 rats had mammary tumors when old animals were started on 2-FAA. Bielschowsky (1947b), and recently Spjut and Eichwald (1957), also observed that the number of mammary tumors depended on the age at which female rats were started on the carcinogen. The last named investigators also observed fewer multiple tumors in the older rats fed 2-FAA. Oophorectomy decreased the incidence of mammary tumors from 64 to 9% if the operation was performed prior to, but not after, the initiation of carcinogen feeding. Thus, induction of mammary tumors appears to be affected by hormonal influences and the age of the animal when first exposed to the carcinogen. A transplantable mammary tumor which in the course of a series of passages took on the characteristics of a sarcoma was reported by Morris et al. (194813). Three transplantable mammary cancers were observed by Bielschowsky (1944). In addition, Dunham and Stewart (1953) list the transplantable Copeland reticulum-cell-like tumor (Engel and Copeland,
1951), the Webster adenocarcinoma, and the IRC 741 adenocarcinoma, which were originally induced by 2-FAA. Ear duct. This type of neoplasm was unknowii prior to its induction by 2-FAA (Wilson et al., 1941), by which it can be produced with considerable regularity. I n the Royal Victoria Hospital hooded strain of rats, the tumors of the external auditory canal were more frequent (57%) than a t any other site (Skoryna et al., 1951a) and were not associated with sex. The lesions appeared to originate in the compound sebaceous glands, and Skoryna and collaborators believed that the tumors arose in the cystic lobules of such glands and might have been associated with a latent middle ear infection. This premise has been questioned, however, by Laws et al. (1955) who obtained tumors in apparently normal lobules and in glands free of otitis media. Indeed, the latter authors remarked that tumor formation in the ear duct was not necessarily accompanied by the hyperplasia or hypertrophy which usually precedes liver tumor induction. Wilson et al. (1947a) suggested that epidermoid tumors of the head occurred only in strains of rats prone to mammary tumors, and Bielschowsky (1946) thought that ear duct tumors appeared at similar rates as hepatomas in male and intestinal tumors in female piebald rats. Brain. This tissue is very rarely affected in experimental carcinogenesis. Nevertheless, a few investigators (Vazquez-Lopez, 1945; Hoch-Ligeti and Russell, 1950; Boyland et al., 1954; Symeonidis, 1954) reported cerebral gliomas and meningiomas following oral administration of 2-FAA. Small intestine. These tumors were especially widespread in female piebald rats (Bielschowsky, 1946) and were found in 50% of male and 25% of female Holtzman stock rats (Miller et al., 1955). Their small size made recognition difficult, and it is possible that intestinal tumors are actually more prevalent than the literature would indicate. Dunn and Kessel (194546) described details of a special type of a cancer found in the Paneth cells. Stomach. The experimental investigation of gastric cancer has been hampered by the fact that neoplasms of the human type cannot be produced readily in animals; the tumors induced are often papillomas of the forestomach and are rarely if ever located in the glandular stomach (Stewart, 1953). Inasmuch as 2-FAA causes tumors in many organs, it might be considered possible to induce gastric cancers with this agent. However, several attempts which combined the administration of 2-FAA with some form of gastric irritation were not successful. The daily intake of emulsions containing 2-FAA and other carcinogens had no effect on gastric ulcers (Denton et al., 1950), and the repeated intraperitoneal injection of 2-FA in a dilute aqueous emulsion yielded erosions and ulcerations in the glandular stomach
356
ELIZABETH R. WEISBURGER AND JOHN H . WEISBURGER
of rats in addition to forestomach papillomas (R.ay el al., 1953). A slightly different approach, that of applying a solution of 2-FAA to an exposed flap of gastric mucosa in rats, led only to hyperplasia (Kobernick et al., 1952, 1953). Richardson (1956) approached the problem of bypassing the mucous barrier of the glandular stomach by the very drastic combination of 2-FAA in the diet and gastric lavage by a 95% ethanol solution containing 2-FAA. The ethanol might denature the proteins of the stomach membrane or change the character of the stomach mucus so that the 2-FAA solution could actually come in direct contact with the gastric wall. In a preliminary account, this treatment was reported to lead to ulceration, gastritis, and epithelial proliferation in a fair percentage of the rats. The as yet unsubstantiated claim was made that 2 of 89 rats had adenocarcinomas of the glandular stomach with metastases to the lymph nodes and liver. Further studies in which the administration of carcinogen is combined with attempts to render the glandular stomach nonfunctional (cf. Hitchcock et al., 1957) might be more successful. Lung. Primary lung tumors have been induced in rats treated with 2-FAA. In general, such neoplasms were multiple and appeared to originate in the bronchial epithelium. Orr and Bielschowsky (1947) have classified them as tubulopapillary cuboidal-celled adenoma or carcinoma. b. Mouse. This species is more resistant to the action of 2-FAA in terms of the amount of compound tolerated, the number of lesions obtained for a given intake of carcinogen, and the latent period on moderate, nonlethal doses of the compound. Tumors may be induced in mice fed diets containing 0.14125% of 2-FAA (Wilson et al., 1947a) or 3 mg. per mouse twice weekly (Armstrong and Bonser, 1947). Tumors may appear after 40-50 weeks or more of treatment. Different strains of mice exhibit varying degrees of susceptibility both in regards to the total incidence of cancers and the organ affected (Table 11). Bladder tumors were more constantly TABLE I1 Effect of Mouse Strain on Tumor Induction by N-2-Fluorenylacetamide" Strain CBA IF R I11 White label Strong A 0
b
Percent Bladder Tumors
Percent Liver Tumors
Percent Mammary Tumorsb
78 70 48 22 22
73 44
11 (0) 73 (0) 91 (71) 58 (3) 39 (30)
Data condensed from Armstrong and Ronser (1947). Spontaneous incidence in parentheses.
16 10 5
2-FLUORENAMINE
A 9 D ItELhTEU COMPOUNDS
357
induced in mice than liver tumors, but in contradistinction to the rat, a sex factor was operative with respect to bladder lesions (males being more susceptible), while liver changes were independent of sex (cf. however, Leathem, 1951). A detailed study of bladder neoplasms in R I11 strain mice showed a variety of histological types, from squamous or transitionalcelled papillomas to spindle or pleomorphic-celled tumors, all of which, however, originated in the transitional epithelium of the bladder (Foulds, 1947, 1950). Other organs affected were the kidney, forestomach, thyroid, renal pelvis, and female reproductive tract. Orally administered 2-FAA alone did not lead to skin tumors, but such lesions were initiated in coiijunction with a painting with croton oil. Oral 2-FAA also seemed to promote skin tumors induced by 7,12-diniethylbenz[a]anthracene(Ritchie and Saffiotti, 1955). The successful induction of transplantable mouse liver tumors in C3H, C57BL, and A strains has been indicated (Morris, 1 9 5 5 ~ ) . c. Fowl. Although tumor induction with certain carcinogenic hydrocarbons, polynuclear heterocyclic compounds, and a stilbene derivative failed, 2-FAA caused neoplasms to develop in the fowl, albeit with considerably more difficulty since higher doses and much longer latent periods were required than in rodents (Peacock arid Peacock, 1954). Almost daily ingestion of 25 mg. 2-FAA for 4 months, with lesser doses thereafter, resulted in tumors after 3 years or later. Kidney lesions of epithelial origin appeared most frequently in this species (Bielschowsky and Green, 1945), but the lung, ovary, and oviduct were also affected. Squamous carcinoma of the crop was produced after 14 and 21 months in 2 of 5 birds injected a t that site with 2-FAA in peanut oil (the local action apparently favored by the trauma due t o the repeated injections; Peacock and Peacock, 1949). Sarcoma formation was, however, not observed. A liver tumor induced by 2-FAA grew on the chorioallantoic membrane of fertile eggs but could not be further transmitted to young chickens (Campbell, 1955). Nevertheless, a malignant lymphoma which arose in the kidney of a fowl fed 2-FAA was propagated, and a tissue (although not cell-free) filtrate of the tumor transmitted the disease. Transplants by grafts or filtrates of other epithelial fowl tumors induced by this carcinogen were, however, not successful (Peacock and Peacock, 1955). d. Dog. Allison et al. (1950) have reported hepatoma induction in dogs by the administration of 2-FAA for only 6 months. Morris and Eyestone (1953) suggested that the lesions observed by Allison et al. may not have been true hepatomas. Morris and Eyestone found no tumors in dogs after continuous intake of 2-FAA for 27 months a t a dietary level of 0.12% and with a total intake of 32 to 37 g. of the carcinogen. Although Bonser (1949) observed no bladder changes in dogs fed 2-FAA for l>i to 435 years, Morris and Eyestone established that liver and bladder tumors did develop
358
ELIZABETH I<. WEISBURGEH AND JOHN H . WICISBURGEIl
in male and female dogs fed a total of 90 to 198 g. of the compound over a period of 68 to 91 months. The liver lesions were classified as carcinoma of the parenchymal cells, while neoplasms of bile duct origin were absent. Metastases to the lungs were noted. The urinary bladder showed multiple papillomas, but the ureter and the renal pelvis were not affected. e. Hamster. The susceptibility of this species to 2-FAA was demonstrated by Miller and Miller (1955), who obtained cystic livers and a low incidence of liver tumors after 10 to 44 months. Paschkis et al. (1957), in attempting to obtain uterine tumors in hamsters treated with a combination of diethylstilbestrol and 2-FAA, observed liver tumors which appeared to be cholangiomas, as opposed to the mixed type of hepatoma-cholangioma normally seen in rats. These cholangiomas could be propagated by subcutaneous implantation in untreated hamsters. f. Cat. The lungs of this species were most susceptible to tumor induction following ingestion of 2-FAA (Harding, 1947; Skoryna et al., 1951b). Histologically the lesions seemed different from those induced in other species in that multiple small nodules were distributed throughout the organ. The cells were poorly differentiated and resembled those of a polymorphous sarcoma. The induction period of the tumors was 16-18 months with a daily dose of 40 mg. of 2-FAA in the first 6 months and 50 mg. per day thereafter. Other organs were unaffected except for liver cirrhosis, found in the early period, and hepatomas, found considerably later, particularly in those animals with retarded lung tumor development. g. Rabbit. Tumors of the urinary tract, especially of the bladder, were produced in 155 to 4 years by the daily ingestion of 50 or 100 mg. of 2-FAA twice weekly. The lesions observed ranged from simple hyperplasia to adenocarcinoma. The total amount of carcinogen administered during the experiment varied from 30 to 52 g. Weight loss of the rabbit was confined to the terminal stages (Bonser and Green, 1950). h. Guinea pig and cotton rat. All attempts to induce tumors in these species by means of 2-FAA failed (Miller and Miller, 1955; Urquhart, 1955). Treatment of the guinea pig with 2-FA was equally unsuccessful (Breidenbach and Argus, 1956; Argus and Ray, 1956) as described later. The quantity of compound ingested and the duration of the experiments appeared adequate compared to more susceptible species. i. Miscellaneous species. McCallion (1954) investigated the effect of 2-FAA on frogs and tadpoles and found that the chemical was quite toxic to these cold-blooded animals. Necrosis and cirrhosis were observed in the livers of frogs surviving after 6 to 8 weeks although no neoplasms could be diagnosed. Both 2-FAA and 2-FA induced hyperplasia at the site of injection (tail) in the crested newt, Molge cristatus (Neukomm, 1957). However, 2-FAA did not lead to neoplasms or accessory limbs in the common newt, Triturus wiridescens (Breedis, 1952).
2-FLUORENAMINE
A N D RELATED COMPOUNDS
359
j. Lower organisms. Bielschowsky and Green (1942) reported that 2-FAA had a definite bacteriostatic action on Staphylococcus aureus while Loveless et al. (1954) found 2-FA reduced the growth rate of Escherichia coli. A certain strain of the mold S. pyogenes aureus could be adapted to grow through several passages in media containing 2-FA, but eventually growth ceased. Changed growth habits, however, were noted for some time after exposure t o 2-FA (Morelli and Misler, 1947a,b). However, this effect was not caused by a change in the structure of the nucleus or of the cell (Parvis and Sirtori, 1953). A Neurospora mutant was adapted to and appeared to utilize 2-FAA and certain azo dyes (Salzberg, 1956). No studies of the metabolism of 2-FAA or 2-FA by bacteria have been carried out, but further investigation is indicated, especially to see whether chemical reactions performed by bacteria could be a source of metabolites of the carcinogen, obtainable only with difficulty by other methods. 2-FAA was reported nonmutagenic to male Drosophila melanogaster by Demerec and co-workers (194849) and also to Neurospora crassa by Barratt and Tatum (1951). Calcutt (1954) has shown that there was a certain degree of correlation between carcinogenicity and the photosensitizing effect of various chemicals on Paramecium bursaria. Roth (1954) found that 2-FA decreased the oxygen uptake of Tetrahymena pyriformis by as much as 44%. The inhibition was reversed by a crude, but not by a purified, preparation of coenzyme A from liver. Succinate oxidation was similarly inhibited by 2-FA, but l-naphthylamine had a n even larger effect, so that a correlation between carcinogenicity in mammals and inhibition of this particular organism was not apparent. Roth assumed that the nonspecific inhibition may have been caused by the toxic nature of the amines. C. Synergistic and Antagonistic Eflects. MacDonald et al. (1952) reported that when minimal doses of the hepatic carcinogens 2-FAA and N,N-dimethyl-4-(m-tolylazo)anilinewere fed simultaneously to rats, the incidence of liver tumors was greater than the sum of the tumor incidences obtained when the carcinogens were fed alone. However, when 2-FAA was administered with N,N-dirnethyl-4-(p-tolylazo)anilinel a weaker carcinogen, there was no synergistic effect. Even if N,N-dimethyl-4-(m-tolylazo)aniline was fed for 4 weeks, then 2-FAA for the next 4 weeks, followed by a 2nd 4 week period of dye feeding, and 12 weeks on basal ration, the incidence of liver tumors was increased over that obtained with the azo dye alone (Rumsfeld et al., 1952). Atabrine was as effective as 2-FAA1 but quinine, auramine, or 3-methylcholanthrene did not increase liver tumors in other experiments. These results were interpreted as consistent with, but not proof of, the hypothesis that there is a phase in the carcinogenic process which is common to both agents (MacDonald et al., 1952). Korpsssy and Mosonyi (1950) reported that rats injected subcutane-
360
ELIZABETH I<. WEISBURGER A N D JOHN H . WEISBURGER
ously with taniiic acid solutions devclopcd livcr cirrhosis with a 56% incidence of tumors. When this treatment was combined with simultaneous intake of 2-FAA, the incidence of rats with liver tumors was %yo,while rats given 2-FAA alone had only a 28.5Y0 iiicidence of liver tumors, most of which were very small (Mosonyi and Korpltssy, 1953). However, the attempts to utilize 2-FAA in addition to chronic irritation (surgically produced gastric ulcer and administration of irritants such as barium sulfate, talc, or powdered glass in the diet) to obtain certain local tumors (stomach) failed (Mosonyi et al., 1954; Korpltssy et al., 1955). Experiments combining croton oil with local painting of 2-FAA have not been very successful in promoting tumor formation in mice (Price, 1947; Salaman and Roe, 1953; Graffi et al.,1953). Similar results with 2-FA and croton oil were noted by Kirby (1948) and Graffi et al. (1953). An explanation for these results may perhaps be found in the fact that 2-FAA did not depress the A’-cholestenol levels of the skin (Kandutsch and Baumann, 1955) and was absorbed rapidly through the skin without being metabolized (Gutmann and Peters, 1957). Ritchie and Saffiotti (1955), however, reported that when 2-FAA was fed (0.024% of the diet) for 21 weeks with twice-weekly application of croton oil, 33% of the mice surviving 53 weeks had skin tumors. Feeding 2-FAA alone or painting croton oil alone caused no skin tumors. With a more sensitive strain of mice (Bar Harbor Swiss), up to 70% of the survivors developed skin tumors. A single application of 7,12-dimethylbenx[a]anthraceneto the skin followed by oral administration of 2-FAA for 41 weeks gave a SO% incidence of skin tumors in survivors. Control mice painted with the hydrocarbon only had an 18% incidence of skin tumors, and those fed 2-FAA alone showed none. Hudali et al. (1952) found that simultaneous oral intake of the carcinogen 4-phenylacetanilide (4-acetylaminobiphenyl) with 2-FAA had no antagonistic or inhibiting effect on the tumor-producing activity of 2-FAA. Even though the structure and reactions of the biphenyl are similar to those of 2-FAA, i t was not able to block the carcinogenic effect of 2-FAA. Furthermore, an even more closely related compound, 2-fluorenol, when fed in large amounts, had no effect on the carcinogenic activity of 2-FAA (Green and Bielschowsky, 1945). Miyaji et al. (1953) noted that feeding the hydrocarbons 3-methylcholanthrene, chrysene, and 7,12-dimethylbenz[a]anthracene along with 2-FAA inhibited tumor formation. J. A. Miller et al. (1954) also reported that simultaneous administration of 3-methylcholanthrene with either 2-FAA or 7-fluoro-2-fluorenylacetamide greatly inhibited the activity of both fluorene compounds so that very few tumors of any organ were obtained. Hill et al. (1954), in a preliminary report, noted that the ability of 2-FAA to induce breast tumors in Sprague-Dawley rats was inhibited by
2-FLUOREKAMINE
AND 11ELATED COMPOUNDS
361
13H-dibenzo[a,q]fluorenewhile simultaneously 2-FAA inhibited the development of liver cirrhosis by the hydrocarbon. In this connection, it was shown recently that the intraperitoneal injection of 3-methylcholanthrene into weanliiig rats enhanced 6- to 10-fold the ability of the liver homogenates from these rats to hydroxylate 2-FAA (Cramer et al., 1958). The increased hydroxylation affected all the isomers studied, i.e., the 1-, 3-, 5-, and 7-hydroxy derivatives. Hoffman (1956) investigated the effect of administration of N-2-fluorenyldiacetamide to rats exposed to tobacco smoke. He found less hepatic damage and less lowering of liver riboflavin than in rats receiving the carcinogen only. Also, intraperitoneal injection of the tar from tobacco smoke condensate protected the livers of rats ingesting fluorenyldiacetamide from precancerous changes and riboflavin depletion. No clear-cut understanding for the antagonistic effect of various FA types of carcinogens has yet been achieved. However, Huggins and Pollice (1958) recently demonstrated an antihypophyseal effect of 3-methylcholanthrene which may have a bearing on the inhibition of 2-FAA carcinogeiiesis by the hydrocarbon (cf. Section IV,7,D). 2. 2-Fluorenamine
Wilson et nl. (1941) observed that rats fed 2-FAA excreted a substance in the urine which colored the pine shavings in the bedding a bright red. By analogy with such a reaction given by pure 2-fluorenamine (2-FA), it was postulated that deacetylation of 2-FAA occurred in vivo and that the substance in the urine was 2-FA which they subsequently demonstrated to be carcinogenic. Wilson et al. (1947~)were of the opinion that 2-FAA was a more active carcinogen than 2-FA in rats, but the latter appeared more potent in mice (see also Morris et al., 1950a; Miller et nl., 1955). Most of the few tumors induced locally were obtained after administration of the free amine. Overall, the carcinogenic tests in the rat, mouse, and the guinea pig parallel those obtained with the acetyl derivative. The same wide distribution of tumors affecting many tissues was observed in the rat and mouse, whereas the guinea pig proved refractory (Breidenbach and Argus, 1956; Argus and Kay, 1956). Further details may be obtained from Hartwell (1951) and Shubik and Hartwell (1957). 3. Related Fluorene Derivatives
A large number of chemicals related to 2-FA have been tested for carcinogenicity by maiiy investigators. Different strains of animals and dosages h a w l)een used, arid frequently only one test of a given substance has beell reported. The carcinogenic potency ascribed to some of these
362
ELIZABETH K. WEISBUBGEH AND JOHN H. WEISBUHGER
compounds must, therefore, be considered preliminary a t this time. The results of the tests are outlined in Tables I11 and IV, and pertinent references are given therein. TABLE I11 Carcinogenic Activity of Derivatives of %FA
Very Active if 2-position = -NHP --NHCOCHab -N(COCHs)zE -NHCOCFsd
Active if 2-position = -NOz' -NHCHa@ -N(CH3)Zh -NHCOCHzNHz~ -NHCOCHzCH&OOHj
Inactive or Slightly Active if 2-position = -NHCOCeHbk -NHSOzCeH&Ha' -NHCOCHzFd -NHCH2CeHam -NHCOCeHrCOOHm
If 2,7-position = 2,7=(NHCOCH,)*e Literature References Cf. Section IV.2. b Cf. Section IV,1. 0 Morris et al., 1950a: Morris and Firminger, 1956. d Morris, 1955c. ' Morris and Dubnik, 1950. Cf. Section IV,3.A. 0 Bielschowsky and Bielschowsky. 1952; Morris. 1 9 5 5 ~ E. ; I<. Weisburger el al., 1956. * Bielschowsky and Bielschowsky, 1952; Miller el al., 1955; Morris, 19550; Schine el al., 1955; E. K. Weisburger et al., 1956. i Hirs, 1949. i Hirs, 1949; Morris, 19558. Morris (as quoted by Dyer, 1955). t Morris (as quoted by Ray and Argus, 1951 ). m Ray el al., 1953.
A. 2-Nitro~Iuorene. Nitroreductases (Green et al., 1956) exist in many tissues so that one might expect 2-nitrofluorene to be reduced in vivo by such enzymes to 2-FA. Green and Bielschowsky (1945) reported that feeding 12 mg. per day of this compound to rats caused liver tumors after 49 to 52 weeks. On the other hand, Morris et al. (1950a) obtained only a few tumors after oral administration (average 2.5 mg. per rat per day), but skin application led to a considerable number of tumors in many organs even though the quantity administered by painting was less than one-tenth that fed. In Holtzman rats, an intake of 2-nitrofluorene a t a level of 1.62 mM./kg. of diet (0.034%) induced tumors of the liver, ear duct, mammary gland, forestomach, and intestine (Miller et al., 1955). B. Derivatives of 2-Fluorenamine (Table 111). N1N-Dimethyl-2-fluo-
TABLE IV Derivatives of 2-FAA-Carcinogenic Activity of Positional Isomers, Halogenated, Hydroxylated, and 9-Substituted Dwivatives
Very Active if:
Moderately Active if:
(7-F)-2-NHCOCHaa
l-NHCOCHab
(7-Cl)-2-NHCOCHab
(3-1)-2-NHCOCH$ (ear duct only)
(7-OH)-2-NHCOCH3e
(9-S-)-3-NHCOCH3Csd
0
b
Miller el al., 1955. Morris. 1 9 5 5 ~ .
0 E. C. Miller el aZ., 1949. d 3-Acetamidodibenzothi~ phene. e Hoch-Ligeti, 1947a.
Morria and Dubnik, 1950. 0 J. A. Miller et al.. 1952; Miller et al., 1955. Miller et a?., 1955; hlorris, 1955c. i 3-.4eetarnidodibenzofuran
Slightly Active if:
4-NHCOCHak (7-OH)-2-NHCOCHZ’ (7-1)-2-NHCOCH$
2,2’-NHCOCH3-9,9’-bifluorcnef 9-NHCOCHsm [9,9-(CHa)&2-NHCOCHEm
2,2’-NHCOCHrAg.g’-bifluoreneJ 3-NHCOCHab (9-OH)-ZNHCOCHa. (9=O)-ZNHCOCH3* (9-0-)-3NHCOCH3‘,* (9-SO-)3NHCOCHace’
r n N H C 0 C H 3 \
Inactive if:
j 3-Acetamidodihenzothiopheneboxide
1 3
& 0
NHCOCH,
\
/
f
*
r\ n N H C / O C H 3
I. hiorris. 1955c; Schinz et d.. 1955. 1 Green and Bielschowsky, 1946; Bielschowsky, 1947h. “1 Schinz et aZ., 19.75.
w
c,
w
364
ELIZABETH K . WEISBURGER A N D JOHN €1. WEISBURGER
renaniine and the monomethyl analog are hoth carcinogenic but somewhat less active than 2-FAA. The N-beazyl derivative and N-2-fluorenylphthalarnic acid were inactive by skin painting. Oral intake of N-2-fluorenyldiacetamide by Miniiesota strain rats gave a 100% incidence of hepatomas in approximately 60 days (Morris et al., 1950a). Fewer hepatomas, but more tumors of other organs such as the mammary gland, were noted after skin application. Morris and Firminger (1956) found a pronounced influence of sex and sex hormones (cf. Section IV,6,A). The progressive changes in the livers of rats fed N-2-fluorenyldiacetamide were studied by means of a liver function test based on the rate of uptake and excretion of radioactivity by this organ after intravenous injection of Rose Bengal labeled with radioactive iodine (Morris et al., 1958). A disturbed clearance of radioactivity was first noted after 4 weeks, whereas the uptake was affected in the 6th week. These factors were lower in 8 t o 10 weeks when the uptake was decreased almost 50% and the clearance very nearly abolished. Correlations between these findings and histological data showed a progressive replacement of hepatic tissue by pseudotubules, connective tissue hyperplastic nodules, and finally hepatoma which occurred simultaneously with a lowering of the hepatic function. On the other hand, fatty changes, degeneration and necrosis, hyaline bodies, and alterations in the amount and location of cytoplasmic basophilic material could not be directly implicated in the altered liver function. Intestinal tumors were induced readily by 2,7-fluorenylenebisacetamide. N-(2-F1uoreny1)2,2,2-trifluoroacetamide1 N-2-fluorenylsuccinamic acid, and N-2-fluorenylglycine were active carcinogens, while the benzamide was a much weaker carcinogen, and the p-toluenesulfonamide was inactive. Thus, it appears that carcinogenic activity of a compound is influenced by the potential availability of a free amino group (cf. Section VI). C. Derivatives of N-2-Fluorenylacetumide (Table IV). a. Positional isomers. The 1- and 3-isomers were moderately to definitely carcinogenic, but N-4-fluorenylacetamide was inactive. In the rat, greater carcinogenic activity results if the amino group is attached to the 2-position of the fluorene ring. b. Halogenated derivatives. N-(7-Fluoro-2-fluorenyl)acetamide was a more active hepatic carcinogen and also more toxic than 2-FAA1 even in female rats which are usually more resistant t o liver tumor induction by 2-FAA. Lesions of the ear duct, mammary gland, small intestine, and of other sites were also observed. The 7-chloro derivative also had some carcinogenic effect on the liver of rats, but the 7-iodo derivative was inactive. On the other hand, the 3-iodo compound was active in inducing ear duct tumors to the exclusion of other tissues. The site-specific action of this
2-FLUOREXAMINE
AND RELATED COMPOUNDS
365
compound is quite unusual and may be related to the easy in vitro dehalogenation of 3-iodo-2-fluorenamine to 2-FA in acid solution (E. K. Weisburger et al., 1951), akin to the loss of halogen by certain other compounds in vivo (Marrian and Maxwell, 1956). c. Hydroxylated derivatives. N-(7-Hydroxy-2-fluorenyl)acetamide appears to be noncarcinogenic (Green and Bielschowsky, 1946; Bielschowsky, 1947b) although Hoch-Ligeti (1947a) obtained a few tumors which she felt were not spontaneous despite a long latent period. Additional carcinogenic tests using higher levels of intake of this metabolite seem indicated. d. $-Substituted derivatives. N-9-Fluorenylacetamide was not carcinogenic. N-(9-Hydroxy-2-fluorenyl)acetamide was weakly active and the 9-keto derivative somewhat more so. N-(9,9-Dimethyl-2-fluorenyl)acetamideand 2,2'diacetamido-9,9'-bifluorene were inactive, while the corresponding A9r9'-bifluorene was slightly carcinogenic. The first two of these molecules are nonplanar owing to the tetrahedral nature of the 9-carbon atom, which may account for their lack of effect. Although the carbon to carbon double bond usually leads to a planar configuration, the A9~9'-bifluorene may not be a true planar molecule because of its possible free-radical nature. On the other hand, the insoluble bifluorene derivatives were poorly absorbed and may not be active for this reason (Dyer, 1955). e. .4-(2!-Fluorenylazo)resorcinol. Because certain dyes are secreted into the stomach after intravenous injection, Ray and Jung (1951) predicted that such chemicals with a pKb of 7.5 to 10 would most likely be thus introduced into the stomach. If a fluorenamine type of residue could be incorporated into such molecules, compounds which might be carcinogenic for the gastric mucosa could be prepared. Such compounds were synthesized by Ray and Peters (1951) by coupling 2-fluorenediazonium chloride or 2-acetaminofluorene-7-diazonium chloride v ith various amines or phenols to give a series of dyes. One of the dyes, 4-(2-fluorenylazo)resorcinol, gave a rather high incidence of erosions and ulcerations in the glandular stomach of rats with no forestomach papillomas (Kay et al., 1953).
4. Some Physical and Chemical Properties and Carcinogenicity A. Ultraviolet Absorption Spectra. Sandin et al. (1952) have correlated the carcinogenic activity of 3 biphenylamines and 2-FAA with ultraviolet absorption maxima a t longer wave lengths and more planar structures. However, among various derivatives of 2-FAA, such relations cannot be established, for the fluorene skeleton is planar and this factor exerts no differential influence. Thus, the noncarcinogenic 7-iodo-2-fluorenylacetamide has an ultraviolet absorption maximum a t 295 mp, a longer wave length than 2-FAA (288 mp), while the carcinogenic 3-iOdO derivative
366
ELIZABETH K . WEISBURGER A N D JOHN H. WEISBURGEH
absorbs maximally a t 219 mp. 7-Hydroxy-2-fluorenylacetamide, which is weakly or noncarcinogenic, and 7-methoxy-2-fluorenylacetamide, which is carcinogenic (this laboratory, unpublished) , both have their maximum absorption a t 293 mp. Of the isomers of 2-FAA1 3-fluorenylacetamide has a maximum a t 245 mp, and l-fluorenylacetamide a t 250 mp, but both are more carcinogenic (Morris, 1955c) than 4-fluorenylacetamide which absorbs a t 266 mp. The noncarcinogenic compound, 9,9dimethyl-2-fluorenylacetamide also has a maximum a t 290 mp (Campbell et al., 1953). These facts lead to the conclusion that a t present the wavelengths of ultraviolet absorption maxima cannot be correlated with carcinogenicity in the fluorene series. This is not an unusual case, for Rondoni (1955a,b) has also discussed the difficulty of relating another physical property which depends on electronic structure, the magnetic susceptibility, with the carcinogenicity of a series of compounds, including 2-FAA. The failure to establish relationships between physical properties reflecting molecular structure and biological effect may be absolute in that no such connection exists. On the other hand, it also may indicate that the compounds compared are not the proximate carcinogens and require metabolic modification to exert their biological action. B. Molecular Dimensions. Calculations with data from tables given by Pauling (1948) and by Wheland (1944) show that the thickness of the molecule of N-(9,9dimethyl-2-fluorenyl)acetamidewould be approximately 4 A. because of the size of the two methyl groups, whereas similar computations impart a cross section of about 2 A. to 2-FAA. Thus, it is possible that the dimethyl derivative would be too large to fit into whatever specific configuration is required for carcinogenic activity. Since the C=O group is planar, there would be no such problem with 9-oxo-2-fluorenylacetamide. Similar calculations indicate that N-(9-hydroxy-2-fluoreny1)acetamide is 2.9 A. thick, while the not yet tested 9-monomethyl derivative requires 3 A. These values may not truly represent the actual absolute size of these molecules but can probably be taken as indicative of the relative dimensions. Pauling stated that the effective thickness of an aromatic molecule in a crystal lattice is 3.7 A. due to van der Waals’ forces. It might be well, however, to check the calculations by determining the thickness of representative molecules of this type. In any event, the 9dimethyl and the 9-hydroxy derivatives of 2-FAA are thicker than the carcinogen itself, which may account for their lower biological effect. However, solubility, ease of conjugation and elimination, metabolism to some other derivative, and other factors enter the picture and contribute, partially a t least, to the carcinogenic activity of a specific compound. The comparison of size can be extended to the 7-halogen substituted derivatives of 2-FAA. Pauling gives the following values for the covalent
2-FI~UOltE"I"iCNAhlINl~
A N D RELATEI) COMPOUNIX3
367
diameters of atoms: Hydrogen 0.60 A.; fluorine, 1.28; chlorine, 1.98; bromine, 2.28; and iodine, 2.68. It can be seen that, fortuitously or not, the fluorine and chlorine atoms do not add to the thickness of 2-FAA1 whereas bromine and iodine do. The 7-iOdO derivative is not carcinogenic, the 7-chloro and 7-flUO1-0are, the latter strongly so. While the 7-bromo compound has not been tested, it might be predicted on the basis of size alone that it would exhibit borderline activity. If any meaning a t all can be attributed t o these relationships, it would signify that size and orientation have an important bearing on the carcinogenic properties of a molecular arrangement. This in turn implies that the compound has to fit into a certain space of restricted dimensions in order to show biological activity. Such data intimate that the carcinogenic process is elicited by the combination of the actual carcinogen with an endogenous body constituent under well-defined and stringent stereochemical conditions. C. Oxidation-Reduction Reactions. One outstanding characteristic of many types of carcinogens is their capacity to function either as simple reducing agents or as an oxidation-reduction system. Aromatic amines or aniinophenols are examples of such substances. The azo dye carcinogens can also be included in this class by virtue of some of the 2-hydroxylated metabolites. Furthermore, an analysis of the significance of a high electron density of the K region of the carcinogenic hydrocarbons leads to the conclusion that in addition to facilitating an easy substitution, the net effect of donating the electrons actually amounts to a reduction of the recipient and an oxidation of the hydrocarbon (Cook and Schoental, 1950; Badger, 1954). An antioxidant property of some carcinogenic compounds was actually demonstrated by Rusch and Kline (1941) and by Bernheim et al. (1953) who also tested 2-FA. Carcinogens might also promote oxidation by acting as oxygen carriers in an oxidation-reduction system (Brown and Tappel, 1957). If one considers that the aminophenol type of molecule also possesses the potential ability of coordinating to certain metals, then such compounds might play a role in interfering with vital oxidation-reduction processes. However, not all molecules containing an ortho-aminophenol structure are carcinogens. A molecule, in addition to carrying the proper substituents, apparently must also fit into a certain pattern of the receptor molecule in order to exhibit carcinogenic properties. D. Michael Condensation and Carcinogenicity. By virtue of the relatively high reactivity of the hydrogens a t the 9-position of fluorene, fluorene compounds were found to enter into Michael condensations rather readily (Pinck and Hilbert, 1946a,b). On the basis of this property, Pinck (1948) proposed that 2-FAA might be subjected to an in vivo dehydrogenation to 2,2'-dia~etamido-A~~~'-bifluorene. This compound was pictured as the actual carcinogen because of its potential ability to undergo repeated
368
ELIZhBETH K. WEISBURGER AND JOHN H . WEISBURGER
Michael c?ondensatioiw with certain cell substances which would lead to cancer. However, very few tumors were induced in Sprague-lhwley female rats by feeding 2,2’dia~etamido-A~~~~’-hifluorene and none by the corresponding bifluorene (Morris and Dubnik, 1950). The experimental results, therefore, apparently contradict Pinck’s hypothesis. Moreover, Miller et al. (1949) have shown that the 9-methylene bridge of fluorene can be or -0- bridges which could not take place removed or replaced by -Sin the condensation reactions proposed by Pinck. The experimental data, therefore, do not seem to support the Michael condensation hypothesis. 5 . In.u,ence of Diets
A. General Considerations. Wilson et al. (1941) noted a reduced weight gain proportional to the 2-FAA content of the diet as shown in Fig. 1. This effect of 2-FAA on body weight was confirmed by Bielschowsky and Green (1942) and has since that time been the constant observation of investigators using this and related compounds in species susceptible to the carcinogenic action. Dunning et al. (1947) have demonstrated that in part this lower weight gain may be related to a decreased food intake of the rats on the carcinogen-containing diet, and that in part it depended on the strain of rats. These effects were studied by Gutmann and Peters (1953b)
I
I
0
25
Death of an animal
50 DAYS
75
I I00
ON D I E T
FIQ.1. Graph showing decreased growth and time of death of rat,s on diets containing N-2-fluorenylacetamide (2-acetamidofluorene). From Wilson el al. (1941).
2-FLIJORENAMINE
AND RELATED COMPOUNDS
369
and Gutmann et al. (195613) who showed by pair-feeding that on a 20% casein diet all of the inhibitioii was due to t,he decreased food intake, but only a part of the inhibition could be ascribed to the lower food intake on an 11% casein diet even with additional glycine, methioninc, or cystine. It can be concluded that other factors play an appreciable role in the observed reduction in the growth rate. That the composition of the diet was of paramount importance for the successful induction of neoplasms by the carcinogenic azo dyes was discovered by the early workers with these compounds. Many of the variables involved in this complex field have received satisfactory explanations on the basis of metabolic experiments which showed that a diet reducing the carcinogenic effect of the dyes promoted their detoxification (cf. Miller and Miller, 1953). I n the light of the knowledge accumulated on the influence of diet in azo dye carcinogenesis, studies were undertaken to determine the effect of diet on the carcinogenicity of 2-FAA. One of the first investigations was that of Harris (1947) who found that rations which profoundly influenced N,N-dimethyl-p-phenylazoaniline (p-dimethylaminoazobenzene) carcinogenesis had but little effect with 2-FAA or 2-FA. Doubling the casein content of the diet from 10 to 20y0 and increasing the riboflavin content more than &fold seemed without effect, and the addition of liver extract retarded tumor development only slightly. Bielschowsky (1947a) reported similar observations but noted that addition of 15y0 dried yeast significantly delayed liver tumor development and diminished the preceding hyperplasia of that organ. Cantarow et al. (1946) also noted a small supplement of yeast appeared to result in fewer tumors. No sizable effect on tumor induction was noted by Wilson et al. (1947b) as a result of fortification of an already adequate diet with 5% yeast. The initial experiments thus indicated that some dietary constituents did not influence carcinogenesis by 2-FAA as readily and in such striking fashion as the induction of liver tumors by the azo dyes. Nevertheless, a number of interesting studies involving dietary effects in carcinogenesis by fluorene derivatives have appeared and will be discussed briefly. It may be noted that the results obtained can be correlated in some cases with the inhibiting effect of caloric restriction on carcinogenesis discussed so admirably by Tannenbaum and Silverstone (1953). B. Stock versus Semisynthetic Diets. Survival of animals was better when fed 2-FAA or related carcinogens in stock than in semisynthetic diets (Miller et al., 1949; Wilson and DeEds, 1950; Engel and Copeland, 1952a; Morris, 1955~).Miller et al. observed better survival on a grain regimen and earlier tumor development; a number of rats succumbed before tumors could develop because of higher toxicity of the compounds tested on a semisynthetic diet. Morris noted that a diet of natural foodstuffs containing
370
ELIZABETII K. WEISRURCER AND JOHN H. WEISBTJRGER
2-FAA favored the survival of rats compared to a certain semisynthetic diet and usually resulted in a greater number and variety of tumors. On the other hand, both Wilson and DeEds, and Engel and Copeland found retarded tumor development, or a lower incidence, with natural foodstuffs at equal time intervals on the comparative diets. Some of the variations in results encountered in the studies of dietary effects may have been due to the different strains of rats used and to differences in dosage. C. Protein Level. Increase of the protein from 13 to 20% had little effect on liver tumor iiiduction by 2-FAA (Harris, 1947), but Morris et al. (1948a) reported more hepatomas on a low-fat, 12% protein diet than on an 18 or 24% protein level. Longer latent periods as well as more mammary and ear duct lesions resulted from the feeding of the higher protein levels. It was noted, however, that the differences between the diets were not clear-cut. An extensive study on the effect of varying casein levels in carcinogeriesis by 2-FAA was published by Engel and Copeland (1952b). The incidence and average tumor induction period of mammary, ear duct, and liver tumors in the Alabama Experiment Station (AES) strain of rats were not affected by diets containing 9, 12, 20, or 27% casein. Mammary tumors were decreased on a 4Oy0 casein regimen, and all three types were decreased on a 60% casein regimen. On the latter, survival was improved and a few animals were tumor-free after 40 weeks. Diets containing 12-27% casein promoted as good growth as that obtained on 60% casein. The protective effect of high protein diets on the induction of mammary, and perhaps other, tumors was largely overcome if food intake during the first 16 weeks of the experiment exceeded 7.2 g. per rat per day (2-FAA intake 2.16 mg. or more daily). Similarly, Leathern (1949a) noted a lowering of tumor incidence on a 78% lactalbumin diet as compared to one containing 18%. The addition of dried beef liver powder to a diet of polished rice containing 0.03% 2-FAA1 which was fed for 6 months with an occasional supplement of greens, reduced the incidence of liver tumors from 44 to 20% (Mori, 1954). Maisin et al. (1957) obtained lengthened survival times and a much lower incidence of liver tumors in rats whose diet (7.5 g./day) of polished, boiled rice, carrots, and olive oil was supplemented with 2.5 g./day of defatted beef liver pulp. A protective effect was also demonstrated in various acetone and/or water-soluble fractions of liver. Further work on the identification and mode of action of these principles will be of interest. A low-casein content, together with minimal vitamin levels, was found by Strijmbeck and Ekman (1949) to favor bladder lesions in Copenhagen strain rats. This strain is very susceptible to bladder tumor induction by 2-FAA (Dunning et al., 1947). Strombeck and Ekman (1949) found the bladder not affected in animals on a more complete semisynthetic diet or a
regimen composed of rice and carrots. However, liver tumors resulted in all three cases. A daily supplement of fresh milk reduced the incidence of hepatomas from 81 and 5oy0 to 50 and 25y0, respectively, for males and females (Hoch-Ligeti and Russell, 1950). However, the number of tumors a t other locations was increased so that 90% of all rats showed neoplasms which were multiple in many cases. D. Amino Acid Supplements. Heimaii and Meisel (1948) reported that no tumors developed in a group of rats given measured doses of aromatic amino acids in amounts ranging from 143 to 416 mg. simultaneously with a peanut oil solution of 2-FAA introduced into the pharynx. One explanation of these results appears to be that of poor absorption of 2-FAA, because many swellings and oily cysts were found in the submaxillary regions extending into the subcutaneous areas of the chest and axilla. The absence of liver damage in the aromatic amino acids-supplemented group requires further study. a. Tryptophan. Substitution of a 26% level of casein by a n equivalent amount of tryptophan-free casein hydrolyiiate and/or variation of the quantities of supplementary m-tryptophan in the 0.06% 2-FAA diet fed to Fischer line 344 rats produced appreciable alterations in the carcinogenic effect (Dunning et al., 1950; Dunning and Curtis, 1954, 1955, 1957) (Table V). The inclusion of 0.14% of the amino acid, which was slightly below the TABLE V Dietary Tryptophan in Cxrcinogenesis by N-2-F1uorcnylacct,amidee Group
(1) Control 2-FAA (2) 0.14% DL-Tryptophan (3) 1.4% DL-Tryptophan (4) 4.3% m-Tryptophan
Initial Final Daily Average Percent Percent Percent Body Weight Ration Survival HepaEar Bladder (Grams) (Grams) (Days) tomas* Cancer Cancer
144 159 130 150
136 100 106 86
6.1 6.2 5.8 6.5
283 352 400 320
37-81 45-55 73 75
19 0 18 0
0 0 100 92
Compiled from the data of Dunning el al. (1950; Dunning and Curtis, 1954. 1955). Controls to Experiment (2) had an 81% incidence of hepatomas; controls t o Experiments (3) and (4) had a 37% incidence. b
amount considered sufficient for growth in rats, resulted in a lower final body weight, increased the average survival by 70 days, lengthened the latent period, and decreased the incidence of liver tumors. However, increasing the tryptophan level to 1.4 arid 4.3% resulted in a bladder neoplasm incidence of 100 and 929.',, respectively. Liver tumors were increased also, and the average survival was prolonged 117 and 37 days, respectively, compared to rats on the control casein-plus-2-FAA diet. Boyland et al. (1954) confirmed the tryptophan effect on the induction of
372
ELIZABETH K. WEISBUHGEH AND JOHN H. WEISBURGEH
bladder tumors using Wistar strain rats. They failed to induce bladder cancers with benzidine or 2-naphthylamine in place of 2-FAA in the tryptophan enriched diet. Dunning and Curtis (1957) reported the induction of bladder tumors when tryptophan was added to a casein diet instead of a casein-hydrolyzate diet, which ruled out the latter as a promoting agent. They also found 0.8% indole and 1.0% indoleacetic acid in the diet were almost as effective as tryptophan in inducing bladder tumors with 2-FAA. Thus, tryptophan, indole, and indoleacetic acid appear to promote the induction of bladder tumors in rats ingesting 2-FAA while a t the same time lengthening the latent period of appearance of the liver tumors. One explanation of the tryptophan-2-FAA effect on bladder tumor induction, suggested by Boyland et al. (1954), involved the modification of the metabolism of 2-FAA by the diet, resulting in a n increase of a n active bladder carcinogen. Examination of this facet of the problem, however, has revealed no appreciable differences in the urinary metabolites or the amount of radioactivity in the livers and kidneys of rats on experimental and control diets containing DL-, D-, and L-tryptophan and fed C14-labeled 2-FAA (this laboratory, unpublished). Dyer and Morris (1957) have extended the metabolism studies to include the effect of 2-FAA ingestion on the degradation of tryptophan. They found an apparent interference between pyridoxine and 2-FAA (or a metabolite) in rats given a 100 mg. oral L-tryptophan load with the usual amounts of this vitamin and 2-FAA incorporated in the diet. This challenging dose of the amino acid in rats ingesting 2-FAA had a pronounced influence on tryptophan metabolism resulting in excretion of much more xanthurenic acid than by the controls not fed the carcinogen. In the absence of a challenging dose of tryptophan, the same strain of rats excreted no detectable xanthurenic acid when fed casein or hydrolyzed casein diets supplemented with either L-tryptophan or indole (this laboratory, unpublished). It is tempting a t this time to postulate the ortho-amino-hydroxy derivatives of fluorene as the proximate agents in carcinogenesis by 2-FAA (cf. p. 412414). If this call be assumed for the sake of thc present argument, it is
.,. - k p m c &*NH( \
c I
@&OH OH - - * \
(
- -,-, ' enzymc
/
oH/!ymc &NHi \
R
OH 2-Amino-1-fluorenolenzyme complex (VII)
Xanthurenic acidenzyme complex (VIII)
3-Hpdroxyanthranilic acid-enzyme complex
(1x1
373
2-FLUORENAMINE AND RELATED COMPOUNDS
not too uiireasonable to imagine that such compounds, and xanthurenic acid (VIII), or other ortho-hydroxylated metabolites of tryptophan such as hydroxykynurenine, or 3-hydroxyanthranilic acid (IX), a bladder-tumorinducing chemical in mice (Boyland and Watson, 1956) are in competitive equilibrium for a crucial site of a liver enzyme. In a greatly simplified picture it might be further thought that occupation of this site by the ortho-aminofluorenols would eventually lead to neoplastic changes in the liver. Competitive displacement of the fluorene metabolites by tryptophan metabolites would thus manifest itself by an increased latent period in the induction of liver tumors. Indole has the same effect as tryptophaii in delaying liver lesions and causing bladder cancers (Dunning and Curtis, 1957). It may be that metabolism would produce the ortho-hydroxylated 2-hydroxyindole (X), or 7-hydroxyindole (XI), structures resembling closely those of the tryptophan metabolites. Some support for these ideas might be derived from the fact that 8-hydroxyquinoline (XII) has recently been shown to be carcinogenic to the bladder of mice (Boyland and Watson, 1956) and, because of the similarity of its structure to the compounds discussed above, inight well fall into the same class.
Q
QQ
7-IIydrosyndole
S-Hydrosyquinoline
(XI)
(XII)
OH
2-Hydrosylndole
(W
E. Fat Level. Further evidence that the site of the carcinogenic action of 2-FAA can be affected by dietary means is derived from a study by Engel and Copeland (1951). The administration of the carcinogen to weanling female AES strain rats on an unrestricted high-fat diet favored the induction of mammary tumors, whereas such lesions were greatly reduced by a restricted intake of this same diet. It appears, therefore, that caloric, restriction played a part in preventing the development of such neoplasms (Engel and Copeland, 1951). In contrast, tumors of the eye, originating in the Harderian gland, were produced with a 32% incidence under exactly opposite conditions, namely the ingestion of a low-fat diet which resulted in extremely poor weight gains. However, such tumors were not obtained in pair-fed rats on the highfat diet. They also noted only 4 of 18 rapidly growing rats had ear duct tumors, while 25 of 44 more slowly growing animals showed this lesion. The induction of the ear tumors may be connected with some function of the sebaceous glands of this duct.
374
ELIZABETH K. WEISBURGER A N D JOHN H. WEISBURGElt
F. Vitamins. Studies on the effect of pyridoxine, riboflavin, vitamin BIZ,ascorbic acid, and biotin on 2-FAA carciiiogenesis have been reported. The ascorbic acid and biotin appeared to bc without influence (Strombeck and Ekman, 1949; Harris, 1955). Some evidence implicating pyridoxine in carcinogenesis can be derived from the abstract of Harris (1955), who found that a 100-fold increase in dietary pyridoxine resulted in an increased rate of formation of liver tumors in both sexes and of ear duct neoplasms in female rats. A 10-fold increase in dietary riboflavin was required to maintain a normal level of hepatic riboflavin in rats fed 2-FAA (Wase and Allison, 1950, 1951). Protection from the toxic effects of 2-FAA was indicated by enhanced growth and raised protein efficiency. According to Engel and Copeland (1949) in a preliminary report, variation of the dietary riboflavin from 1 to 100 mg./kg. of an 11%casein diet had no effect on 2-FAA carcinogenesis in AES strain rats, while Harris (1955) indicated that a 100-fold increase in this vitamin accelerated the development of acoustic sebaceous gland lesions in female rats. Low levels (1 and 2 p g . / g . diet) of riboflavin resulted in poor survival in rats given 2-FAA by stomach tube (Morris et al., 1955). Diets containing 1, 2, and 10 pg. of riboflavin per g. of diet were used and resulted in liver and ear duct tumors on the lowest vitamin level, the same plus mammary tumors on the intermediate, and all of these plus a variety of other cancers at the highest vitamin level. It was suggested at that time that the higher dietary riboflavin promoted mammary, ear duct, and other tumors but had no apparent influence on the incidence of liver tumors. Supplementation of a 60% casein-2-FAA-containing diet with a mixture of vitamin BIZand folacin had a deleterious effect on survival and hastened the induction of mammary tumors in AES strain rats according to Engel and Copeland (195213). G. Miscellaneous Dietary Constituents. The following additions to the diet failed to modify the course of carcinogenesis by 2-FAA: Ventriculin (Green and Bielschowsky, 1947), inositol or choline (Green and Bielschowsky, 1945), methionine (Paschkis et al., 1948), deoxyribonucleic acid, or yeast nucleic acid (Engel and Copeland, 1952b). Administration of a choline-deficient diet, containing 2-FAA, for 5 days and continuation of a complete diet containing the carcinogen did not show any additional effect ascribable to the deficiency (Lau and Baier, 1954). The addition of 0.5% detergent to a 20% protein diet containing 2-FAA caused an earlier appearance of more quickly growing tumors (Engel and Copeland, 1949). Amounts of cystine or glycocyamine which cause liver damage favored liver tumor formation in female rats (Bielschowsky, 1944).
2-FLUORENAMIXE
. 1 S D RELATED COMPOUNDS
375
6. Injluence of Hormonal Factors
A. Steroid Hormones. Wilson et al. (1941) noted female rats tolerated higher dosages of 2-FAA for longer periods of time than males but were not immune t o the carcinogenic effect of the chemical. That the localization of the induced neoplasms was associated with sex was conclusively demonstrated in the experiments of Bielschowsky (1944), who found a higher incidence of liver tumors in males and a lower incidence of mammary neoplasms. Whereas castration only slightly increased hepatic lesions in males (cf. also Read and McGovern, 1953), mammary tumors were almost completely inhibited in spayed female animals. This observation indicated that ovarian secretions were involved in the genesis of mammary tumors and has led t o a number of investigations attempting to direct the carcinogenic effect of 2-FAA to the hormonal target organs. a. Eflect on mammary tumors. Extensive experiments by Cantarow et al. (1948) revealed no influence of estradiol and gonadotropin on the incidence of mammary carcinoma in female rats although testosterone or castration had a n inhibiting effect which was not reversed by progesterone. However, 2-FAA and progesterone did induce mammary lesions in 8570 of intact female rats but proved ineffective in male rats. Supplementary testosterone and estradiol caused a few mammary tumors. It was suggested in view of these results that small amounts of estrogen are required for the induction of mammary carcinoma by 2-FAA and that the level of progesterone may be a limiting factor. Kevertheless, the problem offered considerable intangible complications, for a subsequent experiment (Cantarow et al., 1949) showed that the combined treatment with estradiol and progesterone actually lowered the incidence of mammary tumors, although this may have been due to an excess of estrogen. Implanted pellets of stilbestrol in female rats receiving 2-FAA decreased the percentage of mammary neoplasms from 60-70% (carcinogen alone) to about 30%; estrogen alone caused such lesions in less than 20% of the rats (Bielschomsky and Hall, 1950). In another approach to this question, Bielschowsky (1953) noted that spayed rats bearing ovarian grafts exhibited almost the same incidence of mammary neoplasms as intact rats. Not all of these implants produced corpora lutea but apparently did secrete subnormal amounts of estrogen from which the authors concluded that estrogens are the ovarian hormones required for mammary gland neoplasms. However, such an explanation may possibly present a rather simplified picture of a system involving the mammary gland, the gonads, the pituitary, and perhaps the adrenals, stabilized by multiple and interrelated feedback pathways (cf. Furth, 1957). Administration of a carcinogen such as 2-FAA
376
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
might have widespread repercussions on the animal and shift the hormonal system to a new equilibrium wherein more than one factor may be altered (cf. also Horava and Skoryna, 1955). The distinction made by Cantarow et al. (1946) between a functional hyperplasia of the target organs of the sex hormones with the nonfunctional hyperplasia of the thyroid by goitrogenic agents may be a determining factor in the relationship between simple growth contrasted to neoplastic growth such as induced by 2-FAA. That the mammary tumors induced by 2-FAA could be ascribed to an estrogenic effect of the carcinogen can be ruled out, for such a property was shown to be absent (Strombeck and Ekman, 1949; Bielschowsky, 1944, 1953). The administration of 2-FAA to rats (Hoch-Ligeti, 1948; Danneberg and Schmahl, 1952) and to mice (Leathem, 1949a; Maclagan, 1949) appeared to inhibit estrus in most of the treated animals while regular cycles were observed by Wilson and associates (1947a), Bielschowsky (1953), and Stasney et al. (1947) in animals ingesting 2-FAA. Finally, Hoch-Ligeti found that only 2 rats of a small group fed 2-FAA failed to develop tumors. These two had normal estrus cycles. 2-FAA did not affect the course of pregnancies (Heiman and Meisel, 1946; Bielschowsky, 1947b; Symeonidis, 1954; Leathem, 1955). Symeonidis devoted considerable effort to elucidate the continuing action of the carcinogen on the pregnancy, the embryos, and the resulting litters, as well as the effect of gestation on the development of neoplasia in several strains of rats. Rats of the Marshall M-520 and AxC strains were prone to hepatic neoplasia, while Buffalo, Sprague-Dawley , and Osborne-Mendel rats showed more of a tendency to mammary gland tumors. There appeared to be a reverse relationship between mammary and liver cancer development depending on strain. Symeonidis suggested that hormonal factors may be involved in promoting the carcinogenic effect in one organ, such as the breast, and inhibiting it in another such as the liver. Some evidence on this point can be derived from the fact that breeding with the associated hormonal stimulation increased mammary gland lesions in the two lowmammary cancer strains whereas little difference was noted between virgins and breeders in the genetically responsive types. Lactation was reduced in the last three strains and was completely preveiited in the first two. The reverse was observed in regard to liver tumors: breeding caused only minor changes in the incidence of carcinomas in Buffalo, Sprague-Dawley, and Osborne-Mendel strains but brought about a n appreciable decline in Marshall and AxC rats. Although pregnancy was normal, the number and size of the litters were decreased, and mortality was higher in the newborn due to insufficient lactation. There appeared to be a higher incidence of tumors of all types in the offspring whose mothers had been exposed continuously to the carcinogen although the overall distinguishing response, characteristic of each strain, was maintained.
%FLUORENAMINE AND RELATED COMPOUNDS
377
Bielschowsky (1947b, 1953) observed that while gestation increased the growth rate of mammary tumors induced by 2-FAA1 further growth was often arrest,ed during lactation. Indeed, small tumors tended to regress, and in two cases an apparent disappearance of the lesions was noted. Renewed tumor growth occurred, however, upon weaning. Regressive changes did not take place with large established neoplasms, no more than regression of an already present tumor could be induced by ovariectomy. If lactation can be considered the prime function of the mammary gland, then the restraint of neoplasia during the actual operation of the gland, and the iiicreased growth during the stimulatory, but nonfunctional prepregnancy stages, would supply some evidence for the suggestion of Cantarow and associates mentioned previously. b. Eflect on liver tumors. The larger proportion of liver lesions in males as compared to females has been noted regularly by most investigators in this field. For example, Miller et al. (1949) obtained a 100 and 0% incidence of liver tumors in males and females respectively, a t 8 months; Leathem (1951), after 6 months, found 90 and 10% in that order. However, Stasney et al. (1947), who recorded a similar imbalance in liver neoplasms in most animals examined after less than 250 days of exposure to the carcinogen, suggested that those female rats surviving longer are equally prone t o develop liver tumors. Thus, 88% of the females and 1 0 0 ~ oof the males developed liver tumors after the more prolonged treatment. The liver of female rats appears less sensitive to the action of 2-FAA in the early time periods, as demonstrated by the findings of Skoryna and Webster (1951) and of Farber (1956). More hepatomas were induced faster by the administration of estradiol or pregnant mare serum gonadotropin to females, and of testosterone or chorionic gonadotropin to males (Cantarow et al., 1946; Stasney et al., 1947; Kirby, 1947). However, the experiments of Morris and Firminger (1956) illustrate the sex-associated character of liver tumors. Neoplasms of this organ were produced rapidly and in high yield by the administration of N-2-fluorenyldiacetamide to intact male, and castrated, testosteronetreated female rats. The incidence of such tumors was lowered appreciably in castrated males and decreased still further in castrated males given stilbestrol. Less than 10% of intact females showed liver neoplasms (Fig. 2). Armstrong and Bonser (1947) found male mice more susceptible to bladder but not to liver tumor induction. However, Leathem (1951), in a preliminary report, noted no sex effect on liver tumor induction in Swiss mice on a stock diet but found males more susceptible than females a t the same level of 2-FAA in a semisynthetic diet. Injections of cortisone seemed to accelerate the appearance of liver tumors in Wistar rats receiving 0.07% 2-FAA and increased its toxicity appreciably (Hoch-Ligeti, 1955). In contrast, the direct passage of adrenal
378
ELIZABETII I;. WEISBURGER A N D JOHN H. WEISBURGER
cortical hormones into the liver, achieved by means of adrenal transplantations into the spleen, did not affect the lesions observed in the livers of female Wistar rats fed 2-FAA, but appeared to induce ovarian tumors in a small number of animals (Read and McGovern, 1953). It was suggested that “the adrenal steroids, by direct passage through the liver damaged by the carcinogen, have been able to produce peripherally an adverse effect on the ovaryl without, on the other hand, having any effect on the type of liver damage.” Hormonal effects in liver cancer have bceii reviewed lately (Cnntnrow, 1957s).
-
Castrated
7 Intact d
p + To8tostemm
9
Castrated t Tostosterone
Ca8trated
6 Intact
0 0
4
8
12
16
20
24
28
32
36
40
44
48
52
p
56
60
Weeks FIQ.2. Sex dependence of hepatoma development in rats following the ingestion of N-2-fluorenyldiacetamide. From Morris and Firminger (1956).
B. Thyroid and Hypophyseal Hormones. Direct functional stimulation of target organs by the appropriate hormones in animals treated with 2-FAA rarely causes tumors at the desired site. As a general rule, however, some form of excessive growth has been found to precede iieoplasia (cf. Noble, 1957, for related discussion and references). a. Thyroid. Experimental tumors of the thyroid gland have been produced in a variety of ways (cf. extensive reviews on this subject, Bielschowsky, 1955; Morris, 1955a,b), one of which was the ingestion of 2-FAA simultaneously with one of keveral thiocarbamide type goitrogens or a low
2-FLUORENAMINE
AND RELATED COMPOUNDS
370
iodine diet (Axelrad and Lebloiid, 1955). It can be concluded, however, from the results of a large number of experiments that 2-FAA is unnecessary in the production of thyroid gland tumors but may serve to accelerate the process in the earlier stages. b. Thyroid function and liver carcinogenesis. Cantarow et al. (1946) aud Paschkis et al. (1948) noted a decreased incidence of liver tumors in rats fed both 2-FAA and thiouracil. A protective effect was also found in mice (Gorbman, 1947; Leathem, 1955). According to Paschkis et al. (1948), the goitrogen did not appear to promote the detoxification of the carcinogen because tumors were induced a t other sites. Reduced food intake did not occur. I n an abstract, Leathem and Barken (1950) reported fewer liver tumors in the thiouracil group in tests where varying food consumption was controlled by pair-feeding. A direct action of thiouracil on some enzyme mechanism in the liver cell in such a manner as to prevent the carcinogenic influence of 2-FAA was suggested. Such ail explanation received support when it was found that the protective effect of thiouracil could be overcome by the simultaneous administration of uracil (Paschkis et al., 1951). Hypothyroidism appears to be an important factor inasmuch as the administration of thyroxine also antagonized the effect of thiouracil in rats on a diet containing 2-FAA, and resulted in almost as many liver tumors as those developed on the carcinogen alone (Paschkis et al., 1948). Furthermore, Bielschowsky and Hall (1952, 1953) found that thyroidectomy prevented the development of liver lesions in animals administered 2-FAA or 2-FA but had no effect on tumors a t other sites. It was noted (cf. Bielschowsky, 1955) that the degree of thyroxine deficiency was crucial, for hepatomas were absent in rats with pituitaries void of acidophil cells, provided that the thyroid was extirpated before the beginning of the carcinogen regimen. Liver tumors developed if the thyroidectomy was performed 13 weeks after the initiation of the carcinogen administration, although Skoryna (1955) (cf. Section IV,6,B on hypophyseal hormones) reported that hypophysectomy after similar or even longer periods did not lead to malignancy. This difference may be accounted for by the recent preliminary report of Bielschowsky (1956) that the effects of the thyroid are mediated via the pituitary. Thus, the effect of thiouracil in preventing liver lesions in rats given 2-FAA can seemingly receive satisfactory explanations based on a direct effect on the liver cell and/or on the hypothyroid state induced by it. I n view of the facts a t hand, it is quite possible that both factors play a complementary role. This problem is closely interwoven with the prevention of hepatic neoplasia by hypophysectomy and no clear distinction can be perceived. c. Pituilary. The pituitary is known to exert powerful influences affect-
380
ELIZABETH K. WEISBUHGER A N D JOHN 13. WEISBUIEGER
ing a number of other endocrine glands by its secretions so that there exists a delicately balanced relationship among the various endocrine glands. Excess of hypophyseal hormones. Castration leads to an increased output of gonadotropic hormones by the pituitary owing to the feedback mechanism (see Furth, 1957). A parabiotic union between a castrated rat and a normal rat exposed the target sex organs of the latter to the influence both of his own pituitary secretion, mainly luteinizing hormone, as well as the abnormally high gonadotropins, principally follicle-stimulating hormone, from the castrated partner (Bielschowsky and Hall, 1951a,b). Hence, the gonads of the normal animal hypertrophied and secreted inordinate amounts of sex hormones. Upon administration of 2-FAA to the intact partner, adenocarcinomas of the seminal vesicles were found in males in addition to hepatomas. Female rats were afflicted with tumors of the ovaries, which appeared to have originated in the granulosa cells indicating excess of follicle-stimulating hormone from the castrated partner as an essential factor. These results have been fully confirmed by Sommers and Chute (1956). Of considerable interest was the observation that neoplasia or even hyperplasia of the liver, normally observed after prolonged intake of 2-FAA, was absent in the parabiont litter-mate not receiving the chemical directly. It was noted above that the hypophyseal gonadotropins produced in the castrated partner exerted their characteristic effect in the intact rat. Hence, it must be presumed that the hormone passed from one to the other animal, logically through the common circulatory system. The carcinogen affects a variety of tissues in the treated animal, and it should reach these tissues in the parabiont, yet it does no visible damage. Perhaps a small and insufficient amount of carcinogen passed from one to the other partner, whereas a similarly small but sufficient quantity of gonadotropin was transferred. This point is readily amenable to test. Also the parabiont partner may be exposed only to the metabolites of the carcinogen and thus its liver may be spared. The injection of gonadotropins by Stasney et al. (1947) may have failed to affect the target organs in animals treated with 2-FAA because (1) the injected material did not correspond in composition to the endogenously produced hormones, (2) the quantity available a t the target organ was insufficient, and (3) the duration of the stimulation was insufficient. Periodic injection of hormones may not provide the persistent drive necessary for neoplasia in target organs although it may be sufficient to induce characteristic secondary effects or even accelerate tumor formation at more sensitive sites. Rielschowsky and Hall (1951a) have observed that carcinogenesis in the gonads required higher doses of 2-FAA for longer periods of time than in the organs customarily affected by this compound.
2-FLUORENAMINE A N D RELATED COMPOUNDS
381
Deficiency of h,ypophyseal hormones. Skoryna (1955) showed that hypophysectomy of rats 26 weeks after the initiation of 2-FAA administration prevented the conversion of already-present benign liver tumors and parenchymal hyperplasia into malignant hepatomas. Firminger and Morris (1955), Hoffman (1956), O’Neal and Griffin (1957), and Richardson and O’Neal (1957) reported striking inhibition of liver neoplasia in hypophysectomized rats treated with N-2-fluorenyldiacetamide. Indeed, not even the hyperplasia and cirrhosis normally associated with the precancerous condition could be observed. These findings are thus similar to those noted with the azo dyes. The carcinogenic effect of the dyes was restored to a large extent by the administration of adrenocorticotropin (ACTH), which simultaneously partially restored adrenal morphology, and also by hypophyseal growth hormone, or by a mixture of the two factors (Griffin et al., 1955a,b). In contrast, the ability of N-2-fluorenyldiacetamide to induce hepatic lesions was not reestablished by ACTH, but injections of growth hormone appeared to lead again to livers in a precancerous condition in hypophysectoinized animals, provided that the growth hormone regimen was started within 2 weeks of hypophysectomy. Under such conditions, the atrophied thyroid gave no evidence of returning to a normal morphological state (Hoffman, 1956). It is especially noteworthy that the absence of pituitary hormones not only prevented the induction of liver tumors but also the characteristic symptoms associated with the preneoplastic condition. Malignancy was also avoided even when the surgical removal of t,he pituitary was effected several months after initiation of the carcinogen containing diet. The effect of pituitary ablation on the induction of neoplasms a t a variety of points other than the liver could be studied by the use of fluorene carcinogens but not by the liver-specific carcinogenic azo dyes. Thus, Skoryna (1955) reported a sebaceous gland carcinoma in a completely hypophysectomized rat fed 2-FAA. A more striking preliminary observation of O’Neal and Griffin (1957) was that eye tumors occurred in 65% of the hypophysectomized animals and in 10% of the controls. While the livers appeared completely normal in the hypophysectomized animal, 70% were tumerous in controls. It seems that the prevention of hepatic neoplasia by hypophysectomy is specific since other tumors can still be induced. However, a thorough investigation of the effect on other organs, including the mammary gland, has not been made. C. Pancreatic Hormones, The literature contains only a preliminary unconfirmed report dealing with a deficiency of these hormones (Jacoby and Woodhouse, 1946). Rats rendered mildly diabetic with four injections of alloxan and treated with 2-FAA exhibited one ear duct tumor after 5 to 6 months. The period of observations appeared insufficient to draw any
382
ELIZABETH P. WEISBUI
definite conclusions. Azo dyes have been found t o induce fewer tumors in diabetic rats (Salzberg and Griffin, 1952). 7 . Interaction of Carcinogen and Tissue Constituents
A. I n Viva. Evidence for a direct interaction between a carcinogen and the tissue proteins was presented for the first time in 1947 by Miller and Miller in their study of the carcinogenic azo dyes (cf. Miller and Miller, 1953). The discovery of binding of other carcinogens of diverse types to tissue proteins followed shortly (cf. Heidelberger, 1953; Miller and Miller, 1952, 1955). That the isotope from labeled 2-FAA was combined with liver and other organs was observed by Miller and Miller (1952)) Dyer et al. (1953)) and E. K. Weisburger et al. (1953). Liver proteins isolated 1day, or 1 week after treating a rat with labeled 2-FAA lost by continuous extractions about 30 to 50% of the liver radioactivity; 50 to 70% appeared to be firmly attached. After repeated oral doses of N16-2-FAA the liver protein enrichment was as large after 6 as after 24 hours and could be detected for a t least 2 weeks. Maximal protein binding in rats took place by repeated oral intake of 10 mg. of 2-FAA per 100 g. of body weight. It was seemingly impossible to induce a higher degree of incorporation (Dyer and Morris, 1956). Carbon-14-bound isotope, however, has been detected 32 days after a single intraperitoneal5 mg. dose of 2-FAA, or 1 day after as little as 0.01 mg. of carcinogen intraperitoneally (this laboratory, unpublished). Azo dyes, which exert a carcinogenic effect on the liver exclusively, were bound only to the liver proteins (Miller and Miller, 1953; Salzberg et al., 1951). In rats given labeled 2-FAA or 2-FA orally, the proteins of all organs examined contained isotope although, in general, the liver showed most enrichment (Dyer and Morris, 1956; Weisburger et al., 1954). Such findings are also suggestive of a positive correlation between protein binding and the development of neoplasia. However, tumors have not been obtained in every tissue, e.g., the heart, although isotope was attached to heart proteins. Miller and Miller (1955) showed the highest level of C14labeled 2-FAA was bound to the liver proteins of the most susceptible species, and the lowest to those of most refractory ones (Table VI). Dyer and Morris (1956) also reported less enrichment with N16 in the proteins of the livers from the nonsusceptible guinea pigs fed N15-2-FAA than from rats. This might mean that guinea pig liver does not contain the type of protein required for the binding process. In addition, Dyer and Morris observed enrichment of rat liver proteins with N16to about the same extent after feeding the labeled carcinogens 2-FAA, N-2-fluorenyldiacetamide, and 2-nitrofluorene, although these compounds differ in the rate and degree of induction of liver and other tumors. The liver proteins after the administration of the noncarcinogenic N-4-fluorenylacetamide were not enriched
383
2-FLUORESAMINE ANI) RELATED COMPOUNDS
TABLE VI Relative Amounts of Protein-Bound C14in the Livers of Various Species of Animals After Administration of N-2-Fluoren-9-C14ylacetamidea Species
Sex
Liver (perfused) Counts/min./mg. of Protein
Liver Tumors Percent
Months
5@100 0-10
5-6 6
~~
Rat
M F
92, 112 97, 116
Hamster Mouse Cotton rat Guinea pig
M M M M
30,32
(liver damage) 17, 19 12, 13 9, 10
10 40 0 0
14 10 18 16
a Adapted from b1iller and Miller (1955). Livers were removed 24 hours after a single dose of 22.7 mu. 2-FAA-9-C” (5,000,000 counts/min./mg.) per kg. of body weight. The mice were fed a purified diet containing 0.1% of 2-FAA; all the other species were fed a grain diet with 0.036% of carcinogen.
with Nib. However, radioactivity was attached to the proteins of a variety of tissues of rats given radioactive N-(7-hydroxy-2-fluorenyl)acetamide orally (Weisburger et al., 195713). This is another compound reported noncarcinogenic (Bielschowsky, 1947b), or a t least many times less carcinogenic than 2-FAA (Hoch-Ligeti, 1947a) yet the amount of activity bound was equivalent on a molar basis to that encountered with 2-FAA. A number of other instances of combination of a noncarcinogen with proteins is also known (Woodhouse, 1955). Heidelberger and Moldenhauer (1956) interpreted one such case on the assumption that carcinogens and noncarcinogens affect different binding sites. The rate of increase and disappearance of the protein-bound material could be a factor differentiating carcinogen from noncarcinogen. A carcinogen might be expected to remain tightly attached to the cellular elements for their entire lifetime, whereas a transient combination might be indicative of a noncarcinogen. Examples of this nature have been described in the case of azo dyes (Miller and Miller, 1953) and with polynuclear hydrocarbons (Hadler et al., 1957). The technique of determining protein binding, involving the total protein from one organ, is rather crude, as indicated by isotope present in different concentrations in the protein fractions from the same organ. Thus, a number of investigators have expressed the opinion that several proteins may be affected, some of which may not be involved in the developments leading to neoplasia. Sorof et al. (1957) have sought to refine the methods applied to the study of the protein binding by showing that the azo dyes bound to the soluble proteins of the liver migrated in an electrophoretic field with a minor subclass of proteins classified as “slow-h2” and having an approxi-
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ELIZABETH K. WEISBURGER AND JOHN H . WEISBUNGER
mate molecular weight of 40,000. These proteins increased in the early stages of carcinogenesis by the azo dyes as well as by 2-FAA but appeared to show no change in the liver proteins from rats given a noncarcinogenic azo dye. Induced liver tumors contained but a low concentration of these proteins. Thus, the picture so far obtained strongly suggests an intimate relationship between a relatively select and small group of proteins and the phenomena leading to neoplasia. Further evidence for the multiplicity of the proteins involved can be gathered from the fact that a number of substances were attached to the proteins from rats fed 2-FAA. Upon hydrolysis of the proteins and extraction of the resulting mixture with ether, isotope was found in both the organic as well as the aqueous phase. Actually most of the material was preferentially water-soluble (E. K. Weisburger et al., 1953; Dyer and Morris, 1956). Preliminary experiments indicated that the bulk of the radioactivity carried by the ether-soluble compounds in the protein hydrolyzate was not associated with any of the known metabolites of 2-FAA (this laboratory, unpublished). The protein binding was observed in most cases after isolation of the protein under such conditions that denaturation occurred. The protein examined obviously was not identical with the native kind, and that fact must be considered in evaluating the results obtained by such methods. Apparent artifacts in protein binding have been reported after homogenization of tissue or incubation of heatdenatured homogenates with a carcinogen (Siebert et al., 1952; Dyer and Morris, 1956; Gutmann and Peters, 1957). Dyer and Morris showed that the incubation of rat liver with the noncarcinogen N-4-fluorenylacetamide1 or of guinea pig (resistant species) liver with 2-FAA yielded similarly high levels of isotope per unit weight of protein. On the other hand, the liver proteins isolated from rats fed 4-FAA, or from guinea pigs given 2-FAA showed little evidence of protein binding. It is possible that the contradictory results can be explained by the fact that hydroxylated metabolites of 2-FAA which bind to protein are produced in vitro under all circumstances. In vivo, however, the resistant species may have the ability to convert these hydroxy derivatives to conjugates faster than they can bind to protein. Similarly, the noncarcinogens could be deactivated by conjugative mechanisms. B. In Vitro. Gutmann et al. (Gutmann and Peters, 1954; Gutmann et al., 1956c; Peters and Gutmann, 1956) have made appreciable contributions to the protein-carcinogen interaction by means of in vitro studies. Initially these authors observed that the radioactivity from C14-labeled 2-FAA or 2-FA was bound to the proteins of rat liver slices and of the supernatant fluid but not of homogenates after incubation at 37°C. in an oxygen atmosphere. More sensitive techniques, however, showed that
SFLUORENAMINE AND RELATED COMPOUNDS
385
both slices and homogenates were able to bind radioactivity, but the slices were 3 times more active. Fortification of the homogenates with diphosphopyridine nucleotide, nicotinamide, and sodium succinate resulted in a tripling of the protein-bound activity and simultaneously increased by 9-fold hydroxylation of 2-FAA. These authors concluded that substrate hydroxylation was required for protein binding. The slightly carcinogenic N-2-fluorenylbenzamide was also hydroxylated to an extent comparable to that of 2-FAA and likewise showed similar protein binding. On the other hand, debenzoylation to 2-FA occurred only in small amounts as relative to that found with 2-FAA, demonstrating that protein binding i n vitro was largely independent of free base formation. The major share of the protein-bound radioactivity was not decreased by the removal of the nucleic acids (Gutmann et al., 1956c; cf. Brigando, 1956). However, as was stated in connection with the azo dyes (Miller and Miller, 1953), a combination with nucleoproteins certainly is not excluded. It might be worthwhile to isolate and chromatograph nucleic acids by presently available procedures to see if activity could be found in some concentrated fraction. C. Possible Modes of Combination. The nature of the presently obscure interaction between proteins and carcinogens seems fundamental to an understanding of chemically-induced neoplasia. One method by which combination of carcinogen and cell constituents could occur may involve epoxides. The actual mechanism by which hydroxylation of aromatic compounds takes place is not known but epoxides can be considered as possible intermediates (cf. Boyland, 1950). Such reactive compounds could be conceivably captured by proteins present at the site of their formation. Studies in model systems have suggested that quinone imines derived from the oxidation of certain aminofluorenols (cf. Section 111,3,C) are also compounds which could combine with proteins (Nagasawa and Gutmanri. 1958. The differences in carcinogenic effect of 2-FAA in rats and guinea pigs have emphasized the importance of ortho-hydroxylated amines in the development of neoplasia (cf. Section VI). Perhaps ortho-hydroxy amines could bind to protein through a metal ion (cf. Boyland and Watson, 1956). This conceivably could occur by (1) the metal being linked to the nitrogen (e.g., as in the porphyrins), with the stereochemical arrangement stabilized by a hydrogen bond from the protein to phenolic oxygen, or (2) the metal being attached to the phenolic oxygen and forming a coordinate bond to the nitrogen. I n this connection, Bonser et al. (1956a) reported that stannous ion forms a stable complex with 2-amino-l-naphthol. Inner copper salts were described with ortho-hydroxy or amino monoazo compounds (Mur, 1956; Ueno, 1957), and in addition copper acetate appeared to
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ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
inhibit hepatoma formation by N,N-dimethyl-p-phenylazoaniline (Orr et al., 1955; King et al., 1957). Pinck (1956) studied the reaction of isomers and analogs of 2-FA with montmorillonite, a n inorganic clay mineral containing iron. 2-FA and a number of similar compounds yielded dark green to blue colors, explained on the basis of a zwitterion formation between a positively charged nitrogen atom a t the 2-position and a negatively charged carbon atom on the 9-position. The correlation between this reaction and carcinogenicity was fair. Moreover, iron-free montmorillonites failed to give the reaction. Thus, the colors observed with certain compounds might also be due to an oxidation of the substrate by ferric iron followed by the coordination of the product through the metal to silicate or other constituent of the mineral. In this sense, Pinck’s observations might form a parallel to the protein binding of derivatives of 2-FA in susceptible animals. D. Relation of Binding to Carcinogenesis. While the precise mode of interaction of a carcinogenic agent with a susceptible tissue, including that of combination with protein, is as yet unknown, it appears that some reactions may account for the origin and others for the further development of neoplastic foci, with different conditions favoring or inhibiting these two steps (cf. Berenblum, 1954; Horava and Skoryna, 1955; and many other references). The experimental demonstration of a combination between proteins and the carcinogen or its metabolites is an outstanding finding in the relationship between the chemical and the tissues affected. Miller and Miller (1953) reasoned that proteins possessing some growth-control function might be involved. Their eventual elimination would lead to the unrestrained growth characteristic of cancer. Haddow (1953) , in an excellent review of the whole problem, favored the idea that carcinogens have a more direct action on certain basic genetic processes of which the loss of protein or enzymes is a secondary consequence. Earlier, Haddow (1938) believed that carcinogens interfered in the growth mechanisms of normal cells resulting, in time, in the appearance of new cells not subject to the restraining influence of the carcinogen. This concept retains considerable merit in the light of the discussion on the type of molecule capable of eliciting neoplasia (cf. Section IV,4,C), namely, one potentially interfering in crucial oxidation-reduction processes. Thus, a direct or indirect restraint of metabolic reactions owing to the combination of the carcinogen to cellular constituents may play an appreciable role in the initial conversion of normal to abnormal tissue [cf. latent or dormant tumor cells (Berenblum, 1954; Rusch, 1954)] but is by itself insufficient in generating the chain of events leading to cancer. Somewhat similar views have been expressed by Furth (1957) in discussing the role of hormones in tumor development and growth and by Meites (1957) in suggesting that
2-PLUOItENAMINE
A N D 11ELATE1) C 0 M P O U N I ) S
387
endogenous or hormonal factors may play a part in the induction of skin tumors by 7,12-dimethylbenz[a]anthracene. Thyroidectomy or hypophysectomy, procedures known to delay or prevent carcinogenesis in the livers of rats, have little effect on proteinbound azo dye (Griffin et al., 1955b; Miller and Miller, 1955; Ward and Spain, 1957) or radioactivity from labeled 2-FAA (this laboratory, unpublished). Therefore, the combination of the chemical with certain tissues is in this instance not enough for the carcinogenic process to proceed. Apparently the presence of certain stimulating factors on the liver is also essential for tumor development, those factors presumably being absent in the hypophysectomized animal. In a normal animal, the hormonal stimulation attempts to overcome the restraining effect on cell life due to the presence of the carcinogen (a sort of hormone-mediated homeostasis). It is thought that the continued and long-lasting operation of such a stimulatory factor in the face of an excess of restraint imposed by the carcinogen eventually may result in the production of some cells altered so as to escape the curbs imposed by the presence of the agent. This might occur by slow adaptation, instantaneous mutation, or by the potentiation of some viruslike particle. Such changed cells could have the attributes of tumor cells, with a lower level of differentiation and functionality (cf. Rusch, 1954). They would result, thus, not from the action of the carcinogen alone, but also from the external stimulation of the tissue brought about by an initial restraint of growth. A generalized view consonant with the above concept has been put forward by Crile (1958). This author suggested that the growth of cells is controlled by specific trophic hormones; the level of the latter in turn depends on the needs of the cells, as indicated or transmitted by “signal substances.” Prolonged disturbance by various causes of such a feedbackstabilized system would result in excessive growth leading to tumor formation. Further experimental studies utilizing this concept’ would appear fruitful.
V. METABOLISM-ACTION OF AGENT ON
THE
HOST
The results of investigations on the carcinogenicity of 2-FAA as influenced by various extrinsic and endogenous factors, discussed in the preceding sections, may ultimately lead to an understanding of the mode of action of this compound. At present, however, they provide but a few sparse leads toward that goal. Additional information derived from the studies on the carcinogenicity of analogous chemicals affords some insight on the specificity of the biochemical reactions between tissue components and carcinogen. The knowledge thus gained is, nevertheless, clearly insufficient to reach any conclusions regarding the intimate mechanism involved
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ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
in the conversion of normal cells into malignant ones. Studies on the effect of the carcinogen on various constituents of the treated animal have been performed in many laboratories with the hope of apprehending the system or systems directly affected. The following section will deal with this phase of the work. The effects of the carcinogen on the recipient may not be due to the compound administered but could be caused by one or a combination of several of the metabolites. Griffin (1957) has reviewed certain of these topics. 1. Proteins
In contrast to certain azo dyes which led to changes in serum proteins after only 2 weeks, 2-FAA caused no variations in the proportions of serum components after 2 or 6 weeks of ingestion (Cook et al., 1949). Electrophoretic analyses of the serum over a 24 to 28 week period of 2-FAA intake indicated that a- and P-globulins tended to increase, y-globulin remained the same, and albumin decreased somewhat during the precancerous and tumor stages (Griffin et al., 1949). Leathem (1949b) also reported a decrease in plasma albumin after 100 days. Similarly, Claus et al. (1954) found a tendency for decreased albumin and y-globulin, and increased all a2, and /3-globulin in the liver lymph of rats bearing 2-FAA hepatomas. Griffin et al. (1949) and Leathem (1949b) showed that although the phosphorus and moisture content of the liver remained relatively constant, the protein concentration tended to decrease. However, since the liver weight doubled, the total amount of protein actually increased. The depleting effect of 2-FAA on liver protein and a lower blood albumin/globulin ratio and lower hemoglobin were also observed in the blood of dogs fed the carcinogen (Allison et al., 1950). Laird and Miller (1953) have studied in detail the changes induced by 2-FAA in the liver protein during the precancerous and tumor stages. In the nuclei, mitochondria, microsomes, and supernatant fluid, protein nitrogen decreased during the first 4 weeks and afterwards slowly increased or remained near the minimum value. In the nuclei, the protein level returned to the initial value between the 14th and the 25th weeks but recovered little in the mitochondria. Microscopic examination showed that between 4 and 7 weeks there was a sudden increase in mitoses, so that the liver contained 3 times as many cells of smaller size than the original by the 14th week. The hepatomas produced by the carcinogen also had a 24-fold number of cells as compared to normal liver. The weight of each cell was decreased and had a lower protein concentration, of the order of that observed in the liver 4 weeks after initiation of the 2-FAA treatment. Rutman et al. (1954a) confirmed the precancerous changes and noted that the trend of values continued into the tumor stages, whereas, the data of
2-FLUORENAMINE
AND RELdTED COMPOUNDS
389
Laird and Miller would indicate a reversal. The discrepancy was ascribed to differences in diet, rate of tumor induction, and selection of samples for analysis. In transplanted liver tumors induced by N-a-fluorenyldiacetamide, Maver el al. (1952) observed that although the total nitrogen of the cancerous tissue was less than that of normal liver, the tumor nuclei had a higher concentration of protein than nuclei from normal liver cells. Fritx-Niggli (1956) followed the morphological changes in rat liver cells during the process of feeding 2-FAA or N,Ndimethyl-p-phenylazoaniline. The cells remained approximately normal in size, but the nucleus increased at the expense of the cytoplasm, which seemed to explain the increase in nuclear protein and the decrease in mitrochondrial protein during administration of 2-FAA. Furthermore, paper chromatography of the amino acids of the liver showed that cystine, lysine, histidine, asparagine, arginine, serine, aspartic acid, glycine, glutamic acid, and glutathione increased, that alanine was unchanged, and that cysteine, methionine, phenylalanine, valine, and leucine decreased, which was interpreted by her as evidence for a catabolic displacement of the protein metabolism. 2. Amino Acid Uptake
The uptake of m-methionhe in rat liver slices was inhibited proportionately to the level of 2-FA, while addition of riboflavin reversed this effect (Wase et al., 1952). In rats fed 2-FAA for 56 days followed by a dose of ~ ~ - m e t h i o n i n e - Sthere ~ ~ , was a greater uptake of radioactivity in the livers of the experimental animals than in controls during the first 24 hours. This was followed by a more rapid loss during the next 48 hours, indicating higher utilization of methionine due perhaps to increased growth or tissue regeneration in the carcinogen-fed animals. Rutman et al. (1954b) observed that after rats had been fed 2-FAA for 1 to 2 months, the liver mitochondria showed an elevation of radioactivity of approximately 50% after administering alanine-l-C14. After 3 or 4 months of treatment, there was a sudden increase to 200% of the normal uptake, which persisted in the tumor mitochondria. Thirty-three to 50% of the radioactivity in the protein from normal mitochondria was released as C1402 during ninhydrin treatment which indicated that much of the isotope was not bound in a peptide linkage. In contrast only 10-15% of the C14 of the preneoplastic mitochondria1 protein was freed by ninhydrin. Of the balance, about 50% was in the 2-carbon of amino acids, while the remainder was located in other positions. Thus, 2-FAA appeared to increase the rate of protein synthesis in the liver, to change the composition or configuration of the protein formed, or to cause the amino acid fragments to enter different synthetic pathways.
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ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
3. Nucleic Acids
Griffin et a2. (1949) discovered that 2-FAA gradually decreased the concentration of liver ribonucleic acid (RNA), while the deoxyribonucleic acid (DNA), per gram of liver, as well as the purine and pyrimidine content of liver DNA remained nearly normal (Griffin and Rhein, 1951). The adenine :guanine, cytosine :guanine, and thymine :guanine ratios remained normal in 2-FAA fed rats but decreased in rats fed the azo dye N , N dimethyl-4-(rn-tolylazo)aniline. Laird and Miller (1953) stated that the tumor cells had a DNA content similar to that of normal or pretumor cells, but on a fresh-weight basis the tumor had twice the DNA of normal liver. Rutman et al. (1954a), who had similar results, attributed the increase in tumor DNA to a rise in cellularity and decrease in average cell size. Recently DNA has been implicated in protein synthesis by isolated cell nuclei (Allfrey et al., 1957). The fact that DNA levels are not decreased during the process of 2-FAA carcinogenesis indicates that the ability to synthesize protein should not be greatly affected, other factors being equal. Maver et a2. (1952) found a moderate increase in both DNA and RNA in transplanted liver tumors induced by N-2-fluorenyldiacetamide. Laird and Miller (1953) and Rutman et al. (1954a) noted a decrease in liver RNA caused by 2-FAA that was ascribed by both t o a decrease of this constituent in the mitochondria and a progressive increase in the nuclei but with a net decrease per cell. However, because of the increase in liver size during tumor formation, the total amounts per whole liver were equal to or greater than in the normal liver. Pirozynski and von Bertalanffy (1952, 1955) investigated the changes in cytoplasmic basophilia, representative of RNA, by means of histochemical methods and found that RNA appeared to decrease during the precancerous stages and to increase again as tumor formation occurred. Rutman et al. (1954a) have suggested that the resulting selfaccelerating imbalance between the phenomena required for differentiation and function and those phenomena used for the formation and accumulation of nuclear constituents might lead to a new equilibrium position corresponding to the neoplastic state (cf. Cantarow, 1957b). Stasney et al. (1950a) noted tumors in 2 of 118 rats a t the site of intrahepatic injection of mitochondria from a 2-FAA-induced rat hepatoma. It was suggested that the chromatin material, which may have contaminated the mitochondria, was actually responsible for the tumor-inducing activity. Intrahepatic injection of chromatin, prepared a t 4°C. from a transplantable hepatoma induced by N-2-fluorenyldiacetamide, into 29 rats, gave a 27% incidence of tumors. If the chromatin were prepared a t 32°C. or were injected subcutaneously, no tumors were obtained (Stasney et al., 1955). These observations raise the possibility that the malignant potentialities
2-FLUOHENAMINE
.4ND RELATED COMPOUNDS
391
of neoplastic cells reside in cell-free chromatin material that could be transmitted to normal cells. It would constitute a real advance if further experiments confirm that a nucleoprotein fraction, uncontaminated by whole cells, can indeed transmit tumors. A. Uracil Incorporation. Paschkis et al. (1951) found that the protective effect of thiouracil on hepatoma induction was overcome by simultaneous administration of uracil. Rutman et al. (1953) then observed that uracil-2-C14 was rapidly metabolized by both normal and tumor-bearing rats. The metabolic turnover appeared to be hastened somewhat in tumor rats. Subsequent studies showed that (1) the radioactivity of the uracil was rapidly converted to respiratory C1402and (2) there was an appreciable incorporation of the C14 into the hepatoma nucleic acids in contrast to insignificant amounts in normal rat liver (Rutman et al. 1954~). Hydrolysis of the nucleic acids following administration of uracil-2-C14 revealed a very small amount of radioactivity incorporated into the uridylic acid from normal liver and traces in the cytidylic acid and purine nucleotides. On the other hand, appreciable quantities of C14were in the cytidylic and especially in the uridylic acid of primary hepatoma nucleic acids. Preneoplastic livers from rats fed 2-FAA for 90 days showed the same trend as hepatomas. Thus, in contrast to normal tissue, preneoplastic and neoplastic tissues utilized preformed uracil as a precursor of pentosenucleic acids, which may indicate an alteration of physiological control of nucleic acid synthesis during 2-FAA carcinogenesis (Cantarow et al., 1955). Of interest was the finding that although normal rats did not use uracil for building up nucleic acids, homogenates of normal rat liver did, possibly as a result of a higher effective concentration in the latter. This result emphasizes the importance of studying metabolism in intact animals as well as in isolated systems (Rutman et al., 1954d).
4. Fat and Glycogen The feeding of 2-FAA to rats on an 11% or a 20y0 casein diet for 69 to 82 days decreased the percentages of liver sulfur and nitrogen, while the liver fat appeared unaffected. Since there was usually a decline in weight, the total quantities of liver fat, nitrogen, and sulfur, in per cent of body weight, were, however, greater in carcinogen-treated rats (Gutmann and Peters, 1953b). Later, the effect of adding methionine, cystine, or glycine to an 11% casein diet to provide the same amount of dietary sulfur or nitrogen as the 20% casein diet, was determined after a 100 to 119 day experimental period (Gutmann et al., 1956b). Contrary to their previous findings, 2-FAA caused an increase in the liver fat, except on the 2070 casein diet. The sizable variation in the fat content of the control livers in the two experiments may account for the discrepancy. Gutmann et al. further showed that the liver glycogen was unchanged
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ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
in 2-FAA-treated rats on the 11% casein diet but was increased on such a regimen plus methionine or cystine or on a 20% casein diet. Unfortunately, the livers of rats fed 11% casein and supplementary glycine were not analyzed, so that the apparent effect of the sulfur content of the ration on liver glycogen should be supplemented with further data since the protein of nonsulfur-containing amino acids may have a similar glycogenic action. Cantarow et al. (1946) stated that glycogen granules tended to disappear in the neoplastic liver cells induced by 2-FAA. Woodhouse (1951, 1952) found that rats injected once with 2-FA, or with azo dyes, had very low glycogen levels (stress effect?). 5. Vitamins
A. Riboflavin. Riboflavin has been found to be depleted in the livers of rats (Griffin et al., 1949; Laird and Miller, 1953) and dogs (Allison et al., 1950) following the feeding of 2-FAA. A 10-fold increase in dietary riboflavin was required to overcome the effects of the carcinogen on liver riboflavin (Wase and Allison, 1950). Wase (1956) established that the biological half-life of liver riboflavin was decreased from 6.5 days in normal rats to 1.1 days in 2-FAA fed rats. Thus, the need for more dietary riboflavin is apparent. B. Ascorbic Acid. 2-FAA increased the liver ascorbic acid in CBA male mice (but not in females), in “black and white” rats, and to a smaller degree in Wistar rats. On the other hand, N-(7-hydroxy-2-fluorenyI)acetamide caused no, or a very slight, rise in liver ascorbic acid (Daff et al., 1948). In guinea pigs, 2-FAA decreased liver ascorbic acid to almost onehalf the normal level (Kennaway et al., 1955). Both the rat and the guinea pig oxidize 2-FAA to various hydroxy derivatives. Ascorbic acid has been implicated in the process of aromatic hydroxylation in vivo (Axelrod et al., 1954) and in a chemical system (Udenfriend et al., 1954). Since the rat is able to synthesize ascorbic acid, it might respond to the presence of 2-FAA by increasing the synthesis of the vitamin. On the other hand, the guinea pig, which cannot synthesize its requirements, could have its liver depots depleted. C. Pyridoxine. It was noted by Dyer and Morris (1957) that this vitamin prevented an increased excretion of xanthurenic acid and kyurenine derivatives by rats fed 2-FAA following a challenging dose of tryptophan. This finding has been discussed previously (Section IV,5,D). 6. Enzymes
Comparatively few studies have been performed on the effect of 2-FAA on enzyme systems, either where the carcinogen was actually added to the enzyme mixture or where 2-FAA or its metabolites were present in the
2-FLUORENAMINE -4ND
RELATED COMPOUNDS
393
tissues used for enzyme assays. Most often, enzyme levels have been determined in induced tumors under conditions where there existed no possibility of effects due to the presence of the carcinogen. A. Oxidases. The succinic oxidase activity of a primary rat hepatoma induced by azo dyes was one-third that of normal liver (cf. Greenstein, 1954). Hoch-Ligeti (1947b) reported, however, that rat primary hepatomas induced by 2-FAA had the same succiriic oxidase activity as normal or as uninvolved tissue. Cudkowicz (1952) found that suspensions of 2-FAA did not affect the malic or succinic dehydrogenase activity of a liver homogenate. Since suspensions were used, this result may have been caused, partially a t least, by the insolubility of the chemical in the medium. The data obtained with caffeine solutions of 2-FAA point toward such a n explanation, for malic dehydrogenase was inhibited somewhat more than the control with caffeine alone. The succinic dehydrogenase activity, however, appeared unchanged. Eichel (quoted by Roth, 1954), on the other hand, found that 2-FA and 2-FAA lowered the succinic oxidase activity of rat liver and kidney. Fritz-Niggli (1956) also observed that the ability of a rat liver homogenate to oxidize succinate, ketoglutarate, and citrate was decreased during the period of feeding 2-FAA but that the isolated h e r mitochondria had normal enzyme levels as measured by succinate and pyruvate oxidation. On the basis of her histological findings, the diminution in enzyme activity of the total liver homogenate could be correlated with the decrease of the cytoplasm and the number of mitochondria in the precancerous stages. The effect of 2-FA and related compounds on the oxidation of a number of substrates i n vitro was investigated by Kielley (1956). The oxygen and phosphorus uptake in rat liver homogenates and mitochondria was inhibited by N-2-fluorenyldiacetamide to varying degrees with succinate (very slight), isocitrate, ketoglutarate, P-hydroxybutyrate, and most severely with glutamate. Aged mitochondria were more sensitive than fresh ones, possibly because of an exhaustion of endogenous cofactors. The phosphorus to oxygen ratio was unaffected, showing that phosphorylation was not significantly uncoupled. Glutamate oxidation was interfered with by the compounds studied in proportion to increasing carcinogenic effect, i.e., 4-FAA, < 1-FAA, <2-FA, <2-FAA, and
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ELIZABETH K . WEISBURQER AND JOHN H . WEISBURGER
competitive displacement of diphosphopyridine nucleotide (DPN) from the enzyme (Kielley, 1957a). Curiously, the effect was less pronounced with a crystalline enzyme preparation than with the mitochondria1extract, suggesting that metabolism of the fluorene derivative might indeed be a factor. The inhibition of glutamate oxidation was twice as large with mitochondria prepared from livers of riboflavin-deficient as compared to normal rats (Kielley, 195713). Independently, Emmelot (1957) found that the inhibitory effect of 2-FAA and certain azo dyes, carcinogenic or not, on glutamate oxidation could be reversed by the addition of DPN to a glutamate-mouse liver mitochondria system. Emmelot inferred that 2-FAA released the bound DPN of Gglutamic dehydrogenase in the mitochondria. Certain mitochondrial preparations were more sensitive to the action of 2-FAA than others, suggesting to Emmelot a reverse relationship between tightness of binding of DPN and susceptibility to displacement by 2-FAA, a result perhaps of species and strain differences. This variability was also noted by Kielley. The diverse degrees of inhibition of malic, isocitric, a-ketoglutaric, and 0-hydroxybutyric dehydrogenases by 2-FAA or 2-fluorenyldiacetamide may perhaps be attributed to a greater affinity of these enzymes for DPN than that of glutamic dehydrogenase for DPN. It is also possible that the loss of DPN is due to a higher level of the reduced form, DPNH, induced by 2-FAA or a metabolite. DPNH might diffuse out of the cell more readily than the oxidized form (Glock and McLean, 1957). Additional studies revealed that after 2-FAA had released the bound DPN from mitochondria, it activated the latent adenosine triphosphatase of the mitochondria (Emmelot and Bos, 1957). In this event, a disruption of the energy cycle of the cell under the influence of 2-FAA might result. Another oxidizing enzyme, liver catalase, was depressed for 4 to 6 days after mice had been administered 2-FAA by skin painting (Adams and Roe, 1953). The mechanism by which the carcinogen or its metabolites inhibited catalase in the preneoplastic period has not been determined. The enzyme was greatly reduced in primary liver tumors induced by 2-FAA (Mori e l al., 1954), thus constituting no exception to this general observation for virtually all neoplasms (cf. Greenstein, 1954). Although the liver choline oxidase was slightly higher than normal during the first week of feeding 2-FAA, subsequent weeks showed a decline, followed 3 or 4 weeks later by an increase (Asano, 1955). The enzyme levels of preneoplastic livers were similar to those in normal livers but dropped to one-tenth that value in hepatomas. Both tyramine and monoamine oxidase of rat liver were depressed during the 4th week of feeding 2-FAA. After the carcinogen was discontinued, the activity in the precancerous livers was almost that of normal liver until hepatomas developed when the enzyme activity diminished (Kishi et al., 1956).
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€3. Proteases. The act'ion of 2-FAA on proteolytic enzymes W:~Siiivrstigated by Rondoni and Bassi (1948), Hondoni (1950), and Rondoni and Barbieri (1950). Digestion of gelatin by papain was inhibited to the extent of 50y0 by 2-FAA or 33% by 2-FA. Furthermore these compounds decreased the autoproteolytic effect of cathepsin from it horse liver extract and suppressed the activating function of cysteine and 2,3dimercapto-lpropanol (BAL). The inhibitory action of a series of hydrocarbons and other compounds, including 2-FAA, was found to parallel the carcinogenic activity of the chemicals. The authors concluded that carcinogenic compounds interacted with thiol groups and thus inactivated the enzymes. Rondoni (195513) later found that a number of carcinogenic compounds restrained autoproteolysis of liver, while several noncarcinogenic hydrocarbons showed only weak inhibition. However, the nature of the enzymes actually responsible for the proteolytic effect was not determined. Siebert et al. (1952) reported that addition of 2-FAA had no effect on a purified cathepsin (cathepsin T). Under the same conditions, N,Ndimethyl-p-phenylazoaniline was effective whereas benzo[a]pyrene did not inhibit the enzyme. Siebert et al. pointed out that although their results contradicted those of Rondoni, their respective experimental approaches were entirely different. They measured the amount of tyrosine and tryptophan liberated during incubation, whereas Rondoni used the proteolytic effect as a measure of catheptic activity. Maver et al. (1952) observed that the catheptic activity of two different transplantable liver tumors induced by N-2-fluorenyldiacetamide was increased over that in normal rat livers. The higher enzymatic activit'y appeared to be associated with an increased concentration of pentosenucleic acid. Deckers-Passau et al. (1957) also noted a 2-FAA-induced increase in catheptic activity. C. Esterases. Plasma pseudocholinesterase increased markedly during the administration of 2-FAA before there was any gross evidence of hepatoma induction (Milch, 1950). Later as the neoplasms developed and the liver protein concentration declined, the enzyme activity of the plasma also decreased but was still somewhat above the activity of controls. Sat0 (1956) observed that in the precancerous stages the livers of rats fed 2-FAA had the same acetylcholinesterase levels as normal livers. After hepatomas had developed, the enzyme activity was extremely high. In contrast, the serum cholinesterase levels were not notably different from the normal, even in hepatoma-bearing rats. Carcinogenesis by 2-FAA appeared to have no significant effect on the serum phosphatase of rats (Ichii et al., 195413). The acid phosphatase level of liver tumors was not different from that in normal liver, but the alkaline phosphatase level of tumors induced by 2-FAA was almost 6 times higher than that of normal liver. Serum esterase according to Ichii et al. (1954b) (using tributyrin as substrate) was in-
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ELIZABETH K . WEISBURGER AND JOHN H. WEISBURGER
creased in rats which showed moderate hyperplasia of the livers and continued a t high levels in the presence of liver cancer, while liver esterase values decreased 50% when tumors arose. D. Amidases. Asparaginase activity in the kidney and brain of 2-FAA fed rats showed no significant difference from that of normal tissues (Haruno, 1954), On the other hand, liver asparaginase activity diminished weekly, reaching its lowest level 4 weeks after beginning the 2-FAA. After the carcinogen intake was stopped, the asparaginase activity of the noncancerous liver rose to about two-thirds of the normal value pointing toward some inhibition of the enzyme when the agent was present. In hepatomas, the enzyme activity was very low. Similarly, liver glutaminase was appreciably lowered during the feeding of 2-FAA but increased to twice the normal level in the precancerous livers upon discontinuance of the chemical; it also exhibited the same amount of activity in hepatomas as in untreated controls (Haruno, 1956). However, primary liver tumors induced by N ,N-dimethyl-p-phenylazoaniline showed twice the activity of normal liver. The ability of liver tissue to hydrolyze a-bromo- and chloroamides and simple fatty acid amides generally decreased as the livers of 2-FAAtreated rats passed through the various precancerous stages until tumor tissue showed very little hydrolytic activity (Kishi et al., 1954, 1955). 2-FAA had no inhibitory effect on the arginase activity of an enzyme digest (Siebert et al., 1952). Tumors induced by the carcinogen had very little activity (Takahashi, 1954). However, during the precancerous stages, the enzyme level was almost equal to that of normal liver. E. Deaminases. Takahashi (1954) observed normal liver guanase levels at pH 9 (the optimum for the enzyme) in the various precancerous and cancerous stages induced by 2-FAA. However, a t other pH values, a progressive decrease in enzyme activity occurred until hepatomas had only onehalf the activity of normal liver, suggesting that other enzymes were being measured. In a paper dealing with cathepsin, Siebert et al. (1952) mentioned that 2-FAA had no effect on an enzyme system containing purine deaminase, but no other details were given. F. Miscellaneous Enzymes. As the cancerous changes induced in liver by 2-FAA progressed, the rhodanese activity (Ichii et al., 1954a) and the ability to synthesize p-aminohippuric acid (Ichii, 1955) decreased so that tumors showed little or no activity. However, the uricase activity remained normal or almost normal until liver cancer developed (Mori et al., 1954). At that time, the enzyme activity dropped suddenly to only 2% of the normal value. A preliminary report suggests that urinary 8-glucuronidase activity is increased in rats fed 2-FAA as compared to controls (Dyer and Morris, 1958).
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AND RELATED COMPOUNDS
397
2-FAA had no effect on deoxyribonuclease when the compound was added to the enzyme system (Siebert et al., 1952). There may be some connection between this finding and the fact that the deoxyribonucleic acid content of the liver does not seem to be affected appreciably during carcinogenesis although metabolites of 2-FAA as produced i n wivo should be tested in this system. On the other hand, Roth (1957) in a recent report observed that alkaline ribonuclease activity in the liver mitochondria from rats on 2-FAA was, on the average, depressed during the entire course of a 24-week feeding period, while acid ribonuclease activity was not inhibited. This was interpreted to mean that the carcinogenic processes were riot involved but that the agent had a toxic action on the liver cells. It would certainly be of interest to probe into the actual mechanism for the toxic effect and into the manner by which 2-FAA inhibits alkaline ribonuclease. 7 . Summary During the process of 2-FAA carcinogenesis, the protein, riboflavin, and RNA of liver are lower although DNA and moisture content remain almost normal. The decrease of these constituents seems to be related to a more rapid metabolic turnover in the liver as indicated by a faster uptake and release of amino acids, riboflavin, and uracil. The enzyme levels in 2-FAA-induced tumors follow the pattern found in other primary hepatomas (cf. Greenstein, 1954). Thus, for example, catalase, uricase, and arginase are lowered in the tumor tissue. Acid phosphatase has the same level as in normal livers; alkaline phosphatase and acetyl cholinesterase are increased in liver tumors. I n tumors developed by treatment with 2-FAA, only two enzymes appear to be exceptional. Both the glutaminase and the succinic oxidase levels of such neoplasms are the same as in normal liver. In contrast, glutaminase activity is usually high, and succinic oxidase activity low in primary rat hepatomas resulting from azo dye ingestion. I n addition, 2-FAA or its metabolites appear to possess an inhibiting effect on certain enzymes when the carcinogen is present in the organism. These include liver catalase, choliiie oxidase, tyramine oxidase, moiioamine oxidase, alkaline ribonuclease, and glutaminase. Acetylcholinesterase does not seem to be affected. I n view of the elementary aiid almost inevitable conclusion that carcinogenic processes are preceded and attended by alterations in the activity of cellular enzymes, further studies of this type are a necessity. Such investigations should concentrate on the preneoplastic period both in the presence and in the absence of the carcinogen so that the direct influence of the latter can be evaluated. Once an overall screening has selected certain
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ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
promising enzymes, their additional response to some metabolites of 2-FAA in vivo and in vitro would appear enlightening.
VI. METABOLISM-ACTION OF THE HOBTON THE AGENT The fate of the following compounds has been subjected to extensive scrutiny : N-2-fluorenylacetamide (2-FAA), 2-fluorenamine (2-FA). N-methyl-2-fluorenamine, N-2-fluorenylbenzamide, N-2-fluorenyltosylamide, and N-(7-hydroxy-2-fluoreiiyl)acetamide. In addition, a number of related compounds have been examined in the course of these studies. The rat has served as experimental animal in most cases since it appears to be the most susceptible to the carcinogenic action of these chemicals. A few other species, principally the dog and the guinea pig, have also been used. Unless specifically mentioned otherwise, it will be assumed that the metabolism experiments, usually involving a single dose of the compound, were performed in rats. 1. N-2-Fluorenylacetamide and 2-Fluorenamine
The metabolism of these two substances will be reviewed jointly in view of their close interrelationship. The conversion of ingested 2-FAA to 2-FA was suspected by Wilson el al. (1941) (cf. Section IV) and has been fully substantiated by later investigations. The initial attempt a t unraveling the fate of 2-FAA was carried out by Bielschowsky (1945). On the basis of analogy with the known biological hydroxylation of the carcinogenic hydrocarbons, resulting in phenolic derivatives, this investigator isolated a material from the urine of rats fed 2-FAA which he proved to be N-(7-hydroxy-2-fluorenyl)acetamide.This compound yielded a blue color extractable into amyl alcohol upon treatment with nitrous acid. On the basis of this test, sensitive to 20 pg. of material, it was noted that excretion of the metabolite was undetectable 2-3 days after cessation of carcinogen intake. Thus, Bielschowsky demonstrated that biological hydroxylation of 2-FAA occurred and that the position affected was one of those attacked by chemical substitution with electrophilic reagents. However, other unidentified metabolites must have been produced, for only 54% of the administered compound was isolated as the 7-hydroxy derivative. A. Analysis of Metabolites by Colorimetric Procedures. a. Diazotizable material. Simultaneously, ail important step forward in the problem of the metabolism of 2-FA was achieved by the development of a colorimetric method applicable to the determinatioii of 2-FA and related compounds in biological material (Westfall, 1945). The procedure depended on the aromatic amino group which could be diazotized and the resulting diazo derivative coupled with sodium 2-naphthol-3,6disulfonate (R salt) to form a dye with a peak absorption at 525 mp. The first application of the
method showed that the urine of rabbits injected with 2-FA (Westfall, 1945) and of rats fed 2-FAA (Westfall and Morris, 1947) contained conjugates which were more soluble than the compound administered. I n their pioneering investigations, Morris and Westfall (1948, 1950), after oral administration of 2-FAA, noted diazotizable material in all organs surveyed, which they interpreted as a logical explanation for the induction of neoplasms a t many sites. The highest concentration was reached after 3 to 5 hours but dropped rapidly thereafter. Demonstrable levels persisted for 8 to 16 hours, and the observation was made that the decline in tissue diaxotizable material occurred in spite of the presence of a substantial portion of the administered compound in the stomach. A similar maximum was found in the urine 4 to 6 hours after feeding a single dose, and the material became undetectable after 20 hours. The recovery in the urine after a single dose depended on the quantity administered. A weekly intake of 0.25 mg. in 3 equal portions resulted in a barely distinguishable level of diazotizable material. Chromogenic substances accounted for about 10, 29, and 45y0 of the dose administered when this consisted of 1, 4, arid 16 mg. per week in 3 doses. However, only %yo was recovered when the 16 milligrams were given in 6 rather than 3 portions or when this amount was fed for several weeks. In addition, diazotizable material was detectable for a maximum of 8.5 days after cessation of multiple doses. At that time, this behavior suggested that a considerable proportion of the administered compound was rapidly converted to nondiazotizable derivatives but that initially some diazotizable material was present in the tissues and was excreted in the urine, especially after the larger dosages. However, the total recovery amounted in general to only about one-third of the dose, and metabolites could not be detected in the feces. That the poor recovery of diazotizable material after 2-FAA intake might have been due to the possible combination of the compound or metabolites with tissue constituents and consequent formation of acetoneinsoluble conjugates was investigated by Dyer et al. (1951). A number of tissues were fractionated and saponified ; proteins and lipids were hydrolyzed by acid, alkali, or enzymes. However, this time-consuming and patient search failed to turn up more than 5% additional diaxotizable material. It mas concluded that the administered 2-FAA was not present in combination with tissue proteins or lipids in appreciable amounts and that the nitrogen must be present in nondiazotizable form and/or in extremely water-soluble materials. At this time experiments were started using N16-2-FAA, arrangements having been made between Morris and Ray for the synthesis of isotope-labeled fluorenylacetamide compounds (with C14 and N16) for metabolism studies in the Bethesda Laboratory (cf. Sections I11 and VI). Using a modified spectrophotometric method, Gutmann et al. (1952)
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ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
distinguished between the free and the conjugated, but hydrolyzable, diazotizable material in rat tissues after intraperitoneal injection of 2-FA. While as much as 59% of the dose was free, and about 10% conjugated after 4 hours, only 21 and 9% were detectable, under the corresponding headings, after 12 hours. The better recovery soon after injection could be ascribed to the considerable portion of the dose still present at the injection site. After longer periods of time, however, the drop in yield observed by the earlier investigators was also apparent in this case. Gutmann et al. offered an explanation for this phenomenon in terms of the presence of hydroxylated metabolites of the carcinogen. It was noted that whereas the chromogen from 7-amino-2-fluorenol had a maximum absorption at the same wavelength as the dye derived from 2-fluorenamine, the extinction of the former was only 37% of that of the latter. This weaker color reaction of the hydroxy derivative had already been pointed out by Bielschowsky (1947b). Hence the metabolic conversion of 2-FA to phenolic compounds would be reflected in an apparent decrease of diazotizable amino groups. Dyer et al. (1953) have actually shown by the application of isotopic methods that results obtained by the diazotization procedure, not corrected for hydroxylated metabolites, accounted for 29 to 54% (average 3973 of the N16 in excreta and tissues. b. Nitrite test. However, the conversion to the 7-hydroxy derivative by itself was insufficient to explain the loss in diazotizable material. This was amply demonstrated as a result of the development of an independent color reaction for 7-amino-2-fluorenol by Damron and Dyer (1953). These investigators noted a yellow color upon addition of nitrite to an acid solution of this compound and took advantage of this observation to elaborate a technique for the determination of the chemical in biological material. By means of suitable correction factors to allow for interferences, diazotizable as well as hydroxylated derivatives could be estimated. Pilot experiments showed good recoveries in the analysis of mixtures of 2-FAA and the 7-hydroxylated derivative in varying proportions. Although larger proportions, up to 86% of a given dose, of 2-FAA could be accounted for in biological materials by means of this dual analytical method (Dyer et al., 1953), the procedure is limited in its quantitative aspects since hydroxylated metabolites other than the 7-amino-2-fluorenol have been discovered. Some of these have been found to react in the “diazotization and the nitrite tests” with different color densities from that of the 7derivative. Therefore such “diazotizable amine” actually may represent the composite value for a number of compounds with such a chromogen-yielding function, where each substance gives a dye with a different molar extinction coefficient. An application of these techniques was described by Dyer (1955) in a
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A N D RELATED COMPOUNDS
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comparison of diazotizable and nitrite-reacting materials in the urine and feces of rats treated with 2-FAA and 16 other compounds related to this carcinogen. As shown in Fig. 3 and Fig. 4, hydroxylated metabolites could be detected in both the urine and feces after the administration of some compounds, and in only the urine with others. No hydroxylated metabolites a t all were observed with this test in the case of fluorene, 4,4’-bisacetanilide, and 2,2’diacetamido-9,9’-bifluoreiie. Thus, the production of hydroxylated derivatives was the rule. Apparently 2-fluorenol, which certainly must have been a metabolite in rats fed fluorene (cf. Neish, 1948a), does
FIG.3. Recovery of total diasotizable and nitrite-reacting material from urine. From Dyer (1955).
not respond t o the nitrite test although this procedure probably involves the formation of a nitroso derivative. A noteworthy correlation derived from these studies was that the noncarcinogenic 9,9’-bifluorene derivative (Morris and Dubnik, 1950) was excreted in its entirety in the feces without undergoing metabolism and was similarly recovered from a parenteral injection site. The corresponding unsaturated A9~9‘-bifluorenederivative was absorbed and metabolized to a small but definite extent and was slightly carcinogenic. The insoluble nature of the substituted 9,9’-bifluorene might thus be responsible for the lack of action of this compound.
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ELIZABETH I<. WEISBURGER AND JOHN H. WEISBURGER
Ehrlich’s dictum Corpora non agunt, nisi JEuida appears to apply in this case although it is also possible that steric effects hold the molecule in a nonplanar form, which is too large to fit into whatever specific configuration is required for carcinogenic activity (Section IV,3). B. Analysis of Metabolites by Isotopic Procedures. In contrast to the incomplete accounting of a dose of 2-FAA by the colorimetric methods, the recoveries of C14or N16(cf. Ray and Geiser, 1949, 1950, and Argus and Ray, 1951, Section I11 for syntheses) were uniformly close to 100% (Morris et al., 1950b; J. H. Weisburger et al., 1951; E. K. Weisburger et al., 1953; Dyer et al., 1953; Gutmann and Peters, 1953a). Thus, the data obtained by means of the isotopic techniques could be t,rusted to reveal n reasonably
FIG.4. Recovery of total diazotizable and nitritereacting material from feces. From Dyer (1955).
accurate and complete picture of the modifications undergone by the compounds studied. Furthermore, the anticipated stability of the label at the 9-position of the fluorene molecule was realized, as essentially no radioactivity was detected in the exhaled CO:! of animals given the radioactive 2-FAA. It could be expected, therefore, that this particular tracer would prove to be a suitable guide to locate the carbon skeleton in its journey through the metabolic labyrinth. Experiments with the Nls-labeled compound showed no enrichment of urinary urea and creatinine by the heavy isotope (Dyer et al., 1953), and only a trace was found in the ammonia
2-l<’LUORl!XAMINE;AND
IWL.\TEI) COMPOUNDS
403
aerated from urine (Dyer and Morris, 1956). The latter could have been an artifact resulting from the air oxidation of a labile metabolite present in the urine. In any event the data obtained with the N16-labeledcarcinogen indicated a definite stability of the nitrogen to carbon bond. a. Distribution of isotope. Radioactivity was widely distributed in many organs and tissues of rats after a dose of 2-FAA-9-CI4 (Morris et aE., 1950b; J. H. Weisburger et al., 1951; Gutmann and Peters, 1953a). In most cases, the concentration of activity was higher shortly after administration of the chemical than a t later periods. The presence of radioactivity derived from the carcinogen might suggest a rationale for the induction of tumors in some tissues. It should be kept in mind, however, that the location of activity yields little information on the mode of action of the agent unless further knowledge on the chemical nature of the activity and its interaction with selected tissue constituents can be gathered. The localization of the radioactivity within the tissues has been examined by differential centrifugation of liver and kidney homogenates (Miller and Miller, 1952; E. K. Weisburger et al., 1953). In both cases, radioactivity was associated with the particulate fractions as well as with the supernatant. However, the activities of all fractions were rather similar in a 27-day run. The whole liver, nuclei, large granules, small granules, and supernatant showed levels of 113, 91, 80, 96, and 108 counts/min./mg. of protein. On the other hand, a 3-hour experiment gave values (in the same order) of 2420, 1520, 2640, 3200, and 4280 counts/min./mg. nitrogen. Thus, the specific activity of the small granules was 2 times, and that of the supernatant almost 3 times the activity of the nuclei. It remains to be established whether the differences observed were real or apparent. Nevertheless, the presence of substantial quantities of radioactivity in the particulate fractions suggested some form of tight combination between the carcinogen or its metabolites and the cell constituents. b. Excretion of isotope. The level of radioactivity of the tissues decreased rather rapidly. At the same time, a considerable portion of a dose of the carcinogen was excreted. The highest levels of radioactivity in the urine occurred between 6 and 30 hours after feeding, and lesser amounts were found thereafter for a long time. Easily detectable levels of activity were recorded 32 days after a single dose. In general, however, 60-70% of a dose was excreted via the kidneys in 3 to 4 days (J. H. Weisburger et al., 1951). I n the same interval, about 25-35% of the radioactivity was eliminated with the feces; this constituted an interesting finding in view of the fact that little diazotizable material was demonstrable in acetone extracts of the feces. Difficulties with extraction of fecal material and color interference were avoided by the isotopic procedures. Furthermore, the fecal material still contained the nitrogen atom from the amino group as was
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ELIZABETH K. WEISBUBGER A N D JOHN H. WEISBURGER
proved by the use of isotopic nitrogen (Dyer et al., 1953). Paralleling the results achieved with radiocarbon, the ingestion of N16-2-FAA showed an average distribution of 65% of the isotope in the urine and 35% in the feces in long-term experiments. The concordant results obtained in these and other studies with C- and N-labeled 2-FAA suggested that the carbon atom in position-9 and the nitrogen atom attached to position-2 remained together in most of the metabolic pathways followed by 2-FAA. Although the administered compounds, 2-FAA or 2-FA, are readily soluble or extractable into ether or other organic solvents, partition of the radioactivity or isotopic nitrogen in urine between the aqueous phase and ether transferred only 10-30% to the organic phase (Dyer et al., 1953; Weisburger et al., 1953, 1954). At neutral pH and with freshly collected urine, the lower value was generally found. On the other hand, the experiments of Gutmann and Peters (1953a) indicated little passage of radioactivity into the ether layer a t acidic and neutral pH, while close to 30% of the urinary activity apparently extracted at pH 11-12. The contradictory results are not readily explained, but subsequent work on the actual identity of metabolites revealed their slightly acidic nature. Hence, they should be more ether-soluble a t an acid or neutral pH, for the salt formed with alkali would be hydrophilic (J. H. Weisburger et al., 1956a). Enzymatic hydrolysis of urinary metabolites with bacterial betaglucuronidase, which affects conjugates of glucuronic acid, and with Takadiastase, which cleaves sulfate esters, rendered 50-60%, and &lo%, respectively, of the isotope ether-soluble (J. H. Weisburger et al., 195613; Dyer, unpublished). Accordingly, such conjugates represented a substantial portion of the metabolites in the urine of rats, but the balance of about 20-30a/, must be of a different, preferentially water-soluble, nature. Comparative studies on the metabolism of 2-FAA in guinea pigs showed that only 1-2% of the urinary radioactivity representing the quantity of free metabolites was extractable into ether, while about 80% was conjugated with glucuronic acid, and a t the most 5% with sulfuric acid (Weisburger et al., 1957~). The lower levels of unconjugated metabolites may have some bearing on the lack of carcinogenic effect of 2-FAA in this species. c. Pathway of tracer in the rat after a dose of labeled %FAA. Some understanding of the pathway taken by a dose of 2-FAA was gained by the experiments involving rats with biliary and lymphatic fistulas, with a ligated pylorus (Dyer et al., 1953; E. K. Weisburger et al., 1953), and with a ligated bile duct (Gutmann and Peters, 1957). Upon oral intake, the carcinogen appeared to undergo little if any modification in the stomach. A small amount of absorption across the stomach wall can be presumed to occur in normal animals, but the larger quantities which were transported through the gastric mucosa in pylorus-ligated rats may have been due to
~-FLUORENAMINE A N D HELATEI) C O M P 0 U N I ) S
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the abnormal conditions prevailing during this unphysiological experiment (E. K. Weisburger et al., 1953). Although trypsin had no effect on 2-FAA (J. H . Weisburger, 1055), there are enzyme systems present in the wall of the upper intestinal tract which hydrolyze 2-FAA (Nagasawa and Gutmann, 1957). Nagasawa and Gutmann have found a correlation between the extent of in vitro hydrolysis by rat intestine of substituted 2-fluorenamines, such as acetyl, succinoyl, benzoyl, and p-toluenesulfonyl derivatives, and the carcinogenicity of these compounds. A low susceptibility to the intestinal deacylase corresponded to a poor tumor-inducing power. The absorption of the chemicals from the intestinal tract and their transport across the intestinal wall are not well understood. Kirby (1947) suggested that the carcinogen reached the liver via the mesenteric blood flow, for he noted hepatic neoplasms predominantly in the right lobe, the site of entry of the blood flowing from the intestines, while lesions of the left lobe were less frequent and were usually absent in the caudate lobe. However, Bielschowsky (1949; Bielschowsky and Hall, 1951a) found no distinct localization of the tumors in any of the lobes and indeed on occasion saw the most conspicuous cholangiomas in the left and caudate lobes. I n any case, it seems reasonable to assume that the blood transported the material absorbed from the intestinal tract to the liver since a n alternate path such as the lymphatic system was not involved to any great extent (E. K. Weisburger et al., 1953). Some of the reactions occurring in the liver include deacetylation, acetylation, hydroxylation, conjugation with glucuronic and sulfuric acid, and other reactions unknown a t present. Partial hepatectomy appeared to decrease the capacity of the remaining liver to carry out some or all of these reactions (Gutmann et al., 1952) yet had no effect on the course of tumor induction in this organ (Skoryna and Webster, 1951). Laws (1956), however, found neoplasms earlier in hepatectomized than in intact rats. Some metabolites produced in the liver were distributed via the blood stream throughout the body of the animal. The amount of metabolites transported a t any one time was small as indicated by the relatively low levels of diazotizable material, radioactivity, and isotopic nitrogen per unit volume. Material was present in the blood for a long time. After an iritraperitoneal injection of 5 mg. of labeled 2-FAA (1.25 pc.), the blood had a specific activity of 0.060, 0.052, and 0.031 pc./ml. a t 8, 16, and 32 days, respectively (this laboratory, unpublished). Most of the metabolites were rapidly removed from the blood and excreted in the urine. Some metabolites left the liver via the bile. Studies with both N15 (Dyer et al., 1953), and C14 (E. K. Weisburger et al., 1953) labeled 2-FAA demonstrated that a fairly substantial concentration of metabolites was found
40G
ELIZABETH K. M’EISBURGER AND JOHN €1. WEISBUItOEll
in this fluid shortly after oral intake. About 40% of a dose was excreted in this manner in 3 days. Thirty-three percent of a dose appeared in the urine and only traces in the feces of rats with biliary fistulas, which suggested that the normal fecal metabolites of 2-FAA are contributed entirely by the bile rather than from incomplete absorption of the ingested compound. The decreased urinary excretion in biliary fistula rats implied that the material delivered into the intestinal tract by the bile was in part reabsorbed and recirculated via the blood stream. The data a t hand indicate that roughly one-half of the urinary activity is so involved. Gutmann and Peters (1957) were unable to collect feces from rats with ligated bile ducts administered 2-FAA and 2-FA, so the origin of metabolites in the stools could not be ascertained although rather small amounts of activity in the intestinal tract would suggest again a decreased fecal activity in the absence of bile. After 4 and 21 hours, 9 and 630/,, respectively, of the radioactivity from a dose of labeled 2-FAA was found in the urine, as contrasted to 2.7 and 35y0in a normal animal. Thus, ligation of the bile duct modified the excretory pathway with a shift to renal elimination. Gutmann and Peters interpreted their results in terms of an excretion inversely proportional to the dose since the higher values appeared to have been obtained with a lower dose. However, a series of recent experiments (this laboratory, unpublished) showed the quantity of 2-FAA administered intraperitoneally had only a slight effect on the percentage excreted in the urine. The injection of 10, 5, 1, 0.5, 0.1, and 0.05 mg. of labeled carcinogen per 100 g. of body weight resulted in the excretion of 60, 50, 61, 69, 70 and 60% of the dose in the urine. In conclusion, the following picture can be drawn on the pathway of isotope, i.e., the carcinogen and its metabolites, after an oral dose of labeled 2-FAA. Absorption occurs mainly in the small intestine, whereupon the blood transfers the material to the liver. Considerable metabolic alteration takes place in the liver. A portion of the metabolites leaves the liver by way of the blood, enters the general circulation, and is eliminated by the kidneys. Another part of the metabolites reenters the intestinal tract via the bile. Partial reabsorption transfers some of the metabolites back t o the blood, whereas the remainder is excreted in the feces. These processes proceed very rapidly in rats so that within 3 to 4 days about twothirds of a dose is found in the urine and one-third in the feces while only about 5% remains in the body. In guinea pigs, the urine:feces ratio is approximately 6: 1, and all but traces are excreted. C. Acylation and Deacylation of 2-Fluorenamine. The suspected deacetylation of 2-FAA (Wilson et al., 1941; Bielschowsky, 194713) was proved by a comparative study of the metabolism of 2-FAA labeled with C14 in the acetyl group or in the 9-position (Morris et al., 1950b; J. H. Weisburger
2-FLUOREN.4MINE
.4ND RELATED COMPOUNDS
407
et al., 1951). Ray and Geiser (1950), who first synthesized these C14-labeled compounds remarked: “Radioactive carbon dioxide should be found in the expired air from animals fed 2-FAA with the radioactive side chain but not from those fed 2-FAA with the radioactivity in the fluorene nucleus.” This prediction proved correct, for during 88 hours after administration of a dose of 2-FAA labeled with C14 in the acetyl group, 41% of the radioactivity was exhaled in the respiratory COz of rats, whereas, essentially none was present in the breath of animals given the ring-labeled carcinogen. Furthermore, the distribution of radioactivity in the organs and excreta differed sharply in the two cases (Morris et al., 1950b; J. H. Weisburger et al., 1951). The amine also underwent further metabolism because only small amounts of either 2-FAA or 2-FA were excreted (Gutmann and Peters, 1953a; J. H. Weisburger et al., 1956a). Rat liver slices and homogenates converted 2-FAA to 2-FA in the presence of oxygen but not in a nitrogen atmosphere. Homogenates, in addition, yielded unidentified metabolites with a diazotizable amino group (Gutmann and Peters, 1954). However, J. H. Weisburger (1955) observed that the deacetylation of 2-FAA by liver homogenates proceeded equally well in air as in an inert atmosphere, which might be expected if the reaction involved a simple hydrolytic mechanism for which an energy source is not required. Purification of the deacylase and a study of its properties and requirements would permit a clearer insight into a possible need for oxygen. The homogenate similarly yielded the amines from N-2-naphthylacetamide, acetanilide, N-2-fluorenyldiacetamide, N-4-fluorenylacetamide, N-3-fluorenylacetamide, and N-1-fluorenylacetamide, in decreasing order of SUSceptibility. Three transplantable tumors exhibited a low order of deacetylase activity, and an effective liver acetone powder could be prepared. Deacetylation was found also with other rat tissues such as lung, spleen, heart, kidney, brain, and mouse liver in increasing order of potency. Surface active agents and ethanol inhibited the enzyme (J. H. Weisburger, 1955), which recalls similar effects observed with leucine arninopeptidase (Hill and Smith, 1957). Nagasawa and Gutmann (1957) recently indicated that rat liver slices were rather selective and hydrolyzed only amides of 2-FA derived from alkanoic acids. These authors also discovered that an intestinal deacylase liberated the free amines from the amides, which contributed much to an understanding of the pathway of the carcinogen through the rat. The intestinal enzyme was more active toward a larger number of substrates than the liver enzyme. Thus, the existence of hydrolytic systems in many tissues may account for the considerable deacetylation of 2-FAA noted in the whole animal. Of interest is the related contribution of Greenstein et al. (1955) who prepared some 24 peptide derivatives of 2-FA in which the aromatic amino
408
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
group was acylated by the carboxyl end of D- and L-amino acids (cf. Section 111). A study of the properties of these model compounds involving such a peptide bond may serve as a basis for an understanding of the nature of the observed firm combination of carcinogen and tissue proteins. Several tissue homogenates split to a greater or lesser extent the derivatives of aliphatic type L-amino acids (Table VII). The L-prolyl, L-tryptophyl, TABLE VII Hydrolysis of Amino Acid Derivatives of 2-Fluorennminc by Tissuc Homogenates" 2-FA Derivative Glycyl L-Alanyl D-Alanyl L-Butyrinyl L-Norvalyl L-Norleucyl L-Valyl L-Leucyl L-Isoleuc yl L-Methiony 1
Liver
Kidney
10 22 3 107 87 19 2 27 4 5
28 30 0 108 112 28 1 19 1 0
Small
Spleen
Lung
5 10 0 62 43 10 0 6
4 17 0 49 23 6 0 4
0 20 0 47 19
5
<1
6
2
6
10 0 0
Pancreas 0 11 0 13 14 6 0 0 0 0
-
*The data, from the article by Greenstein el al. (19581, are expressed in terms of micromoles of substrate hydrolyzed per hour per gram of wet tissue.
L-phenylalanyl , and I,-benzylcysteinyl derivatives, as well as the compounds containing D-amino acid residues, were not attacked. Thus the enzyme complexes were rather selective in their action. Hill and Smith (1956) subjected the same peptide derivatives of carcinogenic amines to the action of a purified leucine aminopeptidase derived from hog kidney. The results agreed with the general specificity of this enzyme. Compounds containing D-amino acid residues were refractory. The most susceptible peptide was that derived from L-norvaline, but even this was hydrolyzed a t a rate very much slower than that noted with L-leucinamide. The reaction also proceeded, but to a lesser extent, with the amides containing L-isoleucyl, L-valyl, L-phenylalanyl, glycyl, and L-norleucyl groups. That the deacetylation of 2-FAA to 2-FA might be reversible was indicated by the experiments of J. H. Weisburger et al. (1951), Allison and Wase (1952), and Allison et al. (1954), but the reaction was first demonstrated by Peters and Gutmann (1955). Rat liver slices converted 2-FA to the acetyl derivative a t rates similar to those of the reverse reaction, and added acetate displaced the equilibrium in favor of acetylation. The two reactions appeared to proceed by separate mechanisms, where the
2-FLUORENAMINE
A N D RELATED COMPOUNDS
409
acetylation step probably involved acetyl coenzyme A. Bulky substituents in the ortho position might inhibit the acetylation, for Dyer (1955) reported that, unlike most of the compounds studied, all of the diazotizable material recovered in the urine after administration of 3-iodo-2-fluorenylacetamide was present as the free amine. Peters and Gutmann found about 0.25% of the added 2-FA t o be converted by rat liver slices to N-2-fluorenyldiacetamide in 4 hours. This novel reaction of diacetylation of an aromatic amine has not been previously observed. It might be desirable to explore this interesting point further and extend i t t o other aromatic amines. However, the diacetamide was not excreted in the urine of rats treated with 2-FAA (J. H. Weisburger et al., 19568). D. Hydroxylation of the Fluorene Nucleus Followed by Conjugation with Glucuronic and Sul$uric Acids. a. I n vivo. Hydroxylation of 2-FAA at the 7-position has been demonstrated in rats (Bielschowsky, 1945), rabbits (Neish, 1947), dogs (E. K. Weisburger et nl., 1952), and guinea pigs (Urquhart, 1955). Most likely other species also perform this reaction. The application of Damron and Dyer’s (1953) test led to the conclusion that extensive hydroxylation of the aromatic ring took place during the metabolism of 2-FAA. Further details of this important biochemical transformation could be resolved only by resorting to isotopic tracers and chromatographic methods. Products hydroxylated a t the 1-, 3-, 5-, 7-, and 8-positions were demonstrated in the urine of rats given a dose of labeled 2-FAA (J. H. Weisburger et al., 1956a,b; E. K. and J. H. Weisburger, 1956). From a quantitative viewpoint, hydroxylation appeared to occur mainly at the 5- and 7-positions (Table VIII). These were also the locations substituted chemically by an electrophilic reagent, such as a nitro group, when the 2-position contained an electron-attracting function as -NOz. It will be recalled, on the other hand, that chemical attack took place a t the 3-, 7-, and l-positions, in order of decreasing importance, in the course of nitration of 2-FAA (cf. Section 111). Thus, chemical and biochemical substitution appear to involve, in part, the same positions. I n addition t o hydroxylation, other biochemical reactions might conceivably have occurred with 2-FAA or the derived 2-FA. These include methylation of the amino group, oxidation of the 9-methylene carbon to a ketone, and opening of the ring a t the 9-position to yield derivatives of biphenylcarboxylic acids. However, the urine of rats given 2-FAA contained none of the following compounds : N-methyl-2-fluorenamine1 N , N dimethyl-2-fluoreiiamine, 2-amino-9-fluorenone, N-(9-0~0-2-fluorenyl)acetamide, 4- and 4’-acetamido-2-biphenylcarboxylicacid, and 4‘-amino-2biphenylcarboxylic acid. Furthermore, mercapturic acid excretion was not. increased in rats fed 2-FAA (J. H. Weisburger et al., 1956a).
410
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
TABLE V I I I Urinary Metabolites of N-2-Fluorenylacetamide in Ratso Compound
Percent
I. N-2-Fluorenylacetamide 11. 2-Fluorenamine 111. N-(l-Hydroxy-2-fluorenyl)acetamide N - ( 1-Hydroxy-2-fluoreny1)acetamideglucuronide IV. N-(3-Hydroxy-2-fluorenyl)acetamide
0.4 0.9 0.1-0.9
N-(3-Hydroxy-2-fluorenyl)acetamideglucuronide V. N-(5-Hydroxy-2-fluoreny1)acetamide N-(5-Hydroxy-2-fluorenyl)acetamide glucuronide VI. N-(7-Hydroxy-2-fluorenyl)acetamide N-(7-Hydroxy-2-fluorenyl)acetamide glucuronide N-(7-Hydroxy-2-fluorenyl)acetamide sulfate VII. N-(8-Hydroxy-2-fluorenyl)acetamide N-(8-Hydroxy-2-fluorenyl)acetamideglucuronide
4-6
~1
0.5 0.5 3.5 28
4-10 10-12
5-7 0.15 1.0-1.7
I h t a from -1. H. Weisburger uf nl. (IYRHa).
HO&NHR~ \
/
OH
The knowledge of the composition of the urine in terms of the metabolites of 2-FAA allowed an evaluation of the results obtained by the application of colorimetric tests to urines. The orlho-hydroxylated derivatives of 2-FA, i.e., 2-amino-1-fluorenol and 2-amino-3-fluoreno1, reacted with
2-FLUORENAMINE
AND RELATED COMPOUNDS
41 1
nitrous acid to form diazo oxides and therefore did not couple with R salt to form a dye. The 5-, 7-, and 8-hydroxy compounds yielded dyes with an absorbency of 70, 50, and %yoof that shown by 2-FA a t 525 mp. A value of 34-35y0 was obtained by calculation based on the actual composition of a urine sample containing the compounds in the amounts shown in Table VIII, and using their theoretical color intensities. This seemed to explain the apparently limited recovery possible by the colorimetric technique. Although Damron and Dyer (1953) had corrected the diazotizable material on the basis of 5oy0color density due to the 7-hydroxy derivative estimated by the nitrite test, the values were still unsatisfactory as noted above. Relatively small amounts of unconjugated hydroxylated derivatives of 2-FAA were excreted. Although all of the hydroxy compounds were combined with glucuronic acid, clear-cut evidence for a sulfate ester could be adduced only with N-(7-hydroxy-2-fluoreny1)acetamide (J. H. Weisburger et al., 1956b). The application of the method of Kellie and Wade (1956), involving chromatography of an etherethanol extract of urine on alumina, permitted a separation of the urinary metabolites of 2-FAA into free compounds, sulfuric acid conjugates, and glucuronic acid conjugates (this laboratory, unpublished). As a consequence of the occurrence of deacetylation, all of the corresponding 2-amino-x-fluorenols were also found in rat urine in small quantities (this laboratory, unpublished). In view of the labile nature of these easily oxidized compounds, it is conceivable that the bulk of them undergoes further transformations before excretion in the urine can occur. One of such reactions, of course, would be acetylation, which would tend to stabilize the molecule to some extent. Conjugation with glucuronic or sulfuric acid would also protect the aminofluorenols. These reactions appear to occur to a slightly larger extent in guinea pigs than in rats, as measured by the urinary excretion which averaged 98% conjugates in the former and 88% in the latter. Increased conjugation in the guinea pig might in part account also (1) for the lower proportion of a dose of 2-FAA appearing in the feces, and (2) possibly for the lack of carcinogenic effect. On the other hand, oxidation, e.g., in living cells or in the intestinal tract, might build up insoluble polymeric substances comparable to the brown resins found in an old solution of an aminophenol type photographic developer. Solutions of the pure aminofluorenols undergo similar changes on standing in air, and the ortho-aminohydroxy derivatives are particularly sensitive to oxidation. The production of such oxidized materials, if occurring in viuo, could explain two observations: (1) The excretion of insoluble, unidentified metabolites in the feces, amounting to about 2 0 3 0 % of the isotopic nitro-
412
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
gen or carbon in the excreta, i.e., 6-9yo of a dose of 2-FAA (Dyer el al., 1953; E. K. Weisburger et al., 1953). Even the soluble fecal metabolites have not been identifiable with any presently known compounds such as those in the urine except for a very minor fraction (this laboratory, unpublished). (2) The presence of so-called protein-bound material in most tissues of rats treated with 2-FAA. I n view of the widespread distribution of these substances in tissue fractions, and the heterogeneous nature of the labeled products obtained upon hydrolysis of the proteins, it appears reasonable to suggest that some, but certainly not all, of the protein-bound material might be similar to the solvent-insoluble substances encountered in the feces. Many oxidative processes are present in living cells through which aminofluorenols could react to yield such compounds. It is difficult a t this time to draw any conclusion regarding the possible role of reactions of this type in the course of carcinogenesis. Tha t they might be significant, however, can be inferred from the report of Bonser et al. (195613) that no local tumors were obtained upon injection of a freshly made solution of 2-naphthylamine but that such tumors were found when an aged solution was injected. This kind of conversion might be responsible for discrepancies in the literature regarding the carcinogenicity of the simpler aromatic amines (Hartwell, 1951). Were such oxidatively altered products used in biological tests, positive findings might have resulted while little or no carcinogenic effect could actually be inherent in the pure compound. A comparative study of the metabolism of 2-FAA in rats and in guinea pigs was of considerable interest because of its noncarcinogenicity in the latter species. Damron and Dyer (1953) found that the urine of guinea pigs fed 2-FAA contained increased amounts of a substance reacting like 7-amino-2-fluoreno1, as compared to rat urine. Meade and Ray (1954) also recovered a larger percentage of a dose of 2-FA in a combined form from the guinea pig than from the rat. Urquhart (1955) actually isolated N-(7hydroxy-Pfluorenyl) acetamide from the urine of treated guinea pigs. By isotopic and chromatographic experiments, in agreement with the data of Meade and Ray, Weisburger et al. (1957a,c) found that most of the metabolites from 2-FAA in the guinea pig were of the conjugated rather than the free type. Moreover 70 to 80% of an oral dose was converted to the 7-hydroxy derivative in the guinea pig, as against 25% in the rat. The quantitative estimation of all hydroxylated, ether-extractable metabolites of 2-FAA by chromatographic methods showed even more noteworthy deviations, as shown in Fig. 5 and Fig. 6. Whereas rats produced appreciable amounts of the 1- and 3-hydroxylated compounds, and large quantities of N-(5-hydroxy-2-fluorenyl)acetamide,the urine of guinea pigs contained only traces of the former and rather small amounts of the latter. I n view of the suggested involvement of ortho-hydroxylated derivatives
~ - F L L ~ O I ~ E N A M I NAEW
RELATED COMPOUNDS
413
FIG.5. Effluent diagram of ether-soluble urinary metabolites of N-2-fluoren-9-Cl4ylacetamide in the rat. Peak a contained N-(1-and 3-hydroxy-2-fluoreny1)acetamide which could be resolved further on a higher column. Peaks c, d , and e corresponded to the 5-, 8-, and 7-hydroxylated derivatives of 2-FAA, respectively. From Weisburger et al. (1957~).
FIG.6. Effluent diagram of ether-soluble urinary metabolites of N-2-fluoren-9-Cl'ylacetamide in a guinea pig. Fractions b, c, f, g, and h were the 1-, 3-, 5-, 8-, and 7-hydroxylated derivatives of 2-FAA. From Weisburger el al. (1957~).
414
ELIZABETH K. WEISBURGER AND JOHN 13. WEISBURGER
of other aromatic amines in carcinogenesis (Bonser et al., 1952; Clayson, 1953),the quasi lack of ortho-hydroxylation of 2-FAA in theguinea pig appears significant. The failure to produce the 1- and 3-hydroxylated derivatives in quantities proportionate to those observed in rats may not by itself be the cause of the lack of susceptibility in the guinea pig but may be an effect of deep-seated differences between the species. The results also have a bearing on the mechanism of hydroxylation, for they can be interpreted in terms of several enzyme systems mediating these reactions. Some could affect ortho, others para positions. Similar conclusions have appeared in the recent literature (Udenfriend et al., 1956; Parke and Williams, 1956). b. I n witro. Hydroxylation of 2-FAA has also been demonstrated by means of rat liver slices or fortified liver homogenates in an oxygen atmosphere (Gutmann and Peters, 1954; Peters and Gutmann, 1956; Gutmann et al., 1956~).Homogenates required diphosphopyridine nucleotide, nicotinamide, and succinate to perform this reaction. Hydroxylation of 2-FAA with an enzyme in the microsome fraction of rat liver required triphosphopyridine nucleotide, glucose-6-phosphate and the corresponding dehydrogenase in addition to oxygen (Booth and Boyland, 1957). Reduced triphosphopyridine nucleotide could substitute for the oxidized cofactor and the associated hydrogen donor. Deacetylation was a concomitant reaction in some cases. Unfortunately, the experimental conditions of both groups of investigators precluded the identification of metabolites of 2-FAA hydroxylated at positions other than the 7-position. Gutmann and associates used chromatographic systems which were satisfactory for demonstrating the occurrence of the hydroxylation reaction, but the solvent mixtures employed were unsuitable for discriminating between the various hydroxylated metabolites of 2-FAA. They found an 8% conversion of 2-FAA to N-(7-hydroxy-2-fluorenyl)acetamideby specific carrier isotope dilution. Booth and Boyland (1957) subsequently employed chromatographic separations which were also incapable of resolving the hydroxylated metabolites of 2-FAA. Selective chromatographic methods (J. H. Weisburger et al., 1956b) and the application of specific carrier techniques for each compound should facilitate in vitro studies to obtain further details of the hydroxylation reactions and in particular to determine the enzymes and cofactors involved. Recently, Cramer et al. (1958) were able to demonstrate that fortified rat liver homogenates and microsomal preparations did hydroxylate 2-FAA at the 1-, 3-, 5-, and 7-positions, in agreement with the results obtained in the whole animal. E. Dietary Modijications and the Metabolism of 2-Fluorenamine and Related Compounds. a. 2-Fluorenamine. Allison and Wase (1952) observed that raising the dietary level of either riboflavin or pantothenic acid increased the urinary excretion of conjugated amine and nonvolatile
2-FLUORENAMINE
.4ND RELATED COMPOUNDS
415
phenols in dogs fed 2-FA. The feeding of 2-FAA also resulted in the urinary excretion of a constant level of conjugated material, but the administration of the free amine gave higher initial percentages of conjugated substances, which dropped during 4 hours to the proportion found when the acetyl derivative was fed. 2-FA was more toxic to the dog than 2-FAA, and i t was suggested that acetylation was a form of detoxification. It is interesting that the results indicate that the dog can acetylate 2-FA, whereas sulfanilamide (Marshall, 1954) and other aromatic amines (cf. Stekol, 1941) are apparently not acetylated in this species. The urines of dogs fed 2-FAA and 2-FA were fractionated by solvent partitions by Allison et al. (1954). They noted that increasing the dietary casein and riboflavin raised 2-FA and 2-FAA fractions. Increasing pantothenic acid intake elevated 2-FAA while the ether-insoluble, water-soluble, heat-labile amine was decreased by both treatments. In contrast, Gutmann et al. (1952) using the diazotization method found that riboflavin depletion had no effect on the metabolism of 2-FA in rats. Weisburger et al. (1954) noted a delay in resorption and transport of radioactivity from the upper gastrointestinal tract after oral intake of 2-FAA by riboflavindepleted rats. Such rats converted larger amounts of the ingested carcinogen to water-soluble compounds and excreted less of the ether-soluble compounds as 2-FA, 2-FAA, and the hydroxylated derivatives. I t appears uncertain a t present whether these shifts in metabolism occurred as a result of the slower absorption, or whether the vitamin deficiency was also involved. b. N-Methyl-2-Jluorenamine. Both 2-FA and N-methyl-2-fluorenamine are active carcinogens, in contrast to phenylazoaniline, where methylation of the free amino group is required for carcinogenicity. It was thus of interest to compare the fate of the carbon in the methyl group of the fluorene derivative to that in the azo dye. Dyer (1955) found that N d e methylation and ring hydroxylation occurred. Furthermore, upon oral administration of N-methyl-2-fluorenamine labeled with C14 in the methyl group, this substituent was rapidly removed (E. K. Weisburger et al., 1956). In 3 days, an average of 54% of the dose was exhaled as radioactive CO,, 22% was eliminated in the urine, while 12% was accounted for in the feces. In addition, activity was widely distributed in the tissues, and such constituents as serine, choline, and creatinine were labeled. Only 37& of the urinary activity was accounted for by the administered compound. Whereas the removal of the methyl group was an important metabolic reaction, evidence for the reverse methylation to produce N,N-dimethyl-2-fluorenamine was not found. Performance of these studies in riboflavindeficient rats afforded an entirely different picture which suggested that the demethylation enzyme
416
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
system directly or indirectly depended on a flavin cofactor. For example, the unchanged compound constituted 28% of the urinary radioactivity. Breath, urine, and feces now accounted for 31,45, and 4y0of the dose, and tissue constituents like choline and serine had a lower specific radioactivity. In contrast, a similar vitamin deficiency caused a rise in the radioactivity excreted in the breath and incorporated in many body constituents with labeled N-methyl-p-phenylazoaniline (E. C. Miller et al., 1952). 2. N-2-Fluorenyl-p-loluenesulfonamide
If tumor induction with certain aromatic amines can proceed only when the amino group is free or can be liberated by metabolic processes, then less readily hydrolyzed derivatives as the benzoyl or p-toluenesulfonyl (tosyl) should turn out to be less or not at all carcinogenic. Tests of the former compound showed it to be slightly carcinogenic and the latter not carcinogenic at all (this laboratory, unpublished). Ray and Argus (1951), in metabolic experiments of the ingested tosyl derivative labeled with S35in the side chain, detected radioactivity in the liver, kidneys, blood plasma (but not cells), and the intestinal tract. In 24 hours, the urine contained only 0.3% of the dose, which appeared to be diazotizable like 2-FA, whereas the feces had 64%. After a longer time, practically all of the administered compound appeared in the stools in an unchanged condition. Thus, the chemical appeared to be transported along the intestinal tract with only minor amounts subjected to metabolic alterations. Ray and Argus noted that the level in the liver was as high as that obtained after a dose of 2-FAA. Moreover, this amount persisted for an extended period so that the lack of absorption of the major portion of a dose could not by itself explain the failure to induce tumors. One explanation given for the inability of the compound to induce tumors was the stability of the tosyl-amino bond. Thus, the potential availability of a free amino group appears essential for carcinogenicity in this series of compounds. 3. N-2-Fluorenylbenzamide
Although 2-FAA is an active carcinogen, the corresponding benzaniide is only mildly carcinogenic (this laboratory, unpublished). This suggested that the fate of this compound might differ from that of 2-FAA and prompted a study of its metabolism (Gutmann and Peters, 1953a). After intramuscular administration of the 9-labeled compound, mobilization from the injection site occurred slowly. The latter still contained 20% of the dose after 4 days and almost 6% after 11 days. A t the earlier time period, the carcass accounted for over 50%, most of which was not the injected compound. Radioactivity was quite generally distributed, although
2-FLUORENAMINE
AND RELATED COMPOUNDS
417
a t low levels, in all the tissues examined. No outstanding concentration of activity in any one organ was observed. In a long-term (11 days) study, 16% of the dose was eliminated in the urine and 83% in the feces, in contrast to approximately 48 and 40%, respectively, with intramuscularly administered 2-FAA after 4 days. The benzamide excreted in the feces accounted for 0.10% of the dose suggesting that the balance of the fecal radioactivity was composed of unknown metabolites. The presence of a benzene ring in addition to the fluorene system would provide two aromatic structures in the same molecule open to metabolic degradation. Ether extraction and hydrolysis experiments on the urinary activity suggested that conjugates of hydroxylated and other water-soluble metabolites constituted the bulk of the material eliminated. In this respect, a certain similarity with 2-FAA was apparent. After a dose of the benzamide, the renal elimination of radioactivity was also very slow so that in 4 days only about 8% was excreted in this manner. None of this radioactivity was due to 2-FA, and unchanged benzamide accounted for only 0.88% of the dose. Analysis by colorimetric techniques of urines and feces of rats given oral doses of the benzamide likewise revealed that metabolism of this compound qualitatively resembles that of 2-FAA in that some diazotizable material and substances giving the nitrite test for hydroxylated derivatives were excreted (Dyer, 1955; cf. Section VI,l,A).
L. N-(1-Hydroxy-%$uorenyl)acetamide and N-(3-Hydroxy-2-$uorenyl)acetamide N-(l-Hydroxy-2-fluorenyl)acetamide and the corresponding 3-isomer are produced in the metabolism of 2-FAA in rats, but not in guinea pigs (cf. Section VI,l,D). The further metabolism of these ortho-hydroxylated derivatives of 2-FAA in rats and guinea pigs was, therefore, of some interest (J. H. Weisburger et al., 1958). The labeled compounds required for these studies were isolated from the urine of rats, injected with 2-FAA of high specific activity, by two column chromatography steps (cf. Fig. 5) and several carrier recrystallizations. It was noted that the peak corresponding to the 3-isomer contained only about 4oY0 of N-(3-hydroxy-2-fluorenyl)acetamide; the balance is an as yet unidentified metabolite of 2-FAA. After intraperitoneal injection of N-(l-hydroxy-2-fluorenyl)acetamide guinea pigs excreted mainly the 1,7dihydroxy derivatives as the glucuronide. Rats, on the other hand, eliminated about equal amounts of l-hydroxy, l,Bdihydroxy, and 1,7-dihydroxy derivatives. Similar species differences were observed upon injection of N-(3-hydroxy-2-fluorenyl)acetamide. Guinea pig urine contained 47y0 of the 3-hydroxy and 38% of the 3,7dihydroxy derivatives, while rat urine showed 69y0 of the 3-hydroxy, 8% of the 3,5dihydroxy, and 9% of the 3,7-dihydroxy derivatives
418
ELIZABETH K. WEISBURGER AND JOHN H. WEISBURGER
of 2-FAA. Identification of these compounds was achieved by mobility on paper and column chromatograms, ultraviolet and infrared spectroscopy. Further proof of structure by comparison with synthetic samples should confirm these preliminary findings. In guinea pigs about 85% of the compounds (measured by radioactivity) was conjugated with glucuronic acid, and 2-801, with sulfuric acid; in rat urine it was about 60%, and 7-29%, respectively. Only small amounts were excreted unconjugated. Thus, further hydroxylation a t the 7-position of the fluorene ring was the main reaction in guinea pigs. In rats hydroxylation took place at the 5- and 7-position. The species differences in biochemical hydroxylation, as encountered with 2-FAA itself, also obtained with the ortho-hydroxylated derivatives. 5. N-(7'-Hydroxy-%jluorenyZ)acetamide
This compound is produced in large quantities by several animal species fed the carcinogen 2-FAA although it is very little if a t all carcinogenic (cf. Section IV). After administration of a single dose to 2 rats, Dyer (1955) accounted for 48 and 64% in the urine and 12 and 22y0 in the feces by means of the procedure for diazotizable material and the nitrite test. The further metabolism of the 7-hydroxy derivative of 2-FAA was studied with the radioactive material isolated from the urine of guinea pigs treated with 2-FAA-9-C14 (J. H. Weisburger et al., 1957b). Rats rapidly excreted 55-60y0 of a dose in the urine and 35-40% in the feces. The concordance of values in the urine, but lower recoveries in the feces, with the colorimetric method suggests that the feces may have contained some of the polymeric substances discussed previously (Section VI,l,D). Most of the urinary radioactivity consisted of small amounts of the administered compound and large quantities of its glucuronic and sulfuric acid conjugates. I n addition, the deacetylated product, 7-amino-2-fluorenol1 was present while evidence for other metabolites, possibly polyhydroxylated materials, was obtained. Radioactivity was found in many tissues and was bound to liver proteins (Section IV,7,A). 6 . N-3-Fluorenylacetamide
This compound underwent deacetylation and hydroxylation in rats (E. K. Weisburger, 1955). The occurrence of the first reaction was demonstrated by colorimetric means, and that of the second reaction by the actual isolation of N-(2-hydroxy-3-fluorenyl)acetamide from the urine of the treated rats. It is noteworthy that although ortho-hydroxylation was thus demonstrated in the case of the 3-isomer1 this compound appeared considerably less carcinogenic (Morris, 1955c) than 2-FAA. Hence, a great many variables determine the biological effect ascribable t o a given chemical structure.
VII. SUMMARY A N D CONCLUSIONS The section devoted to the synthesis and chemistry of fluorenamine compounds and their derivatives gives evidence of the success achieved in furnishing materials for investigation of the relationship of structure to carcinogenicity and of the metabolic fate of this class of compounds. Of the isomeric fluorenylacetamides, only N-2-fluorenylacetamide and some of its derivatives can be classed as active carcinogens. The 1- and 3-isomers are much less active, and N-4-fluorenylacetamide is inactive. The carcinogenic effect is maintained in compounds where the hydrogens of the 2-amino group are substituted by groups susceptible to metabolic removal, but the effect is lowered or absent when the free amine cannot be readily liberated. Substituents on the fluorene nucleus which (1) promote the excretion of the compound, or (2) increase the thickness of the molecule above a certain maximum, appear to abolish or reduce the carcinogenicity. Unlike many other chemical carcinogens, fluorenamine compounds induce neoplasms in a variety of tissues, usually remote from the site of application. The most effective method of administration is per os, but skin painting also induces tumors. The ability of these carcinogens to cause cancer varies greatly from species to species, and among different strains in a given species. Rats are most susceptible, while guinea pigs are resistant. The site and tissue affected often are a function of the species or strain. Carcinogenesis by N-2-fluorenylacetamide is influenced by some specific dietary factors. Supplementary tryptophan, indole, or indole-3-acetic acid appear t o promote the induction of bladder cancers in some strains of rats. In one strain, a high-fat diet favored the production of mammary carcinoma while simultaneously lowering the incidence of eye and ear tumors. A high protein diet delayed carcinogenesis and reduced the incidence of tumors a t a number of sites. In some cases a caloric restriction may decrease the carcinogenic effect. Hormonal influences play a considerable role in the production of tumors by N-2-fluorenylacetamide. Male rats are more susceptible to liver, and females to mammary tumor induction. Removal of the thyroid or of the pituitary prevents or delays liver tumor formation and abolishes the preneoplastic symptoms normally observed. Under these conditions the metabolism of the carcinogen appears unchanged. The fluorenamine carcinogens affect a number of body constituents, often reflecting a quickened metabolic turnover of these components. Thus, the half-life of riboflavin in the rat is decreased 6-fold. An apparent interference of the carcinogen with the function of pyridoxine in the metabolism of tryptophan has also been observed. These effects may involve the coenzyme functions of the vitamins. While the tissue levels of a number of enzyme activities are altered by the carcinogen, none of these systems has a t this time been related uniquely to the induction of neoplasia.
420
ELIZABETH K. WEISDURGER AND JOHN 11. WEISBUItGEIl
Studies of the metabolism of N-2-fluorenylacetamide by a variety of techniques have shown that in the rat this compound undergoes (1) deacetylation and acetylation, (2) hydroxylation at 1, 3, 5, 7, and 8-positions, (3) conjugation of the hydroxylated metabolites with glucuronic and sulfuric acids, (4)combination of some metabolites with certain, as yet unidentified, tissue constituents (proteins), and ( 5 ) some unknown reactions. The nitrogen a t the 2-position is apparently not removed from the fluorene ring during metabolism. I n the nonsusceptible guinea pig, hydroxylation at the 1- and 3-positions1 ortho to the amino group, was quasi-absent, a significant difference when compared to the susceptible rat. This finding, and the hypothesis of Bonser and Clayson that ortho-aminophenols may be the proximate carcinogens, focuses attention on the orthohydroxylated derivatives of 2-fluorenamine as directly involved in carcinogenesis. The potential reducing and chelating abilities of these metabolites deserve consideration if they should prove to have a carcinogenic effect. In conclusion, numerous studies on the carcinogenic effect of 2-fluorenamine and related compounds under varying conditions of species, strain, diet, and hormonal environment have defined some of the limits within which the carcinogenic effect can be demonstrated. The data show agreement, in part, with the observations made with the azo dye, with cyclic hydrocarbon, and with other chemical carcinogens in that carcinogenesis is a multi-stage process. The hormonal and dietary effects involved in the induction of neoplasia by 2-fluorenamine provide a wealth of material for further investigation of some of these steps. The fact that the carcinogenicity of 2-fluorenylacetamide was discovered only 17 years ago emphasizes the youthfulness of this branch of the general subject of cancer. ACKNOWLEDGMENTS We wish to acknowledge the benefit of early guidance of Dr. F. E. Ray. We are especially grateful to Dr. H. P. Morris and Dr. Helen M. Dyer for their generous assistance in the writing of this review. We are also indebted to Dr. J. Hartwell who kindly allowed us to see Shubik and Hartwell’s Contribution on survey of compounds (1957) prior to publication. Finally, we express our appreciation for the support and interest shown by Drs. Morris and Greenstein over many years. Thanks are due to Drs. H. Gutmann, J. A. and E. C. Miller, and S. Sorof for allowing us to see recent manuscripts in press.
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426
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Lacassagne, A., Buu-HOT, N. P., Daudel, R., and Zajdela, F. 1956. Advances in Cancer Research 4, 315-369. Laird, A. K., and Miller, E. C. 1953. Cancer Research 13, 464-470. Lau, H.,and Baier, P. 1954. Arch. klin. Chir., Langenbecks 278, 156-172. Laws, J. 0. 1956. Ann. Rept. Brit. Emp. Cancer Camp. No. 34, 301-303. Laws, J. O., Mabille, P., Royer, R., and Rudali, G. 1952. Bull. assoc. franc. itude cancer 39, 450-460. Laws, J. O., Rudali, G., Royer, R., and Mabille, P. 1955. Cancer Research 16, 139-142. Leathern, J. H. 1949a. Proc. Penna. Acad. Sci. 23, 99-103. Leathern, J. H. 1949b. Science 110, 216-217. Leathem, J. H. 1951. Cancer Research 11, 266. Leathern, J. H. 1955. J . Natl. Cancer Znst. 16, 1547. Leathern, J. H., and Barken, H. B. 1950. Cancer Research 10, 231. Little, J. N., and Ray, F. E. 1952. J . Am. Chem. SOC.74, 49554956. Litvinenko, L. M., and Grekov, A. P. 1957. Zhur. ObshcheZ Khim. 27, 234-239; Chem. Abstr. 61, 12866. Lothrop, W. C. 1939. J. Am. Chem. SOC.61, 2115-2119. Loveless, L. E., Spoerl, E., and Weisman, T. H. 1954. J . Bacteriol. 68, 637-644. McCallion, D. J. 1954. Can. J . 2001.32, 225-239. MacDonald, J. C., Miller, E. C., Miller, J. A., and Rusch, H. P. 1952. Cancer Research 12, 50-54. Maclagan, N. F. 1949. Ann. Rept. Brit. Emp. Cancer Camp. No. 27, 128. Maisin, J., Lambert, G., Deckers-Passau, L., and Maldague, P. 1957. Acta Unio Intern. contra Cancrum 13, 804-817. Marrian, D. H., and Maxwell, D. R. 1956. Brit. J . Cancer 10, 739-745. Marshall, E.K., Jr. 1954. J . Biol. Chem. 211, 499-503. Matsuo, H.1954. Med. J . Osaka Univ. 6,763-785. Matmo, H., Shibano, S., Ishida, H., Okarnura, S., Nabeshima, T., and Miyaji, T. 1953. Gann 44, 302-303. Maver, M. E., Greco, A. E., Levtrup, E., and Dalton, A. J. 1952. J . Nall. Cancer Znsl. 18, 687-703. Meade, J. M., and Ray, F. E. 1954. Arch. Biochem. Biophvs. 49, 4 3 4 8 . Meites, J. 1957. Federation Proc. 16, 87; 1958. Cancer Research 18, 176-180. Merkel, E., and Wiegand, C. 1947. Naturwissenschajten 34, 122. Merkel, E., and Wiegand, C. 1948. 2. Naturforsch. Sb, 93-95. Milch, L. J. 1950. Proc. SOC.Exptl. Biol. Med. 73, 321-323. Miller, E. C., and Millcr, J. A. 1952. Cancer Research 12, 547-550. Miller, E. C., and Millcr, J. A. 1955. J . Nall. Cancer Znsl. 16, 1571-1590. Miller, E. C., Miller, J. A., Sandin, R. B., and Brown, R. K. 1949. Cancer Research B, 504-509. Miller, E. C., Plcscia, A . PUT., Miller, J. A., and Heidelbergcr, C. 1952. J . Biol. Chem. 196, 863-874. Miller, J . A., and Miller, E. C. 1953. Advances in Cancer Research 1, 339-396. Miller, J. A., Miller, E. C., Sandin, R. B., and Rusch, H. P. 1952. Cancer Research 12, 283-284. Miller, J. A., MacDonald, J. C., and Miller, E. C. 1954. Proc. Am. Assoc. Cancer Research 1, (2), 32; Miller, E. C., Miller, J. A., Brown, R. R., and MacDonald, J. C. 1958. Cancer Research 18, I n press. Miller, J. A., Sandin, R. B., Miller, E. C., and Rusch, IT. 1’. 1955. Cancer Research 16, 188-199.
Miyaji, T., Moszkowski, L. I., Senoo, T., Ogata, M., Oda, T., I
428
ELIZADlCTH K. WEISBUHCER AND JOHN 1-1. WEISBUltCER
Paschkis, K. E., Cantarow, A., and Stasney, J. 1951. Science 114, 264-265. Paschkis, K. E., Cantarow, A., and Goddard, J. W. 1957. Proc. Am. Assoc. Cancer Research 2, 238. Pauling, L. 1948. “Thc Naturc of the Chemical Bond,” 2nd cd. Corncll Univ. Press, Ithaca, New York. Peacock, A., and Peacock, P. R. 1949. Brit. J. Cancer 3, 289-295. Peacock, P. R. 1947. Ann. Rept. Brit. Emp. Cancer Camp. No. 24, 143. Peacock, P. R., and Peacock, A. 1954. Brit. J . Cancer 8, 147-153. Peacock, P. R., and Peacock, A. 1955. Ann. Rept. Brat. Emp. Cancer Camp. No. 33, 271. Peck, R. M., and Creech, H. J. 1952. J . Am. Chem. SOC.74, 468470. Peters, J. H., and Gutmann, H. R. 1955. J. Biol. Chem. 216, 713-726. Peters, J. H., and Gutmann, H. R. 1956. Arch. Biochem. Biophys. 62, 234-230. Pinck, L. A. 1948. Ann. N.Y. Acad. Sci. 60, 3-17. Pinck, L. A. 1956. Cancer Research 16, 921-925. Pinck, L. A., andHilbert, G. E. 1946a. J. Am. Chem. Soc. 68, 2011-2013. Pinck, L. A., and Hilbert, G. E. 1946b. J. Am. Chem. SOC.68, 2014-2017. Piroeynski, W. J., and von Bertalanffy, L. 1952. A.M.A. Arch. Palhol. 64, 450-457. Pirorynski, W. J., and von Bertalanffy, L. 1955. Exptl. Med. Surg. 13, 261-269. Price, D. E. 1947. Ann. Rept. Brit. Emp. Cancer Camp. No. 24, 110. Pullman, A., and Pullman, B. 1955. “CancCisation par les Substances Chimiqnes et Structure MolBculaire.” Masson et Cie, Paris. Pullman, B., and Berthier, G. 1948. Bull. SOC. chim. France 551-554. Ray, F. E., and Argus, M. F. 1951. Cancer Research 11, 783-787. Ray, F. E., and Barrick, J. G. 1948. J. Am. Chem. Soc. 70, 1492-1494. Ray F. E., and Geiser, C. R. 1949. Science 109, 200-201. Ray, F. E., and Geiser, C. R. 1950. Cancer Research 10, 616-619. Ray, F. E., and Hull, C. F. 1949. J. Org. Chem. 14, 394-396. Ray, F. E., and Jung, M. L. 1951. Brit. J. Cancer 6, 358-363. Ray, F. E., and Peters, J. H. 1951. Brit. J. Cancer 6, 364-369. Ray, F. E., Cambel, P., Jung, M. L., Peters, J. H., and Woislawski, S. 1953. J. Natl. Cancer Znst. 13, 955-962. Read, G., and McGovern, V. J. 1953. Australian J. Expll. Biol. Med. Sci. 31, 283-290. Richardson, H. L. 1956. Proc. Am. Assoc. Cancer Research 2, 141-142. Richardson, H. L., and O’Neal, M. A. 1957. Proc. Am. Assoc. Cancer Research 2, 242. Rieveschl, G., Jr., and Ray, F. E. 1938. Chem. Revs. 23, 287-389. Ritchie, A. C., and Sa5otti, U. 1955. Cancer Research 16, 84-88. Ritchie, E. 1946. J. Proc. Roy. SOC.N . S. Wales 80, 33-40. Rombach, L. H., and MacGregor, I. R. 1954. J . Org. Chem. 19, 428-433. Rondoni, P. 1950. Ricerca sci. 20, 671-672. Rondoni, P. 1955a. Advances in Cancer Research 3, 171-221. Rondoni, P. 195513. Bull. schweiz. Akad. med. Wiss. 11, 274-289. Rondoni, P., and Barbieri, G. P. 1950. Enzymologia 14, 10-14. Rondoni, P., and Bassi, M. 1948. Ricerca sci. 18, 1038-1043. Ross, R. C., Scarf, R. F., and Skoryna, S. C. 1953. A.M.A. Arch. Pathol. 66, 173-180. Roth, J. S. 1954. Cancer Research 14, 346-351. Roth, J. S. 1957. Abstr. 131st Meeting Am. Chem. SOC.,Miami, April p . 9C.; Cancer Research 17, 991-994. Rudali, G., Royer, R., Laws, J. O., and Mabille, P. 1952. Compl. rend. SOC. biol. 146, 1670-1672. Ruiz, C. 1928a. Anales asoc. qufm. arg. 16, 170-186; Chem. Abstr. 23, 4691. Ruiz, C. 1928b. Anales asoc. quim. arg. 18, 225-233; Chem. Abstr. 23, 5179.
2-FLUOKENAMINE A K U RELATED COMPOUNDS
429
Rumsfcld, 13. W., Jr., Clayton, C. C., and Baumann, C. A. 1952. Cancer Research 12, 671-676. Rusch, H. P. 1954. Cancer Research 14, 407-417. Rusch, H. P., and Kline, B. E. 1941. Cancer Research 1, 465-472. Rutman, R. J., Cantarow, A., Paschkis, K. E., and Allanoff, B. 1953. Science 117, 282-283. Rutman, R. J., Cantarow, A., and Paschkis, K. E. 1954a. Cancer Research 14, 111-114. Rutman, R. J., Cantarow, A., and Paschkis, K. E. 1954b. Cancer Research 14, 115-118. Rutman, R. J., Cantarow, A., and Paschkis, K. E. 1954c. Cancer Research 14, 119-123. Rutman, R. J., Cantarow, A., and Paschkis, K. E. 1954d. J. Biol. Chem. 210, 321-329. Salaman, M. H., and Roe, F. J. C. 1953. Brit. J. Cancer 7 , 472-481. Salsberg, D. A. 1956. Proc. Am. Assoc. Cancer Research 2, 144. Salzberg, D. A., and Griffin, A. C. 1952. Cancer Research 12, 294. Salzberg, D. A., Hane, S., and Griffin, A. C. 1951. Cancer Research 11, 276. Sampey, J. R., and Reid, E. E. 1947. J. Am. Chem. SOC.69, 712. Sandin, R. B., Melby, R., Hay, A. S., Jones, R. N., Miller, E. C., and Miller, J. A. 1952. J . Am. Chem. Soc. 74, 5073-5075. Sato, T. 1956. Gann 47, 237-242. Sawicki, E. 1952. J. Am. Chem. SOC.74, 4214. Sawicki, E. 1954. J. Am. Chem. Soc. 76, 2269-2271. Sawicki, E. 1956a. J. Org. Chem. 21, 376. Sawicki, E. 1956b. J. Org. Chem. 21, 271-273. Sawicki, E., and Chastain, B. 1956. J. Org. Chem. 21, 1028-1030. Sawicki, E., and Ray, F. E. 1953a. J . Am. Chem. SOC.76, 2266-2‘267. Sawicki, E., and Ray, F. E. 195313. J. Am. Chem. SOC.76, 4346. Sawicki, E., and Wade, A. 1954. J. Org. Chem. 19, 1109-1112. Sawicki, E., Chastain, B., and Bryant, H. 1956a. J. Org. Chem. 21, 754-756. Sawicki, E., Ray, F. E., and Glocklin, V. 1956b. J. Org. Chem. 21, 243. Schins, H. R., Fritz-Niggli, H., Campbell, T. W., and Schmid, H. 1955. Oncologia 8, 233-245. Schulman, S. 1949. J. Org. Chem. 14, 382-387. Shubik, P., and Hartwell, J. L. 1957. Survey of Compounds Which Have Been Tested For Carcinogenic Activity. Publication #149, Supplement 1. U. S. Dept. of Health Education, and Welfare, Public Health Service, Bethesda, Md. Siebert, G., Lang, K., and Wolf, H. 1952. Biochem. Z . 822, 446-452. Skoryna, S. C. 1955. Proc. Can. Cancer Research Conf., Honey Harbour, Ontario, 1964, 1, 107-113. Skoryna, S. C., and Webster, D. R. 1951. Proc. SOC.Expll. Biol. Med. 78,6 2 4 7 . Skoryna, S. C., Ross, R. C., and Rudis, L. A. 1951a. J. Exptl. Med. 94, 1-8. Skoryna, S. C., Rudis, L. A,, and Webster, D. R. 1951b. Cancer Research 11, 280-281. Sommers, S. C., and Chute, R. N. 1956. A.M.A. Arch. Puthol. 61, 295-304. Sorof, S., Young, E. M., and Knospe, D. E. 1957. Proc. Am. Assoc. Cancer Research 2 , 251; Sorof, S., Young, E. M., and Ott, M. G. 1958. Cancer Research 20, 33-46. Spjut, H. J., and Eichwald, E. J. 1957. J . Natl. Cancer Inst. 18, 473479. Stasney, J., Paschkis, I(. E., Cantarow, A., and Rothenberg, M. S. 1947. Cancer Research 7, 356-362. Stasney, J., Cantarow, A , , and Paschkis, I<. E. 1950a. Cancer Research 10, 775-782. Stasney, J., Paschkis, I<. E., and Cantarow, A. 1950b. Cancer Research 10, 283-288. Stasney, J., Paschkis, K. E., Cantarow, A., and Morris, H. P. 1955. Acla Unio Intern. contra Cancrum 11, 715-720. Stekol, J. A. 1941. Ann. Rev. Biochem. 10, 265-284.
430
ELIZABETH K. WEISIJUltGER .\ND JOHN H. WEISBUItGEIl
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8-FLUORENAMINE .ZND ItELATEI) COMPOUNDS
43 1
Weisburger, J. H., Weisburger, E . K., and Morris, H. P. 1956a. J . Natl. Cancer Inst. 17, 345-361. Weisburger, J. €I., Weisburger, E. K., Morris, H. P., and Sober, H. A. 1956b. .J. Null. Cancer Insl. 17, 363-374. Weisburger, J. H., Weisburger, E. K., and Morris, H. P. 1957a. Science 126, 503. Weisburger, J. H., Weisburger, E. K., and Morris, H. P. 1957b. Abstr. 15fst Meeting A m . Chem. Soc., Miami, April pp. 9C-1OC. Weisburger, J. H., Weisburger, E. I<., and Morris, €1. P. 1957~.Abstr. l S l s l Meeting Ant. Chetn. SOC.,JIzarnk, i l p i d p. 1OC. Wrisbuiger, J. H., Weisburger, E. K., Grantham, 1’. H., :md Morris, H. P. 1958. Proc A m . Assor. Cancer Reseaich, In press. Westfall, €3. €3. 1945. J . Nall. Cancer Inst. 6 , 23-29. Westfall, B. B., and Morris, H. P. 1947. J . Natl. Cancer Insl. 8, 17-21. Wheland, G. W. 1944. “The Theory of Resonance.” Wiley, New York. Wilson, R. H., and DeEds, F. 1950. Arch. I n d . Hyg. Occupational Med. 1, 73-80. Wilson, R. H., DeEds, Y., and COY,A. J., Jr. 1941. Can.cer Research 1, 595-608. Wilson, It. H., DeEds, F., and Cox, A. J., Jr. 1947a. Cancer Re8eaTCh 7, 444-449. Wilson, R. €I., DeEds, F., and Cox, A. J. Jr. 194713. Cancer Research 7, 450-452. Wilson, R. H., DeEds, F., and COY,A. J., Jr. 1947c. Cancer Research 7 , 453-458. Wittig, G., Vidal, F., and Bohnert, E. 1950. Chem. Ber. 83, 359-367. Woodhouse, D. L. 1951. Ann. Hrpt. BriL Emp. Cancer Camp. No. 29, 172-173. Woodhouse, D. L. 1952. Cony. zntern. biorkzm., $e Congr., Paris, Kbsuiuds coviinims. p. 486. Woodhouse, D. L. 1955. Brzl. J . Cancer 9, 418-425. Ziegler, I<., and Wenx, A. 1950. Chem. Ber. 83, 354-358
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AUTHOR IN'DEX Numbers in italics indicate the pages on which references are listed at the end of the article.
A Andre, J., 341, 422 Andrewes, C. H., 188, 197,317, 326 Aas, K., 239, 266, 284 Anfinsen, C. B., 112, 125, 126, 129, 148, Abbot, L. D., Jr., 234, 257, 287 160,162,165 Abelev, G. I., 299, 312, 314, 321, 326, 328, Anissimova, V., 182, 197 329 Annau, E., 36, 44, 46 Sbels, J. C., 15, 28, 46, 222, 223, 288 Antopol, W., 37, 46 Abra.ms, A., 122, 148 Adams, D. H., 5, 7, 8, 39, 46, 160, 163, Apperly, F. L., 192, 197 Appleman, D., 6, 9, 39, 46, 49, 171, 1% 164, 176, 394, 420 Ap Rees, W., 123, 131 Adams, E., 21,46' Aptekman, P. M., 35, 47, 297, 300, 326, Adelheim, R., 75, 90 327 Adelsberger, L., 34, 46, 200, 284 Aqvist, S., 105, f48,150 Adler, S., 60, 90 Argus, M. F., 37, 46, 350, 351, 358, 361, Aezen, F., 23, 42, 46 362, 402, 416, 421, 422, 488 Afifi, M. A., 83,90 Ariel, I., 15, 28, 46 Aiston, S., 297, 526 Ariel, I. M., 70, 90 Akeson, A., 5, 39, 63, 162, 177 Armstrong, E. C., 351, 356, 377, 481 Albaum, H. G., 8,24, 46 Armstrong, M. I., 34, 46 Albert, S., 15, 37, 46, 60 Arnesen, K., 298, 325, 526 Alexander, N., 9, 46 Arnold, H. R., 204, 289 Allanoff, B., 391, 42.9 Artamonova, V. A., 293, 294, 307, 315, Allard, C., 36, $6 323, 326, 328, 529 Allen, R. A., 76, 90 Arvidsson, V. B., 85, 96 Allen, T. H., 208, 284 Asano, B., 394, 396, 421, 426 Allfrey, V. G., 135, 142, 148, 390, 420 Aschaffenburg, R., 99,148 Alling, E. L., 15, 61 Allison, J. B., 26, 27, 43,46, 357, 374, 388, Ashby, W., 228, 284 389, 392, 408, 414, 415, 420, 481, 430 Ashenbrucker, H., 225, 286 Ashikawa, K., 394, 426 Alov, I. A., 324,325 Ashmore, J., 39, 62 AItman, K. I., 112, 160 Ashworth, E., 29, 30, 43, 46 Amatuzio, D. S., 233, 284 Askonas, B. A., 112,113,123,128,148, 149 Amies, C. R., 292, 386 Athens, J. W., 204, 286 Anderson, A. J., 15, 60 Atno, A. J., 15, 62 Anderson, D., 110,149 Aub, J. C., 100, 149, 205, 254, 286, 889 Anderson, D. C., 16, 47, 61 Auerbacb, V. H., 9, 60 Anderson, E. M., 31, 48 Aust, J. B., 204, 284 Anderson, E. P., 23, 46, 105, 148, 150 Austin, P. C., 336, 421 Anderson, J. T., 207, 284 Austin, R. E., 217, 218, 219, 220, 225, 286 Anderson, N. G., 123, 148 Autret, M., 84, 91 Anderson, W., 338,342, 422 Andervont, H. B., 5, 49, 80, 90, 158, 162, Avenirova, Z. A., 316, 388 Avram, M., 347,427 176, 293, 326 433
434 Awapara, J., 118, 120, 161 Axelrad, A. A., 379,421 Axelrod, J., 392, 421, 430
AUTHOR INDEX
20, 21, 23, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 47, 48, 63,164, 172, 176 Behnke, A. R., 207,208, 284, 290 B Belding, T., 316,386 Bablct, J., 62, 90 Beling, C. A., 68, 90 Babson, A. L., 9, 11, 17, 18,27, 33,35, 40, Belkin, M., 14, 64 46, 108, 111, 115, 149 Bell, F., 338, 339, 481 Bach, S. J., 14, 46 Belozersky, A. N., 303, 326 Bachmann, W. E., 342,421 Belyavin, G., 293, 32.9 Bacon, M. O., 17, 62 Bender, C. E., 293,526 Badger, G. M., 367, 421 Bendick, A., 23, 47 Bagg, H. J., 182, 183, 197 Benditt, E. P., 14, 49 Baidakova, 2. L., 299, 329 Hen-Ishai, R. , 164 Baier, P., 374, 426 Bennett, C. W., 336, 421 Baker, H. W., 70, 94 Bennette, J. G., 136, 137, 14.9 Baker, 8. L., 190, 197 Bennison, B. E., 293,326 Baldwin, R. B. T., 60, 96 Bentov, M., 344,421 Bale, W. F., 123, 124, 126, 161, 205, 232, Berenblum, I., 352, 386, 421 234, 284, 286, 286 Bergestrand, I., 69, 90 Balfour, W. M., 205, 232, 886, 286 Bergrnann, E., 335,421 Balis, M. E., 23, 42, 46 Bergmann, E. D., 344, 347,421 Ball, H. A., 29, 31, 46 Berk, J. E., 82, 83, 80 Ballin, J. C., 9, 48 Berkson, J., 202, 287 Bandy, H. E., 30, 63 Berlin, N. I., 35, 47, 205, 206, 211, 215, Barabadse, E. M., 294, 329 216, 234, 238, 242, 244, 257, 258, 284, Barabutes, N. K., 347, 423 286, 888 Barakan, T. H., 24,46 Berlin, R., 228, 232, 236, 237, 257, 265, Barbieri, G. P., 395, 428 266, 286 Bardout, F. E., 345,421 Berman, C., 57, 58, 59, 60, 62, 64, 65, 66, Barken, H. B., 379,426 67,68,70,74,75,79,81,82,84,90,90, Barker, A., 345, 421 91 Barker, C. C., 345, 421 Bernard, J., 245, 886, 302, 310, 329 Barker, G. R., 24, 35, 36, 37, 46, 47, 61 Bernfeld, P., 15, 47, 61, 100, 168 Barker, W. H., 236,284 Bernhard, W., 190,197 Barnes, D. W. H., 203, 205, 284 Bernheim, F., 367,491 Barratt, R. W., 359,421 Bernheim, M. L. C., 367, 481 Barrett, M. K., 292, 298, 326, 328 Berry, M. E., 6,46 Barrick, J. G., 346, 488 Berson, S. A., 205, ,986 Barry, J. M., 113, 120, 149, 168 Bertani, G., 359, @3 Barton, M. X., 342, 421 Berthet, J., 132, 160 Bass, A. D., 23,47 Berthier, G., 335, 488 Baesi, M., 395, 428 Berwick, M., 5, 7, 60, 64, 162, 176 Bateman, J. C., 201, 211, 212, 222, 284 Beskow, G., 145, 161 Bauer, F. K., 111, 149 Best, C. H., 32, 47 Baumann, C. A., 14, 62,80, 93, 359, 360, Beyers, C. F., 60, 91 Besnak, M., 6, 39,63, 162,177 425, 429 Beach, G., 22,61 Bhargava, P. N., 342,422 Becker, B. J. P., 59, 83, 90 Bielschowsky, F., 80, 91, 192, 197, 333, Begg, R. W., 2, 3,4, 5, 6,7, 11, 12, 13, 18, 339, 343, 351, 352, 353, 354, 355, 356,
AUTHOR INDEX
357, 359, 360, 362,363, 365, 368, 309, 374, 375, 376, 377, 378, 379, 380, 383, 398, 400, 405, 406, 409, 421, 424, 427 Bielschowsky, M., 339, 362, 421 Bierman, H. R., 35, 60, 201, 209, 212, 214, 287, 289 Bigelow, N. H., 65, 91 Birjulina, T. I., 294, 295, 329 Birkeland, L., 234, 257, 287 Birnbaum, S. M., 341,407, 408,424 Bishop, J. S., 32, 61 Bittner, J. J., 29, 49, 293, 294, 295, 325, 326 Blanche, P., 204, 286 Bloor, W. R., 3, 18, 19, 20, 21, 22, 26, 28, 29, 32, 41, 42, 49 Blumenau, E., 82,91 Blumenthal, F., 158, 176 Bly, C. G., 27, 61, 123, 124, 126, 151 Bobrinckaja, A. C., 296, 326 Bodansky, O., 10, 11, 47 Bottner, H., 238, 241, 246, 251, 254, 272, 280, 281, 285 Bogden, A. E., 35,47 Bohnel, E., 297, 326 Bohnert, E., 340, 431 Boiron, M., 245, 886 Bolis, L., 351, 481 Bolker, N., 14, 47 Bollman, J. L., 268, 288, 388, 422 Bolyard, M. N., 81, 96 Bonne, C., 63, 65, 81, 82, 91 Bonnichsen, R. I<.,5,6,39,47,63, 162,177 Bonser, G. M., 351, 356, 357, 358, 377, 383, 412, 414, 421 Boorman, K. E., 264, 286 Booth, J., 414, 422 Borges, P. R. F., 16, 62, 120, 162 Borsook, H., 105, 107, 121, 122, 123, 134, 149, 161, 163, 164 Bortner, M. H., 334, 422 Bos, C. J., 394, 423 Bothwell, J. W., 38, 62 Bothwell, T. H., 218, 219, 285 Boussier, G., 256, 287 Boyd, C. E., 20,47 Boyd, E. M., 20,47 Boyland, E., 11, 14, 47, 80, 91, 344, 355, 371, 372, 373, 385, 414, 482 Brachet, J., 131, 142, 149
435
Brackney, E. L., 204, 884 Bradshaw, L., 385,481 Brahn, B., 4, 47, 158, 175 Bras, G., 82, 86, 91 Brashear, D., 192, 198 Braunstein, A., 19, 47 Breedis, C., 69, 91, 3.58, 422 Breidenbach, A. W., 358, 361, 422 Bremer, K., 345, 482 Brennan, M. J., 15, 53 Brigando, J., 385, 42% Brock, J. F., 84, 91 Brodie, B. B., 392, 421, 430 Brook, L., 204, 208, 287 Brown, C. E., 16, 34,47, 51 Brown, G. B., 23, 46, 47 Brown, G. M., 236,247,257,285,334,422 Brown, I. W., Jr., 228, 229, 230, 232, 245, 253, 269, 286, 286 Brown, R. K., 363, 368, 369, 377, 426 Brown, R. R., 14, 62, 360, 426 Brown, W. D., 367,422 Bruce, C., 86, 94 Briickmann, G., 262, 286 Brues, A. M., 80, 91 Brunschwig, A., 70, 91 Bryan, E., 188,197 Bryan, W. L., 293, 325 Bryant,, H., 337, 338, 343, 422, 429 Bucher, N. L. R., 100, 149 Buckley, G. F., 86, 91 Bulbenko, A., 113, 114, 153 Bunting, H., 297, 326 Burgess, L., 31, 53 Burk, D., 10, 15, 19, 27, 43, 47, 54 Burke, W. T., 6, 16, 47, 124, 149 Burmester, B. R., 188, 197, 316, 326 Burnet, F. M., 164 Burns, D. M., 334,422 Burstein, S.,30, 43, 61 Burtle, J. G., 344, 349, 384, 385, 414, 424 Burton, A. F., 12, 23, 39, 47 Burton, K., 164 Busch, H., 16, 17, 18, 40, 47, 50, 61, 105, 106, 107, 110, 149,151 Bush, I. E., 30, 47 Bush, J. A., 206, 225, 286 Butler, C. L., 5, 48 BUU-HoI,N. P., 336, 342, 344, 351, 428, 496,426
436
AUTHOR INDEX
Chodos, R. B., 34, 35, 38, 44,61,220, 246, 251, 264, 265, 281,286,287 Chou, S. N., 204, 284 C Christensen, H. N., 16, 48, 108, 118, 149, 152 Calaresu, F., 321: 326 Chu, C. H. U., 37, 48 Calcutt, G., 359, 422 Chudinova, I. A., 159, 162, lY6 Callender, S., 226, 232, 286 Chute, R. N., 380, 429 Calvery, H. O., 80, 94 Claborn, H. V., 333, 422 Calvin, M., 348, 422 Clark, C. T., 392, 430 Camain, R., 61, 65, 71, 75, 83, 91, 94 Clark, J. H., 201, 209, 210, 211, 285 Cambel, P., 342, 356, 362, 365, 428 Clark, R. L., 221, 286 Campbell, H. W., 15, 62 Campbell, J. G., 80, 91, 192, 193, IOY, Claude, A., 131, 149, 160, 189, 19Y Claus, B. F., 388, 422 351, 357, 422 Campbell, N., 336, 338, 342, 334, 345, 422 Clayson, D. B., 385, 412, 414, 421, 422 Campbell, P. N., 103, 104, 106, 112, 113, Clayton, C. C., 359, 386, 426, 429 115, 125, 126, 127, 136, 137, 138, 139, Cleland, W. W., 21, 48 Clemmesen, J., 65, 91 143, 144, 148,149, 154 Campbell, T. W., 346, 362, 363, 366, 422, Cohen, A., 70, 91 Cohen, B. L., 99, 160 499 Cohen, L., 70, 91 Cannan, R. K., 202,286 Cantarow, A., 14, 15, 61, 80, 91, 354, 358, Cohen, P. P., 388, 422 369, 375, 376, 377, 378, 379, 380, 388, Cohen, S., 37, 48 389, 390, 391, 392, 422, 42Y, 428, 429 Cohn, M., 107, 161 Cole, R. D., 145, 160 Cantero, A., 11, 64 Collens, D. H., 200, 209, 224, 286 Carbone, R., 8, 4 Y Collier, V., 6, 64 Carnahan, D. S., 61,91 Carr, J. G., 189, 190, 194, 195, 197, 292, Collip, J. B., 29, 31, 48, 61 Colwell, L. S., 246, 289 326 Connell, G. E., 112, 160 Carrie, A. W., 19, 4 Y Conrad, E. J., 14, 27, 61 Cartwright, G. E., 206, 225, 286 Castle, W. B., 266, 268, 279, 286,287, %90 Conzelman, G. M., 22, 48 Cook, H. A., 388, 390, 392,422, 4.24 Cavelti, P. A., 292, 326 Cook, J. W., 80, 91, 367,422 Cerecedo, L. R., 22, 37, 42, 4Y, 48, 62 Coombs, R. R. A., 264, 286 Ceriotti, G., 169, 1Y5 Coons, A. H., 112,160 Cervigni, T., 8, 4Y, 48 Cooper, M., 268, 288 Chalfin, D., 261, 286 Coote, J., 145, 160 Chalmers, J. G., 186, 19Y Copeland, D. H., 86,91,94, 354, 369, 370, Chang, C., 208,284 373, 374,423 Chang, F. C., 35, 63 Cori, C. F., 18,48 Chantrenne, H., 122, 142, 149, 161 Cori, G. T., 18, 48 Chanutin, A., 10, 48 Cottrall, G. E., 188, 19Y Chaplin, H., Jr., 202, 206, 285 Counsellor, V. S., 78, 82, 91, 93 Chappell, R. B., 76, 94 Cowdry, E. V., 10, 60, 62, 96 Chargaff, E., 22,48 Cowen, P. N., 192, 19Y Chastain, B., 336, 337, 338, 342,429 Cox, A. J., 80, 96 Cheewer, F. S., 321, 326 Cox, A. J., Jr., 334,351,352,353,354,355, Chen, K. P., 208, 284 356, 361, 368, 369, 375, 398, 406, 422, Chen, P. S., 131, 149 Cheng, H. F., 113,163 431
Buxton, L., 298, 326 Byrnes, W. W., 68, 93
Cox, H. R., 297, 326 Coyne, 13. A., 118,162 Crabh, E. D., 37, 60 Cramer, J. W., 361, 414, 42.2 Creech, H. J., 315, 321, 326, 341, 422, 428 Crile, G., Jr., 387, 422 Crispell, K. R., 204, 286 Crockett, C. L., 238, 251, 289 Crockett, C. S., Jr., 257, 289 Cruz, W. O., 232, 286 Cudkowicz, G., 393, 422 Cuff, J. R., 63, 94 Cummings, E. H. T., 60, 90 Cunningham, L., 388, 390, 392, 424 Curreri, A. R., 14, 62 Curtis, M. R , 353, 354, 36S, 370, 371, 372, 373,42S Cutbush, M., 205, 288 Cutler, S. J., 63, 65, 92
D Dacie, J. V., 227, 236, 264, 265, 286 Daff, M., 392, 422 Dahl, A. W., 339, 340, 342, 349, 423 Dalgliesh, C. E., 112, 160 Dalton, A. J., 29, 31, 48, 80, 92, 133, 161, 389, 390,395,426 Daly, M. M., 135, 148 Dameshek, W., 235, 263, 265, 266, 287, 288,289 Damron, C. M., 400, 402, 405, 409, 411, 412,422,423 Daniels, M. B., 32, 62 Danneberg, P., 376, 422 Darchun, V., 383, 424 Darcy, D. A., 100, 160 Darrach, M., 30,63 Daudel, R., 342, 426, 426 Dausset, J., 245, 286, 292, 326 Davidson, J. N., 22, 42, 48 Davie, E. R., 145, 160 Davies, J. N. P., 59, 60, 65, 75, 83, 8.5, 88, 91
Davis, C. L., 235, 286 Davis, W. E., 23, 48 Day, E. D., 8, 9, 48, 163, 176 Deasy, C. L., 105, 121, 123, 134, 149, 164 Debov, S. S., 308, 309, 329 De Camp, P., 201, 209, 210, 211, 286 Deckers-Passau, L., 354,370,395,422,426
De Duve, C., 132, 160,395, .422 DeEds, F., 80, 96, 334, 351, 352, 353, 354, 355, 356, 361, 368, 369, 375, 398, 406, 422, 431 Degorce, 62, 91 DeGowin, E. L., 226, 228, 229, 239, 246, 248,285, 289 DeJong, J., 82, 91 De La Vergne, L., 234, 284 Delory, G. E., 11, 60 Demany, P., 83, 94 Demerec, M., 359, 423 De Moor, N. C., 70, 91 De Moss, J. A., 145, 160 Dennis, E. J., 196, 1.98 Denoix, P. F., 60, 61, 91 Dent, C. E., 16, 48 Dente, A. R., 34, 64 Denton, R. W., 355, 423 Des Ligneris, M. J. A., 60, 81, 91 Deuchar, E. M., 131,160 Deutsch, H. F., 8,39,49,62, 101,160, 168, 176 Dhar, M. M., 24, 47 Dible, J. H., 89, 91 Dickinson, T. E., 5, 6, 7, 13, 20, 21, 26, 28, 35, 36, 38, 40, 47 Diels, O., 336, 337, 342, 344, 345, 423 Dietrich, L. S., 28, 62 Dillard, G. H. L., 10, 48 Dixon, C. E., 221, 286 Dmorhowski, L., 188, 197, 292, 293, 318, 326
Doan, C. A., 266, 286 Dobriner, K., 31, 48 Dodd, B. E., 264, 285 Doerner, A. A., 67, 92 Doljanski, L., 77, 91 Donati, M., 258, 287 Doniach, I., 351, 423 Donohue, D. M., 218, 219, 286 Donovan, H., 3, 48 Dorfman, R. I., 7, 30, 48 Dorn, H. F., 63, 65, 92 Dornhorst, A. C., 228, 286 Doucet, G., 84, 92 Dounce, A. L., 5, 6, 48, 63, 164 Drake, W. L., 80, 83, 96 Drewry, J., 99, 148 Dreyfus, J. C., 219, 28*9
438
AUTHOR I N D E X
Dubnik, C., 31,48,342,348,351,352,354, 361,362, 363, 368, 370, 401, 427 Duffy, E., 80, 91 Dugan, A., 246, 289 Dulaney, A. D., 298,326, 326 Duncan, H. M., 296,327 Dunham, L. J., 354, 423 Dunn, J. E., 35, 48 Dunn, M. S., 17, 60 Dunn, T. B., 9, 36, 37, 51, 292, 327, 348, 351, 352, 354, 355, 370, 423, 427 Dunning, W. F., 209, 286,353, 354, 368, 370, 371, 372, 373, 423 Duran, S. H., 201, 210, 286 Duran-Reynals, F., 188, 191, 197 Dyadkova, A. M., 293,307,308,311,312, 318,326 Dyer, H. M., 362,365, 372, 382, 384,392, 396, 399,400,401,402, 403,404,405, 409, 411, 412, 415, 417, 418, 422, 423
E Eades, C. H., 14, 48 Eadie, G. S., 228, 229, 230, 232, 245, 253, 269, 286, 286 Ebaugh, F. G., Jr., 230, 232, 286 Eberhart, D. R., 347, 423 Ebert, J. D., 113, 130, 160 Ebert, M. K., 299, 302, 328 Ebert, R. V., 204, 289 Eckert, A., 337, 338, 345, 423 Edlbacher, S., 11,48 Edlingcr, E., 319, 326 Edmondson, H. A., 63, 74, 75, 76, 92 Edwards, J. E., 76, 80, 92 Eerkens, J. W., 83, 96 Eggel, H., 74, 75, 92 Ehrensvard, G., 105,160 Eichwald, E. J., 352, 354, 429 Eisenreich, F., 235, 289 Eitelrnan, E. S., 16, 61 Ekrnan, B., 370, 374, 376, 430 Ekman, C. A., 69, 90 Eliasson, N. A., 105, 160 Ellerbrook, L. D., 296, 326,827 Elliot, S. M., 236, 285 Elman, R., 14, 27, 61 El Mehairy, M. M., 17, 48 Elmes, B. G. T., 60, 92, 96 Elvehjem, C. A., 12,64
Emerson, C. P., 34,35,38,44,61,220, 230, 232,238,246,251,254, 257,265,281, 286, 986,287, 989 Emerson, G. A., 300, 329 Emmelot, P., 394, 433 Endo, H., 9, 48, 166, 169, 176 Engel, R. W., 86, 93, 354, 369, 370, 373, 374,423 Engelbreth-Holm, J., 41, 48 Epstein, M. A., 189, 190, 197 Erikson, C. C., 35, 63 Errere, M., 131, 160 Erwin, C. P., 18, 63 Erwin, H. L., 32, 49 Erxelben, H., 101, 161 Eschenbrenner, A. B., 80, 96 Esser, H., 15, 48, 112, 153 Evans, E. I., 234,257, 287 Evans, E. J., 205, 288 Evans, J. V., 99, 160 Evans, R. D., 205,286 Evans, R. L., 233, 235, ,984, .t?86 Evans, W. A., Jr., 205, 211, 286 Everest, A. E., 334, 423 Everett, M. R., 18, 63 Ewing, J., 7,48, 76, 81, 92 Eyestone, W. H., 357, 467 F Fahey, J. L., 16, 48 Fahraeus, R., 205, 286 Falin, L. I., 183, 197 Fang, H. S., 208,284 Farber, E., 86, 92, 123, 160, 354, 377, 423 Fardon, J. C., 300, 326 Favre, M., 83, 92 Feasby, W. R., 76, 92 Feen, B. G., 207, 284 Feinstein, R. N., 5, 9, 48 Feldman, W. H., 191, 197 Feller, D. D., 208, 286 Fenninger, L.D., 3, 13, 20, 21, 24, 26, 27, 4 4 6 1 , 164 Fenton, S. W., 344,424 Fernando, P. P., 82,9.t? Ficq, A., 131, 160 Figen, J. F., 162, 171, 177 Filbm, D., 369, 391,494 Finch, C . A., 218, 219, 232, 283, 286, 286 Finch, S., 283,,986
Finch, S. C., 257, 2889 Findlay, G. M., 62, 92 Fine, A. S., 28, 63 Fink, M. A., 306, 386 Firminger, H. I., 75, 76, 92, 354, 362, 364, 377,378,423,487 Fischer, B., 76, 92 Fischer, H., 118, 149 Fischer, W. O., 59, 82, 92 Fish, W., 277, 289 Fisher, H., 298, 388 Fisher, R. B., 164 Fitshugh, 0. G., 80, 894 Flavin, M., 112, 129, 160 Fleischer, S., 113, 163 Fleisher, G. A., 10, 65 Flesch, R. N., 347, 426 Fletcher, T. L., 339, 340, 342, 319, 423 Flickinger, R. A., 130, 160 Flores, V., 17, 60 Fluharty, R. G., 218, 286 Foley, E. J., 300, 386 Folkes, J. P., 129, 260, 164 Fong, C. T., 296, 327 Ford, A. B., 205, 888 Forkner, C. E., 200, 286' Forssberg, A., 106, 260 Forster, F. M., 15, 62 Foulds, L., 188, 192, 194, 297, 318, 326, 357,423 Frampton, V. L., 6, 63 Francis, L. D., 34, 63 Francis, M. D., 113, 114, 123, 130, 160 Frank, H. G., 5, 34, 63 Frankel, S., 118, 268 Frantz, I. D., Jr., 124, 125, 163 Frech, M. E., 39, 63 Frederick, G. L., 12, 21, 32, 41, 48 Freeman, L. W., 263, 287 Freidberg, F., 104, 160, 164 Freiman, V. B., 307, 308, 309, 326, 3289 Freinkel, N., 204, 286 Friedberg, F., 16, 18, 64, 140, 163 Friedberg, W., 113, 163 Friedell, H. L., 204, 287, 289 Frieden, E., 128, 163 Friedewald, W. F., 296, 314, 326 Friedland, I. M., 28, 63 Frisch-Niggemeyer, W., 8, 48
Fritz-Niggli, H., 346, 354, 362, 363, 389, 393,483, 429 Frumin, A. M., 266, 989 Fruton, J. S., 164 Fuchigami, A., 161, 166, 176 Fudenberg, H., 247, 2.90 Fujita, Y., 172, 176 Fukai, H., 112, 163 Fukuoka, F., 8, 39, 48, 61,157, 159, 165, 167,169, 170, m , i 7 4 , i r 6 , zrfi Furlong, N. B., 23, 39, 48 Furth, J. 2., 49, 292, 326, 327, 375, 380, 386,423
G Gabrielson, F. C., 8, 9, 48, lg3, 176 Gaebler, 0. H., 7, 49 Gaitondc, M. K., 103, 160 Gale, E. F., 129, 160, 164 Gallant, D. L.. 5, 34, 63 Gallison, D. T., 68, 98 Galuzsi, N. J., 67, 92 Garcia, J. F., 217, 218, 219, 2'20, 225, 286 Gardashyan, A. M., 297,307,309,311,326 Gardner, F. H., 230, 242, 266, 288, 290 Gardner, W. U., 28, 49 Gasson, J. E., 14, 47 Gaucrke, L., 11, 63 Gehrmann, G., 112, 160 Geiser, C. R., 348, 350, 402, 407, 428 Gelfand, M., 59, 60, 82, 9.9 Gelhorn, A, 230, 231, 248, 252, 287 Geller, W., 35, 44, 49 Gelstein, V. I., 307, 309, 315, 319,322,326 Gentil, P., 60, 92 Gentry, R. F., 188, 187 Genuth, S. M., 145, 160 Gerard, P., 60, 95 Gerarde, H. W., 123, 160 Gessler, A. E., 293, 386 Ghadially, F. N., 27, 49 Gibson, J. A., 339, 481 Gibson, J. G., 11, 205, 211, 218, 232, 286' Gilbert, C., 86, 92 Gilliam, A. G., 65, 92 Gillman, J., 68, 83, 84, 86, 92 Gillman, T., 68, 83: 84, 86, 92 Gilmore, J., 338, 342, 422 Ginsig, R., -366, 482 Ginzburg-Kalinina, S. I., 296, 326
440
AUTHOR INDEX
Girges, R., 83, 92 Glaiibach, S., 37, 46 Click, D., 11, 49 Glicksmnn, A. S., 32, 49 Glock, G. E., 394,423 Gloeklin, V., 346, 350, 429 Gluzman, F. L., 326 Glynn, L. E., 86, 93 Goda, I., 170, 176 Goddard, J. W., 3.58, 428 Godin, C., 112, 113, 148, 150 Gold, J. J., 31, 43, 49 Goldblatt, H., 86, 92 Golde, A., 190, I98 Goldenberg, H., 263, 265, 266, 288 Goldfeder, A., 18, 49, 300, 326 Goldsmith, E. D., 7, 48 Goldsmith, Y., 298, 325, 326 Goldzieher, M., 78, 98 Goodman, C. J., 40, 49 Goodman, M., 15, 53 Goranson, E. S., 11, 18, 19, 41, 49 Gorbman, A., 379, 483 Gordon, D., 30,49 Gorer, P. A,, 80, 92, 299, 326 Gorham, A. T., 28,46 Gorodilova, V. V., 293, 308, 319, 326 Gottschalk, R. G., 318, 326 Goulden, F., 343, Grabar, P., 302, 310, 329 Grable, E., 222, 290 Graehe, C., 349, 423 Graf, L., 297, 328 Graff, S., 37, 46 Graffi, A., 360, 423 Graham, J. B., 321, 326 Graham, 0. L., 16, 50 Graham, R. M., 321,326 Grantham, P. H., 349, 417, 430, 431 Gray, C. H., 234, 286 Gray, G. W., 336,424 Grey, S. J., 203, 229, 286 Greco, A. E., 9, 36, 37, 61, 389, 390, 395, 486 Green, H. N., 11, 20, 24,49, 192,197, 351, 357, 359, 360, 362, 363, 365, 368, 374, 421,424 Green, J. W., 14, 49 Green, M. N., 362, 424 Grecn, R. G., 293, 326, 327
Greenberg, D. M., 10, 16, 17, 18, 49, 61, 62, 64, 104, 106, 119, 120, 123, 140, 160,161,168,163,164 Greenberg, M. S., 268, 279, 287 Greene, A. A., 11, 49 Greene, H. S. N., 16,47, 105, 106, 110,149 Greenfield, R. E., 4, 6, 8, 9, 24, 34, 38, 39, 49, 62, 159, 163, 164, 167, 169, 175, 177, 224, 261, 270, 271, 272, 273, 274, 286,288, ,989 Greengard, O., 136,137,138,139,143,144, 149 Greenhouse, S. W., 35, 48 Greenless, J., 17, 27, 40, 49, 108, 160 Greenstein, J. P., 3 , 4 , 5 , 6 , 7 , 8 , 10, 12, 16, 33, 34, 35, 38, 40, 49, 158, 162, 163, 169, 174, 176, 176, 341, 393, 394, 397, 407,408,424 Greenwood, A. W., 183, 197 Greer, M. A., 32, 49 Gregerson, M. I., 203, 205, 206, 209, 286 Greisbach, W. E., 31, 62 Grekov, A. P., 336,426 Griffin, A. C., 23,39,48,49, 381,382, 387, 388, 390, 392, ~$22,424,427, 429 Griffin, G. B., 222,288 Grindlay, J. H., 268, 288 Grinstein, M., 233, 286 Grodsky, G., 128,160 Gromzewa, K. E., 183, 197 Gross, J., 135, 142, 161 Gross, L., 259, 286, 300, S26, 327 Guerin, J., 61, 65, 71, 83, 94 Guerin, M., 81,94, 181, 187,198,292, 294, 328 Guerin, P., 81, 94 Gulland, J. M., 24, 35, 36, 37, 46, 51 Gustafson, E. G., 63, 65, 76, 92 Guthrie, J., 352, 424 Gutierrez, N., 37, 46 Gutmann, H. R., 339, 342, 343, 344, 348, 349,350, 360,368, 369,384, 385, 391, 999, 402, 403, 404, 405, 406, 407, 408, 414, 415, 416,424, 427, 438 Gyorgy, P., 86, 92
H Haagm-Smit, A. J., 105,121,123,134,149 Hack, W., 124,162
AUTHOR INDEX
Haddow, A., 31, 33, 43, 49, 323, 327, 386, 434 Haden, R. L., 265, 886 Hadler, H. I., 383, 424 Haguenau, F., 190,198 Hahn, P. F., 205, 232, 686,286 Haist, R. E., 32, 47, 49 Hakanson, E. Y., 11, 49 Halber, W., 300, 327 Halberg, F., 29, 49 Hale, W. H., 35, 63 Hall, J. W., 84, 92 Hall, W. H., 375, 376, 377, 379, 380, 405, 4.91 Halliday, J. W., 103, 104, 106, 115, 149 Halsted, J. A., 222, 289 Halvorson, O., 164, 293, 327 Ham, A. W., 19, 34, 46, 47 Hamer, D., 386, 427 Hamilton, C. S., 345, 422 Hamilton, H. E., 226, 228, 229, 239, 246, 248, 286, 289 Hamilton, L. D., 70, 94 Hamilton, M. G., 133, 134, 162 Hammarsten, E., 105, 160 Hammarsten, J. F., 204, 2880 Hane, S., 382, 429 Hanot, V., 77, 91 Harding, H. E., 358, 424 Hardy, S., 116, 160, 162 Hargreaves, A. B., 8, 39, 49, 168, 176 Harknew, R. D., 100, 160 Harman, J. W., 78, 94 Harne, 0. G., 235, 286 Hamelson, R. T., 29, 63 Harris, A., 103, 107, 112, 113, 115, 161 Harris, A. S., 348, 349, 424 Harris, H., 99, 160 Harris, J., 355, 371, 372, 422 Harris, J. I., 99, 160 Harris, P. N., 80, 92, 351, 369, 370, 374, 424 Harris, R., 292, 327 Harris, R. J. C., 190, 197 Hartley, J. B., 336, 424 Hartroft, W. S., 86, 91 Hartwell, J. L., 351, 361, 412, 424, 429 Hartz, P. H., 63, 92 Haruno, K., 396, 484,426 Harvey, E. K., 28,60
44 1
Harvey, J., 34, 60 Harvey, J. C., 222, 288 Harvey, J. L., 34, 64, 220, 230, 231, 248, 251, 252, 287 Haskins, D., 218, 283, 286 Hastings, A. B., 39, 62 Hauch, E. W., 70, 92 Haurowitz, F., 113, 163, 164 Hausberger, F. X., 20, 49 Haushka, T. S., 291, 300, 327 Havas, H. F., 341, 422 Haven, F. L., 3, 11, 18, 19, 20, 21, 22, 25, 26, 28, 29, 32, 36, 41, 42, 49, 61 Hawkins, W. B., 226, 234, 286,189 Hay, A. S., 365, 429 Hayashi, M., 336, 337, 338, 424, 42.5 Head, M. A,, 80, 94, 193, 198 Herk, F. J., 221, 286 Heidelberger, C., 17, 23, 40, 60, 63, 107, 161, 321, 327, 348, 382, 383, 416, 422, 424. 426 Heidelberger, M., 102, 160 Heim, W. G., 9, 49, 171, 176 Heiman, J., 351, 371, 376: 424 Heimberg, M., 112, 160 Heinle, R. W., 222, 286 Heins, H. C., 196, 198 Heinz, E., 118, 160 Heinsler, F., 15, 48 Heiselt, L., 31, 63 Heller, B. I., 204, 289 Heller, L., 158, 177 Heller, L. Z . , 5, 64 Helmer, 0. M., 303,328 Helwig, H. L., 34, 61, 216, 288 Henderson, M. E., 16, 48, 118, 149 Hendler, R. W., 139, 140, 160 Hendley, D. D., 5, 48 Henion, W. F., 99, 160 Hennessey, R. S. F., 59, 93 Hennesy. T. G., 217, 218, 219, 220, 225, 286
Herbert, D., 6, 60 Hernandez, A. L., 69, 93 Herxheimer, G., 74, 75, 77, 92 Hew, W. C., 15, 62 Hewson, K., 37, 46 Hicks, D. A., 205,206, 286 Hicks, W., 14, 27, 61 Heiger, I., 81, 93
442
AUTHOR INDEX
Higgins, G. M., 10,63 Higginson, J., 65,94 Hilbert, G.E.,367,498 Hilf, R., 14,27,60 Hill, B. R., 10, 11, 60,62 Hill, J. H., 35,60 Hill, M. S., 15, 63 Hill, R. L., 407,408,486 Hill, W.T., 360,426 Himsworth, H. P.86,93 Hirs, C. H. W., 340,362, 426 Hirsch, G. C., 113, 161 Hirsch, H. H., 8,60, 161,169,176 Hirschfeld, H., 200,236,282,986 Hirzfeld, L.,300,327 Hitchcock, C.R.,16,60,356,426 Hoagland, M. B.,142,144, 145,146,160, 161,153,166 Hobbs, J. H., 15,61 Hoch-Ligeti, C., 355, 363, 365,371, 376, 377,383,392,393,422,426 Hochstrasser, H. T.,22,62 Hoffman, H. E., 361,381,424,426 Hoffmmn, F.,360,423 Hogeboom, G.H., 132, 133,161 Hogness, D.S.,107,161 Hogness, K.R., 15,61 Hokin, L. E.,127,161 Holden, W.D.,204,287,989 Holler, H., 8,48 Holley, R.W., 146,161 Homburger, F.,6,15,18,29,41,47,62, 64, 208, 222,223, 226, 265,268, 283,289 Hope, A., 205,206,986 Horava, A.,376, 386,426 Horel, L.,319,396 Horning, E. S., 31, 33, 43, 49, 351, 355, 371,372,499,496 Horstmann, E.,77,93 Horwitt, B. N.,30,49 Hou, P.C.,63,83,93 Houghton, J. D.,296,327 Howatson, A. F.,189,197 Howell, J. S., 386,497 Hoyle, L.,293, 297,396,327 Hoyne, R. M.,67,74,75,93 Hsieh, K. M.,10,60 Huang, T.F., 208,984 Huff, R. L.,208, 217,218,219, 220, 225, 986
Huggins, C., 26, 63, 164,361,425 Hughes, G. K.,345,426 Hughes Jones, N. C., 229,230,287 Huisman, T.H. J., 100,163 Hull, C. F.,343,498 Hultin, T.,131,134,136,145,161 Humphreys, E. M.,14,49 Hunter, S. W.,16,60 Hurlbert, R.B., 17,40,60,107,161 Hurley, T. H., 229,987 Hurn, M.,202,887 Hurtado, A. V., 218, 219,286 Hutchison, C. A., Jr., 347,426 Hyde, G. M.,35, 47, 205, 211, 216, 216, 984,286,988 Hyman, G . A., 34, 60,64, 220, 230, 231, 247,248,249,251, 252,277, 287,289
I Iacono, J. M., 21,48 Iball, J., 334,429 Ibbotson, A.,336,424 Ichii, S., 394,395,396,426,427 Iijima, N.,172,176 Imagawa, D.,293,397 Imagawa, D.T., 293,326 Inaba, M.,396,426 Ingle, D.J., 14, 17, 19,26, 32,41,60 Inkley, S. R., 204,208,287 Inoue, M.,335,340,427 Ishida, H., 354,426 Ishikawa, N.,336,337,338,424,426 Israels, L. G., 11, 60 Iversen, P.,68,93 Ivy, A. C.,355,493 Iwatsuru, R.,161,176
J Jablonski, J. R., 20,41,60 Jacobs, B. B., 33,60 Jacobs, J. L.,296,397 Jacoby, F.,381,496 J&B, R. H.,200,236,282,,987 Jaffe, W.G.,80,93 Jager, A,, 205,287 James, G.W.,205,288 James, G. W.,111, 234,257,287 Jandl, J. H., 268,279, 987 Janney, C. D.,226,229,239,246,248, 989 Jayle, M. F., 256,987
AUTHOR INDEX
Jehl, J. A., 19, 60 ,Jeliffe, D. B., 82, 86, 91 Jenkins, G. L., 342, 43O Jenrette, W. V., 4, 6, 10, 12, 38, 49, 158, 176 Jensen, D., 123, 161 Jensen, P. K., 131, 161 Jensen, W. N., 206, 225, 286 Jirgensons, B., 15, 60 Johnson, J. M., 342, 351, 352, 354, 361, 362, 42Y Johnson, R. M., 15, 37,46, 60 Johnson, V.. 263, 28Y Johnston. G. A. W., 251, 28Y Jones, H. B., 16, 22, 60, 61, 68, 70, 93 Jones, H. E. H., 136, 137, 138, 139, 149 Jones, M., 123, 160 Jones, R. N., 365,429 Jonmon, U., 223, 224, 289 Jordan, R. T., 10,60 Josimovich, J. B., 362. 484 Judd, 0. J., 219,286 Jull, J. W., 385, 412, 414, 481 Jung, M. L., 342, 356, 362, 365,428 Junqueira, L. C. U., 113, 161 Juranies, E., 254, 889
K Kabara, J. J., 20, 60 Kabat, E. A., 292, 326,32Y Kahler, H., 10, 28, 62 Kalant, H., 123, 161 Kamen, M. D., 233, 886 Kandutsch, A. A., 360,486 Kaplan, H. S., 222, 287 Kaplan, M. H., 112, 160 Kaplan, N. O., 39, 63 Karlina, M. I., 301, 327 Kamofsky, D. A., 41, 60, 70, 94 Kasai, M., 161, 176 Kasten, F., 37, 60 Kato, I., 161, lY6 Kavanau, J. L., 130, 161 Kawai, K., 360,42Y Kawasaki, K., 300, 327 Kawamorita, Y.. 159, 161,176 Keen, P., 70, 91 Keighley, G. L., 105, 107, 121, 123, 134, 149
443
Keller, E. B., 121, 135, 138, 142, 143, 144, 145, 146, 160, 161, 163, 166 Kelley, M. B., 109, 168 Kellie, A. E., 411, 486 Kelling, 311, 32Y Kelly, E. M., 20, 4 Y Kelly, K. H., 35,60,201,209,212,214,2887 Kelly, L. S., 22, 60, 61 Kelsall, M. A., 35, 36, 37, 60 Kelton, D., 306, 386 Kennaway, E. L., 63,81,93,392,482, 426 Kensler, C. J., 6, 18, 41, 64 Kernohan, J. W., 67, 74, 75, 93 Kessel, A. M., 355, 423 Keys, A., 207,284 Khesin, R. V., 136, 161 Khouvine, Y., 22, 60 Kidd, J. G., 293, 296, 297, 314, 316, 318, 326, 32Y, 328 Kielley, R. K., 393, 394, 426 Kiely, G. E., 399, 405, 415,484 Kikawa, T., 159, 176 King, H. D., 297, 326 King, H. J., 396,426 King, J. W. B., 99, 160 Kinosita, R., 76, 79, 93 Kirby, A. H. M., 80,93,351,360,377,405, 426 Kirschbaum, A., 298,329 Kishi, S., 394, 396, 4% Kit, S., 16, 60, 118, 120, 123, 160, 161 Kitamura, Y., 335, 340, 487 Ki-Wei, H., 336, 344, 422 Klatt, 0. A., 7, 60 Klein, G., 106, 160 Klein, M., 399, 405, 415, 484 Kleinenberg, H. E., 81, 93 Klima, R., 236, 287 Kline, B. E., 367, &9 Knight, C. A., 293, 327 Knospe, D. E., 383,429 Knox, R., 188, 197, 318, 326 Knox, W. E., 9, 10, 39, 60, 64 Kobayashi, K., 300,327 Kobernick, S. D., 356,426 Koch, F. C., 81,96 Kochakian, C. D., 7,60 Kogl, F., 101, 161 Kohn, K., 74. 75, 77,82, 93 Koelsch, C. F., 347, 426
444
AUTHOR INDEX
Koler, R. D., 265, 289 Kominos, Z. D., 263, 265, 266, 288 Komiya, H., 336, 424 Kon, G. A. R., 343, 423 Koningsberger, V. V., 145, 160 Konno, K., 9,48, 169, 176 Koritz, S. B., 122, 161 Korn, E. D., 21, 60 Korngold, L., 101, 161, 298, 306, 327 Korosteleva, T. A,, 301, 307,314,315,321, 327 Korphsy, B., 80, 93, 359, 360, 425, 427 Kosuge, T., 8, 61, 159, 166, 170, 176 Kosyakov, P. N., 301, 327 Kouwenaar, W., 63, 65, 82, 83, 91, 93 Kowalsky, A., 347,425 Kratochvil, C. H., 101, 160 Kremen, A. J., 16, 60 Kreyberg, L., 182, 197 Krieger, H., 204, 208, 287, 289 Krogh, A., 205,287 Kruh, J., 122, 161, 219, 289 Kuff, E. L., 133, 161 Kuhn, R., 347,426 Kuhn, W. E., 336, 426 Kullmann, 258, 287 Kuain, A. M., 159, 162, 176 Kuznetsova, N. Y., 301, 327
L Lacassagne, A., 342, 351, 425, 426 Lacour, L., 190, 198 Laird, A. K., 388, 390, 392, 426 Laird, H. M., 190, 19'7 Lamasters, T. J., 37, 46 Lambert, G., 354, 370, 426 Lamson, B. G., 112, 113, 165 Lan, T. H., 10,60 Landsteiner, K., 180,198 Lang, K., 384, 395,396, 397, 429 Lange, L. B., 183, 198 Langecker, E., 337, 338, 345, 423 Laskowsky, I., 300, 327 Latham, E., 196, 198 Lau, H., 374,426 Lauenstein, K., 112, 160 Laurell, C. B., 256, 287 Law, L. W., 41,60, 291, 327 Lawler, A., 207, g84
Lawrence, E. A., 33,60 Lawrence, J. H., 35, 47, 70, 93, 205, 211, 215, 216, 217, 218, 219, 220, 225, 238, 242, 244, 257, 284, 286, 286, 288 Laws, J. O., 123, 161, 354, 355, 360, 405, 426,428 Leathem, J. H., 357, 370, 376, 377, 379, 388, 392, 420, 426 Leavitt, D., 218, 219, 289, 290 Leblond, C. P., 379, 421 Ledentu, G., 60,93 Lediic, E. H., 112, 160 Lee, H. C., 238, 242, 244, 257, 284,286 Lee, K., 383,424 Lehman-Facius, H., 300, 327 Lehmann, F. E., 131,161 Lehninger, A. L., 10, 63 Leise, E. M., 28, 60 Leiter, J., 23, 64 Leitner, S. J., 34, 60 Lemmer, K. E., 76, 93 Lemon, H. M., 68, 93 Lepage, G. A., 17, 23, 27, 40, 49, 60, 65, 107, 108, 160,161 LeRoy, G. U., 20, 60 LeRoy, G. V., 229,230,246,288,290 Levi-Montalcini, R., 37, 48 Levina, D. M., 293, 307, 308, 314, 323, 326,527 Levine, B., 105, 163 Levy, H. M., 17, 60 Lewis, M. R., 297,300,326,327 Ley, A., 222, 290 Ley, A. B., 221, 222, 223, 224, 269, 282, 287, 290 Libby, R. L., 69, 70, 96, 222, 277,289 Lichstein, J., 70, 92 Lieber, M. M., 82, 83, 90 Lietze, A., 113, 163 Light, A., 128, 161 Lin, E. C. C., 9, 60 Linden, L. W. F., 201, 207, 213, 215, 287 Lindquist, T., 205, 286 Lions, F., 345, 425 Lipari, R., 101, 151 Lipkind, 3. B., 8, 9, 48, 163, 176 Lipmann, F., 145, 160, 164 Lippincott, S. W., 296, 326, 327 Lisa, J. R., 76, 90 Little, J. N., 339, 349, 426
AUTHOR INDEX
Littlefield, J. W., 121, 135, 138, 142, 143, 145, 161, 166 Litvinenko, L. M., 336,426 Liu, Y., 63, 83, 93 Lloyd, T. W., 227, 287 Lockey, E., 15, 60 Loehlein, W., 75, 78, 93 Loewy, A., 263, 287 Loftfield, R. B., 103, 107, 112, 113, 115, 124, 135, 161, 163,165 Lombard, L. S., 364, 42Y Lombardo, M. E., 22, 42, 4Y, 4 8 London, I. M., 123,152,219,233,234, 28Y Longini, J., 263, 88787 Longmuir, I. S., 123, 151 Lorentz, F. H., 212, 213, 214, 215, 216, 217, 28Y Lorenz, E., 80, 90 Lothrop, W. C., 335,426 Lotz, C., 206, 211, 234, 238, 258, 284 Lotz, F., 11, 13, 21, 41, 42, 4Y, 60 Loubeyre, J., 83, 93 Loutit, J. F., 203, 205, 264, 267, 284, 286, 28Y Loveless, L. E., 359, 426 Lovtrup, E., 389, 390, 395, 426 Low-Beer, B. V. A., 70, 93 Lowy, P. H., 105, 121, 122, 123, 134, 149 Luck, J. M., 388, 422 Lucke, B., 5, 7, 60, 54, 162, lY6 Ludogovskaya, L. A., 294, 319,327 Lund, H.. 297,327 Lutz, J. F., 235, 286 Lyons, C., 201, 209, 210, 211, 285 Lyons, W. R., 70, 93
M Mabille, P., 354, 355, 360, 426, 428 McAfee, M., 209, 289 McBride, J., 11, 18, 19, 49 McCallion, D. J., 358, 426 MacCallum, W. G., 77, 93 McCarthy, P. T., 22, 42, 48 McCook, J., 69, 93 McCoy, J. R., 27, 46 MacDonald, J. C., 359, 360, 426 MacDowell, E. C., 291, 298, 317,327, 328, 329 McEuen, C. S., 29, 31, 60 McEwen, H. D., 25, 36, 61
445
McFarlane, A. S.,154 MacFarlane, 60, 93 McGovcrn, V. J., 375, 378, 428 MacGregor, I. R., 336, 341, 428 McHenry, E. W., 10, 17, 24, 26, 61, 54 McIndoe, A. H., 78, 82, 91, 93 McIntosh, J., 185, 197 McIver, F. A., 14, 62, 196, 198 McKee, R. W., 19, 60 MacKensie, I., 296, 328 Maclagan, N. F., 15, 60, 376, 426 McLean, J. R., 136, 163 MacLean, L. D., 3.56, 426 McLean, P., 394, 423 McNeei, G., 68, 93 McNeil, D., 348, 349, 424 McPhee, J. G., 63, 96 Maculla, E. S., 297, 328 MacVicar, N., 60, 93 Madden, S. C., 107,163 Madqen, M. E., 353, 368, 370, 423 Magath, T. B., 202, 287 Maggio, R., 145, 162 Mahler, H. R., 113, 114, 153 Maisin, J., 354, 370, 395, 422, 426 Makari, I. D., 308, 328 Malbica, J. O., 22, 52 Maldague, P., 354, 370, 426 Malmgren, R. A., 36, 39, 44, 61 Mandel, H. G., 22, 23, 42, 46, 48, 64 Mandelstam, J., 86, 92 Manginelli, A., 36, 44, 46 Mann, L. S., 301, 328 Manoilov, S. E., 297, 328 Manson, D., 344, 422 Marchand, F., 258, 2887 Marchant, J., 386, 427 Marchello, A., 263, 2887 Marks, P. A., 32, 61 Marnie, J. G., 63, 93 Marrian, D. H., 365, 426 Marschelle, H. P., 221, 286 Marshall, E. K., Jr., 415, 426 Martens, J., 162, 177 Maruya, H., 80, 93 Marvin, J. F., 204, 284 Masaarelli, A., 8, 47 Mathies, J. C., 7, 49 Matsuo, H., 354, 426 Matsuura, K., 172, 176
446
AUTHOR INDEX
113, 123, 124, 126, 149, 161, 163, 234, Maun, M. E., 354,371,423 Maver, M. E., 9,36,37, 61, 389, 390, 395, 284 Miller, T. R., 69, 94 426 Milne, L. S., 76, 78, 93 Mayer, J., 19, 60, 218, 219, 290 Milstein, S. W., 20, 49 Mayer, M. E., 298, 328 Miner, D. L., 80, 9.3 Mayerson, H. S., 201, 209, 210, 211, 2886 Mirsky, A. E., 135, 141, 142, 148, 163,297, Maxwell, D. R., 365, 426 303, 328, 390,420 Meacham, G. C., 222,286 Misler, G., 359, 427 Meade, J. M., 412, 426 Mitoma, C., 414, 430 Medvedev, N. N., 308,329 Miyaji, T., 354, 360, 426, 4%' Mehl, J. W., 11, 63 Miyajima, S., 161, 176 Meinken, M. A., 32, 63 Miyake, A., 101, 116, 162 Meisel, D., 351, 371, 376, 494 Meister, A., 8, 9, 24, 39, 49, 159, 163, 164, Mizen, N. A., 133, 134, 152 Moir, T. W., 205, 288 167, 175 Meites, J., 386, 426 Moldave, K., 108, 161, 162 Moldenhauar, M. G., 383, 424 Melby, R., 365, 429 Molkov, J. N., 317, 328 Melchior, J. B., 140, 163 Mollison, P. L., 202, 203, 205, 206, 226, Mellanby, E., 188, 197 227,228, 229,230, 253, 264,267,286, Mendom, 0. R., 8 2 , W 887,988 Merkel, E., 334, 486 Moloney, J., 193, 197 Meyer, F., 116, 162 Monod, J., 107, 161 Michalowsky, L., 182, 197 Monroe, S. E., 222, 288 Micheli, F., 258, 287 Mider, G. B., 3, 6, 10, 12, 13, 15, 17, 20, Montpellier, J., 83, 93 21, 22, 24, 25, 26, 27, 28, 36, 40, 41, Moore, A. E., 35, 44, 63 42, 43, 48, 49, 61, 63, 107, 161, 262, Moore, C. V.,233, 886 Moore, G. E., 16, 60, 204, 284 287 Migliarese, J. F., 9, 27, 61, 389, 408, 415, Moore, J. W., 205, 288 Moore, S., 117, 163 421,430 Milanes, B., 69, 93 Moosey, M. M., 293,326 Morales, M. F., 207, 288 Milch, L. J., 395, 486 Morelli, E., 300, 328, 329, 359, 427 Millar, J., 5, 47 Miller, A., 34,35,38,44,61,220,246,251, Moreschi, C., 264, 288 254, 265, 272, 280, 281, 286,287, 289 Morgunova, T. D., 294, 319,327 Mori, K., 370, 394, 395, 396, 426, 497 Miller, E., 298, 328 Miller, E. B., 235, 287 Morris, H. P., 31, 48, 333, 342, 346, 348, 350, 351, 352, 354, 357, 361, 362, 363, Miller, E. C., 78, 80, 93, 94, 321, 328, 333, 364, 366, 368, 369, 370, 372, 374, 377, 339, 340, 342, 351, 355, 358, 359, 360, 378,382,383, 384, 390, 392, 396, 399, 361, 362, 363, 365, 368, 369, 377, 382, 400,401,402,403,404, 405,406,407, 383, 385, 386, 387, 388, 390, 392, 103, 408,409,410,411,412,413,414,415, 414,416,492, 426, 429 417, 418, 4S3, 4E7, 429, 430, 431 Miller, E. E., 16, 61 Mortimer, R., 219, 288 Miller, G. L., 16, 61 Miller, J. A., 78, 80, 93, 94, 101, 161, 321, Morton, J. J., 6, 13, 15, 17, 20, 21, 25, 26, 388,333, 339, 340, 342, 351, 355, 358, 36, 40, 51, 63, 107, 161, 262, 287 359, 360,361,362,363,365,368,369, Morton, T. V., Jr., 201, 208, 211, 212, 288 377, 382, 383, 385, 386, 387,403, 414, Mortreuil, M., 22, GO Mosbeck, J., 222, 288 416, 4 9 ,426, 429 Miller, L. L , 6 , ~ 2 , 1 6 , 3 8 , 4 7 , 6 1 , 1 0 0 , 1 1 2 , Mosonyi, M., 80,93, 359, 360, 426, 487
447
AUTHOR INDEX
Mmzkowski, L. I., 360, 427 Mouchet, R., 60, 93 Mourant, A. E., 264, 286 Muir, H. M., 233, 288 Muir, R., 78, 93 Mulford, D. J., 35, 60 Mulholland, D. B., 338, 339, 421 Muller, H., 82, 91 Muller, W., 83, 93 Mur, V. I., 385, 42Y Murdock, M. E., 20, 47 Murphy, E. A , 17, 60 Murphy, J. B., 180, 182, 185, 186, 191. 195, 198, 303, 328 Muwazi, E. M. K., 59, 93
N Nabeshima, T., 354, 426 Nachman, H. M., 205, 288 Nadel, E. M., 30, 43, 61 Nagasawa, H. T., 348, 385, 405, 407, 42Y Nagase, K., 394, 396, 427 Nair, M. G. R., 342,421 Nakagawa, S., 8, 61, 160, 163, 166, 176 Nakahara, W., 8,39,48,61, 157, 159, 165, 167, 169, 170, 173, 174, 176, lY6 Nakaide, Y., 159, 170, 176 Nakayama, A., 345,424 Namkung, M. J., 339, 423 Narancio, M. M., 68, 93 Narcissov, N. V., 293, 294, 295, 299, 302, 312, 314, 316, 321, 328, 329 Naughton, M. A., 99, 160 Neber, M., 11, 48 Nechelew, T. F., 229, 230,288 Neish, W. J. P., 15, 61, 344, 345, 346, 401, 409, 427 Nelson, A. A., 80, 94 Nelson, W., 201, 209, 210, 211, 286 Neniteescu, C. D., 347, 427 Nesmith, J., 34, 62,261, 269, 988 Neuberger, A., 105, 162, 219, 233, 234 258,286, 288 Neufarh, S. A., 81, 93, 94 Neukomm, S., 358,427 Nevler, A. I., 297, 328 Newson, S. E., 30, 65 Nickerson, J. S., 206, 286 Nickson, J. J., 70, 94 Nielsen, S. S., 182, 197
Nieset, R. T., 204, 286 Nisselbaum, J. S., 15, 61, 100, 162 Niven, J. 5. F., 219, 258, 288 Noble, R. L., 2, 28. 29! 61, 378, 42Y Nogue, 60, 94 Norberg, B., 234, 257, 287 Norberg, E., 17, 18, 61, 106, 168 Norcross, J. W., 222, 288 Norman, L., 222, 290 Norman, T. D., 18, 61 Norris, E. R., 11, 63 Novelli, A., 342, 427 Novelli, G. D., 146, 160 Noyes, W. A., 337,421 Nungester, W. J., 298, 328 Nyhan, W. L., 16, 61 Nyma.n, M., 256, 287
0 Oherling, C., 181, 187, 190, 197, 198, 292, 294, 328 Ochsner, A., 63, 94 Oda, T., 360, 427 Oettle, A. G., 65, 94 Ogada, M., 360,427 Ogata, Y., 335, 340, 42Y Oginsky, E. L., 321, 326 Ohashi, M., 395, 426 Okamura, S., 354, 426 Okita, G. T., 20, 60 Okushima, D., 159, 162, 164, 17Y Olfelt, P. C., 69, 94 Olson, M. E., 119, 120, 138, 162 Olson, R. E., 20, 41, 60 O'Neal, M. A., 381, 387, 424, 42Y, 428 Ono, T., 9, 34, 48, 63, 161, 166, 167, 169, 170, 171, 173, 176, 177 Oomen, H. A. P. C., 84, 94 Opie, F. L., 76, 79, 80, 94 Oppenheim, A., 222, 223, 288 Orchin, M., 335, 491 Orenstein, A. J., 60, 94 Orr, J. W., 79, 80, 94, 356, 386, 427 Orr, S. F. D., 81, 93 Osawa, Kyoko, 159, lY7 Osawa, S., 142, 148, 390, 420 Osgood, E. E., 265, 289 Osserman, E. F., 16, 61 Ott, M. G., 383, 429
448
AUTHOR INDEX
Owen, C. A., Jr., 268,288 Ozawa, G., 10,64
P Pace, N., 207, 288 Pack, G. T., 15, 28, 46, 68, 69, 70, 93, 94, 222, 223, 288 Palade, G. E., 132, 133, 135, 162 Palatine, I. M., 118, 149 Palay, S. L., 133, 168 Pallade, G. E., 132, 161 Pan, H., 339, 423 Panzacchi, U. G., 258,288 Paoletti, L., 245, 286 Papanicolaou, G. N., 68, 94 Pappenheim, A., 200,288 Pareira, M. D., 14, 27, 61 Parfentjev, I. A., 35, 61 Parke, D. V., 414, 427 Parker, T. G., 30, 61 Parkinson, M. C., 293,826 Parnes, V. A., 308, 309, 310, 328, 329 Parsons, L. D., 24, 35, 36, 37, 46, 47, 61 Parsons, R. J., 35, 47, 205, 211, 215, 216, 284, 286, 288 Parvis, D., 359, 427 Paschkis, K., 236, 288 Paschkis, K. E., 14, 15, 61, 80, 91, 354, 358, 369, 375, 376, 377, 379, 380, 388, 389, 390, 391, 392, 422,487, 428, 429 PasSey, R. D., 293,326 Pastor, R. C., 347, 426 Patt, D. I., 35, 63 Paul, K. G., 5, 39, 63, 162, 177 Pauling, L., 366, 428 Paulson, M., 222, 288 Payet, M., 61, 65, 71, 75, 83, 94 Payne, A. H., 22,61 Peacock, A., 180, 184, 187, 188, 191, 192, 193, 194, 195, 198, 357, 428 Peacock, P. R., 80,94, 180, 183, 184, 186, 187, 188, 190, 191, 192, 193, 194, 195, 197,198,351, 357,428 Peacock, W. C., 205, 218, 232, 286 Peamon, 0. H., 223, 224,269, 282, 290 Peck, R. M., 341, 422, 428 Pene, P., 61, 65, 71, 75, 83, 94 Peng, M. T., 208, 284 Pentimalli, F., 191, 195, 198, 294, 328 Perrone, J. C., 105, 162
Petermann, M. L., 15, 61, 133, 134, 168 Peters, J. H., 342, 349, 350, 356, 360, 362, 365, 368, 369, 384, 385, 391, 402, 403, 404, 406, 407, 408, 414, 416, 424, 428 Peters, J. P., 205, 888 Peters, T., Jr., 125, 126, 16.2 Pfeiffer, H., 311, 328 Pflatzer, R. F., 228, 290 Pfutrer, W., 8, 60, 161, 169, 176 Phillips, R., 70, 94 Pickels, F. G., 189, 197 Pierson, J. C., 68, 93 Pinck, L. A., 347, 367, 386, 428 Pinkus, H., 15, 37, 46, 60 Pinsent, J., 6, 60 Pirie, J. H. H., 60, 75, 76, 83, 94 Pirofsky, B., 262, 265, 288, 289 Piroeynski, W. J., 390, 428 Pisciotta, A. V., 263, 265, 266, 288 Pitts, H. H., 63, 96 Place, E. F., 23, 47 Plaza de 10s Reyes, M., 102, 177 Plazy, P., 83, 94 Plescia, A. M., 416, 426 Plescia, 0. J., 35, 68 Polnitz, W., 235, 289 Pollack, M. A., 34, 63, 209, 289 Pollack, R. L., 14, 48 Pollice, L., 361, 426 Pollister, A. W., 297, 303, 328 Pollycove, M., 219, 288 Ponder, E., 34, 62, 261,262, 263, 264, 266, 267, 269, 270, 276, 288 Ponder, R. V., 34, 62, 266, 269, 270, 276, 288 Popper, H., 86, 94 Porter, K. R., 189,197,205,218,219,284, 288,289, 290 Posner, H. S., 414, 430 Postnikova, 2. A,, 294, 316, 323, 328, 329 Potter, J. S., 317, 327, 328 Potter, V. R., 8, 17, 40, 46, 60, 107, 161 Powell, E. O., 226, 232, 286 Powers, O., 23, 64 Praag, D. V., 23,42,46 Prates, M. D., 61, 75, 94 Pratt, A. W., 26, 62 Pratt-Thomas, H. R., 196, 198 Prentice, T. C., 205, 288 Presnov, M. A., 297, 328
AUTHOR INDEX
Pressman, D., 298, 306, 327 Prestrud, M. C., 14, 60 Price, D. E., 360, 428 Price, J. M., 14, 69,78, 94 Price, V. E., 4, 6, 34, 38, 39, 49, 63,169, 177, 224, 261, 270, 271, 272, 273, 274, 986,288, 289 Prigmore, J. R., 38, 62 Prince, J. E., 300, S26 Pritchard, W. H., 205, 288 Prosky, L., 27, 46 Prusoff, W. H., 222, 286 Pugh, E. J., 32, 49 Pullman, A., 337, 428 Pullman, B., 335, 337, 428 Purr, A., 9, 62 Purves, H. D., 31, 62 Putnam, F. W., 16, 62, 101, 116, 162 Pyfrom, H. T., 9, 49, 171, f76 Pyrah, L. N., 412, 414, 421
Q Quappe, G., 162,177 Quattlebaum, J. K., 70, 94 Quinland, W. S., 63, 94 Quinlin, P. M., 32, 65, 339, 430 Quinsenberry, W. B., 63, 94
R Rabinovitz, M., 119, 120, 138, 162 Race, R. R., 264,285 Radin, N. S., 233, 288 Radzikhovskaya R. M., 293,303,316,328 Rafferty, J. A., 228, 290 Rajasuriya, P. K., 82, 92 Ramachandran, L. K., 112, 168 Randall, C., 18, 20, 21, 29, 42, 4.9 Ranney, H. M., 123, 162 Rao, M. V. R., 77, 82,94 Rashkoff, I. A., 218, 219, 290 Rapport, M. M., 297,328 Rath, C. E., 218, 286 Rathbun, E. N., 207, 288 Ratner, S., 102, 150 Rawson, R. A., 203,286, 288 Rawson, R. W., 32, 49 Ray, F. E., 37,46, 334, 338, 339,342,343, 346, 347,348, 349, 350, 351, 356, 358, 361, 362, 365, 402,407, 412, 416, 421, 424,426,498, 499,430
449
Read, G., 375, 378,498 Read, R. C., 230, 242,288 Reddy, D. V. N., 10,22,42,47,48, 69 Reeve, E. B., 203, 284 Reeves, E. B., 205,288 Regan, F. D., 67, 92 Reich, C., 209, 286 Reichard, P., 105, 160 Reid, E., 133, 162 Reid, E. E., 336, 429 Reid, J. C., 16, 17, 62, 348, 492 Reif, A. E., 101, 160 Rekers, P. E., 15, 28,46 Reilly, W. A., 34, 69,216, 288 Renfer, H. R., 246,248,250,254,272,280, 281,988 Renold, A. E., 39, 62 Resegotti, L., 170, 177 Reynolds, E. F., 7,47 Rhees, M. C., 296, 396,S27 Rhein, A., 390, 494 Rhoads, C. P., 15, 28, 35, 44, 46, 65, 80, 96, 222, 223, 988 Ribbert, H., 75, 78, 94 Rice, K. L., 14, 60 Richardson, H. L., 356, 381, 387, 424,428 Richmond, S. C., 35, 63 Richter, D., 103, 160 Ridout, J. H., 32, 47 Riegel, B., 360, 496 Rieke, H. S., 348, Rieveschl, G., Jr., 334, 347, 428 Rigdon, R. H., 192,198 Riggs, T. R., 118, 149, 162 Rigler, L. G., 69, 94, 222, 987 Ritchie, A. C., 349, 357, 360, 428 Rittenberg, D., 16, 62, 102, 106, 160, 165, 219, 226, 233, 234, 287, 288, 289 Rivkind, T. L., 299, 399 Rivlin, R. S., 10, 39, 64 Roberts, B. M., 217, 218, 219, 220, 225, 286
Roberts, E., 16, 62, 118, 120, 162 Roberts, S., 109, 159 Robertson, C. H., 381, 387, 424 Robertson, W. van B., 10, 28, 62 Robinson, B. W., 63, 94 Roboz, E., 15,612 Robscheit-Robbins, F. S., 200, 990 Rodriguez, N. M., 22, 37, 62
450
AUTHOR INDEX
Roe, F. J. C., 360, 394, 420, 429 Roholm, K., 68, 93 Roll, P. M., 23, 47 Rombach, L. H., 336,341,428 Rondoni, P., 323, 328,366,395,428 Rose, G. A., 16,48 Rose, N . R., 301,329 Rose, W. M., 200, 209, 224, 286 Rosenherg, D. M. L., 63, 94 Rosenfeld, S., 16, 61 Rosengard, J . L., 63, 94 Rosenthal, E., 158, 177 Rosenthal, M . C., 263, 265, 266, 988 Rosenthal, N., 266, 267, 289 Rosenthal, S. R., 82, 94 Ross, E., 30, 48 Ross, G. A. L., 10,64 Ross,H . E., 382,399, 404,423 Ross, J. F., 34,35,38,44,61,205,220,230, 232, 238, 246, 251, 254, 257, 265, 272, 280, 281, 286, 286, 287,289 Ross, R. C., 354,355, 428,429 Rosset, J., 60, 94 Roth, A., 36, 44, 46 Roth, J . S., 359, 393, 397, 428 Roth, K. L., 266,289 Rothenberg, M . S., 80, 91, 369, 376, 377, 379,380, 392,428, 429 Rothlauf, M., 306, 326 Rothschild, H . A., 113, 161 Rothwell, J. T., 118, 149 Rowller, C., 190, 198 Roulet, F. C., 77, 83, 93, 94 Rounds, D. E., 130, 160 Row, P., 2, 62, 179, 183, 187, 190, 191, 195,198, 297,316, 327, 329 Rovnova, 2. I., 297, 38.9 Royer, R., 351,354,355,360,496, 426, 428 Rudali, G., 351, 354, 355, 360, 426, 426, 428 Ruddock, J. C., 68, 94 Rudis, L. A., 355, 429 Ruiz, C., 343,428 Rumsfeld, H. W., Jr., 14, 62,359, 429 Rundles, R. W., 223, 224, 889 Rusch, H . P., 27, 62,80, 93, 339,342,351, 355,359, 361, 362, 363, 367, 382, 386, 387,426, 429 Russel, M . A., 10, 64 Russell, D. S., 355, 371, 426
Rutman, R. J., 20, 49, 388, 389,390, 391, 499,499
S Saakov, A. K., 301, 329 Sack, T., 205,286 Saeki, R., 112, 163 Sachs, H., 146,162 Saffiotti, U., 357, 360, 428 Salaman, M . H., 260, 289, 360, 499 Salmon, W . D., 86, 91, 94 Salzberg, D. A., 359, 382, 429 Sampath Kumar, K . S. V., 112, 162 Sampey, J. R., 336,489 Sample, A. B., 246, 989 Samuels, A. J., 208, 289 Samuels, L. T., 26, 29, 31, 46, 62 Sandin, R. B., 339,342,351,355,361,362, 363, 365, 368, 369, 377, 382, 426, 429
Sanger, F., 99, I60 Sannie, C., 81, 94 Sarason, E. L., 30,& Sarma, P. S., 112, 166 Sasaki, T., 79, 94 Sassenrath, E. N., 10, 17, 44,68 Sato, H., 160, 161, 177 Sato, J., 112, 163 Sato, T., 395, 429 Sava, 60, 96 Savard, K., 29, 62,172, 177 Sawasaki, C., 163, 177 Sawicki, E., 336, 337, 338, 340, 342, 343, 346, 350,&?9
Sayama, Y., 360,427 Sayers, G., 29, 30, 31, 62, 63 Sayers, M. A., 29, 62 Sayre, F. W., 11, 62 Scarano, E., 145,169 Scarf, R. F., 354,428 Schabad, L. M., 81, 9.3 Schade, A. L., 10,47,62 Schade, R., 6,64 Schaefer, A. E., 86, 94 Schapira, G., 219, 289 Scharf, V., 222,288 Schatzki, R., 69, 94 Schill, E., 337, 423 Schinz, H. R., 346, 362, 363, 429 Schiadt, E., 289
AUTHOR INDEX
Schlamowitz, M., 99, 162 Schlegel, B., 238, 241, 246, 251, 254, 272, 280,281, 286 Schlumberger, H. G., 16,64 Schlumberger, J. R., 60, 61, 91 Schmlihl, D., 376, 4.92 Schmid, H., 346, 362, 363, 366, 422, 42.9 Schmid, R., 162, 171,177,256, 289 Schmitt, L. H., 10, 64 Schneider, W. C., 132, 161 Schoenheimer, R., 102, 107, 160.162 Schoental, R., 80, 91, 94, 193, 198, 367, &2
Scholler, J., 10, 47 Scholler, K.L., 347, 426 Schottenfeld, L. E., 70, 94 Schreier, K., 124, 162 Schreiner, G. E., 204, 286 Schrek, R., 25,62 Schulman, M. P., 164 Schulman, S., 339, 346,429 Schulte, H. A., 201, 208, 211, 288 Schultz, A. L., 204, 889 Schulz, I., 360, 423 Schupbach, H. J., 76, 94 Schwanfelder, A. B., 28, 60 Schwartz, L., 219, 247, 389, 390 Schwartr, S., 162, 171, 177 Schwarz, 5. O., 321, 326 Scott, J. F., 100, 146, 149, 163 Scott, K. G., 32, 34, 62, 216, 288 Seabra, A., 8,158 Seaman, A. J., 265, 289 Sears, R. A., 118, 149 Seegers, W. H., 14, 62 Segaloff, A., 30, 49 Seguchi, Y., 161, 177 Seibert, F. B., 15, 62 Seibert, M. V., 15, 62 Seidel, W., 235, 289 Seki, L., 6, 8, 41, 64 Selbie, F. R., 185, 197 Seligman, A. M., 205, 286, 362, 424 Seligmann, M., 302, 310, 389 Selye, H., 29, 31, 37, 60, 62 Senoo, T., 360,427 Severina, I. S., 127, 162 Seward, F. C., 164 Shabad, L. M., 81, 94, 293, 308,326, 529 Shanewise, R. P., 5,48
4.51
Shanmugaratnam, K., 63, 96 Shapiro, D. M., 28, 62,69 Shapiro, M. P., 70, 91 Sharney, L., 219,289 Sharoukhova, K. S., 159, 162, 176 Sharpe, L. E., 222, 287 Sheets, R. F., 226, 228, 229, 239, 246, 248, 286, 289 Sheffield, F. W., 293, 329 Sheldon, J. H., 82, 83, 96 Sheldon, P., 355, 423 Sheldon-Peters, J. C. M., 120, 162 Shemin, D., 16, 62, 106,163,219,226,233, 234,287, 288, 289, 290 Shen, S. C., 208, 222, 223, 226, 265, 266, 268, 283, 289, 290 Sherman, C. D., 13, 17, 36, 63 Sherman, J. D., 35, 63 Shershulskaya, L. V., 307, 308, 309, 311, 313, 319, 326, 929 Shetlar, M. R., 18, 63 Shibano, S., 354,426 Shils, M. E., 28, 62, 63 Shimkin, M. B., 35, 60, 201, 209, 212, 214, 287 Shimura, K., 112,165 Shipley, R. D., 30, 48 Shiroshita, K., 159, 170, 176 Shubik, P., 351, 360, 361, 426, 429 Shulman, S., 301, 329 Sibley, J. A., 10, 63 Siebert, G., 384, 395, 396, 397, 429 Siegel, R. I., 201, 208, 211, 288 Siekevitz, P., 132, 133, 135, 140, 141, 142, 162, 163 Silverman, I., 68, 96 Silverstone, H., 27, 63, 369, 430 Simbonis, S., 16, 47, 110, 149 Simkin, J. L., 112, 128, 136, 142, 149, 169, 164 Simonscn, D. G., 16, 62 Simpson, M. V., 109, 112, 128, 136, 161, 163 Simpson, W. L., 15, 63 Sims, P., 344, 42.2 Singer, K., 235, 265, 287, 289 Sirlin, J. L., 131, 163 Sirtori, C., 359, 427 Skavinski, E. R., 6, 39, 46 Skinner, D., 68, 92
452
AUTHOR INDEX
Skoryna, S. C., 354, 355, 376, 377, 379, 381, 386, 405,426, 428, 429 Slack, H. G. B., 105, 162 Smith, A. B., 18, 61 Smith, E. C., 60,96 Smith, E. L., 10,63,407, 408,426 Smith, H. P., 204, 289 Smith, K. J., 75, 96 Smith, L. E., 333, 422 Smith, N. C., 31, 33, 43, 49 Smith, P., 306, 326 Smith, P. K., 22, 23, 42, 46, 48, 64 Smith, R. E., 207, 888 Smith, W., 293, 329 Smith, W. E., 187, 198 Smithies, O., 99, 163 Smyth, I. M., 15, 64 Sneath, P. H. A., 234, f286 Snell, G. D., 306, 326 Snell, K. C., 76, 96, 354, 430 Snijders, E. P., 63, 75, 79, 91, 96 Sober, H. A., 404, 409, 411, 414, 431 Sohier, W. D., Jr., 254, 289 Solovieva, N. J., 294, 316, 328 Sommers, S. C., 30, 61, 380, 429 Sorof, S., 383, 429 Southam, C. M., 35, 44, 63 Southcott, C. M., 30,63 Spacek, M., 14, 63 Spain, J. D., 381, 386, 387, 424, 426, 430 Spendrio, L., 169, 176 Spiegelman, S., 164 Spjut, H. J., 352, 354, 429 Spoerl, E., 359, 426 Sprunt, D. H., 35, 63 Spurrier, W. A., 321, 326 Sribhishaj, K., 234, 289 Staehlin, O., 344, 345, Stafford, W. H., 344, 345, Stanger, D. W., 81, 96 Stanger, W., 360,426 Stasney, J., 14, 15, 61, 80, 91, 354, 369, 375, 376, 377, 379, 380, 390,391, 392, 424
4 . ~ 4 . 429 m~
Stats, D., 247, 266, 267, 889, 890 Staub, H., 86, 96 Stavinski, E. R., 5, 63 Steele, J. M., 124, 163 Steele, R., 81, 96 Stein, A. M., 5, 6, 11, 39, 46, 63
Stein, W. H., 117, 163 Steinberg, D., 109, 112, 129, 148, 163 Steiner, M. M., 65, 96 Steiner, P. E., 63, 74, 75, 76, 79, 81, 92, 96 Steinfeld, J. L., 206, 289 Stekol, J. A., 415, 429 Stelos, P., 101, 162 Stepantschenok, G. I., 316, 328 Stephenson, M. L., 124, 145, 146, 161, 163 Sterling, K., 203, 229, 286 Sterling, W. R., 224, 261, 289 Stern, H., 141, 163 Stern, K., 27, 63 Stetten, M. R., 102, 163 Stevens, C. D., 32,63 Stewart, A. G., 11, 13, 21, 26, 28, 33, 30, 37, 40, 41, 42, 43, 47, 63 Stewart, H. L., 76,96, 354, 355, 423, 430 Stewart, J. D., 209, 289 Stewart, M. J., 82, 96 Stewart, P. H., 32, 65 Stickland, L. H., 123, 161 Stindt, H., 349,423 Stirret, L. A., 69, 70, 96 Stitch, W., 289 Stoerk, H. S., 300, 329 Stone, N. E., 106, 115, 126, 127, 14.9 Stone, R. S., 70, 93 Stoner, H. B., 24, 49 Storaadi, J. P., 204, 287, 289 Stowell, R. E., 35, 60 Strachan, A. S., 59, 96 Strasburger, J., 336, 430 Straub, M., 63, 75, 79, 91, 96 Straube, R. L., 15, 63 Streicher, J. A., 118, 149 Strombeck, J. P. 370, 374, 376, 430 Strong, G. F., 63, 80, 96 Strong, L. C., 34, 63, 96 Strumia, M. M., 228, 246, 289 Stuart, K. L., 82, 86, 91 Sturm, E., 180, 182, 185, 186, 191, 198, 303, 328 Sugimura, T., 9, 34, 48, 63, 166, 167, 169, 170, in, 173, in,177 Sugiura, K., 23, 47, 80, 96 Sullivan, A. D., 356, 426 Sumner, J. B., 6, 63 Sun, S. C., 84, 98 Sundararajan, T. A., 112, 168
453
AUTHOR INDEX
Sunteeff, V., 10, 60 Sure, B., 29, 63 Surmont, T., 60, 96 Sutherland, E. W., 99, 160 Susuki, S., 161, 176 Svedres, E. V., 342, 430 Swartz, M. N., 39, 63 Sweat, M. L., 31, 63 Swendseid, M. E., 222, 289 Sydnor, K. L., 31, 66 Symeonidis, A., 353, 355, 376, 430 Syverton, J. T., 298, 329 Sztanojevits, A., 360, 426, 427
T Takahashi, T., 396,430 Takano, G. M. V., 26, 63 Talalay, P., 26, 66 Tallan, H. H., 117, 163 Tamaki, H., 161, 176 Tanaka, K. K., 16, 62 Tanaka, T., 16, 62 Tannenbaum, A., 27, 63, 369,430 Tappel, A. L., 367, 422 Tarver, H., 104, 123, 128, 160,164 Tatum, E. L., 359, 421 Taylor, A., 7, 34, 60, 209, 210, 289 Taylor, D. R., 209, 289 Taylor, L., 246, 289 Taylor, M. E., 339, 340, 342, 349, 423 Taylor, M. J., 291, 317, 628, 329 Temmer, 0. S., 17, 62 Temple, A. F., 336, 339, 345, 422 Terry, R., 68, 96 Tesluk, H., 6, 13, 25, 26, 40, 61, 107, 161, 262,287 Theis, R. M., 29, 63 Theorell, H., 4, 5, 39, 63, 162, 277 Thomas, H. B., 111,149 Thompson, J. W., 14,64,164,158,162,176 Thomson, D. L., 31, 48, 60 Thorell, B., 105, 160 Thornton, H., 296, 326, 327 Tilden, I. L., 63, 94 Tilser, G. J., 19, 41, 49 Tipler, M. M., 392, 422, 426 Todd, C., 229, 289 Tokunaka, H., 8, 61, 166, 176 Tolbert, B. M., 348, 422 Tolson, S., 337, 423
Tomlinson, W. J., 65, 96 Toovey, E. W., 356, 426 Torralba, G., 3, 17, 34, 60, 63 Traub, A., 360, 426, 427 Travers, J. J., 22, 42, 47, 48 Treffers, H. P., 102, 160 Trew, J. A., 20, 41, 47, 63 Troescher, E. E., 11, 63 Trotter, A. M., 64, 96 Trowell, H. C., 59, 84, 93, 96 Truhaut, R., 81, 94 Tscheburkina, N. V., 301, 327 TSOU, K.-C., 362,424 Tubiana, M., 245, 286 Tubis, M., 111, 149 Tull, J. C., 63, 67, 75, 96 Turba, F., 112, 163 Turnbull, A. L., 205, 206, 286 Turner, J. E., 113, 163 Tyagoraja, S., 82, 96 Tyner, E. P., 23, 63 Tytler, W. H., 191, 195, 1Y8
U Uchida, K., 62, 96 Udenfriend, S., 392, 414, 421, 430 Ueno, K., 385, 460 Ultmann, J. E., 34, 64, 277, 289 Umeda, M., 34,63,161,166,167,170, 171, 173, 176, 177 Urquhart, M. E., 358, 392, 409, 412, 426, 460 Usuda, H., 62, 96
V Vadova, A. V., 294,329 Vanags, E., 340, 430 Vanags, G., 340, 430 Van Daele, G., 84, 96 Van der Horst, G. A., 83, 96 Van der Schaaf, P. C., 100, 16s van der Sheer, J., 297, 326 Van Heukelom, S., 78, 96 Vasquez-Lopez, E., 355, 430 Vaughan, M., 109, 112, 129, 163 Vaughn, J. M., 224, 289 Veall, N., 205, 288 Veglens, 205, 289 Velat, C. A., 351, 362, 374, 415, 427, 430 Velick, S. F., 112, 160,163
454
AUTHOR INDEX
Verel, D., 205, 206, 286 Verloop, M. C., 238, 245, 290 Vermund, H., 29, 49 Vetter, H., 206, 286 Victor, J., 317, 327 Vidal, F., 340, 431 Videback, A., 222, 288 Vigier, P., 187, 198 Vint, F. W., 59, 60, 82, 83, 96 Viollier, G., 86, 96 Vlamynck, E., 360,423 Voegtlin, C., 14, 64 von Bertalanffy, L., 390, 428 Von Bokay, Z., 78, 92 von Euler, H., 5, 8, 64, 158, 170, 177 von Kress, H., 200, 236, 290 Vorlaender, K. O., 292, 329 Vos, J. J. T., 82, 91
W Waddington, C. H., 131,163 Wade, A., 340,429 Wade, A. P., 411,426 Waelsch, H., 146, 162, 164 Wagley, P. F., 266, 290 Wagner, B. P., 351,364,374, 487 Wagshal, R. R., 15, 60 Wahi, P. N., 82,96 Wainio, W. W., 357, 388, 392, 420 Waldvogel, M. J., 10, 64 Waley, S. G., 112, 163 Walker, A. R. P., 85, 96 Walker, N. F., 99, 163 Wall, R. L., 16, 64 Wallace, B., 359, 423 Wallace, D. M., 11,47 Walshe, J. M., 83, 96 Walter, H., 113, 114, 163 Walters, J. H., 85, 96 Wania, A. F., 23, 39,48 Wannemacher, R. W., Jr., 27, 46, 408,415, 421 Waravdekar, U. S., 23, 64 Ward, D. N., 387,430 Warren, F. 209, 289 Wasren, F. L., 99,160 Warren, S., 80, 83, 96 Wartman, W. B., 360,426 Warvi, W. N., 67, 76’96 Wase, A. W., 357, 374, 388,389,392,408, 414, &O, 430
Wasserman, L. R., 218,219,247,266,267, 289, 290 Waterlow, J. C., 82, 84, 85, 96, 96 Watkins-Pitchford, W., 60, 96 Watson, C. J., 236, 290 Watson, E. J. P., 23,48 Watson, G., 373, 385, 422 Watson, M. L., 126, 133, 161, 163 Watson, R. W., 112, 160 Way, J. L., 23, 42, 64 Webb, A. C., 63,96 Weber, G., 11, 18, 19, 49, 64 Weber, L. G., 296, 3.96 Weber, R., 131,161 Webster, D. R., 354, 356, 377, 405, 426, 489 Wegelin, K., 75, 96 Weigand, C., 334, 426 Weill L., 10, 64 Weil, R., 258, 290 Weiler, E., 102,163,302,309,310,315, 389 Weil-Malherbe, H., 6, 64 Weingarten, W., 67, 92 Weinhouse, S., 10, 64 Weinstein, I. M., 229, 230, 246, 288, 290 Weisberger, A. S., 105, 163 Weisburger, E. K., 334, 336, 338,339,343, 344, 346, 347, 350, 362, 365, 382, 383, 384, 402, 403, 404, 405, 406, 407, 408, 409,410,411,412,413, 414,415, 417, 418,427, 430, 431 Weisburger, J. H., 334, 336, 338, 343, 344, 346, 347, 349, 362, 365, 382, 383, 384, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 417, 418, 427,430, 431 Weisman, R., Jr., 229, 287 weisman, T. H., 359, 426 Weiss, S. M., 321, 387 Welch, A. D., 222,886 Welch, C. S., 266, E86 Welham, W. C., 207, 284, 290 Welker, W. H., 301, 328 wenz, A., 340, @l Werder, A. A., 298, 329 Werthemann, A., 86, 96 West, C. D., 223, 224, 269, 282, 290 West, R., 219, 233,234, 287 Westfall, B. B., 348, 351, 352, 370, 398, 399,487,431 Westheimer, E., 202, 886
455
AUTHOR INDEX
West-Watson, W. N., 265, 290 Wheland, G. W., 347, 366, 426, 4.31 Whipple, G . H., 107, 112, 113, f63, 200, 205, 226, 234, 283, 284, 286, 289, 290 White, A. V., 7, 32, 35, 36, 47 White, E. N., 348, 349, 424 White, F. R., 13, 14, 40, 64, 80, 96 White, J. 4, 10, 12, 38, 49, 158, 176 White, J. E., 76, 92 White, J. M., 10, 64 White, M. R., 22, 51 White, R. G., 112, 123, 128, 148, 229, 289 Wiener, A. S., 228, 290 Wilbur, D. L.,63, 65, 74, 75, 79, 96 Wilbur, K. M., 367, 421 Wild, H., 15,48 Willett, F. M., 63, 65, 74, 75, 79, 96 Willheirn, R., 27, 68 Williams, C. D., 84, 96 Williams, D. C., 11, 14, 47 Williams, J. N., 12, 38, 62, 64 Williams, R. T., 414, 427 Willis, R. A., 3, 64, 64, 76, 79, 96, 277, 280, 290 Wilson, G. W., 230, 242, 288 Wilson, R. H., 80, 96, 334, 351, 352, 353, 354, 355, 356, 361, 368, 369, 375, 398, 406, 422, 431 Wiltshire, G. H., 101, 163 Winitz, M., 341, 407, 408, 424 Winnick, T., 16, 17, 18, 46, 64, 108, 111, 112, 113, 114, 115, 123, 130, 140, 149, 160, 162, 163,164 Winternitz, M. C., 79, 96 Wintrobe, M. M., 202, 206, 225, 285, 290 Winzler, R. J., 15, 27, 43, 44, 47, 64, 101, 163 Wirth, K., 124, 162 Wiseman, G., 27,49 Witebsky, E., 300, 301, 32.9 Witkin, E. M., 359, 423 Wittenberg, J., 233, 290 Wittig, G., 340, 431 Witts, L. J., 226, 232, 286 Woislawski, S., 342, 356, 362, 365, 428 Wolf, H., 384, 395, 396, 397, 429 Wolff, E., 65,96 Wolff, H. H., 83, 95 Wolff, S. A., 218, 686 Wood, D. A,, 63,65, 74, 75, 79, 96 Wood, S., 10, 39, 64
Woodhouse, D. L., 381,383,386, 392, 426, 427, 431 woods, M., i 9 , 4 r Woodward, H. Q., 12, 64 Woolley, D. W., 28, 64 Work, T. S., 112, 113, 128, 136, 146, 149, 160, 163, 164 Wright, A. W., 65, 91 Wright, L. E. A., 345, 426 Wrobel, C. J., 70, .93
Y Yalow, R. S., 205, 286 Yarnigiwa, K., 65, 75, 76, 78, 96 Yankwich, P. M., 348,422 Yeakel, E. H., 7, 36, 64 Yeh, S., 62, 96 Yen, C. Y., 23, 46 Yonemato, R. H., 268, 279, 287 Yoshida, H., 170, 176 Yoshida, T., 79, 94 Younathan, E. S., 128, 163 Young, C., 69, 91 Young, C. J., 265, 290 Young, E. M., 383,429 Young, F. G., 123, 151 Young, I. M., 226, 288 Young, L. E., 228, 290 Young, N. F., 6, 18, 41, 54 Young, W. h., 236, 285 Yuhl, E. T., 69, 70, 96 Yuile, C. L., 112, 113, 165, 234, 284 Yun, I. S., 62, 96 Yunoki, K., 160, 161, 277
Z Zahl, P. A,, 24, 46 Zahn, H., 347, 426 Zajdela, F., 342, 351, 425, 426 Zamcheck, N., 222,290 Zamecnik, P. C., 16,64, 124, 125, 135, 142, 144, 145, 146, 160, 161, 163, 166 Zbarsky, B. I., 308, 309, 323, 329 Zeckwer, I. T., 7, 60, 64, 162, 176 Ziegler, D. M., 140, 163 Ziegler, K., 340, 481 Zilber, L. A., 294, 295, 299, 308, 309, 312, 317, 323, 328, 829 Zimmerman, H. M., 34, 46, 260, 284 Zimmermann, G. I., 235, 286 Zitsu, Y., 336, 424
SUBJECT INDEX A synthesis in vilto, 128 tumor, specificity, 306 AAF, see N-2-Fluorenylacetamide 2-Acetamidofluorene see N-2-Fluorenyl- Antigens of tumors, see Tumor antigens acetamide 2-Acetylaminofluorene see N-2-Fluorenyl- Aortography, abdominal, in diagnosis of primary liver cancer, 69 acetamide Ascites, 119-121 Adrenals incorporation of amino acids into, 119of tumor-bearing animals, 29-31, 37, 38 121 Albumin effect of glutamine on, 120 serum, synthesis in normal and tumort,umor, amino acid incorporation into, ous liver tissue, 125-127 136-138 Allylisopropylacetylcarbamide (Sedormid) toxohormone in, 161 effect on liver catalase, 171 Autoimmunization Amines diseases caused by, 292 aromatic, carcinogenicity of o-hydroxyAzo compounds lated derivatives, 412, 414 induction of liver cancer in experimental D-Amino acid oxidase animals by, 79-80 activity in tumor-bearing animals, 10 Azo dyes Amino acids binding of proteins by, 382-385 ability of cells to concentrate, 117-119 incorporation into subcellular particles, 131-147 B in t h o , 134-136 Balanitis, cancer of penis and, 196 rate of incorporation, 102-107 Bence-Jones protein, synthesis of, 115-1 17 in toxohormone biosynthesis, 165-166 Bile pigment excretion uptake by bone marrow cells, 123 erythrocyte destruction and, 234-235 3-Amino-1,2,4-triazole, effect on liver in leukemia, 236 catalase, 171 Bittner virus, 293 Amylase, synthesis in vitro, 127-128 Blood, effect of tumors on 33-36, 44 Anaphylaxis, passive, 311-312 Blood volume Anemia body measurements and, 206-208 of cancer, 33-35, 44, 199-290 changes in cancer patients, 209-217 incidence of, 201-217 standard, 209 increased erythrocyte destruction and, Body weight 226-284 of cancer patients, blood volume and, role of decreased erythrocyte forma210 ff., 216-217 tion, 217-226 Brain pernicious, gastric cancer and, 222-223 rat, tumorigenic action of 2-FAA in, 355 Animals, primary liver cancer in, 64 Brown-Pearoe rabbit carcinoma Antibodies antigen of, 296-297 formation, carcinogenesis and, 323 induced, 314 C neutralizing, for sarcoma virus, in normal birds, 195 C F , fate in tumor-bearing rats, 274 456
457
SUBJECT INDEX
Cachexia of cancer, toxohormone activity and, 172 malignant, 3 Caloric requirement of tumor-bearing animals, 26, 43 Cancer, immunological aspects, 35, 44 Cancer tests, 35 Cancer toxin, see also Toxohormone effect on h e r catalase activity, 7-9, 13, 39,158,169-170,173-174 newer concept of, 157-198 thymotropic, 173 Carbohydrate metabolism in tumor-bearing animals, 18-19, 41 Carboxypeptidase activity in tumor-bearing animals, 10 Carcinogenic substances effect on cellular proteins, 292 in human livers, 80-81 induction of hepatic tumors by, 79-80 interaction with tissue components, carcinogenetic activity and, 386-387 oxidation-reduction reactions, 367 role in primary liver cancer, 79-80 Cat carcinogenic activity of 2-FAA in, 358 Catalase inhibitors of, 168-169, 171 liver, effect of cancer toxin on, 158, 169-170, 173-174 normal levels, 163-164 hormonal factors regulating, 164 purification, 6 Cathepsins activity, in tumor-bearing animals, 9, 10 liver, of rats with and without livcr tumors, 298 Cell-free systems protein synthesis in, 147--148 Cells antigenic structure prior to nialignization, 315 fractionation of, 132-134 Chemotherapy of primary liver cancer, 69-70 Chick embryo protein synthesis in, site of, 131 Cortisone, antimitotic activity, 324
D Diabetes tumor growth and, 19 Dibenzanthracene, 196 carcinogenicity, effect of solvent on, 185-187 Diet liver cirrhosis and, 84, 86 primary liver cancer and, 83-84, 86 Dog carcinogenic activity of 2-FAA in, 357358
E Ear duct r a t , carcinogenic action of 2-FAA in, 355 Eggs protein synthesis in, 130-131 Ehrlich ascites tumor antigen of, 297 biologically active agent of, 37 Electronmicroscopy of subcellular components, 132-134 Enzymes, see also individual enzymes of carbohydrate metabolism, effect of tumors on, 9-10 of lipid metabolism, effect of tumors on, 11 of protein metabolism, effect of tumors on, 9-10 proteolytic, protein synthesis and, 131 tissue, response to presence of tumor, 4-13, 38-39 Erythrocytes of cancer patients, destruction of, 236255 anemia and, 226-284 bile pigment excretion and, 234-235 criteria of increased, 228-236 erythropoiesis and, 235-236 mechanism of, 226-228 by tumor toxins and hemolysins, 258 extrinsic defects of, 263-268 injury by extrinsic factom, 258 intrinsic defect in, 257-258 destruction in neoplasms other than leukemias and lymphomas, 247-255 pathways of, 255-257 due to vascular defects, 268-278
458
SUBJECT INDEX
survival, methods of measuring, 234 Erythropoiesis increased, as response to erythrocytc destruction, 235-236 iron metabolism and, 218-219 limited, in cancer patients infection and, 225-226 iron deficiency and, 221-222 role in anemia, 217-226 state of bone marrow and, 223-225 vitamin Ble deficiency and, 222-223
F 2-FA, see 2-Fluorenaminc Z F A A , see N-2-Fluorenylacctamidc Fat carcinogenic activity of 2-FAA and, 373, 419 Fcrritin synthesis in rat liver, 103 1-Fluorenamine chemistry of, 335-336 2-Fluorenamine N-acyl derivatives, pharmacological properties, 341 acylation and deacylation by tissue homogenates, 406-409 carcinogenic activity, 361 of derivatives, 361-365 chemistry of, 336-337 derivatives, chemistry of, 337-350 carcinogenesis, 350 ff. pharmacological properties, 342 metabolism, diet and 414415 3-Fluorenamine, preparation, 345-346 4-FluorenamineJ preparation, 346 Fluorenamincs derivatives of, chemistry, 336-346 Fluorenc chemistry of, 334-335 Fluorene quinones preparation, 347-348 N-2-Fluorenylacetamide carcinogenic activity, 333, 350 ff. in amphibia, 358 compounds increasing, 359-360 diet and, 368-374, 419 effect of hormonal factors on, 364, 375, 382, 419 of species on, 352, 419
of strain on, 352, 419 in fowl, 357 inhibitors of, 360-361 in mammals, 352-358 in mice, 366-357 Michael condensation and, 367-368 in microorganisms, 359 mode of administration and, 351-352, 419 physico-chemical properties and, 365368 sex differences in, 375 combination with tissue protcins, possible modes of, 385-382 derivatives, carcinogenic activity, 364365 structural requirements for, 419 molecular dimensions, 366-367 preparation of 2,7-substituted, 34G347 of O-substituted, 347 synthesis of isotopically labeled, 348350 ultraviolet absorption spectra, 365-366 effect on host metabolism, 387-398 on amino acid uptake, 389 on enzymes, 392-397 on fat and glycogen, 391-392 on protein metabolism, 388-389 on nucleic acids, 390-391 on vitamins, 392 interaction with tissue components, 382-387 lymphosareoma induced by, 193-194 metabolism of, 398, 420 analysis of metabolites, 398-406 biochemical reactions occurring during, 409-414, 419 condensation reactions during, 344345 pathway of, from isotope studies, 402-406 metabolites of, 342-343, 417 ff. preparation, 343 urinary, of rats, 412, 413 mo1ecular dimensions, 366 toxicity, 334 ultraviolet absorption spectra, 365 N-3-FIuorenylacctamide, metabolism of, 418
450
SUBJECT INDEX
N-2-Fluorenylbenzamide, metabolism of, 416-417 N-2-Fluoreny l-p-toluenesulfonamide
metabolism of, 416 Fowl carcinogenic activity of 2-FAA in, 357 chemically induced tumors of, 179-198 differences between virus sarcomata and, 189-190 resemblance between induced tumors in mammals and, 195 species and tissue susceptibility to, 194 transmissibility, 180, 185 influence of host factors on, 181-183 domestic, spontaneous sarcoma in, 195 tumors of, effect of radiation on, 190191 epithelial, 191-193 spontaneous, 179, 195 Fujinami myxosarcoma, 180, 181
G Gastric juice of gastric cancer patients, toxohormone in, 161-162 Glycolysis of cancer cells, 157 Glycoprotein from plasma of tumor-bearing animals, 100 GRCH series of chemically induced tumors, 180 chance infection with viruses, 180-181 Gonads of tumor-bearing animals, 33, 37, 38 Guinea pigs, noncarcinogenicity of 2-FAA in, 412, 419
H Hamster carcinogenic activity of 2-FAA in, 358 Hematocrit values, 202 in cancer patients, 208 determination, 202 Hemoglobin concentration in cancer patients, 208-209 determinat,ion of, 201-202 Hemosiderin anemia of cancer and, 200
increase in cancer patients, 236 Hemoglobin synthesis, in isolated leucocytes, 121 in tumor-bearing animals, 38, 44 Hepatomas, antigens of, 302, 314-315 Hormones, see also individual hormones effect on carcinogenic activity of %-FAA, 364, 375, 382 on liver catalase, 7, 39 in tumor-bearing animals, 43-44 Hydrocarbons carcinogenic, susceptibility of fowls to, 194 N- (1-Hydroxy-2-fluorenyl) acetamide metabolism of, 417-418 preparation, 343 N-(7-Hydroxy-2-fluorenyl)acetamide metabolism of, 418
I Indole carcinogenic effect of 2-FAA and, 373 Insulin synthesis i n vilro, 128, 129 Ioshida rat sarcoma, ascitic, antigen of, 297-298 Iron distribution in normal and tumor-bearing animals, 270-273 Iron metabolism, in cancer patients, 22G 221 erythropoiesis and, 2 18-2 19 toxohormone and, 170-171
K Kwashiorkor, 84-85 effect on liver, 85-86
L Larvae protein synthesis in, 130-131 LQucob 1asts antigenic structure, 302 Leucocytosis in tumor-bearing animals, 35 Leukemia anemia in, 200-201 bile pigment excretion in, 236 increased erythrocyte destruction in, 236-245
460
SUBJECT INDEX
plasma and red cell iron turnover in, 219 Lipid metabolism in tumor-bearing animals, 19-22, 4 1 4 2 Liver effect of kwashiorkor on, 85-86 human, carcinogens in, 80-81 morphological changes in tumor-bearing animals, 36-37, 38 perfused, protein synthesis in, 123-124 primary carcinoma of, 55-97 anaplastic, 76 in animals, 64 cholangiocellular, histogenesis, 75 histopathology of, 73-74 clinical classification, 66 clinical manifestations, 65-71 diagnosis, 61-69 differentiation between hepatocellular and cholangiocellular, 75 distribution, geographic, 57-64 etiology, role of environmental factors in, 79-89 hepatocellular, frequency of, 73 histogenesis, 75 histopathology of, 71-71 histogenesis, 75-79 embryology and, 77-78 multicentric versus unicentric theories of, 78-79 incidence, age and sex, 65 limitations of available statistics, 64-65 liver cirrhosis and, 81-84 massive, 71 metastases, 74-75 nodular, 71 onset, mode of, 66 pathological anatomy, 71-74 symptomatology, 66-67 treatment suggested for, 69-71 vesicular, 75 rat, carcinogenic action of 2-FAA in, 354 sex differences in, 377 steroid hormones and, 377-378 synthesis of ferritin in, 103 Liver catalase activity in tumor-bearing animals, 4-6, 38-39 cancer toxin and, 7-9, 158, 169-170, 173-174
hormones and, 7 Liver cirrhosis factors producing, 83 kwashiorkor and, 85 malnutrition and, 84-87 primary liver cancer and, 81-84 Liver function tests in diagnosis of primary liver cancer, 67-68 Liver xanthine oxidase activity, in tumor-bearing animals, 12, 38 Lung, carcinogenic activity of 2-FAA in, 356 Lymphosarcoma uptake of amino acids by cell suspensions of, 123
M McIntosh sarcoma No. 5, 180 Malignization loss of normal cellular antigens during, 302, 311 Malnutrition, liver cirrhosis and, 84-87 Mammals, carcinogenetic activity of 2-FAA in, 352-358 Mammary gland rat, carcinogenic action of 2-FAA on, 354-355 steroid hormones and, 375-377 Metabolism intermediate, in tumor bearing animals, 16-18 response to presence of tumor, 13-24, 39-42 Methylcholanthrene tumor induction in fowl by, 187 N-Methyl-2-fluorenamine metabolism of, 415-416 Microsomes incorporation of amino acids into, 142147 Mitochondria protein synthesis and, 136 Morphology, in tumor host, 36-38, 44 Mouse carcinogenic activity of 2-FAA in, 356357
461
SUBJECT INDEX
Myeloma, multiple increased erythrocytc dcstruction in, 239 ff.
N 2-Nitrofluorene carcinogenic activity, 362 Nitrogen balance in tumor-bearing animals, 13-15 Nitrogen trap, 14, 17, 40, 108 Nucleic acid(s) carcinogenic activity of 2-FAA and, 374 role in morphogenesis and protein synthesis, 131 of toxohormone, 166 Nucleic acid metabolism in tumor-bearing animals, 22-24, 42 Nucleolus protein metabolism and, 131 Nucleoproteins of tumor tissue, antigenic difference between those of normal tissue and, 297 association of antigen with, 303, 323 Nucleus incorporation of amino acids, 141-141 role in protein synthesis, 142 Nutrition of tumor-bearers, 24-28, 43
P Pancreas of tumor-bearing animals, 32, 37 Pancreatic hormones carcinogenic activity of 2-FAA and, 381-382 Penis cancer of, balamitis and, 196 Peptides as intermediates in protein synthesis, 111-112 Peritoneoscopy, in diagnosis of primary cancer of liver, 68 Phospholipid tumor, effects of, 22, 24, 42 Pituitary of tumor-bearing animals, 31-32 Pituitary hormones carcinogenic activity of 2-FAA and, 379-381
Plasma proteins in tumor-bearing animals, 15-16 Plasma volume in cancer patients, 210 Porphyrin metabolism, toxohormone and, 170-171 Portal splenic venography in diagnosis of primary liver cancer, 69 Protein factor, of sarcoma, 180, 37 Protein metabolism in tumor-bearing animals, 13-18 Protein requirements of tumor-bearing animals, 26-27, 43 Protein synthesis in carcinogenesis, 322, 323, 324 growth processes and, 97-155 inhibition by ribonuclease, 131 intermediates in, 111-112 microsomes and, 135-136 mitochondria and, 136 in tissue slices, 124-125 in tumor and normal tissues, 124-125 in whole cells, 117-131 Proteins cancerous, 301-302 difference between normal and, 100102, 323 interconversions of, 112-115 in normal tissue, 112-114 in tumor tissue, 115 iron-containing, effect of toxohormone on synthesis and metabolism of, 171-172 liver, incorporation of glycine into, 105 plasma, metabolism in normal and tumor tissues, 109 as precursor of tissue proteins, 109111 structure, mitotic rate and, 99 specificity of, 99 tissue, effect of growth on nature of, 99100 metabolism in Vz'uo, 102-117
R Rabbit, carcinogenic activity of 2-FAA in, 358 Races incidence of primary liver cancer among different, 57-63
462
SUBJECT INDEX
Radiation effect on fowl tumors, 190-191 Radioisotopes in treatment of primary liver cancer, 70 Rat, carcinogenic effect of 2-FAA in, 3628. Red cell volume determination of, 202-204 plasma volume and, 204-206 Reticulocytes, incorporation of amino acids into, 105, 121-122, 138 ribonucleic acid content and, 122 Ribonucleic acid cellular, types of, 148 of reticulocytee, 122 Rous sarcoma effect of radiation on, 190-191 fractionation of tumor homogenates, 190 specific antigen of, 318 Rous virus aggregates of, 293 antigenic properties, 292-293
s Sarcoma chemically induced in fowls, 180 Sarcoma virus, potential, 195 Serum phosphatase activity, in tumor-bearing animals, 12 Sex hormones carcinogenic activity of 2-FAA and, 364 Smegma human, carcinogenic activity, 196 Spleen morphological changes in tumor-bearing animals, 37, 38 Steroid hormones carcinogenic activity of 2-FAA and, 375-378 Stomach rat, attempted induction of tumors with 2-FAA in, 355-356 Surgery results in primary cancer of liver, 70
T Thymus involution, in tumor-bearing animals, 172
morphological changes in tumor-bearing animals, 37, 38 Thyroid of tumor-bearing animals, 32-33 Thyroid hormones carcinogenic activity of 2-FAA and, 378-379 Tissue cultures uptake of amino acids by, 123 Tissue homogenates incorporation of amino acids into, 140141 Tissue slices incorporation of amino acids into, 138140 Tiesues antigenic differences between tumor and normal, 306, 309 components of, interaction of 2-FAA with, 382-387 differences between proteins of normal and neoplastic, 100-102 leukotic, antigenic structure, 308-309 of normal and, 298 metabolism of nonmalignant and malignant, 163 proteins, metabolism in uiuo, 102-117 tumor, resemblance between embryonic and, 310 uptake of amino acids by, 106 Tobacco smoke as inhibitor of 2-FAA, 361 Toxohormone, 157 amino acid composition, 166 cancer cachexia and, 172 carrier substance for, 169 chemical nature of, 164-168 effect on liver catalase, 7-9,13,39, 158, 169-170, 173-174 mechanism of, 170 effect on synthesis and metabolism of iron-containing proteins, 171-171 on thymus of normal animals, 173 inhibitor of, 170 isolation, 169-162 from cancer tissues, 159-160 from other material, 160-162 mode of action, 168-171 nondialyzable and dialyzable forms of, 167-168
463
SUBJECT INDEX
nucleic acid of, 166 occurrence in normal tissues, 163 purification, 164 synthesis, possible mechanism, 174 role of amino acids in, 165-166 terminology, 158 Trimethylcolchicine acid methyl ester tartrate of, antitoxohormone activity of, 170 Tryptophan carcinogenetic activity of 2-FAA and, 371-373 Tumor antigens in chick, 318 difference between, 308 of filterable tumor, 292-296, 315-317 of human tumors, 300-303 of mitochondria and microsomes, 312, 314 of nonfilterable tumors, 296-300 specific, 291-329 adsorption on red cells, 318-321 detection by anaphylaxis following desensitization, 303-31 1, 313 by reaction of passive anaphylaxis, 311-312 localization in cell, 312, 314 nature of, 315-318 time of appearance, 314-315 of transplantable tumors, 296, 297 Tumor cells proteins of, 148 Tumor-host relations, 1-96 definition, 2 general, toxohormone in, 171-173
Tumors rtnimal, immunological behavior, 299300 antigenic structure, localization and, 307 antigens of, see Tumor antigens chemically induced, of fowls, 179-198 immunological aspects, 187-188 transmissible, 180 cpithelial in fowls, 191-193 hormone-dependent, 28 properties, variability of, 41 spontaneous, chemical carcinogens and, 196-197 etiology of, 196
U Ultraviolet rays effect on Row sarcoma, 190-191 IJrine of cancer, toxohormonc in, 100-161
V Vascular defects erythrocyte destruction and, 268-278 Viruses of filterable tumors, 291 antigenic properties, 292 tumor, adsorption on red cells, 294-295, 296 association with normal cell components, 293 Vitamin requirements of tumor-bearing animals, 27-28 Vitamins carcinogenic activity of %FAA and, 374, 419
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