ADVANCES I N CANCER RESEARCH VOLUME 10
Contributors to This Volume J. M.
Barnes
J. S. Harington
Daniel E. Bergsagel
A. Haut
H. V.
W. Eugene Knox
Gelboin
A. Clark Griffin
P. N. Magee
K. M. Griffith
W. J. Stuckey, Jr.
ADVANCES IN CANCER RESEARCH Edited by ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital, London, England
SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania
Volume 70
@ ACADEMIC PRESS
1967
NEW YORK AND LONDON
COPYRIGHT @ 1967, BY ACADEMIC PRESS INC. ALL RIGHTS RESERVED. NO PART OF T H I S BOOK MAY BE REPRODUCED I N ANY FORM,
BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITIIOCIT WRITTEN PERMISSION FROM T H E PUBLISHERS.
ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003
United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W . l
LIBRARY O F CONGRESS CATALOG CARD
NUMBER: 52-13360
PRINTED I N T H E UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME 10 Numbers in parentheses rcfer to the pages on which the authors’ contributions begin.
J. M. BARNES, Toxicology Research Unit, Medical Research Council Laboratories, Carshalton, Surrey, England (163)
DAKIEL E. BERGSAGEL,* University of Texas M . I). Anderson Hospital and Tumor Institute, Houston, Texas (311)
H. V. GELBOIN,National Cancer Institute, National Institutes of Health, Departrnent of Health, Education and Welfare, Bethesda, Maryland (1) A. CLARK GRIFFIN,T h e University of Texas M . D . Anderson Hospital and Tumor Institute, Houston, Texas (83)
K. M. GRIFFITH,Department of Biomathematics, University of Texas M . D . Anderson Hospital and Tumor Institute, Houston, Texas (311) J. S.HARINGTON,? Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London, England (247) A. HAW, University of ilrkarisas School of Medicine, Little Rock, Arkansas (311)
W. EUGENE KNOX,Department of Biological Chemistry, Harvard Medical School and the Cancer Research Institute, N e w England Deaconess Hospital, Boston, Massachusetts (117) P. N. MAGEE,Toxicology Research Unit, Medical Research Council Laboratories, Carshalton, Surrey, England (163)
\V. ,J. STUCICEY, JR.,Tulane lrniversit y School of Medicine, N e w Orleans, LOU~S~U (311) WL
* Present, Address: t PreserLl
Princess Margaret Hospital, Toronto, Ontario, Canada.
Atlclress: Canwr Rcsearch Unit of the National Cancer Association of
South Africa, South African Institute for Medicaal Research, P. 0. Box 1038 Johannesburg, South Africa. V
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CONTENTS CONTHIBUTORS T O VOLU\lE 10 . CONTENTS OF PRIWIOUS Vor.u\ifi>s
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Carcinogens. Enzyme Induction. and Gene Action
H . V . GBLBOIN I . Introduction
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I1. Polycyclic Hydrocarbons and Enzyme Induction . . . . . I11. Tlie Effect of Drugs and Insecticides on Liver Microsomal Enzymes
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IF’. The Nature of the Microsomal Drug-Metabolizing Enzyme Systems . . . . . . V . Effect o f Oncogrnic Viruses on Enzyme Intlnction V I . Enzyme Induction in Rat Hepatoma . . . . . . . . . VII . Pharmacological and Physiological Significance of Polycyclic Hydro. . . . . carbons and Drug-Induced Enzyme Activity . VIII . The Mechanism of Polycyclic Hydrocarbon and Drug-Induced Increase . . . . . . . . . . . . in Enzyme Activity . . . . . . . . . IX . Carcinogenesis and Gcne Action . References . . . . . . . . . . . . . . .
36 51 66 76
In V i k o Studies on Protein Synthesis by Malignant Cells
A . CLARKGRIFFIN
I . Introduction . . . . . . . . . . . . . . . I1. Current Concepts of Protein Biosynthesis . . . . . . . . I11. Protein Synthesis by in Vitro Systems Derived from TLUIIO~ Cells . . I V . Comparison of Protein Gynt.licsis in Tiimor with Microbial and Normal Mammalian Systems . V . Summary and Conclusions References . . . .
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83 84 92 97 111 113
The Enzymic Pattern of Neoplastic Tissue
W . EUGENE KNOX I . Gencral Ideas about Ncoplasia . . . . . . . . . . I1. The Measurement of Enzymes in Tissues and the Prediction of
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. . . . 111. Glycolysis and the Enzymrs of Glycolysis I V . G l y c e r o l ~ ~ l i o s p l ~Dcliydrogcmm at~~ Levels and Glycolysis .
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Metnbolir Behavior
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V . The Enzymes of the Pentosc Pathway . . . . . . . . VI . Enzymes of Gluconrogcnesis and Glycogen Formation . . . . . V I I . Tentativcb Formilltition of the Pat.tern of Enzymes in Neoplastic Tissues VIII . Discussion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .
vii
117 123 125 133 134 137 143 155 158
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CONTENTS
Vlll
Carcinogenic Nitroso Compounds
P. N . MACEEA N D J . M . BARNES I . Introduction . . . . . . . . . I1. Chemistry . . . . . . . . . 111. Acute Toxic Effccts . . . . . . . IV . Neoplastic Changes . . . . . . . V . Some Special Features of Nitroso Compounds as VI . Mutagenic Action . . . . . . . VII . Metabolism of Nitroso Carcinogens . . . VIII . Biochemical Effects . . . . . . . . . . I X . Reactions with Cell Constituents X . Possible Mechanisms of Action . . . . X I . Public Health Aspects . . . . . . References . . . . . . . . .
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. . . Carcinogens . . . . . . . . .
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164 165 171 175 191 193 202 209 220 227 234 238
The Sulfhydryl Group and Carcinogenesis
J . S. HARINGTON
I . Introduction . . . . . . . . . . . . . . . 248 . . . . 249 I1. The SH Group and Normal Cell Division and Growth 111. The Stimulation of Cell Division by SH Groups . . . . . . 250 IV . SH-SS Cycles in Cell Division . . . . . . . . . . 252 . . 255 V . Growth Inhibition and Stimulation by Carcinogenic Substances VI . The Interaction of Carcinogens with SH Groups . . . . . . 259 VII . The Interaction of SH-Reactive Substances of Unknown Carcinogenic Activity or without Carcinogenic Activity . . . . . . . 277 VIII . The SH Group in Carcinogenesis . . . . . . . . . . 280 I X . SH Metabolic Systems Possibly Involved in Carcinogenesis . . . 285 X . Other SH Systems and Carcinogens . . . . . . . . . 289 X I . Discussion . . . . . . . . . . . . . . . 292 References . . . . . . . . . . . . . . . 300
The Treatment of Plasma Cell Myeloma
DANIELE . BERGSAGEL. K . M . GRIFFITH. A . HAUT.A N D
I . Introduction . . . . . . . . . . I1. Plasma Ccll Neoplasms . . . . . . . 111. Antineoplastic Treatment of Plasma Cell Myeloma IV . Summary . . . . . . . . . . References . . . . . . . . . . AUTHOR INDEX .
SUBJECTINDEX
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w. J . STUCKEY. JR. . .
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311 312 329 353 354
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38 1
CONTENTS OF PREVIOUS VOLUMES Volume 1
Electronic Configuration and Carcinogcnesis C . A. Coulson Epidermal Carcinogenesis E . V . Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Asprcts of Experimental Tumorigenesis 7'. U . Ga h e r Prolwrtics of the Agent of Rous NO. 1 Sarcoma it. J. C . Harris Applications of Radioisotoprs to Studies of Carcinogcnesis and Tumor Me tab olism Charles Heidelberger The Carcinogenic Aminoazo Dyes James A . M i l l e r wid Elizabelh C . Miller The Chemistry of Cytotoxic Alkylating Agents M . C . J . Ross Kutrition in Relation to Cancer Albert Taiznenbaitm and Herbert Silverstone Plasma Proteins in Cancer Iiicliartl J . W i n d e r A U T l I O I I INDEX-SUBJECT
INDEX
Volume 2
The Reactions of Carcinogens with Macromolecules Peter A l e x a d e r Chmiical Constitution and Carcinogenic Activity G . M . Badger
Carcinogenesis and Tumor Pathogenesis I . Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D . Fenninger and G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards Calvin T . Klopp and Jeanne C. Bateman Genetic Studies in Experimental Cancer L. W . Law Tlir Role of Viruses in the Production of Cancer C . Oberling and M . Gueiin Experimental Cancer Chemotherapy C . Chester Stock AUTHOR INDEX-SUBJECT
INDEX
Volume 3
Etiology of Lung Cancer Richard Doll The Exprrimental Development and Metabolism of Thyroid Gland Tumors IIarold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pullman and B. Pullman Some Aspects of Carcinogenesis P. i2 ondoni Pulmonary Tumors in Experimental Animals Michael B. Shimkin
ix
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CONTENTS O F PREVIOUS VOLUMES
Oxidative Met,abolism of Neoplastic Tissues Sidney Weinhouse AUTHOR INDEX-SUBJECT
INDEX
Volume 4
Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manwing Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A . G. Gallon The Employment of Methods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian Organism Abraham Goldin Some Recent Work on Tumor Immunity P. A . Gorer Inductive Tissue Interaction in Development Clifjord Grobstein Lipids in Cancer Fmncss L. Haven and IY. R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . Lacassagne, N . P. Buu-Hoi, R . Daudel, and F . Zajdela The Hormonal Genesis of Mammary Cancer 0. Miihlbock AUTHOR INDEX-SUBJECT
INDEX
Volume 5
Tumor-Host Relations R . W . Begg Primary Carcinoma of the Liver Charles Berman Protein Synthesis with Spccial Reference to Growth Processes both Normal and Abnormal P. N . Campbell
The Keuer Concept, of Cancer Toxin Waro Nalcahara and Fumako Fukuoka Chemically Induced Tumors of Fowls P. K. Peacock Anemia in Cancer Vincent E . Price and Robert E . Greenfie Id Specific Tumor Antigrns L. A . Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth I<. Weisburger and John H. Weisberger tUT1IOIt INDEX-SUBJECT
INDEX
Volume 6
Blood Enzymes in Cancer and Othcr Diseases Oscar Bodansky The Plant Tumor Problem Arrnin C. Braun and Ilenry N . JC-ootl Cancer Chernothc7ral)y by Pcrfusion Oscar Creech, Jr. and Edward T. Krementz Viral Etiology of Mouse Lculcemia Ludwik Gross Radiation Chimeras P. C. Koller, A . J . S. Davies, and Sheila M . A . Doak Etiology and Pathogenesis of Mouse Leukemia J . F. A . P. Miller Ant,agonists of Purim. and Pyrimidine Metabolites and of Folic Acid G. M . Timmis Bchavior of Liver Enzymes in Hepatocarcinogeneais George Weber AUTHOR INDEX-SUBJECT
INDEX
Volume 7
Avian Virus Growths and Their Etiologic Agents J . W . Beard
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Agciits 11'. 1Y. Biocktnnn
Cross licsistancc and Collateral Scnsitivity Studic,s in Canwr Chcmotherapy 1)orri.s J . H u k h i s o n
Cytogcmetic Studies in Clironic Myeloid I~cukomia 1V. M . Cout-6 Broirti a11d Ishbt.1 M .
. A I T I I O R ITI)l3S-StiIiJICC1'
7'011gll
1':Lliioninc~ C;ircinogc'nr& E inma it rrr,l 1'(i rlw r
INI)ES
Volume 9
Atmosplic~ric:Factors i n h t Iiogc,iwsis of Lung Caiiccr P a d Kotin and Ilans L. Talk Progress with Sonic Tumor Viruscs of Cliickcns and Maiiinials: The Problem of Passenger Virusrs
G. Negroni AUTIIOII INDEX-SUBJECT
Kncleolar Ctironiosomc~s: Structures, Intcmrtioiis, and Perspcctivcs M . J. Kopac nntl Gladys 211. M a l ~ y k ~ Carcinogenesis Relatcd to Foods Contaminatcd by Proccssing :tiid Fniig;iI Mvtabolitcs 11. P. Kraybill and hf. B . Shimkin Expc.riiiicnta1 Tobacco Carcinogcnesis Eruc,st L. Tl'gnder and Dietrich Hogmann
INDEX
Volume 8
Thr Structurc of Tumor Viruses and Its Braring on Their Ilc1:ition to Viruscs in General A . F . Howatson Nuclcar Proteins of Ncoplastw Cells Harris Busch a d Walliam J . Sleele
Urinary Enzyinrs and Tlrcir 1)iagnnstic Value in Huiii:tn C'anwr Ricliard Stambuiigh u d Sitltrc y Weinhouse The Relation of tlic Inininnc Reaction to Cancer Louis V . Caso Amino Acid Transport in Tumor Cells I < . AI. Johnstone nxd P. G. Scholefield Stuc1ic.s on the Dcvclopmr~nt,Biochcmistry, and Biology of Ifxpc.rinrent:~l Hc,patoinas Ilarolcl P. Morris Bioclieniistry of Normal and Leukcmica Lrucocytes, Tlironihocytes, and Bonn Marrow Cells I. F. Seik . + ~ ; T I I O H INDEX-SUBJECT
ISDEX
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CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION H. V. Gelboin National Cancer Institute, National Instituter of Health, Department of Health, Education and Welfare, Bethesda, Maryland
I. Introduction . . . . . . . . . . . . . 11. Polycyclic Hydrocarbons and Enzyme Induction . . . . . A. Hepatic Enzymes in Young and Adult Rats . . . . . B. Hepatic Enzymes in Fetal, Neonatal, and Immature Rats . . C. Species Other than the Rat . . . . . . . . . D. Nonhepatic Rat Tissue . . . . . . . . . . E. Activity as an Enzyme Inducer and as a Local Carcinogen . 111. The Effect of Drugs and Insecticides on Liver Microsomal Enzymes IV. The Nature of the Microsomal Drug-Metabolizing Enzyme Systems V. Effect of Oncogenic Viruses on Enzyme Induction . . . . VI. Enzyme Induction in Rat Hepatoma . . . . . . . . A. The Effect of Polycyclic Hydrocarbons and Phenobarbital on Microsomal Drug-Metabolizing Enzymes . . . . . . B. The Effect of Dietary Factors . . . . . . . VII. Pharmacological and Physiological Significance of Polycyclic Hydrocarbons and Drug-Induced Enzyme Activity . . . A. Effect of Polycyclic Hydrocarbons on Carcinogenesis . . . B. Hydrocarbon-Induced Protection against Adrenal Necrosis . . C. The Effect of Polycyclic Hydrocarbons and Drugs on the Duration of Drug Action . . . . . . . . . . D. Effect of Carcinogenic Polynuclear Hydrocarbons on Ascorbic Acid Metabolism . . . . . . . . . . . VIII. The Mechanism of Polycyclic Hydrocarbon and Drug-Induced Increase in Enzyme Activity . . . . . . . . . A. Effects of Inhibitors on the Hydrocarbon Induction of Enzyme Activities . . . . . . . . . . . B. Stimulatory Effect of Polycyclic Hydrocarbons and Drugs on Protein Synthcsis . . . . . . . . . . . C. The Effect of 3-Methylcholanthrene on Nuclear RNA Metabolism IX. Carcinogenesis and Gene Action . . . . . . . . . References . . . . . . . . . . . . . .
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I. Introduction'
A large segment of this review concerns the effect of polycyclic hydrocarbons and drugs on the level of certain enzyme activities. It is, 'The following abbreviations have been used in this chapter: DNA, deoxyribonucleic acid ; DNase, deoxyribonuclease ; RNA, ribonucleic acid ; RNase, ribo1
2
H . V . GELBOIN
therefore, important to summarize briefly some of the factors which may affect the observed activity of a n enzyme system. Enzyme profiles of animal tissues are generally obtained by the use of prcparations of tissue slices, homogenates, subcellular fractions, or extracts. Any given enzyme activity is usually determined in the presence of what arc believed to be optimal levels of the required substrates and cofactors. I n crude tissue preparations, however, there are a multiplicity of factors which influence the observed activity arid which frequently are not or cannot be controlled. Since the majority of citations in this review rcfcr to work in which crude preparations were used, one should be aware of the largely operational nature of the description of the enzyme content of a givcri tissue. Although the use of unpurified preparations may have limitations in dcscribing the enzyme profile of a tissue the method of purification and isolation of spccific enzymes from tissues also has its shortcomings when this information is used to extrapolate t o the functional i n vivo content of enzyme. Thus, enzymes may be activated or inactivated cithcr rcversibly or irreversibly during their isolation and purification. I n addition, it is evident that both approaches, i.e., the asswy of crude preparations and the isolation of purified enzymes, involve a dcstiuction of cell architecture and normal intracellular or interccllular control mechanisms involving a host of factors such a s pH, ionic strength, substratc, cofactor, and product localization, all of which may affect cnzynic activity. Enzyme control mechanisms may be classified into two broad categorics. First, there arc those factors that affect the enzymatic activity of spccific protciin molcculcs, and sccontl, those factors that regulate the riiiiount of cnzynie protcin. I n respect to the former, the activity of a n onzymc ni.&y I)c affected by the concentration of either substrate or product. A prime cxample of substrate inhibition is the inhibitory effect of high lcvelh of acctylcholine on acetylcholinesterase (Nachmanson and Wilson, 1945). Typical product inhibition is exemplified by the inhihition of liexokinasc by its reaction product glucose-6-phosphate (Crane an(l Sols, 1953). The latter may be an example of a n “isosteric inhibition.” The inhibitor is sterically related to the substrate or cofactor and competes with either of the latter for the catalytic site on the enzyme. Thus, isosteric inhibition may take place if the tissue preparation contains a compound which competes with substrate or cofactor nuclease; niRNA, messenger RNA; sRNA, soluble RNA ; ATP, adenosine triphosphate ; ATPasc, adcnosinetriphosphatnse; CMP, cytidine monophosphate ; GTP, guanosine triphosphate ; DPN, diphosphopyridlne nucleotide (NAD) ; DPNH, redurrd DPN(NADH) : FDPasr, fruc~tosr-1,6-diphosphatils~ ; T P N , tri1,tiosphopyridinc nucletidc (NADP) ; T P N H , reduccd T P N (NADPH).
CARCINOGESS, E S Z T h f E INDUCTION, AND G E N E ACTION
3
for the catalytic site on the enzyme. Other enzynic controls may involve either a stimulation or inhibition of the eiizyiiie by an “allosteriC” modification. In this type of inhibition or activation, the molecule modifying enzyme activity is not a sterie analog of the substrate but rather exerts its effect on tlie enzyme by binding to a site other than the binding site of the substrate. Feedback inhibition, in which a distal product of a metabolic p:tthway inhibits an early enzyme in the pathway, is an example of this type of inhibition. A typical example of an enzyme modified in this way is aspartate transcarbamylase which catalyzes a n early step in pyrimidine synthesis and wliicli is inhibited by one of the end products of pyrimidine Liosynth , cytidine triphosphate (Gerhardt and Pardee, 1962). Anotlier exaniplc of a feedback inhibition is that of tlic enzyme ribosyl-5-phoipliatc-ATP-pyropliosphorylasewhich catalyze, the first step of histidine biosyiithesis and is inhibited by histidine (Anics et a1 , 1961; Martin, 1963). Anotlier example of alteration of enzyme activity which is not related to an altered protein amount is the activation of the digestive enzymes. These are prime examples of inactive enzynies which are activated subsequent to their synthesis (Neurat11 and Dixon, 19.57). The zymogcns are converted to the active enzyme foim by structural modification, :tnd this type of activation can casily mimic enzyme induction, i.c., any iiicreased rate of de ~ L J synthesis O of enzyine protein. Thcre are also sevcr:il examples of irreversible inactivation of niainmnlian cnzynies. For example, the reaction products of mouse fibroblast DPNase irreversibly inactivate this enzyme (Licberman, 1957). Also, glucose-6-phosphate dehydrogenase is inactivated when T P N is not present. Thus, the enzymatic destruction of T P N results in a loss of glucosc-6-phosphntc dehydrogenase activity (Marks, 1961). Other cases of activation and inactivation are those that occur through a n association and dissociation of peptide subunits of an enzyme. Tliis has been called “molecular conversion” by Monod and ,Jacol, (1961). Phospliorylase is inactivated when phosphate is split from the enzyme by a phosphatase (Wosilait and Sutherland, 1956). This inactivation apparently occurs because the enzyme is split into two subunits. Phosphorylation of the protein reverses this process and activates the enzyme. Epincphrine and glucagon stimulate the activity of phosphorylase by increasing the formation of cyclic 3’,5’-adenylate which increases the activity of the enzyme that phosphorylates the subunits (Ra11 and Sutherland, 1961). Acetyl-CoA-carboxylase is assembled from three subunits in tlie presence of citrate (Vagelos et al., 1962). Liver glutainic dehydrogcnase dissociates into subunits upon the addition of a nuinher of compounds including certain steroids or A T P plus TPNH. Reassociation occurs in thc presence of ,4DP, D P N , or T P N .
4
H. V. GELBOIN
Glutamic dehydrogenase activity is present only in the associated form while the dissociated form is active as alanine dehydrogenase (Tompkins and Yielding, 1962). Other factors which affect enzyme activity are those which modify the level of cofactors required for the reaction. The maintenance of the cofactor in a specific form may require the activity of other enzyme systems. Thus the conversion of phenylalanine to tyrosine by phenylalanine hydroxylase requires the presence of a pteridine cofactor in the tetrahydropteridine form (Kaufman, 1962). The reduced cofactor is oxidized during the hydroxylation t o an inactive form and requires reduction by an additional enzyme, dihydropteridine reductase. This reaction, in turn, requires T P N H which must be converted back to the reduced form by a TPNH-generating system involving still other enzyme systems, e.g., glucose plus glucose dehydrogenase. Thus, one would obtain an accurate value for phenylalanine hydroxylase only in the presence of stoichiometric amounts of the tetrahydropteridine cofactors if the additional enzyme systems were not present. If the latter enzyme systems were present, catalytic amounts of cofactor would be adequate for maximum activity. In other cases the presence of competing enzyme systems utilizing common substrates or cofactors may lower the apparent activity of the assayed enzyme by successfully removing limiting amounts of substrate or cofactors. I n this case substrate and cofactor concentrations and the relative affinities of the latter for the competing enzyme systems would determine apparent enzyme activity. The amount of enzyme protein may be regulated by factors affecting either or both the rate of enzyme synthesis or the rate of enzyme degradation. The term “enzyme induction” has been specifically used to describe the process which increases the rate of synthesis of a spccific enzyme relative to its rate of synthesis in the uninduced organism. Enzyme repression is the process in which the rate of enzyme synthesis is decreased relative to the normal rate. In a large number of studies investigating mammalian systems the terms “induction” and “repression” are used in situations where enzyme activity is altered but where enzyme amount has not been determined. In almost all of thc reports cited in this review, a strict demonstration of an increase in the amount of enzyme protein has not been established. Nevertheless, a number of indirect studies suggest that many of the increased activities are due to an increased rate of synthesis, and for the purpose of this review the terms “enzyme induction” and “enzyme repression” will be used, although direct evidence for “induction” or “repression” are lacking.
CARCINOGENS, E K Z T M E INDUCTION, AND GENE ACTION
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Berlin and Schimlie (1965) have pointed out the importance of both enzyme synthesis and degradation in any studies on cnzyme induction. Thus, a stabilization of an enzyme which prevents its degradation may mimic enzyme induction. Examples of this are the 10-fold increase in thymidylatc kinase activity when thymidine is administered. This increase is not due to an increased rate of enzyme synthesis, i.e., to enzyme induction, but rather to a thymidine stabilization of thymidine kinase (Bojarski and Hiatt, 1960). Also tryptophan administration increases tryptophan pyrrolasc activity hy increasing its affinity for a cofactor hematin (Fcigelson and Greengard, 1962) which stabilizes the enzyme and prevents its degradation (Schimke et al., 1964). Thus, in the absence of tryptophan, the enzyme has a half-life of about 3 hours; in its presence enzyme degradation is not detectable. Another example of enzyme stabilization mimicking enzyme induction is the 2-fold increase in rat liver arginase when the rats are deprived of food for 6 days. During this time, however, the rate of arginase synthesis is constant while the degradation of the enzyme is halted. On c1i:mging from a high to a low protein diet the rate of degradation is increased and the rate of synthesis decreased. This mimics but is not true enzyme repression, which can be defined as a decrease in thc rate of enzyme synthesis (Schimkc, 1964). II. Polycyclic Hydrocarbons and Enzyme Induction
A single in vivo administration of certain polycyclic hydrocarbons to rats increases a large number of liver microsomal enzyme activities (Table I ) . Among the many enzyme systems which are enhanced by polycyclic hydrocarbons are N-dernethylation (Conney et al., 1956 ; Conney et nl., 1959; von tlcr Decken and Hultin, 1960), 0-demethylation (Henderson ant1 Nazel, 19641, S-demethylation (Henderson and Maze], 1964), aromatic ring C- and N-hydroxylation (Conney et al., 1959; Dao, 1964; Gillette, 1963; Cranier et al., 1960), reduction of the azo linkage of aminoazo dyes (Conney et al., 1956; Conney et al., 1959; Jervell et al., 1965; von der Decken and Hultin, 1960), and the glucuronide conjugation of hydroxy compounds (Inscoe and Axelrod, 1960). Many of the latter enzyme systems are also enhanced by a variety of drugs of different types and of varied clinical use. The literature reveals t h a t almost all of the enzyme activities increased by hydrocarbon or drug pretreatment that have been localized iiitracellularly have been found in the microsonial fraction. Certain other “inducible” enzymes of rat liver such a s tryptophan pyrrolase, serine dehydrase, and arginase are not significantly affected by treatment of the rat with hydrocarbons or drugs. Although a large number of compounds of different molecular types may
TABLE 1 THE EFFECT
Pretreatment
Species
3.4-Uenzpyrene (UP) 3-hlethylcholanthrene (MC) 1,2,5,6-Dibenzanthracene 4’-Methyl-l,2-Benzanthracene 9.10-Dimethylbenzanthracene (DMBA) 2-1Methylbenzo[o]phenanthrene 3.4-Benzpyrene-5.&qumone hLC 31C hIC
hlC hIC MC hlC MC hfC hlC hlC Benzpyrene
7,12-Dimethylhenzanthracene Naphthacene Phenanthrene Anthraeene Xaphthalene Uenz pyrene 1,2,5.6-Dibenzantbracene AfC Anthanthrene Kaphthacene Nanhthalene. anthracene 2‘,l-Anthra-l,2-anthracene, 3.9-dimethylanthanthrene
OF POLYCl-CLIC
Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat
HYDROCARHONS I N D REI.ITED
Tissue Liver Liver Liver Liver Liver Liver Liver Small intestines Liver Kidney Lung Small intestine Liver Kidney Thyroid Testis Lung Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver
Enzyme system H ydroxylation Hydroxylation Hydroxylation Hydroxylation H ydroxylation Hydroxylat ion Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation JTydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation
COMPOUVDS O N VkRIOUS
Substrate 3 ,l-Benzpyrene 3.4-Benzpyrene 3,4-Benzpyrene 3,4-Benzpyrene 3,CBenepyrene 3,4-Renzpyrene 3.4-Benzpyrene 3,4-Benzpyrene 3.4-Brnzpyrene 3,CBenzpyrene 3.4-Benzpyrene 3.4-Benzpyrene 3.4-Benzityrene 3.4-Benzpyrene 3.4-Benepyrene 3.4-Benzpyrene 3.4-Benzpyrene 3.4-Benzpyrene 3,4-Benzpyrene 3,4-Benzpyrene 3,4-Uenzpyrene 3.4-Benzpyrene 3.4-Benzpyrene 3.4-Benzpyrene Zoxazolamine Zoxazolamine Zoxazolamine Zoxazolamine Zoxazolamine Zoxazolamine Zoxazolamine
ESZKME SYSTEMS
Product Mixed bydroxylation Mixed hydroxylation Mixed hydroxylat ion Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation AIixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation hIixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation Mixed h kdroxylat ion hlixed hkdroxylation AMixedhydroxylation Mixed hydroxylation Mixed hydroxylation Mixed hydroxylation 6-OH-zoxazolamine 6-OH-zoxazolamine 6-OH-zoxazolamine 6-OH-zoxazolamine 6-OH-zoxaeolamine 6-OH-zoxazolamine 6-OH-zoxazolamine
Activity ratio treated/ untreated
10-12 10 10 10
+-
6-20 9 60 6 70
+ + + + +
30
12
7 2 2 1 1 6 8 8 6 4.5 2 5
1.5
References Conney et al. (1957.1959) Conney et al. (1957) Comiey ct al. (1957) Conney et al. (1957) Conney el al. (1957) Couney et al. (1957) Conney st al. (1957) Wattenberg et al. (1962) Gelboin and Blackburn (1963) Gelboin and Blackburn (1963) Gelboin and Blackburn (1963) Gelboin and Blackburn (1963) Wattenberg and Leong (1962) Wattenberg and Leong (1962) Wattenberg and Leong (1962) Wattenberg nnd Leong (1962) Wattenberg and Leong (1962) Dao and Yogo (1964) Dao and Yogo (1964) Dao and Yogo (1964) Dao and Yogo (1964) Dao and Yogo (1964) Dao and Yogo (1964) Dao and Yogo (1964) Gillette (1963) h c o s el al. (1961) Arcos et al. (1961) Arcos et al. (1961) Arcos el al. (1961) Arcos el al. (1961) Arcos et al. (1961)
Acenaphthene, 5-methyl3.4.8,9-dihenaol,yrenc, \ iolanthrene, pyranthrene Eenzpyrene Eenzpyrene 13enzpyrene Henzpyrene
Rat
Liver
Ilydroxylation
Zoxazolamine
6-OH-aoxazolamine
1 .o
Arcos el aI. (1961)
Rat Rat Rat Rat
I.iver Liver Liver Liver
Hydroxylat ion H ydroxylation Hydroxylation Hydroxylation
Chlorzoxazone Quiiioline Xaphtlialene Naphthalene
1.4 2 .o 2.0 1 9
Conney Coniiey Conney Conney
Eenzpyrene 1:enzpyrene I3enzpyrene
Rat Rat Rat
Liver Liver I.ivcr
Hydroxylation Hydroxylation Hydroxylation
Acctanilide Acetanilide Hexobarbital
AIC
Rat
Liver
Hydroxylation
Phcnylbutazone
RlC
Rat
Liver
Hydroxylation
Testosterone
hlC
Rat
Liver
H ydroxylation
Estrogen
RIC RlC
Rat Rat
Liver Liver
Hydroxylation Hydroxylation
RIC
Rat
Liver
Hydroxylatio~i
RIC
RIC
Rat Rat
l
Hydroxylation Hydroxylation
RIC
Rat
Liver
Hydroxylation
Perylene 1,2-Benzantliracene 9,lO-Dimethyl1.2-benzanthracene 3,4-Benzpyrene 2-.kcetylamincfluorene (AAF) 2-Acetylaminofluorene
6-OH-Chlorsoxazone 3-OH-rruinoline 1-Naphthol Naphthalene-l,2dihydrc-1.2-diol 4-OH-acetanilide 4-OH-acetanilide Hexoharbital disappearance Hexobarbital disappearan ce Hydroxylated products Water-soluble products Mixed hydroxylst ion Mixed hydroxylation
hlC
Rat
Liver
Hydroxylation
RIC
hlouse
Liver
Hydroxylation
-a
nrc
Mixed hydroxylation
0.5 17 1
~t al. (1959)
et al. (19%) et al. (1959) et al. (1959)
Conney f t al. (1929) Gillette (1963) Conney ~t al. (1959)
1.2
Conney and Iilutch (1863)
1
Conney and Klutch (1963)
1
Conney and Iilutcb (1963)
+ i-
Wattenberg and Leong (1962) Wattenberg and Leone (1962)
+
Wattenberg and Leong (1962)
+ 9
Cramer rt al. (1980) Cramer rt al. (1960)
B-OH-.LAF
11
Cramer et nl. (1960)
7-OH-AAF
11
Cramer et al. (1960)
I-OH-.kAF 3-OH-AAF
WAF) 2-Bcetylamincfluorcne (AAF) 2-Acetylaminofluorene
Mixed hydroxylated
1.8
Cramer et al. (1960)
AAF
(AAF) RlC
Guinea
IJk
Liver
Hydroxylation
2-Acetylamincfluorene
(.k.4F )
31ixed hydrox ylat ed
A.4F
1.7-1.9
Cramer et al. (1960)
TABLE I (Continued)
Pretreatment
Species
Tissue
Enzyme system
nic
Cotton rat
Liver
Hydroxylation
RIC
Hamster
Liver
Hydroxylation
hlC
Hamster
Liver
Hydroxylation
MC
Rat
Liver
N-Demethylation
Benzpyrene
Rat
Liver
1.2,5,6-Dihenzanthracene
Rat
Liver
Pyrene, fluorene acenophthene. fluoranthrene, benzene, phenanthrene, 9,10-dimethyl-1,2benzanthracene &hlethyl-l,2-henzanthracene
Rat
Liver
N-Demethylation h'-Demethylation N-Demethylation
Rat
Liver
4-Methyl-l,2-benzanthracene
Rat
Liver
lO-Methyl-l,2-benzanthracene
Rat
Liver
1,2,3.4-Dibenzanthracene.L23.6dibenzanthracene, 20-methyleholanthrene anthanthrene, naphthacene
Rat
Liver
m
N-Demethylation N-Demethylation N-Demethylation N-Demethylstion
Substrate
Product
Activity ratio treated/ untreated
References
Mixed hydroxylated AAF
1.4-2.0
Cramer et al. (1960)
Mixed hydroxylated AAF
1.4-1.5
Cramer et al. (1960)
2-.4cetylaminofluorene (AAF) 2-Bcetylaminofluorene (AAF) 2-Bcetylaminofluorene (AAF) 3-Methyl-monomethylammoazobenzene (I\IAB) 3-Me-MAB
3-Me-AB
3-8
3-hIe-hl AB
3-Me-AB
3
Conney et al. (1956)
3-Me-hlAB
3-Me-AB
1-1.3
Conney et al. (1956)
3-Me-MAB
3-Me-AB
4
Arcos et al. (1961)
3-Me-MAB
3-Me-AB
4
Arcos et al. (1961)
3-Me-MAB
3-Me-AB
3
Arcos el al. (1961)
3-Me-MAB
3-Me-AB
3
Arcos et al. (1961)
N-OH-AAF
3-Methyl-aminoazobenzene (AB)
10
Lotlikar et al. (1965)
3
Conney el al. (1956)
Conney et al. (1956, 1959)
(o
3,4-Benzopyrene, 2'-methyl-1.2henaanthracene. 9-methyl-l,2benaanthracene, chrysene, 3,4,8,9dihenzopyrene. 1,2.4,5-dibenacpyrene, 1'-methyl-1 2,-benaanthracene, 3,4,9,10-dibenzopyrene, l,Z-benaanthracene, 6'-methylnaphtho (2',3': 3,4) pyrene picene 9,lO-Dirnethyl-l,2-benzanthracene. perylene, 2,3-benzofluorene, 5Methyl-3,4,8,9-dibenaopgrene 1,Z-Benaofluorene, pentscene, retene. 2'-methyl-l,2.3,4-dihenzopyrene, 6',7'-dimethylnal~htho (2',3': 3-4) pyrene, 9- (I-naphthyl)-l,2benaanthracene. 4-aaaflnorene, 2azafluoranthene, 1,IZ-dimethyl3,4-beniophenanthreiie, violanthrene Benzene, naphthalene. aaulene, anthracene, Uenzo[cleinnoline. aeenaphtnene. drrrene, fluorene phenanthrene, pyrene, fluoranthene, 3.4-benzofluorene, 3,4henzophenanthrene, triphenylene, 1.2-benzopyrene. coronene. 5,sdimethyl-3,4-benzophenanthrene, 3.9-dimethylanthanthrene. 2',1'anthra-1,2-anthracene. 9,IO-diphenylanthracene, lO-phenyl-l.2henzanthracene, pyranthrene, 10(2-naphthyl) 1,2-henzanthracene Benzpyrene
Rat
Liver
N-Demethylation
3-Me-hfAB
3-Me-AB
3
Arcos et al. (1961)
Rat
Liver
N-Demethylase
3-Me-MAB
3-Me-AB
2
Arcas et al. (1961)
Rat
Liver
N-Demethylase
3-Me-MAB
3-Me-.4B
1.5
Arcos et al. (1961)
Rat
Liver
N-Demetbylase
3-Me-MAB
3-Me-AB
1.3
Arcos el al. (1961)
Rat
Liver
A'-Methylaniline
Aniline
2 .O
Conney et al. (1959)
Benzpyrene
Rat
Liver
Meperidine
Normeperidine
0.3
Conney et ~ l (1959) .
Benzpyrene
Rat
Liver
N-Demethylation N-Demethylation N-Demethylation N-Demethylation
Benadryl
2 (Benahydryloxy )
0.7
Conney et al. (1959)
ethylamine Aminoantipyrine
3.3
Conney et al. (1959). Gillette (1963)
Benapyrene
Rat
Liver
Monomethyl, 4aminoantipyrine
TABLE I (Continued)
Pretreatment
Substrate
Product
Activity ratio treated/ untreated
Species
Tissue
Enzyme system
References
Benzpyrene
Rat
Liver
0-Demethylation
4-Methoxyacetanilide
4-OH-Acetaniline
1.4
Conney el al. (1959), Henderson and Maze1
Benzjryrene
Hat
Liver
S-Demethylation 4-Methoxyacetanilide
4-OH-Acetaniline
1.4
Conney et al. (1959). Henderson and Maze1
MC
Rat
Liver
AZOreduction
2.55
Jer\.ell et al. (1965)
Benzpyrene
Rat
Liver
1 .o
Conney et al. (1859)
Benzpyrene Benzpyrene
Rat Rat
Liver Liver
1.6 1.6
Conney et al. (1959) Conney et al. (1959)
MC
Rat
Liver
hIC
Rat
Liver
MC
Rat
Liver
Hnlfoxide formation C-oxidation Glucuronide formation KADH diaphorase NADH diaphorase NADH cytochrome c reductase NADPH cytochrome c reductase NAD cytochrome hl reductase NADPH disphorase NADPH cytochrome bs Mcnadione reductase
Aniline and .V-dimethyl-phenylenedionine Chlorpromazine snlfoxide p-Nitrobenzoic acid pSitrobenzoic acid
(1964)
(1864)
E
h1C
Rat
MC
Rat
Liver
Liver
MC
Rat
Liver
ILIC
Rat
Liver
9,I0-Dirnethyl-12-benzantk1racene
Rat
Liver
(DXIBA)
4-Dimethylaminoazobenzene Chlorpromazine pNitrotoluene pNitrotoluene
.o
von der Decken and Hnltin
1.0
von der Decken and Hultin
1 .o
von der Decken and Hilltin
1
(1960) (1960) (1960) 1
.o
von der Decken and Hultin (1960)
1
.o
von der Decken and Hultin (1960)
1.2
von der Decken and Hultin
1.5
yon der Decken and Hultin
2. I
Huggins et 02. (1064a)
(1960) (1960)
DMBA
i\.Iouse
Liver
DMB.4
Rat
Liver
DhI13.A
Rat
Liver
DMBA
Rat
Liver
DMB.4
Rat
Liver
hienadione reductase Lactic dehydrogenase Malic dehydrogenase Isocitric dehpdrogenaPe Glucose-6phosphate dehydrogeiiave
1.1
~IrlnginsPI al. (1964a)
1. O
Huggins rt al. (1964a)
1 .O
Huggins et al. (1964a)
1. O
Huggins et al. (1964a)
1 .O
Huggins rt al. (1964a)
12
H. V. GELBOIN
increase the same enzyme system, there are a number of enzymes which are increased by one class of compound and not another; e.g., microsoma1 androgen hydroxylases, although inducible by phenobarbital, are not affccted by 3,4-benzpyrene (BP) treatment. I n contrast, the enzyme system that hydroxylates 3,4-benzpyrene is induced by pretreatment with either B P or phenobarbital. Thus, the profile of enzyme systems induced by a particular compound may be unique. I n other cases, however, two inducers may increase a similar profile of enzymes. An explanation for these differences and for the molecular basis of hydrocarbon or drug-induced alterations in enzyme activity await knowledge in several areas. Questions must be answered concerning the transport and availability of the inducer to its initial cellular site of action as well as to the nature of the receptor. Other problems are that of the metabolic conversion of an inactive administered compound to an active form; finally, there is only little understanding of the mechanism of the induction process. Is the increased cnzyine activity due to activation of preexisting enzyme molecules or to an increase in amount of enzyme protein? If due to the latter, are the increased amounts of enzyme protein due t o a greater synthesis of enzyme or to a less rapid degradation? Further, if there is increased enzyme synthesis, does i t occur on a stable messenger RNA and is i t perhaps due to a decreased destruction of specific messenger RNA, or does the increased protein synthesis depend on activation of specific genes a t the DNA level with subsequent messenger RNA synthesis? I n order to answer some of the latter questions, more information is needed about the stabilities and turnovcr rates of the enzyme as well as messenger RNA for each enzyme. Only when each of these major factors is understood will one have adequate insight into the structure-function relationship between the vast variety of inducing agents and the enzyme systems induced. A. HEPATIC ENZYMES IN YOUNG A N D ADULT RATS 1. N-Demethylases
One of the early reports suggesting an effect of exogenous factors on enzyme level was that of Brown e t al. (1954), who found that a number of dietary factors enhanced the activity of rat and mouse liver aininoazo dye N-demethylase. The lowest level of enzyme activity was observed in animals fed a semisynthetic purified diet or a grain diet. The feeding of commercial chows doubled mouse liver aminoazo dye N-demethylase activity, and increased the level of this enzyme in rat liver by 30%. Diets supplemented with either aged animal products or crude tissue extracts also stimulated liver aminoazo dye N-demethylase activity, In
CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION
13
addition, cyclic organic peroxides and several aromatic hydrocarbons were also active when fed in the diet a t the low level of 0.05%. Reif e t al. (1954) suggested that the increased arninonzo dye N-dernethylase in rats fed the chow diet was due to tlic presence of peroxides. This conclusion was based on the finding t h a t the addition t o synthetic diets of aged or oxidized cholesterol, but not purified cholesterol, increased the ability of the liver t o N-dcmethylate 3-methyl-4-monomethylaminoazobenzene (3-Me-RIAB) . Thc enzyine level also was increased when the
50 D
E * 40 m
a
) .
5
30 m (3
3.
20
10
e
e Control
I
I
I
I
I
I
I
I
2
3
4
5
6
7
Days
FIG. 1. Hepatic demethylase activity at various times after a single intraperitoneal injection of MC. Activity is expresscd a t pg. 3-methyl-.4B formcd/50 mg. liver/30 minutes (Connep et nl., 1956).
diet contained pcroxide or oxidized steroids, such as oxidized dihydrocholesterol and ergosterol. Gillette ( 1963) reported t h a t liver rnicrosomes froni rats fed chow diets were inore active than normal rnicrosomes in the S-demethylation of monoinethyl-4-aminoantipyrene. I n an earlier study, Conncy e t ul. (1956) (Fig. 1) found t h a t a single intraperitoneal injection of 0.1 to 1.0 mg. of 3-mcthylcholnnthrene (MC) markedly increased the amount of aminoazo dye N-demethylase in rat liver homogenates. Twenty-four hours after 0.1 mg. of hfC was given a %fold increase in enzyme activity was observed. An increase in enzyme
14
H. V . GELBOIN
activity was detected as early as 6 hours after the M C was given. The highest level of activity was observed a t 24 hours and the value observed a t 7 days was the same as that of control rats. The administration of larger amounts of MC caused a greater increase in aminoazo dye Ndemethylase activity a t earlier times, and the level of enzyme activity persisted for longer periods before returning to the normal values. The return of enzyme amount to the levels of activity of untreated controls indicates that the hydrocarbon does not irreversibly affect those factors controlling enzyme amount. A number of polycyclic hydrocarbons other than M C were tested for inducing activity (Conney et al., 1956). I n addition to MC, those compounds exhibiting significant inducing activity were 3,4-benzpyrene (BP) , 1,2,5,6-dibenzanthracene-9,10-quinone, and 1,2,5,6-dibenzanthracene-3,4-quinone.Many closely related polycyclic hydrocarbons were inactive us inducers of liver aminoazo dye N-demethylase activity. Among these were pyrene, fluorene, acenaphthene, fluoranthrene, benzene, phenanthrene, and 9,10-dimethyl-1,2-benzanthracene. The hydrocarbon-induced increase in enzyme activity is not mediated through hormonal factors since similar effects of M C were observed in adrenalectomized or hypophysectomized rats (Gillette, 1963). von der Decken and Hultin (1960) investigated the effect of partial hepatectomy on MC-induced aminoazo dye N-demethylase and on aminoazo dye reductase and found that M C caused similar increases in both normal and partially hepatectomized adult rats. Coriney et al. (1959) reported that a single administration of 1 mg. of B P caused a 5.5-, 2.0-, and 3.3-fold increase in the deniethylation of 3-methyl-MAB, N-methyl-aniline, and monomethyl-4-aminoantipyrene. von der Decken and I-Iultin ( 1960) confirmed the MC-induced increase in aminoazo dye N-demethylase. Conney et al. (1959) reported that B P pretreatment decreased meperidine and Benadryl N-demethylation by 70 and 30010, respectively. Henderson and Mazel (1964), however, reported that the B P pretreatment caused a slight increase in meperidine N-demethylation. Fouts and Rogers (1965) found that B P and M C increased the N-dealkylation of aminopyrene by 40 to 100%. 2. 0-Demethylases and S-Demethylases
Gillette (1963) reported that microsomes from rats fed chow diets showed increased activity in the 0-demethylation of methoxyacetanilide. Conney et al. (1959) reported a 40% increase in the 0-demethylation of methoxyacetanilide after young rats were pretreated with BP. Henderson and Mazel (1964) studied the effect of B P on S-demethylation and 0-demethylation, as well as N-demethylation, and found a substantial BP-induced increase in the 0-demethylation of methoxyacetanilide and
CARCINOGESS, E N Z Y M E INDUCTION, A K D GENE ACTION
15
the S-demethylation of inethylthiopurine. They found either a small or no difference between control and BP-treated rats in the N-demethylation of Demerol, methylbarbital, and codeine.
3. Aminoazo Dye Reductnse The prior administration of MC or BP increases hcpatic aminoazo dye reductase. Conney et al. (1956), Conney et al. (1959), and Jervell et al. (1965), using dimethylaininoazobenzene (DAB) as the substrate, found an MC stimulation of the reduction of the N=N bond. von der
50
D
30
20
10
I
I
1
I
I
2
3
4
5
Days
FIG.2. Hepatic reductae activity at various times after a single intraperitoneal injection of MC. D a t a are expressed as pg. DAB reduced/30 mg. liver/30 minutes (Conney et al., 1956).
Decken and Hultin (1960) uscd MAB as a substrate and found a 3-fold increase in hepatic aminoazo dye reductase activity after a single dose of MC. The rate of increase of hepatic aminoazo dye reductase after MC treatment (Fig. 2) differed from the rate of increase of aminoazo dye X-demethylase. The maximum reductase activity was observed 3 days after 1 mg. of MC was administered, whereas aminoazo N-demethylasc approached a maximum level a t less than 2 days. The different rates of increase suggest that these two activities represent different enzymes
16
H. V. GELBOIN
rather than a single enzyme with either two active sites or a single active center with different affinities for the different substrates.
4. Hydroxylases Conney et al. (1957) first reported an increase in the in vitro rate of benzpyrene hydroxylation in liver homogenates from rats pretreated with BP. After pretreatment with 1 mg. of B P liver homogenates from treated rats showed a 10-fold greater BP hydroxylase activity than did
\
11
\ \
\ ‘1
\ \ \1 \
b 8
l
-
I
x
1
3 6
+X
n
b I
I2
I
I
24
48
I
144
Hours
FIG.3. Hepatic 3,4-benzpyrene hydroxylase after a single intraperitoneal injection of 3,4-benzpyrene (Conney et al., 1957).
similar preparations from control rats. A doubling of enzyme activity was detected as early as 3 hours after B P administration (Fig. 3 ) . Other polycyclic hydrocarbons, for example, MC, l12,5,6-dibenzanthracene, and 4-niethyl-1,2-benxanthracene,were as effective as B P ; 9,10-dimethyl-l,2-benzanthracenewas one third as active; 2-methylbenzo [ c] phenanthrene, 1,2-benzanthracene, and 3,4-benzpyrene-5,8-quinone and 3,4-benzpyrene-5,10-quinoneexhibited little or no inducing activity. The intracellular distribution of the increased enzyme activity was primarily in the microsomes (Conney et al., 1957). The nuclear fraction showed about one third the activity of the microsomcs. These studies were performed prior to the development of procedures for obtaining
CARCINOGENS, ENZYME INDUCTION, A K D GENE ACTION
17
rat liver nuclei of high purity, and thus the nuclear activity reported is at least partially due to cytoplasmic contamination. The hydroxylation reaction occurs a t a number of different carbon atoms of the polynuclear hydrocarbon substrate. Thus, B P was converted to 8-hydroxy BP, 5,8dihydroxy BPI 5,8,-BP-quinone and 5,lO-BP-quinone. However, it is possible that some of the above products may have formed nonenzymatically from unstable bcnzpyrenols. The study did not indicate which of these hydroxy products were increased by B P pretreatment. Falk e t al. (1964) investigated the temporal relationship between the appearance of the different hydroxylated metabolitcs of B P in the bile. During the first hour after an intravenous injection of BPI 5-OH B P glucuronide constituted the major metabolite. I n addition, certain dihydroxy compounds appeared during this interval. Only subsequently did %OH B P and its derivatives appear in the bile. 1-OH BPI a metabolite reported by Conney e t al. (1957), was not detected in the bile by these investigators. The temporal relationship between the appearance of the 5-OH B P and the later appearance of 8-OH B P suggests the possibility that the formation of these metabolites is catalyzed by different enzyme systems whose levels are altered a t different rates. Other studies reporting a polycyclic hydrocarbon-induced increase in the hydroxylation of benzpyrene are those of Strand and Wattenberg (1962), Gelboin and Blackburn (1963, 1964), and Dao and Yogo (1964). Wattenberg and Leong (1962) developed histochemical methods for the detection of hydroxylated products of a number of hydrocarbons and used these techniques to demonstrate an increase in enzyme activity in the liver as well as in other tissues after the rats were pretreated with 9,10-dimethyl-1,2henzanthracene (DMBA) . The prior administration of the latter compound was found to stimulate its own hydroxylation. Cramer et al. (1960) found an MC-induced increase in the ability of rat liver honiogcnates to hydroxylate 2-acetylaminofluorene (AAF) . This activity was increased 3- to 4-fold 6 hours after 1 mg. of MC was administered; 24 and 48 hours after the M C was given there was a 10- to 12-fold incrcase. The level of activity had declined considerably a t 7 days and was back to normal levels by 9 days. The young rat was the most sensitive to the MC induction of AAF hydroxylation activity. Thus there was a 10-fold increase in AAF hydroxylase activity in young 60-g. rats, and only a 2-fold increase in this activity in the adult rat. The increased hydroxylation occurred a t each of the positions of the fluorene ring which are normally hydroxylated. Thus M C pretreatment increased the formation of the 1-, 3-, 5-, and 7-hydroxy derivatives of the aminofluorene. This hydroxylase activity was localized primarily in the microsomes and displayed cofactor requirements similar to that reported for
18
H.
V.
GELBOIN
benzpyrene hydroxylase by Conriey et al. (1957). Cramer et al. (1960) found that rats pretreated with M C increased not only C-hydroxylation of AAF but also the N-hydroxylation of aminofluorene. Thus, these investigators isolated the N-hydroxy metabolite of aminofluorene only from the urine of rats that were pretreated with M C and then fed AAF. When the rats were not pretreated with M C the AT-hydroxy metabolite of AAF was not dctccted. Subsequent studies (Lotlikar e t al., 1965) showed that MC pietrcatment of rats caused a 5-fold increase in the Nhydroxylation of AAF and lo-fold increase in the ring hydroxylation of this substrate. Conney et al. (1959) surveyed the effect of B P on the hydroxylation of several different substrates. B P pretreatment induced large increases of 12-, 6.5-, and 3.8-fold in the hydroxylation of RP, acetanilide, and zoxazolamine respectively. The same pretreatment causcd a doubling in quinolinc and naphthalenc hydroxylation and only a 40% increase in chlorosoxazonc hydroxylation. Jellinck and Irwin (1963) reported an increase in estrogen metabolism, presumably hydroxylation in MCtreated rats. Coniicy and Klutch (1963) reported no effect of M C on the mctaholism of A4-androstenc-3,17-dione and only a very slight stimulation, less than 15%, in the metabolism of tcstostcronc by rat liver microsomes. This is in sharp contrast to the marked stimulatory effect of phenobarbital pretreatment which enhances the microsonial metabolism of these steroids by 3- to 8-fold. Gillette (1963) studied the effect of both B P and MC when administered individually and together on the hydroxylation of acetanilide and zoxazolamine. When the hydrocarbons were administered individually, thc hytlroxylating enzymes were stimulated t o about the same extent. When both hydrocarbons were given the amount of stimulation of hydroxylase activity was similar to that ohserved when either hydrocarbon was given alone. Thcsc observations support the view that both B P and M C have similar or identical receptor sites in the liver and are stimulating the same microsomal enzymes by thc same mechanisms. 5. Microsomal Electron Transport Enzymes Von der Decken and Hultin (1960) and Conney and Burns (1962) found an MC-induced increase of 50% in the activity of microsomal T P N H cytochrome b, reductasc. Both reports showed that M C had no effect on D P N H oxidation by liver microsomes, and the former authors found that RiIC had essentially no effect on microsomal D P N H diaphorase, T P N H diaphorase, DPNH cytochrome c reductwe, T P N H cytochromc b, reductase, and microsomal cytochrome b,. This is in contrast to the marked stimulation of some of these enzymes by compounds such
CARCINOGEKS, ESZI'ME INDUCTION, A N D GENE ACTION
19
as phenobarbitid, chlorcyclizine, phenylbutazonc, and orplienadrine (Conney e t al., 1961a; Conney and Klutch, 1963).
6. Miscellaneous Enzymes Iiiscoe and Aselrod (1960) showed :in increase in U D P glucuronyl transferase in liver after MC or B P injection. They found t h a t these hydrocarbons stimulntcd U D P glucuronyl transferase in both fetal and neonatal rat liver. They found no cnliancement of this enzyme system, however, in fetal rat liver when the hydrocarbon was administered t o the pregnant mother. Arias et al. (1963) confirmed these findings and also found that B P prctre:itment increased the activity of UDPG dehydrogenase in 3-day-old ratb approximately 2-fold. W'hen the prcgnant mothers were pretreated with BP, their livers showed a n increase ill UDPG glucuronyl transferase activity, but the newborn, 3 days after birth, showed no effect of B P pretreatment of their mothers. When chloroquine or chlorcyclizine were given to the pregnant mother, an increase in these enzyme systems was observed in the newborn. The data suggest the inability of the polycyclic hydrocarbon or an active metabolite thereof to pass the p1:xentnl membranes into the fetus. Murphy and Dubois (1958) reported that polycyclic hydrocarbons stimulated the onzyme which t1c~ulfuryl:ited thiopliosphonates such as ethyl-p-nitrophenylthiohenzene phosphonates. Conney e t al. (1959) reported that BP pretreatment caused a 60% increase in the side chain oxidation of p nitrotoluene t o p-nitrobenzoic acid. Fouts and Rogers (1965) found no effect of M C or B P on thc reduction of p-nitrohenzoic acid. Huggins cf nl. (1965) found that tlic ntliiiinistrntion of 7,12-dimethylbenzanthracene (DMBA) doubled menadione rcductase activity in rat liver. The saine treatment had no cffcct on rat livw lactic dehydrogenase, malic dehydrogcnnsc, isocitric deliytliogeniwe, or glucose-6-phosphate dehydrogenase. Although 1iydroc:irhon treatment increased nienadione reductasc in rat liver, it had essentially no effect on the level of this enzyme in either (3.57 black mice or in C F l mice. Conney et al. (1959) reported no effect of B P pretreatment on the formation of chlorpromazine sulfoxide from c1iloiprom:izine. von dcr Dcckcn and Hultin (1960) reported no effect of RIC on niicrosoninl glucose-6-phosphatase. E:NZT\lES B. HEPATIC
I N FET4I., XEOK'ATAL, AND IMMATURE
RATS
Cranier et al. (1960) reported t h a t of several species tested the young rat rx1iil)itcd the greatest sensitivity to the induction of AAF hydroxylase by M C pretrentment. In the mouse, guinea pig, cotton rat, arid lramster the d c g r c ~of stimulation by M C was similar in both the young and adult animal. Ho~vever, with rats, M C treatment of the
20
H. V. GELBOIN
young animal caused a 10-fold rise in AAF hydroxylating activity. I n adult rats and both the young and adults of the other species tested the MC-induced increases ranged from 40 to 100%. In each species, however, thcre may bc certain ages during which the enzyme level will be particularly sensitive to induction. I n this study, the induction process was only studied at a single age of the young animal. It is possible t h a t a t an earlier age the enzyme system may be more readily inducible.
C. SPECIESOTHER THAN THE RAT Inscoe and Axelrod (1960) reported that M C caused a n increase in glucuronide formation in guinea pig liver microsomes. Dutton and Stevenson (1962) found an increase of 2- to 3-fold in UDP glucuronyl TABLE I1 HYDROXYLATION O F 2-ACETYLAMINOFLUORENE B Y LIVER HOMOGENATES FROM DIFFERENT SPECIES A F T E R TRE.4TMENT W I T H M E T H Y L C H O L A N T H R E N E 'in V'kJO'
SI'IMULATION O F
2-Acetylaminofluorene converted to phenolic metabolitesb
IJntrested (pg.) Species Rat Mouse Guinea pig Cotton rat Hamster 4
Young 3 8 12 13 16
Methylcholanthrenet,reated ( p g . )
Methylcholanthrenetreated/untreated
Adrilt
Young
Adrilt
Young
Adult
4 10 8 9 17
30 14 20 18 24
8 18 15 18 24
10 1.8 1.7 1.4 1.5
2.0
1.8 1.9 2.0 1.4
Cramer et al. (1960).
* The young rats, mire, guinea pigs, cotton rats, and hamsters weighed approximately 60, 10, 120, 50, and GO g. respectively; the adult animals, in the same order, weighed approximately 300, 30, 500, 140, and 100 g.
transfernse activity in the skin of mice pretreated with 3,4-benzpyrene for 3 successive days. Cramer et al. (1960) (Table 11) showed that MC pretreatment increased the C-hydroxylation of AAF in both young and adult mice, guinea pigs, cotton rats and hamsters. The stimulation ranged from 40% increase to a doubling of activity. Lotlikar e t al. (1965) reported an MC-induced increase in the N-hydroxylation of AAIC in hamster liver. Gelboin (1965) found an incrcased benzpyrene hydroxylase activity in the livcrs of mice pretreated with MC. Wattenberg et al. (1962) found benzpyrene hydroxylating activity in the duodenal mucosa of mouse, guinea pig, rabbit, hamster, dog, and man. However,
CARCINOGENS, EKZPME INDUCTION, AND GENE ACTION
21
the inducibility of this enzyme in the duodenum of these species was not testcd.
D. NONHEPATIC RAT TISSUE Gilman and Conney (1963) found that M C pretreatment increased amionazo dye N-demcthylase activity in lung and kidney, as well as liver. They detectcd no effect of MC on the low level of activity in the brain or testes. I n the same report they found that phenobarbital did not cause any significant changes in the N-demethylation of 3-methyl-MAB in any of the nonhepatic tissues tested. The same pretreatment with phenobarbital incrcascs A'-demethylasc activity in liver. Huggiiis and Fukunislii (1964) found that M C increased nienadione reductase in livcr, lung, atlrcnal, m:mmary gland, and mammary tumors of rats. The increased meiiadionc reductase activity of the mammary gland, mammary tumor and liver was 214, 275, and 22470, respectively. Lesser increases werc observed in the lung. Strand and Wattenberg (1962) found that the oral administration of 7,12-dimethyl-1,2-beneantliracene(DMBA) to rats stimulated its own metabolism in kidney and stomach, as ell as in liver. Wattenberg and Leong (1962) demonstrated by histochemical techniques the presence of benzpyrene liydroxylating activity in rat liver, kidney, aclreml, and small intestine. When rats werc pretreated witli MC, perylene hydroxylase or benzpyrcne liydroxylnse activity was increased in liver, small intestine, and kidney. The activity of this enzyme system in the adrcnals was not i n c r c a d by RIC pretiwtmcnt. In addition, this enzyme system was found in the follicular cells of the thyroid, lung, and the intcrstitial cells of the testes of hlC-trcated rats, although the same enzyme system was not detected in the latter tissues of control rats. I n addition, the enzyme system was not detccte(1 in the following tissues of either control or MC-treated rats: heart, brain, spleen, pancreas, skclctal muscle, pituitary, salivary gland, thymus, lymph node, ovary, fallopian tube, breast, stomach, esophagus, small intestine, and lnrgc intestine. I n another report, Wattenberg (1962) reported that tlic skin of rats painted with MC showed enhancccl BP hytlroxylnse activity. Based on their histochemical studies of bcnzpyrenc liydroxylasc this group subsequently developed sensitivc methods for the assay of BP hydroxylaee and were able to determine quaiititativcly this enzyme activity in various segments of the gastrointestinal tract. They found BP hydroxylating activity in each of the scgincnts examined (Fig. 4).The greatcst activity was observed in the anterior scction of the small intestine and lowest levels of activity in the posterior end of the small intestine. I n control rats no activity was observed in the stomach, cecum, and colon. Pre-
H . V. GELBOIN
22
treatment for 36 hours with l12-benzanthracene increased hydroxylase activity throughout the gastrointestinal tract. After pretreatment, B P hydroxylase activity was also observed in the stomach, cecum, and colon. Wattcnberg et al. (1962) also found that either starvation or the feeding of a low-fat diet markedly reduced the B P hydroxylase activity of the gastrointestinal tract. Dao and Yogo (1964) found that bcnzpyrene hydroxylase activity was increased in the liver but decreased in the adrenals after treatment with MC, BP, and DMBA. Phenanthrene and anthracene had esscntially no effect on benzpyrene hydroxylase activity Stomach
Sequential Segments of Small Intestine
Fore- Glandular
'plil"
I Cecum
colon Right
Left
Untreated Rats Rats fed 1,2 Benzonthracene
Segment of Rat Gastrointestinal Tract
FIQ.4. Distribution of benzpyrene hydroxylase activity along the gastrointestinal tract of untreated rats and rats given 20 mg. of 1,2-benzanthracene by mouth 36 hours prior to being sacrificed. The small intestine has been divided into seven segments so that differences in activity in sequential portions of the structure may be depicted (Wattenberg et al., 1962).
a t the dose levels used. Gelboin and Blackburn (1964) found tliat the prior administration of MC caused increases of about 7-, 30-, 60-, and 3-fold in rat liver, kidney, small intestine, and lungs.
E. ACTIVITYAS A N ENZYME INDUCER AND AS A LOCAL CARCINOGEN The relative activity of a compound as an enzyme inducer and as a local carcinogen does not correlatc. Arcos et al. (1961) examined the effect of more than 50 polycyclic hydrocarbons on thc induction of
CARCINOGENS, EKZYME INDUCTION, AND GENE ACTION
23
aminoazo dye A~-deniethylase and zoxazolamine hydroxylase. They found no correlation betweeii thc ability of L: polycyclic hydrocarbon to induce enzyme activity in the liver and its reported activity as a local carcinogen. The latter activity in most cases was measured by the ability of thc compound to induce bkin tumors in mice or s:trcomas in various species. Arcos et nl. (1961) founcl that naplitliacenc and antlianthrcnc, which are both inactive as carcinogens, and the potent carcinogen MC are all strong inducers of the two liver microsoinal ciizymes studied. On the other hand the carcinogen 3,4-benzphenanthrcne did not incrcase liver N-demetliyl:w or hydroxylasc. Anothcr polycyclic hydrocarbon, coronene, which is inactive as a carcinogen, is also inactive as an inducer of microsoiiial cnzymcs. Tlie data presented by Arcos et al. (1961) suggest a lack of positive relationship bctwcen carciiiogenic activity and microsonial enzyme-inducing activity. This conclusion, however, iieeds further investigation, since in tlieir study they utilized a single dose of 1.86 pmolcs of the hydrocarbon. In most cases this is considerably greater than the niinimurn required to ($licit a response. With only three of the compounds tested was a dose-response curve determined, and in the case of two of them, 1,2,3,4-diben~antliraceneand hlC, 1.86 pmoles was found to be in considerable excehs of the saturating level. With the third coinpound tested, anthanthrenc, lower levels of the compound, e.g., 0.4 pnioles, caused very little change ill aminoazo dye A7-demethylasc, while high levels were almost as effective as &lC. With 1,2,3,4-dibenzanthracenc, a compound not carcinogcnic to mouse skin, greater inducing activity was observed a t cach of the levels tested than was observed with the potent carcinogens testcd. Although these studies suggest the molecular recluimiicnts for enzyme inducibility a n ~ lcnrcinogcnicity arc not the snmc, :t host of factors KiluSt br nsscssed before this conclusion can be drawn. ill. The Effect of Drugs and insecticides on Liver Microsomal Enzymes
A large number of drugs of pharmacological value have been found to stimulate either their own metabolism or the metabolism of other compounds by liver niicrosomcs. These stimulators of enzyme activity may alter the rate of detoxification of subsequently administered drugs and thereby altcr the duration of drug action. Conney (1965) has rcviewed the effects of drugs on enzyme systems and discussed their pharmacological significance. Table I11 is his compilation of sonic eompounds which liave been tested a s stimulators of enzymc activity. T o cite somc typical exaniples, hypnotics ant1 scdativcs, including certain barbiturates, glutethimitie, chlorobutnnol, urethane, carl)roniaI, pyridione, :~nd mcthyprylonc; trmquilizers such as phenaglycotlol, chlorpromazine;
24
H. V. GELBOIN
TABLE I11 COMPOUNDS REPORTED TO STIMULATE T H E ACTIVITY O F DRUG-METABOLIZINQ ENZYMES~ Pharmacological action Hypnotics and sedatives
Anesthetic gases
Hypoglycemic agents and related sulfonamides
Antiinflammatory agents Muscle relaxants
Analgesics
Antihistamines Insecticides
Drug tested as enzyme stimulator Barbiturates Glutethimide (Doriden) Chlorobutanol (Chloretone) Carbromal (Adalin) Urethane Pyridione (Persedon) Methyprylone (Noludar) Ethinamate (Valmid) Ethanol Chloral hydrate Hydroxydione (Viadril) Thalidomide Paraldehyde Nitrous oxide Ether Chloroform Divinyl ether Halothane Chlorpromazine Triflupromazine Promasine Chlordiazepoxide (Librium) Tolbutamide (Orinase) Carbutamide Snlfathidole Sulfanilamide Phenylbutazone Orphenadrine Carisoprodol Zoxaeolamine Aminopyrine Narcotics (morphine, Levorphan) Chlorcyclieine Diphenhydramine Chlordane, DDT, hexachlorocyclohexane, dieldrin, aldrin, heptachlor, heptachlor epoxide Pyrethrums, piperonyl butoxide
Effect
+ + + + + + +0 5
f 0 0 0
5 5
+ 0 0
+ + 0 0
+ + 0 0
+ + + 0 + Decrease + + + 0
25
CARCINOGESS, ENZYME INDUCTIOS, AND GENE ACTION
TABLE I11 (Conlinmed) Pharmacological action Steroid hormones and related substances
Thyroid hormone Central nervous system stimulators
Anticonvulsants
Tranquilizers
Drug tested as enzyme stimulator Phndrostene-3,17-dione Testosterone Testosterone propionate 4-Chlorotestosterone 19-Nortestosterone Methyltestosterone 4Chloro-19-nortestosterone acetate (SKF 6611) Cortisone Prednisolone ACTH Estradiol Progesterone Thyroxin Nikethamide (Coramine) Bemegride Pentylenetetrazol (Metrazol) A4mphetamine Me thylphenylethylhydantoin (Mesantoin) Diphenylhydantoin (Dilantin) Parame thadione (Paradione) Trimethadione (Tridione) Phenaglycodol (Ultran) Meprobamate
EBect
+ + + + + + + + + + Decrease Decrease 0 or decrease
+ +0 0
+ +
-
+ 0
+
*
This table was compiled in an excellent review by Conney (1965).
hypoglycemic agents such as tolbutamide ; analgesics such as aminopyrine ; muscle relaxants like orphenadrine and carisoprodol ; antiinflarnmatory agents, e.g., phenylbutazone ; insecticides such as chlordane, DDT, hexachlorocyclohexane, dieldrin, aldrin, heptachlor, heptachlor epoxide, and anticonvulsants like methylphenylethylhydantoin,all have been found to exhibit activity as enzyme inducers. I n addition, steroid hormones, for example, testosterone, 19-nortestosterone, and cortisone, have been found to alter microsonial enzyme activity. I n addition to the findings in rat liver, phenylbutazone, diphenhydramine, chlorcyclizine, tolbutamide, and phenobarbital have been found to stimulate their own metabolism when chronically administered to dogs. The kinds of enzyme
26
H. V. CELBOIN
activities that are stiniulatcvl by various drug pretreatment include Cand N-hydroxylation, N - , S-, and 0-demethylation, and glucuronide formation. I n addition to induction in hepatic tissue there have been several reports of drug-induced increases in enzyme activity in tissues other than the liver. Reininer (1962) reported that phenobarbital stimulates barbiturate-metabolizing enzymes in kidney as well as liver. Although several investigators have reported a polycyclic hydrocarbon stiinulation of hydroxylase activity in lung, kidney, and intestine, there have been no rcports of a phenobarbital stimulation of the same enzyme system in thcse tissues. IV. The Nature of the Microsomal Drug-Metabolizing Enzyme Systems
Gillette (1966) has recently reviewed the biochemistry of drug oxidation and reduction by enzymes in the cndoplasmic reticulum of liver. Each of the rnicrosonial drug-metabolizing enzyme systems, whether they involve hydroxylation, demcthylation, or reduction, require NADPH and atmospheric oxygen for activity. These requirements and a number of other studies, suppo,t the theory that these enzymes function by a “mixed oxygenase” nieclianism of oxidation. This is the mechanism that has been found to be operative in the hydroxylation of phenylalanine to tyrosine in thc soluble fraction of liver (Kaufman, 1957). The mixed oxygenase mechanism can be described as follows: NADPH reduces a coinponcnt “A” in the microsomes that reacts with oxygen to form an active oxygen intermediate. The active oxygen is then transferred to the drug substrates.
+ A + H+ AH2 + NADPf + O2 -+active oxygen 3. Active oxygen + drug oxidized drug + -4+ H,O NADPH + 0% + drug + N h D P ‘ + H?O + oxidized drug 1. NADPH
+
2. AH2
--$
This mechanism requires the utilization of equivalent amounts of NADPH, oxygen, and substrates during the reaction. The stoichiometric relationships in this type of mechanism have been demonstrated for the liydroxylation of phenylalanine by liver (Kaufman, 1957) and for the hydroxylation of steroids by adrenal inicrosomes (Cooper e t al., 1963). In liver microsomes, however, definitive evidence supporting this mechanism has been lacking since the stoichioinetry of the drug-metabolizing reactions has not been established. The difficulty in quantitating the relationship between reactants arid cofactors is due to the fact that liver microsomcs contain enzymes which rapidly utilize oxygen (Hochstein
CARCISOCnESS, E S Z T M E I S D U C T I O S , A S D GEKE ACTION
27
and Ernster, 1963) iiiicl oxitlizc NADI'H (Gillettc e t al., 1957) in the absence of drug substrate. A pigment, P-450, which is reduced by NADPH, is found in liver rnicrosomes and is thought to be an important component of the steroid and drug-metabolizing enzymes (Cooper e t al., 1965). This pigment has bccn found only in microsomes and forms a complex with carbon monoxide under anaerobic conditions. When complexed with carbon monoxide P-450 has a n absorption maximum a t 450 mp, and a minimum a t about 405 mp. Under anaerobic conditions the reduced form of P-450 binds carbon monoxide tightly. Omura et al. (1965) found the association constant t o be about 2 x lo-' M . Under aerobic conditions, the concentration of carbon monoxide required to observe the absorption band a t 450 nip must be considerably higher (Omura and Sato, 1964a). There is substantial but not direct evidence suggesting that P-450 is a component of the electron transport system in liver microsomes and may be part of the active oxygen complex described above. First, treatment of liver microsomes with dcoxycholate, sleapsin, or certain snake venoms inactivates the NADPH-requiring oxidation of drugs by liver microsomal enzymes. These treatments also change the absorption maximum of the carbon monoxide complex from 450 mp to an inactive state having an absorption maximum a t 420 mp (Omura and Sato, 1964a,b). Also, carbon monoxide inhibits the metabolism of drugs by microsomes. A summary of this supportable view of microsomal hydroxylation is a s follows: NADPH
+ oxidized P-450 NADPH-cyt NADP + reduced P-450 reductase Reduced P-450 + -+ [P-450-oxygen] [P-450-oxygen] + drug drug-OH + 1/2 O2 c
0 2
+
The following is the suggested mechanism for drug reduction such as that involved in the reduction of p-nitrobenzoic acid to p-aminobcnzoic acid and for the reductive cleavage of ili-Ar-dimethylaminoazobenzene: c + oxidized P-450 NADPH-cyt NADP + reduced P-450 reductase Reduced P-450 + drrig reduced drug + oxidized P-450
NADPH
-i
Part of the reduction of aminoazo dyes may proceed through mechanisms not involving P-450. The reason for this is the fact that carbon monoxide blocks aniinoazo dye reduction by only 40%. Similarly, treatment with steapsin, which converts P-450 to the P-420 inactive form, also decreases aminoaeo dye reduction by 40%. These treatments, however, do riot reduce the activity of microsomal T P N H cytochrorne c rcductase. Other evidence for two mechanisms for aniinoazo dye reduction is based
28
H.
V.
GELBOIN
on the findings that solubilization causes a decrease of both aminoazo dye reduction and P-450 activity by 40%. Upon purification after s o h bilization, the ratio of aniinoazo dye reduction to T P N H cytochrome c reductase activity remains constant throughout the purification. Thus, it is thought that 40% of azo dye reduction is carried out through the P-450 enzyme, and the other 60% is carried out through a mechanism directly involving T P N H cytochrome-c reductase. Microsomal demethylation reactions may bc viewed as follows: NADPH
-
+ oxidized P-450 NADPH-cyt NADP + reduced P-450 Reduced P-450 + O2+ [P-450-oxygen] [P-450-oxygen] + drug-CHz drug-CHzOH Drug-CHzOH demethylated drug + HCHO 0
-+
-+
V. Effect of Oncogenic Viruses on Enzyme Induction
Weil et al. (1965) and Dulbecco et al. (1965) have demonstrated a polyoma virus induction of cellular DNA synthesis as well as of polyoma DNA in mouse kidney cell cultures. Dulbecco e t al. (1965) examined the effect of polyoma virus infection on a number of the enzymes involved in DNA synthesis. Twelve hours after infection there was a rise in the activities of thymidine kinase, deoxycytidinemonophosphate deaminase, and DNA polymerase. The increased activity of each of the three enzymes followed the same course until 28 hours after infection when a maximum was reached. Two other enzymes studied, deoxycytidinemonophosphate (dCMP) kinase and deoxyadenosinemonopliosphate (dAMP) kinase, showed no change upon viral infection. The observed enzyme changes are somewhat similar to those observed in liver regeneration (Davidson, 1962). Dulbecco et al. (1965) also investigated the role of the infectious particle and the viral capsid on the induction of thymidine kinase and dCMP-deaminase activities and found that the increased activities were relatcd to the infectious particle and not to the viral capsid. These interesting results with polyoma virus seem unique among the animal viruses thus far studied, since the DNA-containing vaccinia and pseudorabies viruses do not stimulate host DNA synthesis (Kit et al., 1962; Kaplan and Ben-Porat, 1962). Frearson e t al. (1965) examined the effect of polyoma virus infection on the thymidine lcinase and deoxythymidylate synthetase of niouse embryo cells. Both activities were not significantly altered a t 10 to 24 hours after infection. At later periods, however, the thymidine kinase activity was 3 to 6 times higher than the activity of noninfected cells, and the deoxythymidylate synthetase activity was about twice that of the control cells. The effect
CARCINOGESS, EKZYME INDUCTIOK, A K D GENE ACTIOK
29
of oncogeiiic viruses on enzynie or protein synthesis may also be reflected by the well-known induction by polyoma virus of new cellular antigens in transformed hamster and niouse cells (Sjogren et al., 1961). Rogers (1959, 1962) had reported that Shope virus infection of rabbit epithelium induced an extremely high level of arginase activity. The arginase activity was found in the papilloma but not in any significant amount in either normal rabbit skin or in the virus. Subsequently, Rogers (1962) reported that purified arginase from papillomas contains a different peptide pattern from purified liver arginase, suggesting that the arginase of the papillomas is a virus-induced enzyme. Winocour et al. (1965) examined the synthesis and transmethylation of DNA in polyoma-infected cultures and found the amount of transmethylation to be proportional to the amount of DNA synthesized. There was no evidence that the polyoma virus induced a hypermethylation of cellular DNA. Silber et al. (1964) investigated the effect of the Friend leukemia virus on the level of three enzymes of one carbon metabolism, thymidylate synthetase, deliydrofolic reductase, and formate-activating enzyme in the spleen of animals infected with this virus. They reported a 3- to 10-fold increase in the initial low level of activity of each of these enzymes during the first two weeks of the disease. The alkaline phosphatase level of leukemic spleen decreased to one-half of its initial activity. I n these in vivo experiments it is difficult to assess whether the virus is directly involved in the induction of those enzymes or whether the increased levels are caused by other factors affected by the virus. VI. Enzyme Induction in Rat Hepatoma
A. THEEFFECTOF POLYCPCLIC HYDROCARBONS A N D PHENOBARBITAL ON MICROSOMAL DRUG-METABOLIZING ENZYMES Several studies (Conney and Burns, 1962; Conney et al., 1957; Smith et al., 1958; Adamson and Fouts, 1961) have shown the absence of TPNH-dependent drug-metabolizing enzymcs in rat hepatoma, although adjacent liver tissue generally shows activity comparable to that found in normal liver. Conncy and Burns (1962) studied the effect of MC on the level of aminoazo dye iV-demethylase in a highly malignant tumor induced by aminoazo dye feeding and in the Hepatoma 5123, a relatively slow-growing hepatoma of the “minimal deviation” type (Table IV). I n vivo treatment with MC increased aminoazo dye N demethylase activity in liver by about 4- to 8-fold but did not alter the low level of aminoazo dye N-demethylase activity in hepatomas
30
H . V. GELBOIK
TABLE IV ADMINISTRATION ON THE LEVELO F N - D E M E T H Y L A S E I N HEPATOMA*
E F F E C T OF 3-METHYLCHOL.4NTHRENE
DYE
AZO
Demethylase activity Tissue
Azo dye hepatoma 5 21 2 2
Liver control Liver, MC-treated Tumor, control Tumor, MC-treated ~
a
_
_
_
_
_
_
_
_
~
(2-8) (20-23) (1-2) (1-2)
Hepatoma 5123 2 16 0 13
(2-2) (14-19) (0-0) (7-16)
~
Conney and Burns (1962).
produced by feeding carcinogenic aniinoazo dyes. I n control rats bearing the Morris Hepatoma 5123 no aminoazo dye N-demethylase activity was detected in the turnor tissue but a high level of this activity was found after pretreatment of the rat with MC. Although Hepatoma 5123 did not contain detectable amounts of enzynic, it nevertheless contained the components of the enzyme-forming system necessary t o synthesize enzyme since M C administration induced the appearance of enzyme activity. I n contrast to these findings, other studies (Pitot and Morris, 1961; Pitot and Cho, 1961; Pitot e t al., 1961; Potter and 0110,1961) with Hepatoma 5123 showed that the level of tryptophan pyrrolase activity was not responsive to tryptoplian or cortisone administration. I n addition, the normally inducible enzymes of rat liver, thrconine dehydrase, serine dehydrase, and glucose-6-phosphate dehydrogenase, were not inducible in Hepatoma 5123. H a r t e t al. (1965) examined the effect of phenobarbital, a potent inducer of niicrosomal drug-metabolizing enzymes in liver, on the induction of various drug-metabolizing enzymes in a number of rat hepatomas. The enzyme systems studied were those involving the side-chain oxidation of hexobarbital, the N-dcalkylation of aminopyrene t o 4-aminoantipyrene, the reduction of the aromatic nitro group of p-nitrobenzoic acid to p-aminobenzoic acid, the hydroxylation of aniline to p-aminophenol, and the reductive cleavagc of the azo linkage of neoprontosil to sulfanilamide. Phenobarbital pretreatment significantly enhanced all of the enzyme activities in the livers of either control rats or rats bearing hepatornas (Table V) . Hart e t al. (1965) also showed that phenobarbital pretreatment of rats bearing the highly malignant DAB-induced hepatic tumor caused a substantial increase in the amount of hexobarbital metabolism of the tumor. The salne treatment did not affect aminopyreiie N-dealkylation in the tumor. With the Novikoff Hepatoma, phenobarbital pretreatment stimulated liexobarbital metabolism but did not affect neoprontosil metabolism. The Novikoff
TABLE V EFFECT OF PHENOBARBITAL PRETREATMENT ON DRUGMETABOLIZING ENZYME ACTIVITY OF VARIOUSHE PAT OM AS^^^ Enzyme system Hexobarbital Aminopyrene Aniline p-Xtrobenzoic acid
Phenobarbital pretreatment
-
+ + + +
DAB
Novikoff
Morris 5123 D
Morris 7800
Reuber H 35
Morris 7316
Morris 5123 B
0.00 1.01
0.00 0.34
0.00 0.51
1.35 3.21
-
1.31 2.07
0.60 0.77
0.02 0.03
0.00 0.00
0.32 0.34
0.03 0.16
0 05 0.18
-
-
-
-
-
0.00 0.00
0.08 0.30
1.23 2.57
0.15 0.50
0.25 0.49
0.19 0.24
-
0.00 0.01
0.35 0.59
0.61 1.06
0.29 0.44
0.45 0.55
0.55 0.55
-
Neoprontosil Compiled from data of Hart et al. (1965). The numbers shown represent the units of each enzyme activity. They are comparable with respect to a given activity, but :lot comparable to the figures of a different enzyme system. a
b
32
H. V.
GELBOIN
tumor in either control or phenobarbital-treated rats showed no enzyme activity with respect to the N-dealkylation of aminopyrene, hydroxylation of aniline, or reduction of p-nitrobenzoic acid. However, with the minimal deviation Hepatoma 5123-D, phenobarbital pretreatment caused an increase in hexobarbital metabolism, aniline hydroxylation, and p-nitrobenzoic acid reduction but did not affect the level of aminopyrene N-dealkylation or neoprontosil reduction. Thus the response of the different tumors to phenobarbital pretreatment varied considerably. The DAB-induced and the Novikoff Hepatomas, both of which are highly malignant “multiple deviation” hepatomas, showed sensitivity only t o the induction of hexobarbital metabolism by phenobarbital pretreatment. All of the other enzyme activities in these two tumors were not inducible by phenobarbital. With the slow-growing “minimum deviation” 5123 B Hepatoma, the intermediate levels of three enzyme activities in the tumors of untreated rats were not affected by pretreatment with phenobarbital. With Hepatoma 5123 D, phenobarbital pretreatment of tumor-bearing rats increased hexobarbital, aniline, and p-nitrobenzoic acid metabolism of the tumor but had no effect on aminopyrene or neoprontosil metabolism. Hepatomas Morris 7800, Reuber H 35, and Morris 7316 were all sensitive to phenobarbital pretreatment with respect to the enzyme systems with which they were tested. Thus, certain tumors, e.g., the Novikoff or DAB-induced hepatomas exhibit very low or nondetectable levels of activity with respect to the drug-metabolizing enzyme systems and are not inducible by phenobarbital. On the other hand, the minimum deviation tumors may exhibit either low or intermediate levels of enzyme and may either respond to phenobarbital or be insensitive, as is Hepatoma 5123 B. The latter is of particular interest since the enzyme-forming systems examined are present but uninducible. This is in contrast to the Novikoff hepatoma where the enzyme is neither present nor inducible.
B. THEEFFECT OF DIETARY FACTORS The studies of enzyme induction by MC and phenobarbital show the variability of responses of different tumors with respect to the different enzyme systems studied. Thus there are tumors which do not exhibit certain enzyme systems or the capability for the induction of these enzyme systems. Other tumors contain a moderately high level of endogenous enzyme activity which are, however, not inducible to higher levels; another class of tumors contain either very low or moderately high levels of endogenous activity and are inducible to higher levels by administration of polycyclic hydrocarbons or drugs. These results are similar to those obtained in studies on the regulation of enzyme syn-
CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION
33
thesis in a variety of rat hepatomas by dietary factors. Ono et al. (1963) studied the cffect of dietary protein on glucose-6-phosphate dehydrogenase, thymine reductase, and uracil reductase activity of rat liver and of Morris Hepatoma 5123. All three enzymes in normal rat liver were found to increase upon feeding a high protein diet and to decrease on a low protein diet. The magnitude of these changes in enzyme level were somewhat suppresscd in the livers of tumor-bearing rats. However, in the Morris Hcpatoma 5123 both pyrimidine reductases were completely insensitive to thc dietary content of protein. Glucose-6phosphate dehydrogenase activity showed a similar response to dietary protein in both normal liver and in the tumor. Weber (1963) reported no significant effect of fasting or refecding on the level of glucose-6phosphate dehydrogenase activity in the minimal deviation Hepatoma 7794 A. Weber (1963) also examined the effect of cortisone adniinistration on glucose-6-phosphatasc, fructose-l,6-diphosphatase (FDPase) , ant1 lactic dehydrogenase. Cortisone markedly increases glucose-6-phosphatase activity in liver, fails to elicit a response in the rapidly growing Novikoff tumor or Hepatoma 3924 A, and increases slightly the initially low levels of enzymes of Hepatomas 5123 D and 7800. FDPase induction by cortisonc was not observed in any of the four hepatomas studied. Lactic dehydrogenasc (LDH) activity was increased slightly by cortisone in Hepatoma 5123. Cortisone injection did not affect L D H activity in the other tumors. Pitot and hlorris (1961) examined the induction of tryptophan pyrrolase and tyrosine a-kctoglutarate transaminase and found that Hepatoma 5123 showed high endogenous levels of both enzymes. Neither tyrosine nor cortisone administration significantly changed the level of these enzymes in this tumor. Pitot and Morris (1961) suggest that the high level of tyrosine-a-ketoglutarate transaminasc in Hepatoma 5123 is due to a constant stimulation of the enzyme-forming system by endogenous cortisone, since in adrcnalectomized hosts the endogenous lcvcl of enzyme was low but was markedly stimulated by the administration of cortisone. Pitot e t al. (1961) found that dietary protein did not affect the level of thrconine and serine dehydrasc activity in Morris Hepatoma 5123, although these activities were markedly affected by dietary protein in the livers of control rats and tumor-bearing rats. I n another study Pitot and Morris (1961) found that tryptophan or cortisone did not affect the low level of tryptophan pyrrolase activity in the Morris Hepatoma 5123. I n a subsequent study, Cho et al. (1964) found that tryptophan pyrrolase was inducible in the Reuber Hepatoma H 35 and in the Morris Hepatoma 7793. This induction occurred by administering either tryptophan or cortisone. Cho et al. (1964) suggested that the
34
H. V. GELBOIN
inability to induce tryptophan pyrrolase in the majority of the hepatomas studied was due to a loss of their ability to maintain a stable RNA template for tryptophan pyrrolase synthesis. This was based on their findings that substrate induction in the hepatomas did not occur unless the synthesis of the RNA template was stimulated by corticosteroid administration. Bottomley e t al. (1963a) examined the effect of dietary protein content on threonine dehydrase and serine dehydrase in other hepatomas. The capacity of the hepatomas to respond to a high protein stimulus varied considerably. I n a series of seven tumors, a high protein diet increased the level of threonine dehydrase in two of the tumors, and caused no change in the moderate level of activity of one tumor or in the very low level of enzyme activity in three of the tumors. I n one tumor the moderate level of activity was decreased with the feeding of a high protein diet. I n another series of four tumors a high protein diet did not appreciably affect the level of activity of serine dehydrase in the tumors, although the same high protein diet greatly enhanced enzyme activity in the host liver. I n a subsequent study Bottomley e t al. (1963b) investigated the levels of glucose-6phosphate dehydrogenase and threonine dehydrase in normal liver and in hepatomas. They found what appeared to be a reciprocal relationship bctween thc induction of glucose-6-phosphate dehydrogenase and threonine dehydrase in normal liver. Using various combinations of diets and cortisone, they found that when one of these enzymes was induced to high levels the other remained a t a low level of activity, and vice versa. However, in their examination of a series of eight tumors this relationship was not maintained. A wide range of values was found for these two enzymes. Some tumors showed high levels of threonine dehydrase and low levels of glucose-6-phosphate dehydrogenase. I n other tumors the reverse was true, and in one tumor high levels of both cnzynies were observed after the feeding of a high protein dict. Bottomley e t al. (1963a) suggest that the “minimal deviation” hepatomas studied rcpresent a spectrum of tuniors with thc cndogenous enzyme I e ~ c l sand their inducibility varying within the range found in normal liver under different dietary and hormonal conditions. Pitot e t al. (1965) used actinomycin as a tool to investigate the messenger RNA template stability for certain enzymes of rat liver and hcpatoma. Their data suggest that in normal liver the template lifetimes for serine dehydrase, ornithine transaminase, and tyrosine transaminase are 6 to 8 hours, 18 to 24 hours, and less than 3 hours, respectively. The template lifetime for serine dehydrase in the Reuber H 35 was found to be less than 2 hours and in the Morris 5123 hepatoma greater than 2 weeks. Compared t o normal liver the template stability
CARCINOGEKS, E S Z T M E INDUCTION, A K D GENE ACTION
35
of serine dehydrase was thus diminished in the Reuber H 35 and considerably increased in the Morris 5123. I n other studies, thymidine kinaze (Pitot et al., 1964) and cleoxycytidine monophosphate (dCMP) clcairiinase (Iioth, 1964) messenger RNA templates mere reportedly more stable in tumors than in normal livcr. T h a t messenger RNA stability iiiay be altered in tuiiiors seems clear. Pitot (1964) has suggested a relationship between nicsscnger RNA tcriiplate stability and cancer. This possibility merits further investigation. Several studies (Greengnrd and Feigelson, 1961 ; Greengard e t al., 1963 ) suggest that tryptophnn pyrrolase induction by cortisone is mediated by the activation of genes transcribing messenger RNA for tryptophan pyrrolasc. Pitot (1963) has suggested that tryptophan pyrrolase induction by tryptophan is due to a n induction of synthesis a t the translational level or a t the messenger RNA-directed enzyme synthesis step. More recently Schimke et al. (1964) and Berlin and Schimke (1965) have examined the significance of both enzyme synthesis and degradation in respect to the kinetics of induction of rat liver tryptophan pyrrol:ise. Their findings, babed on an analysis of the time course of changing enzyme levels and the results of isotope incorporation into purified enzyme, indicate that hormone administration increases the rate of enzyme synthesis while the administration of tryptophan decreases the rate of degradation of the enzyme. Thus, tryptophan does not appear to act as an inducer of enzyme synthesis a t the translational level, but rather as a st:i tiilizcr of preexisting enzyme. Their findings indicate that tryptophan administration decreases the rate of degradation of tryptophan pyrro1:isc to zero, wliile 1iydrocortiFone increases the rate of synthesis of tlic enzyme 6- or 7-fold without altering the rate of degradation. Thcir studies suggest a half-life of 2 to 3 hours for tryptophan pyrrolase when tryptophan is not present. In contrast, when tryptophan is administered, there is essentially no degradation of the enzyme. Berlin and Pchiinke (196.5) (Tablv V I ; Fig. 5 ) liavc cxaniined tlie responsc to cortisone in respect to thc half-lives of four cnzymcs, tryptophan pyrrolase, tyrosine-glutaniic transaminase, glutamic-alanine transaminasc, ant1 nrginase. Thcse enzymes have half-lives ranging from 2 t o 96 hours. Four hours after hydrocortisone injection the increased activities ranged front a 9.G-fold increase in tryptophan pyrrolase activity to only a 1.1fold increase in arginase activity. Thus, it might appear superficially that tryptophan pyrrolase is considerably more responsive to cortisone atlininistration than is arginase. However, when the basal level of enzyme, tlie half-life of the enzyme, and the level 4 hours after cortisone :idministration were all taken into account, the ratio of enzyme synthesizml in the cortiFoiic-tt,e:ttecl rat to tlitit *ynthchizcd in the nonnnl rat
36
H. V. GELBOIN
T-4BLE VI UNDER BASALCONDITIONS AND SYNTHESIS COMP.IRISON OF RATESO F ENZYME DURING CORTISONE TREATMENT^ Basal
Enzyme Tryptophan pyrrolase Tyrosine-glutamic transaminase Glutamic-alanine transaminase Arginase
Enzyme synthesized
Half-life (hours)
Enzyme activity (units)"
2.5c 2.0d
0.05 1.3
0.014 0.45
0.056 1.9
4.0 4.2
4.2
0.034
0.24
7.0
2.3
9.9
4.3
841 96f
33
Normal (units/ hour)b
Cortisone Itatio of (units/ cortisone: hour)* normal
Berlin and Schmike (1965). A unit here refers t o specific activity; unit per gram wet liver. c As determined by following a decay curve from high levels induced with cortisone (Feigelson et al., 1959), by decay of normal levels after administration of puromycin (Nemeth, 1962) and by isotope decay under basal conditions (Schimke et al., 1965). d Determined by achieving high levels with two hydrocortisone injections a t 0 and 4 hours, and then sacrificing 3 animals every 2 hours during the decay (6-12 hours after second injection). The value obtained was 2.0 hours. 8 As determined by following a decay curve from high levels induced with cortisone (Segal and Kim, 1963). f As determined by following specific activity decay of prelabeled arginase (Schimke, 1964). Q
were similar. These ratios were 4.0, 4.2, 7, and 4.3 for the four enzymes studied. These results point out thc very important role of enzyme degradation on interpretation of changes in enzyme level induced either by polycyclic hydrocarbons, drugs, or hoi niones. Similsrly, they point out the difficulty in assessing messenger RNA templatc half-lives if one does not know the basal level of enzyme, its half-life or the rate of degradation of the cnzymc with the various treatment conditions. VII. Pharmacological and Physiological Significance of Polycyclic Hydrocarbons and Drug-Induced Enzyme Activity
A number of pharmacological and physiological effects of polycyclic hydrocarbons appear related to their activities as enzyme inducers. These phenomena includc the inhibition of liver carcinogenesis by the simultaneous feeding of polycyclic hydrocarbons, a reduction in the duration of drug action caused by polycyclic hydrocarbons or drugs and a polycyclic hydrocarbon-induced protection against adrcnal necrosis, atrophy of the testes or death in rats caused by the administration of 9,10diiiiethylbenzanthracene (DMBA) . The cvidencc that relates the indue-
37
CSRCINOGENS, ENZYME INDUCTION, AND GENE ACTION
irig ability of a compound and its physiological or pharmacological effect is greatest for the inhibition of carcinogcnesis and the altered duration of drug action. The relationship between enzyme induction and protection against acute toxicity, adrenal necrosis, and atrophy of thc tcstcs is uncertain. Finally, the administration of AlC reverses some of the symptoms of scurvy in guinea pigs being maintained on ascorbic acid-free diets. This may be rclated to the formation of prccursors of the vitamin induced by MC, since the latter is known to induce ascorbic acid synthesis in the rat.
Glutamic -alanine tronsaminase
transaminose
OO
1
I
I
2
3
1
4
Days of cortisone administration
FIG.5. Time course of the increase in tryptophan pyrrolase, tyrosine-glutamic transaminasc, glutamic-alanine transaminme, and arginase with cortisone adminietration. Animals received 10 mg. cortisone acctatc every 8 hours intramuscularly. Each value is the mran of thrce animals (Bcrlin and Schimltr, 1965).
A . EFFECT OF POLYCYCLIC HYDROCARBONS O N CARCINOGENESIS
It is well known t h a t certain polycyclic hydrocarbons induce tumors when applied to the skin of mice or when injected subcutaneously or intramuscularly (Hartwell and Stewart, 1942) in a number of species. Adult rats, however, are not susceptible to hydrocarbon-induced liver carcinogenesis. To the contrary, the siniultancous administration of polycyclic hydrocarbons with certain liver carcinogens abolishes or greatly rcduccs carcinogenesis by the liver carcinogens. Richardson et al. (1952) found that simultancous feeding of AIC with the potent liver carcinogen 3’-i~iethyl-4-dimethylaniinoazobenzcnc ( 3’-Me-DAB) greatly reduced the ability of the latter compound to inducc hepatomas. This finding was confirmed and further cxtcnded by Meechan et al. (1953), who investigated some of the time relationships involved in the
38
H . V. GELBOIN
inhibition of aminoazo dye liver carcinogenesis by MC. They fed a basal diet containing 0.06% of the potent liver carcinogen 3’-Me-DAB and a t intervals of 3 t o 18 weeks MC was added to the diet at a level
Tali:
EFFECTS O F
V.\RIOUS
LIVER TUM ORS
Series
Comporinds added to dictb
I
BY
TABLE VII HYDROCARBONS O N THE INDUCTION 3’-8fEl7HYL-4-DIMETHYL\MINO \ZOBENZENE”
POLYCYCLIC
No. of rats with Wt. increment No. liver tumors a t 1 rats at 3 5 month start months months (g.)
None -9 Me thylcholanthrene 24 Benep yrene 27 1,2,5,6-Dibenzanthracene 35 11 None 1 Methylcholanthrene 25 1,2-Benxanthracene 30 Pyrene -16 111 None -10 30 R/Iethylcholanthrene 20 (3,10-Dimethyl-l ,2-benzanthracene 25 9,10-Dimethyl-1,2-benzanthracene photoxide 0
OF
Gross cirrhosis
15
16 16 13 18 16 14 17 17
12 0 0 0 8 0 0 6 9 0 5
16 1 7 16 14 0 15
Moderate-severe None None None Moderate-severe None None-mild Moderate-severe Severe None Mild-moderate
17
3
16
Mild-moderate
16
16 16
1 1
Miller el al. (1958).
* Each of the hydrocarbons was added to the diet a t a level of 0.123 rnmoles per kg.
All rats were fed 0 0 3 % 3‘-methyl-L).lB in the diet. Each group contained 16 rats a t the start of the experiment.
of 0.0067%. When the hydrocarbon was added t o the diet prior to the sixth week of the 3’-R/Ie-DAB feeding, liepatoma formation was preventetl. When the hyclrocarbon was atldcd to the dict aftcr the sixth week but before the tenth week tlicrc was a partial inhibition of hepatoma formation, and wlieri it was added after the tenth week, no inhibition was observed. Miller e t al. (1958) investigated the effect of a nuniber of different polycyclic hydrocarbons on liver carcinogenesis by a variety of liver carcinogens of the aminoazo dye and the 2-acetylaminofluorene (AAF) class. Miller e t al. (1958) showed (Table VII) that the formation of hepatomas induced by thc feeding of 0.054% of 3’-Mc-DAB in the diet was strongly inhibited by the simultaneous fccding of 0.0033% of MC. I n addition, thcy found that benepyrene and 1,2,5,6-dibenzanthracene markedly inhibited arninoazo dye-induced hepatic carcinogcnesis. M’cak inhibitors werc 9,10-din~ethyl-1,2-bcnz-
CARCINOGENS, ENZYME INDUCTION, AKD GENE ACTION
39
anthraccne and its photoxidc ; pyrenc had no cff cct on 3’-Me-DABinduced tumor formation. The administration of MC also inhibited the liepatocarcinogenic action of 4’-fluoro-DAB and 2’,4‘-difluoro-DAB. I n addition to the inhibition of liver carcinogenesis induced by carcinogens of the aminoazo dye type, M C also inhibited carcinogenesis induced by AAF or 7-fluoro-2-acetylaminofluorcnc in several sites other than the liver. Thus, M C inhibited tumor formation induced by the AAF class of carcinogens in rat mammary gland, ear duct, and small intestine as well as liver (Table V I I I ) . Miller et al. (1958) investigated the mechanism of the polycyclic hydrocarbon inhibition of hepatocarcinogenesis and found t h a t the feeding of the hydrocarbons a t low levels caused the liver to maintain high levels of certain niicrosomal enzyme systems which metabolize the csrcinogcn to either less active or inactive forms. The feeding of 3’-methyl-DAB ordinarily causes a progressive reduction in the ability of the liver to N-clemethylate and to cleave reductivcly the aminoazo linkage of aminoazo dyes. The feeding of a protective polycyclic hydrocarbon, with 3’-Me-DAB, appreciably prevented the degree of reduction in enzyme activity observed when the dye only was fed. I n addition, when tlie protcctivc hydrocarbon was fed simultaneously with 3’-Me-DAB the levels of free aminoazo dye in the livcr and blood, and protein-bound aminoazo dye in the liver were similar to those observed in rats which were fed one half the amount of 3”-Me-DAB. These levels of free and protein-bound aminoazo dyes were altered to a lesser extent by the feeding of the less protective hydrocarbon 1,2benzanthracene and were not affected by feeding the nonprotective polycyclic hydrocarbon pyrene. Cramcr et al. (1960) (Table 11) showed in a subsequcnt study that pretreatnicnt with M C increased thc ability of rat liver t o hydroxylate the carcinogen AAF. These findings suggest that the protective action of M C toward carcinogcncsis by AAF may be due to enhancement of the hydroxylating enzyme systems which metabolize the carcinogenic AAF to noncarcinogenic phenolic metabolites. Other studies from the Millers’ laboratory indicate that the proximate carcinogen of AAF is the N-hydroxy derivative. Thus any agent which increases ring hydroxylation to a greater extent than N-hydroxylation would be expectcd to reduce the carcinogenicity of AAF. Lotlikar et al. (1965) reported an AlC-induced increase in hydroxylation in the ring positions and in the formation of N-hydroxy AAF in r a t liver. Furthermore, M C pretreatnicnt also increased reductase activity, i.e., the formation of AAF from N-hydroxy AF, in r a t livcr homogenatcs. N-Hydroxylation was increased 5-fold, whereas C-hydroxylation was stimulated 10-fold by pretreatment with MC. Since the N-hydroxy compound is closcr to the proximate carcinogen, and the C-hydroxylated
THE INHIBITION
BY
TABLE VIII METHYLCHOLANTHRENE O F TUMOR INDUCTION
BY
2-ACETYL.4MINOFLUORENE
A N D 7-FLUORO-2-ACETYLAMINOFLUORENE"
Group
Id
Compounds added to dietb AAF
16
+ MC
2d
AAF
3 4
7-Fluoro-AAF 7-Fluoro-AAF
5
MC
No. of KO. of rats with tumors in rats Wt. tumorincrement No. of rats Liver Ear Mammary Small free Time a t end of compounds compound duct gland intestine at 32 fed (wk.) Sex feeding (g.) Initial 23 wk.c 23 wk. 32 wk. (32 wk.) (32 wk.) (32 wk.) weeks
16
+ MC
14.5 14.5 16
M F M F M M
M F
98 49 133 58 6 136 200 72
30 30 30 30 18 18 6 6
26 29 27 29 7 17 6 6
13 0 0 0 7 0 0 0
22 0 0 0 7 4 0 0
11 14 1 0 2 0 0 0
0 22 0 4 0 0 0 0
3 2 0 0 1 0 0
21 20 0 12 6
0
5
1 2
Miller et al. (1958). AAF and 7-flUOrO-Akki refer to 2-acetylaminofluorene and its 7-fluoro derivative; these were fed as 0.03 and 0.02% of the grain diet, respectively. MC was added as 0.0036% of the diet. Alive a t 23 weeks plus those dead with a tumor prior to 23 weeks. d The data for groups 1 and 2 are the combined results of two experiments. b
F 5 0
M
r m
s2
CARCINOGESS, ENZYME INDUCTION, AND GENE ACTION
41
products are inactive as carcinogens, their results explain the reduced carcinogenicity of AAF in MC-treated rats and are consistent with the hypothesis previously developed in the Millers’ laboratory that the niechanisni of the M C protection of AAF carcinogenesis is through an induced enzymc formation resulting in a preponderance of the inactive C-hydroxylated products. Huggins et al. (196413) showed that the feeding of any of 6 polycyclic hydrocarbons inhibited mammary tumor formation induced by 7,12DMBA. The inhibition represented a decreased yield of mammary cancer, a delay in their appearance, and a complete suppression of tumor formation in a proportion of the protected rats. Lacassagne et al. (1934) applied mixtures of hydrocarbons to the skin of mice and found that the weakly carcinogenic hydrocarbons, for example, chrysene and dibenz [ a,g] fluorene, inhibited the production of skin tumors by the potent carcinogen MC. A number of subsequent reports (Riegel et al., 1951; Hill et al., 1951, 1952; Stanger et al., 1952; Steiner and Falk, 1951) demonstrated the inhibition by some polycyclic hydrocarbons of carcinogenesis induced by potent polycyclic hydrocarbon carcinogens. Falk et al. (1964) studied the effects of a number of hydrocarbons found in pollutcd urban air and cigarette smoke on sarcoma production by 3,4-benzpyrene. Figure 6 (Falk et al., 1964) shows the cffect of different polycyclic hydrocarbons given a t the ratios indicated on sarconiagenexis induced by benzpyrene. Benzo [ a ]fluorene, perylene, perinaphthoxanthene, benzo [ a ]carbazole, and chrysene were potent anticarcinogens. These compounds when given in the ratio of 0.10 to 0.15 of anticarcinogen to 1.0 of B P inhibited sarcomagenesis by more than 70%. Less active were benz [ k ]fluoranthene and benz [m,n,o]fluoranthene, and 2-naphthol. There is no evidence which relates the inhibitory effect of these hydrocarbons to their possible activities as enzyme inducers. The inhibition may be clue either to a stimulation of enzyme activity capable of detoxifying the carcinogenic agent, or may be related to an antimetabolite type of competition of the inhibitor and the active carcinogen for a relevant reccptor site in the cell. 1. Possibility of Enzyme Induction in Cancer Prophylaxis
It is clear that the activity of an organism’s detoxification mechanisms plays an important role in the ability of the organism to withstand insult froin a variety of foreign compounds. Experimentally, the induction of high levels of liver microsomal enzymes parallels the protective activity against aminoazo dye and AAF carcinogenesis. Many reports have shown that some of these detoxification mechanisms, eg., the enzyme system involved in aromatic hydroxylation, is present in a
42
H. V. GELBOIN
variety of organs and species. Wattenberg and Leong (1965) have discussed the intriguing possibility that the induction of these enzyme systems may be useful :is a protective measure against chemical carcinogenesis. They have suggested the possibility that the maintenance of artificially high levels of detoxifying systems may protect against INHIBITION OF CARCINOGENESIS CARCINOGEN 4 0 0 ~ gBENZO(a)PYRENE VEHICLE TRICAPRYLIN DURATION 15 MONTHS ANTICARCINOGENS ENCOUNTERED IN AIR POLLUTION
EENZO(a)FLUORENE 01 I
PERYLENE 0 I I
P P P P
PER1 NAPHTHOXANTHENE 01.1
BENZ(a)CARBAZOLE 0 15 I
CHRYSENE 0.15;l
BENZO(k)FLUORANTHENE I:I
BENZ(rn,n,o)FLUORANTHENE I:I
P
2-NAPHTHOL 5:l
L
ANTHRACENE PHENANTHRENE PYRENE 10 10 10.1
0
10
20 3 0 4 0 50 60 70 80 90 PERCENT T B A
F I ~6.. Inhibition of carcinogenesis by related polycyclic hydrocarbons. TBA = tumor-bearing animals (Falk et al., 1964).
specific types of carcinogenesis. In pursuing the feasibility of this approach, Wattenberg and Leong (1965) have examined the effects of a large series of compounds of different types and structure on the level of benzpyrene hydroxylase in the liver. The type of compounds tested ranged widely from a variety of oils, fats, and vitamins to various
CARCINOtiENS, E S Z Y M E INDUCTION, A X D GENE ACTION
43
drugs used clinically. Among the many compounds tested, phenothiazine and a number of its derivatives were found to be potent inducers of Lenzpyrerie hydroxylase in both r a t liver and in the niucosa of the m a l l intestine. Some of tliesc results arc shown in Table IX. The phenothiazine-type compounds, although of low toxicity for a number of species, have been found t o produce anemia and photosensitization, and niiglit be expected upon chronic administration to have detrimental effects on the organism. This docs not preclude the possibility that agents with potent inducing activity might be developed that could be chronically administered and would b e l ~ v cas protective agents toward specific types of carcinogenesis. Although this approach might be useful in specialized situations, the chronic administration of drugs in general should be approached with extreme caution. It would be of interest to investigate the relationship, if any, between a n individual’s susceptibility to lung carcinogenesis and the level of benzpyrene hydroxylase activity in the lungs and other tissues. I n other studies, Gelboin et al. (1965) have shown the marked inhibitory effect of actinomycin D on the initiation of mouse skin tumorigenesis by DRIBA. This inhibitor of DNA activity markedly inhibits the initiation process, suggesting that simultaneous D N A activity is required for “initiation.” The various studies cited suggest t h a t anticarcinogenesis might be approached in three different ways. Carcinogenically inactive compounds might be clcveloped which may ( 1) compete with carcinogens for active sites in the cell, (2) stimulate thc detoxifying mcchanisms of the cell which converts carcinogens to iiiactivc mctaholites, or ( 3) temporarily inhibit cellular processes which are requisites for carcinogenesis. Hypothetically, any or all of tliesc approaches might yield compounds capable of anticarcinogenic activity.
B. HYDROCARBON-INDUCED PROTECTION AGAINST ADRENALNECROSIS The administration of massive doscs of DMBA t o rats causes adrenal necrosis and induces death within one d:iy (Huggins and Fukunishi, 1964; Huggins et ,aZ., 1964a). Dao and Tanaka (1963a) found that a number of polynuclear hydrocarbons, including carcinogens and noncarcinogens, prevent DMBA-induced adrenal necrosis. Other studies iDao and Tanaka, 1963b) showed that the histological changes in the :Idrenal induced by DMB-4 were prevented by the prior administration of XIC. I n addition to MC, benzpyrene, benz [ a ]anthracene, anthracene, and phenanthrcne protect against DMBA adrenal necrosis. Anioiig the compounds tested MC was the most effective protector. As little as 1 mg. of M C prevented adrenal necrosis in 70% of the rats. The same pretreatment with 1 mg. of DMBA, 3,4-benzpyrene, and benz [ a ]anthracene
I+
TABLE IX EFPECTS OF PHENOTHIAZINES A N D CERTAINOTHERCOMPOUNDS ON THE BENZPYRENE HYDROXYLASE ACTIVITY O F THE RAT LIVER A N D THE MUCOSAO F THE SMALL INTESTINE'
I+
Benzpyrene hydroxyhse activity (units/mg. wet weight)"
Compound administered* None None Phenothiazine 1-Methylphenothiazine 2-Chlorophenothiazine 2-Trifluorophenothiazine 10-Acetylphenothiazine 10-Phenothiazine propionic acid Methyl-(10-phenothiazine) propionate 2-Chloro-lO-(3-dimethylaminopropyl) phenothiazine [chlorpromazine] 2-Chloro-lO-(3-dimethylaminopropyl)phenothiazine HCI [chlorpromazine HCl] 2-Chloro-lO-(3-dimethylaminopropyI) phenothiazine sulfoxide HCl [chlorpromazine sulfoxide HCl] 2-Chloro-lO-(3-trimethylaminopropyl)phenothiazine iodide [chlorpromazine methyl iodide] lO-(3-Dimethylaminopropyl) phenothiazine HCl (promazine HC1) 10-[2-(l-Pyrrolidyl)ethyl]phenothiazine HCl (pyrathiazine HC1) 2-Methylmercapto-lO-[2-(N-methyl-2-piperidyl)ethyl] phenothiazine HCl [thioridazine HCl] 2-Chloro-10-[3-(1-[2-acetoqethyl]-4 piperaziny1)propyll phenothiazine HCI [thiopropazate HCl]
Liver
Small intestined
16+4 14fl 311 +39 360 19 232 k 18 156 +40 374 23 18 +3 18fl 310f29 314 +45 300f35
13f3 16f4 55f9 81 5 8 56 k 19 23 +8 77 +25 17f7 16+4 144f23 105 k30 108 30
Water
13 +3
13f5
Water Water Water
188k29 268 k35 48 f19
49f23 117525 77 f28
Water
52 f 9
451
Vehicle Water Sesame oil Sesame oil Sesame oil Sesame oil Sesame oil Sesame oil Sesame oil Sesame oil Sesame oil Water Water
+
+
E < 0
8m sz
2-Trifli1oromethyl-l0-[3’-(l-methyl-4-piperazinyl)prop~l~phenothiazine [trifluoperazine] Acridan Phenazine Phenoazine Thianthrene 2-Chloro-9-(3dimethylaminopropylidene)thioxanthrene HCl [chlorprothixene HCl] 3,7-Bis(dimethylamino) phenazathionium chloride [methylene blue] Sodium 5-ethyl-5-phenylbarbiturate [phenobarbital sodium]
Sesame oil
22+3
7+4
Sesame oil Sesame oil Sesame oil Sesame oil Water Water Water
17+4
13+3 14+1 16 + 3 14+1 21 5 3 18+l 1x51
18+3 19+4 3958 48 f.14 34 + 3 34-13
U’attenberg and Leong (1965). 0.03 mmoles of each compound in 4 ml. of sesame oil or water (or only the vehicle) was administered postoperatively to 50-day-old female Sprague-Dawley rats 48 hours prior to sacrifice; 4 rats were in each group except for the sesame oil vehicle control, which contained 20 animals. Trivial names are in brackets after the chemical name. c Mean: S.D. Mucosa of the proximal 12 cm. of the small intestine. a
c)
+
m
46
H. V. GELBOIN
gave virtually no protection. The ininha1 protective dose nccded to induce 100% protection against DMBA-induced adrenal necrosis was 10 mg. with any of the four carcinogenic polycyclic hydrocarbons. When the noncarcinogens were used, the niininium protective dose was 25 mg. for anthracene and 100 mg. for phenanthrene. Huggins and Fukunishi (1964) investigated the time relationship of protection and found that 2 mg. of M C induced protection within 2 or 3 hours which lasted for 2 days. Ethionine administered prior to the MC or 2 hours aftcr M C prevented the protective effect of the hydrocarbon. With a higher dose of MC, 5 mg., the protection lasted for 6 days, and larger and repeated doses of ethionine were necessary to block the protective effect. Huggins and Fukunishi (1964) also investigated the protective effects of a large series of hydrocarbons and found all of the following to be active when given a t doses ranging from 0.25 mg. to 2 mg.: MC, 3-aminochrysene, benzo [ a ] pyrene, 3,9-dimethylbenz [ aI anthracene, 2-methylbenx [ a ] anthracene, 3-methylbenz [ a ]anthracene, 4-methylbcnz [ a ] anthracene, 5-methylbenz [ a ]anthracene, 6-methylbenx [a]anthracene, 7-niethylbenz [ a ]anthracene, 9-methylbenz [ a ] anthracene, 10-methylbenz [ a ] anthracene, 12-methylbenz [ a ]anthracene, benz [ a ]anthracene, 7,12-diethylbenz [ a ]anthracene, 6,8-dimethylbenz [ a ]anthracene, 6-aminochrysene, Diels hydrocarbon, cyclopentenophenanthrene, 7,12-dimethylbenz Ia ] anthracene, and 7,12-dimcthylbenz [ a ]anthraccne-16d. Although the protective effect of hydrocarbons on DMBA-induced adrenal necrosis is dependent on protein synthcsis, as suggested by the inhibition of protection by ethionine, Huggins and Fukunishi (1964) suggest that the protection is not due to the induction of enzymes, since large amounts of DMBA are not rapidly inactivated as a result of prior treatment with MC. Dao and Yogo (1964) found that benzpyrene hydroxylase activity was increased in the liver but decreased in the adrenals after treatment with methylcholanthrene, benzpyrene, or D1LIBA. Phenanthrene and anthracene had essentially no effect on benzpyrene hydroxylase activity but nevertheless were effective protectors of the DMBA-induced adrenal necrosis a t the dose levels used. Methylpyrone [2-methyl-l,2-bis (3pyridyl) -1-propanone] , an inhibitor of ll/3 hydroxylation of adrenocortical steroids, did not inhibit benzpyrene hydroxylation ; on the contrary, it stimulated benzpyrene hydroxylase induction and protection against DMBA-induced adrenal necrosis. Certain compounds were found to be inactive as inducers of benzpyrene hydroxylase activity when given a t low levels and only slightly active a t high levels. The high levels, however, exhibited a marked protection against adrenal necrosis. Dao and Varela (1966) found that puromyciii and ethionine prevented thc
C.4RCINOGES.C;, ENZTME INDUCTIOS, A S D GENE A C T I O N
47
adrenal protection induced by aromatic hydrocarbons. Actinomyciii D, under conditions where it prevented hydroxylase induction in the liver, did not affect the adrenal protective activity induced by aromatic hydrocarbons. The results suggest that protection of adrenal necrobis by hydrocarbons is dependent on protein but not RNA synthesis. Tanaka and Dao (1965) found that liver injury induced by the feeding of high-fat diet increased the susceptibility of the adrenal cortex to the induction of necrosis and hemorrhage by DMBA. On the other hand, liver injury induced with carbon tetrachloride did not affect the susceptibility of the adrenal cortcx to injury by DRIBA. Similarly, the induction of inaiiiniary cancer was ni:trltedly retarded in rats with fatty liver but not affected by liver injury induced by carbon tetrachloride. Thus carbon tetrachloride-induced injury did not affect either the sensitivity of the adrenal cortcx to damage by DRlBA or the incluctioii of mammary cancer by polynuclear hydrocarbons. Kotin et al. (1962) have shown that CCl, treatment markedly reduces the ability of liver to hydroxylate BP. It would be of interest to investigate the effect of CC1, liver damage on the hIC protection of DMBA-induced adrenal damage. Huggins e t al. (1964a) reported that very large doses of 7,12-DMBA induced the death of rats within one day after administration; MC, 3,9DRIBA, 6-aniinochryscnc, and Benz [ a ]anthracene all prevented the rapid death and allowed the survival of the rats for more than 2 months. I n addition, the atrophy of the testes induced by large doses of 7,12DMBA was prevented w1ic.n the latter compounds were administered prior to the 7,12-DhlBA. When ethionine was given just prior to the protective agent, MC, tlic protective effect was abolished. However, ethionine given 8 hours after the M C had no effect. Dao (1964) and Huggins and Fukunishi (1964) investigated the relationship between the molecular structure of the polynuclear hydrocarbon and its activity as a protector against DMBA-induced adrenal necrosis. Dao (1964) studied each of seventeen conipountls a t three dose levels. The data suggest that the most effective hydrocarbons are those with four or five benzene rings, and any significant reduction of the molecular size of the hydrocarbon reduces its protective activity. Tht shape of the hydrocarbon does not correlate well with its protective activity. Thus, naphthacene, a planar compound, is as active as bcnzanthracene, which is angular. Huggins and Pataki (1965) investigated a large number of aromatic azo dye derivatives, as well as a nuniber of derivatives of pyridine and aromatic ethylene derivatives, as protective agents. Over 40 compounds were tested, and a large proportion were found active in protecting against adrenal necrosis induced by DMBA. I n addition, many of these compounds induced meiiadione reductase activity in liver.
48
H. V. GELBOIN
The most active of the compounds tested was 1-(p-phenylazo) -2naphthol (Sudan 111).This compound was the most efficient in the protection against adrenal damage, in the induction of liver nienadione reductase, and in preventing hydrocarbon-induced mammary cancer.
C. THEEFFECTOF POLYCYCLIC HYDROCARBONS AND DRUGS ON DURATION OF DRUGACTION
THE
Conney et al. (1960) examined the effects of 3,4-benzpyrene and phenobarbital on zoxazolamine metabolism in vivo. Microsomes from 100
80
60 40
W
u 3 )
:2 0 W \
c W
-
E 5
10
2
8
2
N
W
3 - 6 4
2
0
60
120
180
240
MIN
FIQ. 7. The effect of benzpyrene and phenobarbital administration on the disappearance of eoxazolamine from rat tissues in vivo (Conney et ul., 1960).
either phenobarbital- or benzpyrene-treated rats hydroxylate this compound to a 3- to 4-fold greater extent than microsonies from control rats. Figure 7 (Conney e t al., 1960) shows the rapid disappearance of zoxazolamine in vivo; when the animal is pretreated with 3,4-benzpyrene the
49
CARCINOCESS, ENZYME INDUCTION, AND GENE ACTION
rate of drug disappearance is considerably greater than that in the control rats. Phenobarbital pretreatment, which affects microsomal activity to a lesser degree, also has a lesser effect on the disappearance of zoxazolamine in vivo. These results parallel those shown in Table X (Conney TABLE X EFFECT O F ])RUGS
O N THE I)liR\TION
OF n R U 0
ACTION
A N D O N TIIE l i C T 1 V I T Y
OF r)RUO-hIET \BOLIZING E N Z Y M E S '
Zoxazolamine
Pretreatment
Metabolism Metabolism by liver by liver Daily 1)iiration of microsomes Duration of microsomes dose paralysis (pmo1elg.l sleep (pmole/g./ (mg./kg.) (minutes) hours)b (minutes) 1iours)c
Control Zoxazolamine Phenylbutazone Diphenhydramine Aniinopyrine Barbital Orphenadrine Phenobarbital 3,4Benzpyrene 3-hlethylcholanthrene a
Hexobarbital
50 125 50 125 125 50 75 25 125
730 555 307 303 263 181 158 102 17 12
0.53 0.59 1.05 1.43 1.6 4 1.64 2 . 0'2 2.63 -
216 18 26 36 23 11 302 -
0.34 1.24 1.02 1.15 1 .20 1 47 0.33 -
Conney et al. (1960).
* Male rat,s ( 3 5 4 0 g.) were injected intraperitoneally
twice daily with drug for 4 days, except that the animals receiving 3,4-benzpyrene and 3-methylcholanthrene were injected only once 24 hours before a n intraperitoneal injection of zoxazolamine (100 mg./kg.) or hexobarbital (125 nig./kg.). Duration of drug action was estimated by determining when the rats regained righting reflex. In zdro enzyme assays were carried out wit,h liver microsomes fortified with a system that, generated excess reduced triphosphopyridine nucleotide. c Expressed as pmoles metabolized by microsomes from 1 g. of wet weight liver.
and Burns, 1960) in which the effect of various drugs on the duration of drug action a,nd on the activity of microsomal drug metabolizing enzyiiies was cxaiiiincd. With no pretreatment, zoxazolamine caused a paralysis lasting 730 minutes. With phenobarbital pretreatment the paralysis time was reduced to 102 minutes; and with benzpyreiie pretreatment, the paralysis t,ime was reduced to 17 minutes. I n a parallel manner, the metabolism of zosazolamiiic by liver microsomes was increased by phcnobarbitnl %fold and by bcnzpyrene approximately 4fold. That benzpyrene :tnd plicnobarbital do not have identical modes of action is shown by thcir effcct.s 011 hexobarbit,al metabolism anti on hcxobarbital sleeping time. Thus BP has essentially no effect on hcxobarbital
50
H. V. GELBOIN
metabolism and increases sleeping time slightly. On the contrary, phenobarbital pretreatment greatly enhances hexobarbital metabolism by liver microsomes and markedly decreases the duration of its action. Fujimoto et al. (1960) found that urethane pretreatment of mice accelerated hexobarbital slccping tinic. Fujinioto and Plaa (1961) found that both urethane and phenobarbital sliortcned duration of hexobarbital action but did not exert this action when either ethionine or carbon tetrachloride werc givcn simultancously. Studies of Remnier (1959) and Conney et al. (1960) suggest that barbiturates induce tolerance to themselves in part by increasing the activity of liver inicrosoiiial enzymes that metabolize barbiturates. Thus, if rats were pretreated with barbiturates or other stimulators of barbiturate-metabolizing enzymes, they became resistant to the hypnotic action of hcxobarbital or pentobarbital. Kato (1959a,b, 1960a,b) showed that pretreatment of rats by a riumbcr of drugs, such as phenobarbital, thiopental, chlorpromazine, or meprobamate shortened the duration of drug action of pentobarbital. The metabolism of these drugs in vivo was also accelerated by the indicated pretreatments. Ethionine was found to prevent the cffect of thiopental prctreatment on meprobamate nietabolism. The pretreatment of animals with foreign compounds may enhance thc metabolism of a drug from an inactive to an activc form and may thereby increase the activity of the drug. Murphy and Dubois (1958) showed that polycyclic hydrocarbons enhanced the toxicity of the diniethoxyester of benzotriazine dithiophosphoric acid and ethyl-p-nitrophenylbenzenephosphonatc. The examples of altered drug-action time induced by hydrocarbons or drugs are representative of many such reports. For a revicw of this subject, sce Conney and Burns (1962).
D. EFFECTOF CARCINOGENIC POLYNUCLEAR HYDROCARBONS ON ASCORBIC ACIDMETABOLISM Longenecker et al. (1940) reported an increased ascorbic acid excretion induced by tlic administration of certain narcotics, such as urethane, chloretone, and barbiturates. Although urethane is of known carcinogenicity, barbiturates have not bcen found to exhibit significant carcinogenicity. Several investigators have reported polycyclic hydrocarbon-induced increases in the excretion of ascorbic acid (Allen and Boyland, 1957; Conney and Burns, 1959; Boyland and Grover, 1961; Boyland and Jondorf, 1962). Conney and Burns (1959), Burns et al. (1960), and Conney et al. (1961b) demonstrated that M C caused a marked increase in the synthesis of L-ascorbic acid in rats. Dao et al. (1963) found that MC caused a significant increase in ascorbic acid synthesis in rat adrenals, but DMBA, which causes a necrosis of adre-
51
CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION
nals, greatly reduced the ascorbic acid content. Conney et al. (1961b) (Table XI) have shown that MC, chloretone, and pentobarbital markedly affect the body pool of ascorbic acid, its turnover rate, its excretion in the urine, and its catabolic metabolism, as measured by the appearance of C1400,from C' '-L-ascorbic acid. Although there are soiiic studies indicating a drug-induced stimulation of the pathway from glucose or galactose to L-ascorbic acid, these effects have been reported t o be only slight in
Drug pretreatment None Chloretone 3-Methylcholanthrene Pentobarbital
Turnover Body pool rate of Ah of A i (mg./100 ( w . / 1 0 0 g.) g./day) 10.7 I!). a 22.5 19.6
2.6 21.5 19.0 1l.G
Excretion of AA (mg./100 g./day)
Metabolism of AA (ing./100 dday)
0.40 10.2 7.1 2.4
2.2 11.3 11.9 9.2
Conney et al. (19Glb).
* Pentobarhital (30 mg.) or Chloretone
(45 mg.) was administered orally for 4 to 7 days prior to the administration of L-ascorbic arid-1-CIS. Riethylcholanthrene (10 mg.) was injected intraperitoneally daily for 4 days, and the L-ascorbic acid-l-CI4 was admiriistered 5 days later. A11 values are given on a milligram per 100-g. body weight basis.
MC-treatcd rats. Martin (1961) investigated the effect of MC on scorbutic guinea pigs and found that some of the pathology of scurvy was reversed by the administration of MC. MC caused a marked reduction in hemorrli:ige, a reduction of bone absorption, a preservative effect on dentiogcncsis, and prevented the death of odontoblasts. These effects did not appear t o be mediated through a n induction of L-ascorbic acid synthcsis in the guinca pig or by an increased rctention of the vitamin. Touster et nl. (1960) and Boyland and Jondorf (1962) reported t h a t ethionine prevented the hfC stiniulation of L-ascorbic acid excretion in thc rat. VIII. The
Mechanism of Polycyclic Hydrocarbon and Drug-Induced Increase in Enzyme Activity
The primary question relating to the mechanism of the induced increases in enzynie activity is wlicthcr they are due to an activation of preexisting enzyme or to an increase in the number of enzyme molecules. Conclusive evidence for an incre:tsed amount of enzyme protein can be obtained by an immunochcmical determination of increased enzyme protein or by extensive purification of the enzyme, coupled with isotopic
52
H. V. GELBOIN
analysis. Most, if not all, of the microsomal enzymes which are markedly stimulated by drugs or polycyclic hydrocarbons have not been solubilized and hence have not been purified to any appreciable extent. Further, there has been no immunological study showing that the increased activities represent increases in enzyme protein. Thus, there is no conclusive evidence that the hydrocarbon and drug-induced increascs in enzyme activity are due to net enzyme synthesis. There is, however, a considerable body of evidence which supports the hypothesis that the increased enzyme activities are due to a protein Synthesis which is dependent on a DNA-dependent RNA synthesis. These conclusions are bascd primarily on the findings that various inhibitors of either protein or DNA-dependent RNA synthesis prevent or markedly inhibit the MCor phenobarbital-induced increases in niicrosomal enzyme activity. In addition, there is evidence that M C and phenobarbital alter the protcinsynthesizing activity of the microsomes by changing their content of messenger RNA and their capacity to bind messcnger RNA. Other studies have also suggested that MC alters the RNA metabolism of the nucleus and, in particular, stimulates RNA synthesis. OF INHIBITORS ON TIIE HYDROCARBON INDUCTION A. EFFECTS OF ENZYME ACTIVITIES
Ethionine has been found to block protein synthesis by being incorporated in S-adenosyl ethionine and thereby preventing the synthesis of A T P (Villa-Trevino et aZ., 1963). Thus, the prevention of protein synthesis by this inhibitor may occur a t several different levels. Amino acid activation may be blocked, or this inhibitor may prevent the synthesis of nucleic acid precursors needed for either messenger RNA or for transfer RNA formation. Thus, ethionine may be acting at any of several sites where an inhibition should cause a subsequent decrease in amino acid incorporation. Several investigators have examined the effect of ethionine on the induction of enzymes by MC. Ethionine completely inhibits the induction of aminoazo dye N-demethylase activity by MC (Conney e t al., 1956). Ethioninc had no effect on the levels of this enzyme in control rats, and the simultaneous administration of methionine completely reversed the inhibitory cffect of ethionine on the induction (Fig. 8) (Conney et al., 1956). Ethionine also inhibited the induction of DAB reductase. Conney e t al. (1957) obtained similar results when they cxamined the effect of ethionine on the induction of benzpyrene hydroxylase by MC pretreatment. Ethionine completely prevented the induction, and the inhibition was not observed when methionine was given simultaneously. I n another study, Cramer et aZ. (1960) examined the effect of ethionine on the MC stimulation of micro-
CARCINOGEXS, E N Z Y M E INDUCTION, AND GENE ACTION
53
soma1 AAF hydroxylation. Here, also, etliioninc completely inhibited the MC induction of liydroxylase activity. The inhibition was reversed by mcthionine. Puromyciri blocks protein synthesis a t the microsomal level by preventing the transfer of soluble RNA-bound amino acid into polypeptide chains (Yarmolinsky :tnd D e la Haba, 1959). Gelboin and Blackburn (1963) (Fig. 9) examined the effect of puroinycin on the MC induction of benzpyrene hydroxylase in liver, kidney, small intestine, and lungs. To avoid the problem of chronic toxicity of the inhibitor these investigators used a short 7-hour period of induction.
20 -
15 -
T mJ
g
.+.
10-
m
a
Ethionine ( ~ M M ) Methionine ( 3 u M ) MC ( I M g )
0
0 0
+ 0 0
0
0
+
+
0
0
0
+
+
+ + +
FIG. 8. Antagonistic action of methionine on etliioninc inhibition of MCstimulated increase in demethylnse activity (Connep ct nl., 1956).
Puromycin completely inhibited the hIC-induced increase in activity in liver, kidney, and sniall intestine. I n lung the stimulatory effect of MC was inhibited by 50%. Dao and Varela (1966) found that puroniycin prevented both the induction of hydroxylase activity in liver and small intestine and the MC-induced protection against adrenal necrosis induced by DMBA. Kato and Gillette (unpublished observations) found that puromycin prcvcnted the plicnobarbital-induced increase in the activity of p-nitroanisol-0-demethylase and aminopyrene-N-demethylase. Orrenius et al. (1965) reported that puroniycin prevented the phenobarbital-induced increase in N-demethylation, T P N H cytochrome c
54
H.
V.
GELBOIN
reductase activity, and the CO-binding pigment of the microsomes. Conney and Gilman (1963) reported a complete inhibition by puromycin of the increase in aminoazo dye N-demethylase induced by either MC or phenobarbital. Puromycin alone had no effect on enzyme activity. When the puromycin was given a t various times after the MC or phenobarbital, thc induced level of enzyme was not lowered, but further increases in enzyme activity were prevented. Thus, when puromycin was given 10 hours after the enzyme had been induced by MC, the higher level of activity was not affected for 10 hours subsequent t o puromycin administration. This suggests that the enzyme in the control as well as in the
300
-2 U 2 4 6 7
'
250
p ppp pp
Q Normal
a
M~
YCtAchncmycmD IM tPuicmyctn
=
Y
2 200 I-
. = "
150
In
h z
= 100
so 0
LIVER
KIDNEY
SMALL INTESTINE
LUNGS
FIG.9. The inhibition of MC induction of benzpyrene hydroxylase by puromycin and actinomycin (Gelboin and Blackburn, 1964).
MC-treated rats does not have an extremely short half-life and that MC induction of enzyme activity is not due to an inhibition of the degradation of the cnzyme. Jervell et al. (1965) have reported that MC-inducible DAB reductase is also increased in rats by starvation. The induction by starvation is prevented by the simultaneous administration of either puromycin or ethionine but not by actinomycin D. This enzyme appears to have a shorter half-life than that suggested by the studies of Conney (1965) for aminoazo dye N-demethylase. The administration of puromycin 10 hours after the start of the fast decreased the level of enzyme so that it returned to its normal level within 48 hours. These investigators suggest that carbohydrate represses rat liver DAB reductase formation. A possibly related effect is that reported by Pitot and Peraino
CARCINOGENS, E N Z Y M E INDUCTION, AND GENE ACTION
55
(1964) of a glucose or fructoPe repression of hepatic threonine dehydrase :ind ortiithine-D-transaniinase. In each cabe reported, puromycin was found to be an effective inhibitor of polycyclic hydrocarbon or phenobarbital-induced enzyme activity. The question arises as to the dependence of new enzyme synthesis on newly formed messenger RNA. The induction may require activation of specific genes producing specific messenger RNAs or may involve the activation of stable messenger RNAs at the microsomal or translational level. To examine this aspect of the problem, several investigators (Conney, 1965; Gelboin and Blackburn, 1963) have used actinoniycin D, an inhibitor of DNA-dependent RNA synthesis (Reich e t al., 1961; Tamaoki and Muoller, 1962; Merits, 1963; Korner and Munro, 1963) on the drug-induced enzyme synthesis. Gelboin and Blackburn (1963) examined the effect of actinomycin on the MC induction of benzpyrene hydroxylase in liver, lung, kidney, and small intestine (Fig. 9 ) . During a 7 - 1 1 0 ~period of induction and a 9-hour period of exposure to actinomycin, the inhibitor was found to prevent completely the induction of bcnzpyrene hydroxylase in liver and small intestine and to prevent partially the induction of this enzyme activity in kidney and lungs. It is possible that in liver, small intestine, and kidney the messenger RNA for this enzyme is rapidly turning over, and a blockage or increase of enzyme synthesis depends on newly synthesized messenger RNA. On the other hand, the failure of actinomycin D to block the bIC induction completely in lung may reflect either an inability of thc inhibitor to reach its site of action in adequate concentration; or alternatively, the stability of the messenger RNA for these enzymes niay be greater in lung, and the induction process may take place a t least in part a t the level of microsonial translation. Cooney (1965) reported that actinomycin D inhibited the MCinduction of arninoazo dye il~-deniethylase in the livers of male rats. This study also suggested that in the liver the messenger RNA for this enzyme may be turning over rapidly, since actinomycin D administered 10 hours after methylcholanthrene prevented any further increase in the lcvel of enzyme. Actinomycin L> also prevented the MC-induced increase in rat liver DAB reductase activity and in the phenobarbitalinduced increase in the niicrosomal content of CO-binding pigment (.Jervell et al., 1965; Orrenius e t al., 1965). There h a r e been no reports of failure of nctinomycin D to prevent induction of microuomal enzymes hy methylcholantlirenc or by phenobarbital. Jervell et nl. (1965), however, reported that actinomycin failed to prevent the increake in DAB rcductase activity induced by starvation. This suggests that there niay be ii second nicch:inisni by which microsomal enzymes can be induced which
56
H. V. GELBOIN
is independent of DNA-dependent RNA synthesis. This may be similar to thc actinomycin D-insensitive enzyme synthesis reported by Grcengard e t al. (1963) for tryptophan pyrrolase induced in rat liver by tryptophan. This has been shown to be due to a stabilization by the substrate of tlic rapidly turning over tryptophan pyrrolasc (Berlin and Scliimkc, 196.5). The stiriiulatory cffcct of polycyclic hydrocarbons arid drugs on certain liver niicrosonial enzymes appears not to be mediated through the endocrine system, since the stimulation is observed in gonadectomizcd, adrcnalcctoiiiized, hypophyscctomizcd, or thyroidectoinized rats. Conricy (1965) reported that M C increases azo dye N-demcthylase in either hypopliy~ectomized~ or adrcnalectoinized rats.
B. STIMULATORY EFFECT O F P O L Y C Y C L I C HYDROCARBONS AND DRUGS ON PROTEIN SYNTHESIS Arcos et al. (1961) showed that many polycyclic hydrocarbons increase liver size, with a proportionate incrcase in total liver protcin. Within 4 days after the administration of MC, naphthacene, or anthantlirenc there was a 20 to 30% increase in liver protein contcnt. Although M C increased total protein content of the liver, it did not change the amount of protein per grain of liver. On the other hand, the adniinistration of phenobarbital and relatcd drugs caused an increase in both the total liver protein and in the amount of protein per gram of wet weight of liver. Conney et al. (1960) and Conney and Gilnian (1963) have found that phenobarbital, chlorcyclizinc, orphenadrine, or phenylbutazonc caused increases in microsoinal protcin per gram of wet liver ranging from 22 to 39%. Orrenius et al. (1965) rcportcd an increase in both protcin and RNA content of the microsomes of liver treated with phcnobarbital for a 120-liour period. T h e latter investigators fractionated the microsomes and reported an increase in both the agranular and granular endoplasmic reticulum 120 hours after the rats were treated with phenobarbital. The phenobarbital effect was considerably greater in the agranular endoplasmic reticulum where a 2-fold increase was observed ; the granular endoplasmic reticuluni increased only 20% upon Phenobarbital treatment. These results are consistent with the reports from several laboratories of a proliferative effect of phenobarbital on the agranular endoplasmic reticulum. Thus, Remmer and Merker (1963) and Fouts and Rogers (1965) have demonstrated by electron niicroscopy an increased content of the agranular endoplasinic reticulum of liver after rats or rabbits were treated with phenobarbital. The studies demonstrating a n increased protein content, a proliferation of the agranular endoplasmic reticulum, and an induction of a variety of microsoinal enzymes raise the question as to the effect of poly-
57
CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION
cyclic hydrocarbons and drugs on the protein-synthesizing systems of the microsomes. Kato et al. (1965) studied the effect of phenobarbital on the in vivo incorporation of Cl4-leucine into the various subcellular fractions of rat liver. Table XI1 (Kato et al., 1965) shows that phenobarbital affected only the labeling of the inicrosomal fractions. The proteins from a highly purified nuclear fraction, iiiitochondria, or the soluble fraction of the cell sap were labeled equally in control and in phenobarbital-treated rats. Microsomes, however, froiii phenobarbital-tre:ted rats showed approximately a 25% increase in the incorporation of leucineC14. This increase was observed both in thc deoxycholate (DOC) -soluble TABLE XI1 THEEFFECTS OF
P H E N O B A R B I T l L ON T H E I N C O R P O R 4 T I O N
L-LEUCINE-C'4
I N S U B C E L L U L k R F R A C T I O N S O F RAT
in
VtUO OF
LIVER"' *
Specific activity (c.p.m./mg. protein) Fraction Nuclei Mitochondria Microsomes Ribosomes DOC-soluble microsnies Supernatant
PB
Control
69 121 216 84 239 55
f. 8 f. 14 f 21 f. 6 f 36
f. 4
71 115 265 104 290 57
f4 k 11 k 10 f0 12 7
* *
%
1'
+3 -5 +23 +24 +21 +3
>0.05 >0.05 <0.001 <0.001 <0.01 >0.05
Kato et al. (1965). Groups of 6 female rats of the Sprague-Dawley strain and weighing about 180 g. were injected intraperitoneally with 80 nig. phenobarbitallkg. a t 42 and 18 hours prior to decapitation. The rats were fasted from food during the entire 42-hour interval. Each rat was injected intraperitoneally with 10 pc. L-leucine-C14/kg. 20 minutes prior to death. a
proteins of the microsomes, that is, in the lipoprotein component a s well as in the ribosomal coniponeiits of rat liver iiiicrobonies. Von der Decken and Hultin (1960) reported an increase of 22% in the in vztro incorporation of 1euci1ie-C~~ into microsoiiial protein. Gelboin and Sokoloff (1961, 1964), using two different types of in vitro amino acid-incorporating systems, found a 60% and 30% increase in Icucinc-C14 incorporation after the rats were pretreated with MC. Figure 10 shows the rate of incorporation over a 30-minute period in the preparations from normal and MC-treated rats. The increased incorporation of DL-leucine-C1' observed a t 15 minutes in preparations from MC-treated rats appears not to be due to any possible preservative effect by the M C pretreatment. Thus, both the initial rate and the total amount of amino acid incorporated a t 30 minutes was higher in the preparation from the MCtreated rats. If the effect of M C was to preserve the activity of the incorporating system, one might expect identical or similar initial rates.
58
H . V. GELBOIN
Pretreatment with phenobarbital causes a 163% increase in the leucineC14 incorporation. Gelboin and Sokoloff (1961) also observed the stimuwas latory effect of MC when sRNA-leucine-C14 or sRNA-pr01ine-C~~ used as a precursor in place of the free amino acid. This result suggests that a t least part of the stiinulatory effect of RIC on amino acid iricorporation is not due to soluble factors which may influence the formation of sRNA-bound amino acid. I n a subsequent study, Gelboin and Soltoloff (1964) found evidence for increased levels of GTP in rat liver supernatant of animals treated with MC. Supernatant from MC-treated rats was more effective in supporting microsonial amino acid incorporation than rat liver supernatant fluid from control rats. I
I
n "
1
I
/
CONTROL
I
I
15
30
, I 60
lNCUBATlON TIME (MINUTES)
FXQ.10. The effect of MC on in vitro incorporation of leucine-C" into protein (Gelboin and Sokoloff, 1964).
Gelboin (1964) and Kato et al. (1965, 1966) have examined the mechanism of the MC and phenobarbital stimulation of protein synthesis. They used techniques which enabled them to examine amino acid incorporation directed by endogenous messenger RNA and that directed solely by polyuridylic acid. The microsomal incorporating system can be operationally described as consisting of two parts, the messenger RNA and the microsomal amino acid incorporation site, i.e., the microsomal RNA protein complex on which messenger RNA "programs" amino acid incorporation. Preincubation of the rnicrosomes in the presence of an energy source, GTP, and Mg++removes phenylalanineincorporating activity, normally directed by messenger RNA. Phenylalanine incorporation can then be restored by the addition of poly-
CARCINOGEXS, ENZYME INDUCTION, A N D GENE ACTION
59
uridylic acid. Figure 11 shows that the relative rate of loss of phenylalanine-C14-incorporating activity in the microsomes from MC-treated rats parallels the loss of activity observed in the initially less active microsomes from normal rats. The loss of activity observed during the preincubation is not due to an irreversible inactivation of the microsornes but rather to a release of endogenous messenger RNA, since microsomes so incubated are reactivated by the addition of polyuridylic acid. The results showing similar rates of loss of messenger RNA activity suggest that the increased incorporation in microsomes from MC-treated rats is not due to an MC-induced inhibition of the loss of endogenous messenger RNA activity or to an MC-induced stabilization of the microsomal-bound inessenger RNA.
PREINCUBATION T I M E ( M I N U T E S )
FIQ. 11. Rate of removal of messenger RNA activity from microsomes from normal and MC-treated rats (Gelboin, 1964).
Figure 12 shows that with the nonpreincubated microsomes, i.e., those containing endogenous RNA, the addition of polyuridylic acid stimulated incorporation to the sanic extent in microsomes from control and MCtreated rats. This suggests that the number of messenger-free binding sites for poly-U is not affected by M C treatment. However, after thc removal of endogenous messenger RNA activity, the addition of polyuridylic stimulated phenylalnnine incorporation to a greater extcnt in the preparation from MC-treated rats than in normal preparations. This suggests that after preincubation there are more messenger RNA-free niicrosomal sites for the binding of polyuridylic acid in the preparations from MC-treated rats than there are in the microsomes from normal rats. Also, with no preincubation, the addition of 100 pg. of polyuridylic
60
H.
V.
GELBOIN
acid was sufficient t o saturate the binding sites of microsomes from normal and MC-treated rats. After preincubation, however, 100 pg. was sufficient to saturate the active sites of the normal microsomes but was insufficient to saturate the active sites of the microsomes from MCtreated rats. If one assumes that the newly available sites are those that were previously occupied by the released messenger RNA, then the results are interpreted to mean that the M C microsomes contained, prior to preinrubation, a greater amount of messenger RNA and, hence, upon preincubation more active sites became available in the M C preparation than in the normal preparation.
NONPREINCUBATED
PREINCUBA TED
.
MC,,'
?"
/
I
0
I 100
I
I
200
NORMAL
0
100
200
Kato e t al. (1965, 1966) found that phenobarbital caused similar but quantitativcly greater differences in the microsomal protein-synthesizing activity. They showed that the rate of removal of messenger RNA during a preincubation is not affected by phenobarbital pretreatment. They also showed that with no preincubation, added polyuridylic acid stimulates L-phenylalanine incorporation to the same extent in microsomes from control and phenobarbital-treated rats. Thus, microsomes froin phenobarbital-treated rats are more active in endogenous L-phenylalanine-C1* incorporation but are equal to microsomes from control rats in thcir sensitivity to exogenous messenger RNA, polyuridylic acid. After the removal of endogenous messenger RNA, however, the microsomes
CARCINOGESS, ENZYME INDUCTION, AKD GENE ACTION
61
from phenobarbital-treated rats are much more sensitive than control microsomes to polyuridylic acid- directed L-phenylalanine-C14 incorporation. This is probably due to a phenobarbital-induced increase in both the endogenous microsomal messenger RNA content and the total num-
Fro. 13. Phenylalanine incorporation in microsomes and ribosomes from normal and phenobarbital-treated rats (Kato et al., 1965).
ber of microsomal binding sites for messenger RNA. I n further examining this effect, Kato et al. (1965, 1966) found the increased activity of microsomes from phenobarbital-treated rats in respect to both endogenous mRNA-directed and polyuridylic-directed phenylalanine incor-
62
H. V. GELBOIN
poration was not observed in the ribosomes isolated from the same experimental animals. Figure 13 from Kato et al. (1966) shows that with nonpreincubated microsomes, L-phenylalanine-C*4 incorporation remains constant a t two levels of added microsomes, and that in each case the niicrosomes from phenobarbital-treated rats are about twice as active as controls. After preincubation, however, both control and phenobarbital microsomes are inactive unless polyuridylic acid is added to the system. With the addition of either 100 or 200 yg. of polyuridylic acid the phenobarbital microsomes are about twice as active as the control preparations. The lower half of the chart shows a similar experiment performed in ribosomes isolated from the same group of rats. Although there are marked differences in microsomal L-phenylttlanine-C" incorporation, there is only a negligible difference between ribosomes from control and phenobarbital-treated rats. This is true when ~-phenylalanine-Cl~incorporation is directed either by endogenous messenger RNA or by polyuridylic acid. Hence, the factors responsible for the stimulatory activity of phenobarbital are removed by deoxycholate treatment during the preparation of the ribosomes and are likely coniponents of the endoplasmic reticulum. Gelboin (1964) found that actinomycin D not only inhibited the M C induction of benzpyrene hydroxylase but also prevented the MC-induced changes in inicrosomal protein synthesis. Figure 14 shows the effect of actinomycin D and MC on L-phenylalanine-C** incorporation in the nonpreincubated microsomes and in the preincubated system with polyuridylic acid added. The inhibition by actinomycin D in the nonpreincubated system suggests that the maintenance of normal microsomal-incorporating activity depends on continued DNA-dependent RNA synthesis. When this process was blocked over a 16-hour period the amino acid-incorporating activity of the microsornes was lowered by 30%. M C given alone caused a 50% increase in incorporating activity. When MC and actinomycin D were both given the M C effect was prevented and the observed activity was similar to that of microsomes from rats given actinomycin D only. When the assay system utilized preincubated microsomes with added polyuridylic acid, the results were similar but more pronounced than that observed with the nonpreincubated system. Actinomycin D given over a 16-hour period decreased the number of available microsomal incorporation sites for polyuridylic acid activity. When MC was given alone there was an increase in the number of these sites, and this increase was prevented by the simultaneous administration of actinomycin D. These results suggest that the synthesis of microsomal binding sites for polyuridylic acid is a function of an actinornycin D-sensitive reaction and that there is appreciable synthesis of these sites during a 16-hour period. Yankofsky
63
CARCINOGEXS, ENZYME INDUCTION, AND GENE ACTIOK
and Spiegclman (1962) have shown the presence in Escherichia coli of a sequence in DKA complementary to ribosomal RNA. Presumably niicrosomal RNA is synthesized through a n RNA polymerase reaction, using this sequence as information. If this is so, actinomycin D would be expected to inhibit microsoinal RNA synthesis as well as messenger RNA synthesis.
c. THE EFFECTO F 3-hfETHYLCHOLANTHRENE
ON
NUCLEAR
RNA h'IETABOLISM Loeb and Gelboin (1964) and Hishizawa e t al. (1964) reported an incrwised orotic acid incorporation into RNA after M C treatment. MC ACT n0 .
*
n n
M C STIMULATION OF EP HYDROXYLASE AND - PHENYLALANINE- U - C - 14 INCORPORATION:
L
0 4 8 12 16 Hours
EFFECT OF ACTINOMYCIN D
L-Phenylolonine-U-C- 14 lncorporotlon
Benzpyrene Hydroxvlase
I
MC ACT-D
-
-
-
+
+ + - +
I
No Preincubafion
-
+
+ +
-
+
+
Preincubafed Po&urdylic Acid
I
-
-
+ +
-
t
-
+
FIG.14. The effect of actinomycin D on the MC stimulation of benzpyrene hydroxylase and endogenous mRNA-dircctcd and polyuridylic acid phenylalanine incorporation (Gelboin, 1964).
Jcrvcll e t al. (1965) obtained the same results using liver slices. The latter determined the specific activities of acid-soluble uridine nucleotides in the preparation from MC-treated rats and found them greater than those of the control rats. They pointed out, however, that the higher specific activities of the acid-soluble uridine nucleotides from the MC-treated rats did not account for the greater increase in the specific activity of the RNA. The above iiivestigators suggest that the increased specific activity of the RNA indicates a MC-induced increase in the
64
H. V. GELBOIN
rate of RNA synthesis. This conclusion must be viewed with caution, since the intracellular and intranuclear pool size of precursors of RNA, as well as the rate of degradation of RNA, may markedly affect the specific activity of the RNA. Bresnick et al. (1966) reported a failure to detect increased orotic acid incorporation after M C pretreatment. The latter did not invesbigate the specific activity of the RNA precursors. 1. R N A Content of Isolated Nuclei
An accumlation of RNA in the nuclei of MC-treated rats may be expected if M C increases the rate of nuclear RNA synthesis and docs not concomitantly increase the rate of RNA degradation of the transfer of RNA out of the nucleus. Loeb and Gelboin (1963, 1964) reported that nilC has no effect on the amount of DNA per gram of wet liver; furthermore, 4 hours after M C treatment the RNA content of isolated nuclei was the same as in the controls. However, 16 hours after MC treatment they found a significant increase in the amount of nuclear RNA. I n four experiments, there was a 15 to 50% increase in the RNA content of the isolated nuclei, as shown by an increase in the RNA to DNA ratio (Table XIII) . TABLE XI11 LIVERNUCLEAR RNA CONTENT OF CONTROL AND MC-TREATED RATSG Nuclear RNA-P/DNA-P
a
Expt.
Control
MC (4 hours)
Diff. (%)
1 2
0.20 0.20
0.20 0.20
0
Control
MC (16 hours)
0.20 0.20 0.24 0.25
0.23 0.30 0.33 0.32
0
Diff.
(7%)
Loeb and Gelboin (1964).
2. Characterization and Labeling o f Nuclear R N A from Normal
and MC-Treated R a t s Loeb and Gelboin (1964) reported that the sedimentation profile of nuclear RNA from control and MC-treated animals was grossly similar. These findings were confirmed by Bresnick et al. (1966). Normal nuclear RNA showed three peaks of RNA which corresponded to 33, 19, and 6s (Svedberg units). The stimulatory effect of M C on orotic acid incorpora-
65
CARCINOCESS, ENZYME INDUCTION, AND GENE ACTION
tion was unequal in the various RNA fractions analyzed but was largcst in the 9 to 23 S region (Loeb and Gelboin, 1964). This area corresponds roughly to some of the RNA fractions reported to have the greater amount of activity in stimulating the incorporation of amino acids into protein in a cell-free E . coli system. 3. Specific Stimulntory Activitu of Nuclear RiVA f r o m Normal and 111C -Treated X u t s
Loeb and Gelboiri (1964) tested the nuclear R N h obtained from norn1:tl and MC-treated rats for its activity in directing the incorporation of L-plienylalariine-I-C*~into protein in the cell-frce E . coli system of hlatthaei and Nirenbcrg (1961 ) . In this system, isolatcd nuclear RNA had 5-10 times the stimulatory activity of microeoinal RNA isolated by a similar procedure. Soluble RNA showed no activity. Table XIV T.IBLE XIV S T I M U L I T I O N O F PIIENYL I L l N I N E - C ' 4
ItNA
FROM
I N C O R P O R \TION
NORM \L
.\Ni)
lIC-'I'RE
I N T O P R O T E I N BY \TED
NUCLE\R
R \TS"
Hpecsific stirnulatory activity (ppmoles phenylalanine incorporated/lOO pg. nuclear RNA) Nuclear
RNh Expt. I Expt. I1 Expt. 111 a
(pg.)
S 11 25
Norm21
JIC
I1 3 14 8 12 :I:
16 2 23 2 15 9
lliflerence
(XI
+43 +57 +29
Loeb and Gelboin (1963).
shows the stimulation by nuclwir RX-4 from iiorinal and RIC-treated rats. The nuclear RKA from thc AIC-treated rats was more active than were equivalent amounts of RNA obtained from normal rats. Thus, a t each of the three levels of adtlcd RNA, the MC preparation was from 29 t o 57% more active. Recently Holland e t al. (1966) h a w shown that heated ribosomal RNA, as well as DNA, is able to act as template in the incorporation of amino acids in an E . coli system. These results suggcst the possibility that the increased template activity may not be due to a greater number of messenger RNA molecules but may be due to a n MC-induced alteration in the physical state of the RNA. Thus, the various studies on the effect of M C on nuclear RNA metabolism have shown that: (1) MC causes an increase in the uptake of orotic acid into nuclear RNA which suggests increased RNA synthesis; (2) MC increases the amount of RNA in liver cell nuclei; and (3) RNA isolated from the liver cell nuclei of MC-treated rats has
66
H. V . GELBOIN
greater stirnulatory activity in an E . coli phenylalanine-incorporating system, which suggests a greater messenger RNA content in the nuclear RNA from MC-treated rats. IX. Carcinogenesis and Gene Action
I n very general terms, carcinogenesis may be described as a series of changes that occur in a tissue when it is exposed to the appropriate stimulus, and which results in the phenotypic traits that characterize a tumor. A number of different agents or conditions may effectuate the carcinogenic transformation-viruses of both the DNA and RNA type, ultraviolet and X-radiation, chemical agents, including literally thousands of different structural types, films of metals, plastics, or glass, hormonal stress of the appropriate severity, and finally normal cells become malignant when they are simply propagated in tissue culture in vitro. Are there common features to all these carcinogenic transformations? The multiplicity and heterogeneity of causal agents strongly suggests that a t least the initial cellular sites of carcinogen action are quite different. On the other hand, although there are indeed a variety of carcinogenic conditions, they often antagonize or complement cach other’s carcinogenic action. Examples of this are the synergistic and antagonistic effects of hormones and chemical carcinogens (Clayson, 1962) and the synergism between X-radiation and carcinogens, such as urethane in experimental leukemogenesis (Kawamoto et al., 1958; Berenbluin and Trainin, 1960). Although the diversity of carcinogenic agents implies that there is no single common, initial site of action for all carcinogens, the ability of these agents to act concertedly or antagonistically suggests their action to be the result of their impingement on common cellular systems altered during carcinogenesis. This is more strongly suggested by the fact that although cach tumor has uniquc characteristics, there is nevertheless a tendency toward a convergencc of biological and enzymatic character during carcinogenesis. Thus, tumors arising from different tissues tend to lose their specializcd characteristics and become more similar to one another than are the tissues from which they originated. Based on enzyme analyses of a number of transplantable tumors, Greenstein (1954) formulated the “convergcnce” hypothesis of cancer in which he concluded that “tumors tend to converge enzymatically to a common type of tissue.” Some of the phenotypic deviations from the normal that are seeii in tumors are a loss of growth control, a loss of contact inhibition, greater invasiveness and mctastnscs, a loss or gain of spccializcd func-
CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION
67
tion, a n increased aerobic glycolysis, a deletion or appearance of specific antigens, an alteration in the enzyme profile primarily involving the loss of enzyme activity, and finally a loss in responsiveness to environmental stimuli as seen by a decreased enzyme inducibility. The degree of change of a given character from normal is variable. I n some cases the property may remain similar or deviate only minimally from the normal tissue, and in other cases the deviation may be gross. The phenotypic change may be present in a stable state or may progress away from the normal either slowly or rapidly and thus reflect the relative malignancy of the tumor. This is often observed with successive tumor transplantation. I n addition, a relatively stable condition may bc modified to the progressive state as in the case of two-stage skin tumorigenesis (Berenblum, 1963). Here the carcinogen-induced first stage may represent an alteration from normal which remains stable and unrecognized until it is modified to the recognizable tumor state by treatment with croton oil, a noncarcinogenic or weakly carcinogenic irritant. Since phenotype is the end product of genetic activity and the alterations observed in tumors are inherited by successive generations of its cells, it is necessary to relate the observed changes in the tumor either to changes in the genetic information, i.c., DNA or changes in the regulatory rncchanisrns controlling the c~xpressionof genetic information into specific phenotypic character, i.e., the gene action system. I n other words, the phenotypic changes observed in carcinogenesis must be related either to modifications in the chemical structure of the gene itself or to changes in the system that transcribes information from the gene into particular messages, which are then translated into specific enzyme proteins. Thus carciriogenesis involves either mutational or genetic regulatory changes. For this discussion wc shall neglect the problem of the conversion of a distal or inactive carcinogen to its active or proximate carcinogen form, and consider the earliest relevant event between the proximate carcinogen and the cell during carcinogenesis. The first possibility is that the carcinogen interacts with the gene or DNA itself or that it interferes with genc reduplication in a manner which causes a change in the primary structure of the gene, that is, a deletion, modification, or addition of nucleotidc bases in DNA. DNA structure is known to be altered by radiation, and this may be the primary event in radiation carcinogenesis. Chemical carcinogens also bind covalently to DNA. Brookes and Lawley (1964) have demonstrated a positive correlation between the carcinogenicity of a series of polycyclic hydrocarbons and their binding to DNA, and Magee and Farber (1962) have shown the alkylation of DNA by nitrosamine. In addition, nitrogen and sulfur mustards that are carcinogenic alter DNA structure (Brookes and Lawley, 1960).
68
H. V. GELBOIN
Recently Sporn and Dingman (1966a,b) have detected a binding of the carcinogen AAF t o highly purified rat liver DNA. It is evident, however, that covalent binding to D N A is not the only type of interaction that may changc D N A structure or activity. Thus potent mutagens, e.g., manganese or acroflavine dyes, do not covalently interact with DNA and induce mutations. Furthermore, actinoniycin D is not covalently bound to DNA, but rather, is complexed by weaker bonds (Reich and Goldberg, 1964), and yet exhibits a profound effect on DNA-dependent DNA synthesis and DNA-dependent RNA synthesis. Thus, thc transitory contact of a compound with the appropriate steric and electronic structures might induce base alteration in D N A or drastically alter D N A activity without being covalently linked. I n the case of viral carcinogenesis i t has been suggested that the virus genome may be incorporated into DNA much as in prophage. Thus, Axelrocl et al. (1964) have dcrnonstrated an increased complementarity between polyoma virus D N A and the D N A of polyoma virus-induced tumors. T h a t oncogciiic viruses may be incorporated into host cell D N A is also suggested by the continued presence in sonie virally induced tumors of virus-specific antigens. Although it is clear that many carcinogens alter the structure of DNA, is altered DNA structure an obligntc of carcinogencsis? The answer to this question is unknown, since there is no adequate means for the analysis of the primary structure of DNA. At the present one cannot detect the prcscnce of a single or even multiple alteration in the stiucture of D N A except in microorganisnis by genetic analysis. Sincc one cannot answer the question of altered D N A structure as a rcquisite for carcinogenesis, one might consider the altcrnativc possibility : that carcinogens affect thc activity of genes without changing the structure of the genetic material and that thc altered pattern of genetic activity is hcritable. I n this model the structure of the gene remains intact but thcrc is an altered pattern of gene cxpression which may be then amplified be subsequent alterations to yield eventually a pattern of messenger RNA and enzyme synthesis t h a t characterizes the tumor. Current concepts of embryonic differentiation invoke this type of model. Thus, cells of widely varied phcnotype dcvclop from a common ancestor and have no detectable differences in either their D N A content, in the ratio of nucleotides in their DNA, the hybridizing properties of the DNA, or in their chromosome morphology. All of these facts plus the exactness of the D N A replicative process suggest the identity of the D N A of somatic cells of differing phenotype. Based on hybridization studies RlcCarthy and Hoyer (1964) have estimated t h a t a relatively small proportion of a cell’s DNA, about 776, is active in messenger RNA synthesis. Thuq, differentiation and the diversity of phenotype among tissues is thought
CARCINOGENS, ENZYME INDUCTION, A E D GENE ACTION
69
to be the result of the tlcvclopnient of differing loci of activity on identical DNA molecules. Although the early events of carcinogenesis may be alterations in either gene structure or in gene expression, it s e e m t h a t subsequent events always involve a large nurnbcr of progressive alterations in the expression of cliffcrciit genes. The rcasoii for this conclusion is t h a t a single or even several mutations, if taken as isolated events, would delete or alter a singlc or scvcral enzymes. Fuitliernioi,c, even in the case of viral carcinogcncsis, i t lins t m n cstiniatcd that tlicre arc no more t h a n 5 t o 10 genes present in the vir:iI genome. T h i s is based on tllc finding by Wcil and Vinogratl (1963) of a molecular w i g l i t of 3 x 10, for polyonin virus DKA. Tlius, tlic+c genes woul~limpart perhaps 5 t o 10 new proteins to tlic infected cell. Tlic lwgc plicnotypic tliffcrenccs between normal and tumor cells cannot be explained by such a relatively sniall number of changes antl must involve :t large number of events subsequent to the initial cliangc~s.I n other words, although tlic initial changes m a y involve alterations iii either structure or activity of one or a few genes, this event must he nniplificd into v:iriutions froiii tlie normal in t h e activities of a large number of g c w s . This arnplifictttion would result in a, pattern of gene expres~iont h t evcntunlly charncterizes the tumor cell. This concept can acconiniodatc many of the apparent inconsistencies in t h e different liiiitls of cnrcinogeiiic progressions. In certain cases, the initial altcrations in gene activity niay progress t o the event ua 1 turn or st R t e wit 11out c o n t i nu ous nppl ic a t ion of cii r cinogen or promoter. Tllis would bc tlic ca>c wlicii :I single (low of carcinogen induces tumor formation. 111 othcr c:tses, tlic initial alteration in t h e pattern of gene cxprcsion may require further progression by the imposition of either iiior(~ carciiiogcn, tlic propw liornional conditions, or a so-called pronioting ngciit. Once tlic pattcim of g t w cxlwession has reached a particulnr state, further progrcssion niay proceed w ~ t h o u t tlic presence of carcinogen antl might I)c incxorablc, since tlie cell with tlic iicw pattern of iiiewngcr RN.4 and cnzymc synthesis m:ty have the advnntagc over Its noriiial neighbors i f it protluccs a cell morc active in cell division. I n this type of m c c h a n i ~ mcilcli species and tissue llas a spccific resistmce to a given rnrcinogcn since cncli tis\uc is operating with a highly specific gcnc-action system, i c , p:ittcm of RNA a n d protein synthesis. Also this motlcll accommodates the well-known genetic factors in tumor susceptibility. I n order to account for the progressivc nature of the normal to neoplastic conversion after transitory contact with the carcinogen, this hypothesis requires t h a t the activity of different sets of genes be intcrlocked in some manner. This interloclting may occur cithcr a t tlie cytoplasmic or at t h e genetic level, b u t since the progression is licritablc, it must in all cases impinge in some manner
70
H . V. GELBOIN
on the genetic level. Thus, in an interlocking system the turning on of one set of genes would concomitantly switch off another set. Figure 15 shows a model of an interlocking gene-action system, which is based on the Monod-Jacob repressor model with the added suggestion of Waddington (1962) that there is more than one regulator gene for each structural gene. This has been called “cascade regulation” and brings more attention to bear on the potential interlocking nature of the gene-action system. Thus, a n active operori is controlled by regulator gene R , and contains a second regulator gene R,, which produces a molecule P, which is repressing a second set of genes. I n this way sets of genes can be interlocked so that when one set is active in RNA and protein synthesis, the other is inactive. This model is based on repression and on the model of Monod and ,Jacob (1961) for genetic regulatory mechaNORMALLY ACTIVE
RG
,
I
0
SG,
SG2
NORMALLY INACTIVE RG2’
rO
SG3
11
I
I
’
I
I
I
I
RNA
‘
RNA PROTEINS
-- _ - _ - - - - - - - - - - - - - - - -
i
t
I
I I
NORMAL ENVIRONMENT
SGq’
T
I
tPROTEiNS I
1
I
I
_t--L-
-ACTIVE
---- INACTIVE
FIG.15. Interlocking gene-action systems. RG = regulatory gene; 0 = operator; SG = structural gene; P = repressor.
iiisins and would predict an altcrcd iiiesscnger RNA metabolism. This has h e n suggcsted a s an early event in carcinogenesis (Loeb and Gelboin, 1963, 1964; Kidson and Kirby, 1965). This model has been elaborated upon in its possible relationship to carcinogenesis by Pitot and Heidelbergcr (1963). Although little is presently known about the inechanism of gene regulation, any number of models with interlocking gene-action systems may be proposed if one accepts two premises; first, that certain genes affect the activity of other genes, and second, that erivironmcntal conditions alter this gene-gene regulation. The first premise has been well established in microorganisms by both classic genetic studies as well as studies in enzyme induction (Monod and .Jacob, 1961). I n higher organisms this is suggested by studies of p glucosidase in yeast by Halvorson (1961) and by McClintock (1956)
CARCINOGEKS, ENZYME INDUCTION, AND GENE ACTION
71
in maize. The second premise, that envirorimcntal conditions can alter gene regulation, is supported by a number of studies on the effects of nutritional and hormonal factors on mammalian enzyme induction as well as by the classic studies of enzyme induction in microorganisms. With an interlocking system, the phenotype of a cell may be permanently altered by a transitory exposure to a n environmental agent. This can occur with no change in genotype. Beale (1950) has shown t h a t the transitory exposure of paramecium to a specific temperature induces the formation of a specific antigen. The production of this antigen continues for more than 50 generations after the original organism’s single experience with the new temperature. The basic fact of carcinogenesis is similar. The phenotype of a tissue is altered by its transitory exposure to a carcinogen. If carcinogenesis proceeds through this kind of mechanism, then what are the events that might alter gene activity and initiate this type of redifferentiation. In considering the multiplicity and variety of carcinogenic stimuli one may concludc t h a t the initial site of carcinogen action may be different in each situation. Within the broad framework of this concept one could speculate on how any number of events might induce carcinogenesis. First, the deletion of a rcprcsbor protein by a carcinogen may have a direct effect on gene activity. R.liller and Miller (1955) observed a good correlation between the binding of a series of aminoazo dyes to soluble liver proteins and their carcinogenic activities. Heidelberger and Moldenhauer (1956) and Abell and Heidelberger (1962) have demonstrated a positive correlation between the binding to certain skin proteins and the carcinogenic activities of a large series of polycyclic hydrocarbons. Thc studies of soluhle proteins during liver carcinogenesis suggest t h a t a certain class, the 11 proteins which interact to the greatest extent with aniinoazo dyes, may be implicated in carcinogenesis (Sorof e t al., 1951, 1958, 1963). This class of proteins is almost totally lacking in the tumor (Sorof and Cohen, 1951). I n other studies, interactions between carcinogens and RNA have becn demonstrated by Magee and Farber (1962). More recently reports have accumulated dcrnonstrating a positive correlation between the carcinogenicity of a series of polycyclic hydrocarbons and the degree of their binding to mouse skin DNA (Brookes and Lawley, 1964). I n other recent studies the liver carcinogens, AAF, 3’-hIe-DAB, and aflatoxin, liave hcen found to bind to liver D N A and to reduce the RNA to D N A ratio (Sporn and Dingman, 1966a,b; Sporn e t al., 1966; R o l m t s and Warwick, 1966). The functional significancc of the interactions with DNA may be reflected in altered template activity. Thus aflatoxin and the carcinogenic aminoazo dyes
72
H. V. GELBOIN
reduce RNA synthesis as measured by DNA-dependent RNA polymerase (Gelboin et al., 1966; Gclboin, 1967). Thus, chemical carcinogens may interact with any or all of the informational macromolecules of the ccll DNA, RNA, or protein. Similarly, radiation or hormones may interact with any of these components. The question arises concerning carcinogenic stimuli which do not interact directly with cellular components; for example, inert metals or plastics which are carcinogenic, or in vitro cell culture conditions in which normal cells become malignant. Here the agent may affect the normal environment of the cell or the cell-ccll interactions which maintain genetic regulatory substances a t nornial conccntrations. It is of interest t h a t plastic films induce sarcomas, whcrcas idcntical quantities of plastic powdcrs do not (Oppenlieimer e t al., 1955). This may suggcst the requirement for a n appropriate molecular interchange between cells in order for them to continue their existence in a normal differentiated state. This is, of course, suggested by many of the studies of Eagle (1965), which have shown a number of population-dependent nutritional requirements in mammalian cells grown in tissue culturc. For example, cells grown without cystinc necd a population density of 200,000 to 500,000 cells/ml. to permit growth. Cells grown in the prescnce of cystathionine could be grown a t a density of 10 cclls/ml. (Eagle and Piez, 1962; Eagle et al., 1961). That cell-cell interchange may be disturbed in populations of tumor cells is also suggested by the apparent differences in the membranc structure of tumor cells which exhibit rclativcly little contact inhibition. This may be a n indication of altered membrane components needed to regulatc the transfer of metaholites required for thc integrity of tissue specific gene-action systems. Thus, any event such as a direct carcinogcn interaction with cellular macromolecules or an altered level of a substrate affecting gene activity may alter gene-action systems and hence initiate carcinogcnesis. A number of studies have shown that RiIC affects gene activity, i t . it is affecting the DNA-dependent production of RNA. It is not known that the primary interaction of MC is with the gene, but it has been cstablishcd that soon after MC is adniinistercd there is an alteration in gene activity. Howcver, other compounds that are noncarcinogenic such a s hormones and some drugs such as phenobarbital also induce changes in the gene-action system. Furthermorc, carcinogens may alter gene activity in tissues which are resistant to malignant transformations like MC in rat liver as well as in those tissues which are susceptible. It may be t h a t in tissues not undergoing malignant transformation thc alterations in gene expression revert back to the normal state as in a target tissue’s response to a hormone. I n tissues susceptible to the carcinogen’s action, these changes may be irreversible and may progress in the direc-
73
CARCINOGENS, ENZYME INDUCTION, AND GENE ACTION
tion of malignancy. I n exainining this hypothesis one might ask: “DO the early stages of transformation require alterations in gene action?” If they do, transformation would not occur in the absence of gene activity. The studies reported by Gclboin e t al. (1965) used specific inhibitors of gene action to investigate this question. They studied the effect of actinomycin D, an inhibitor of gene function, applied a t various TABLE XV A N D PUROMYCIN O N DRIB.\-CROTON OIL SKINTUMORIGENESIS“
EFFECTOF ACTINOMYCIN D
Expt. Group 1
1 2 3
Treatment (compound in p g . )
DMBA (12) DMBA (12)
+ Act. D (84) DMBB (12) + Puroniycin
Avg. Effec- AIicc tumors DXBA tive with Total per control total tiimors tumors mouse (70) 32 32
20 9
89 19
2.8 0.6
100 21
30
17
71
2.4
86
42 43
26 1.1
121 39
2.9 0.9
100 31
44
34
152
3.5
121
40 40
34 22
165 58
4.1 1.5
100 35
(1328) 2
1 2 3
3
1 2
DMBA (12) DMBA (12) (98) DNBA (12) (1666)
+ Act. D + Puromycin
DMBA (20) DiMBA (20)
+ Act. D (15)
~~~
a In experiment 1 the IIMBA\was applied in 0.2 nil. of acetone to groups 1 and 2 a t 8 hours and to group 3 a t 6 hours; 14 pg. actinomycin L) in 0.2 nil. of acetone wits applied a t 0, 3, 6, 10, 13, and 16 hours. Group 3 received 1.338 nig. of puromycin (0.166 pg. in 0.2 ml. of acetone at 0, 1.5, 3, 4.5, 7.5, 9, 11.5, and 13 hours). Groups not receiving in hibitor or carcinogen received acetone a t corresponding times. In experiment 2 DLIBA was given to groups 1 and 2 a t 7 hours and to group 3 a t ci hours. Actinomycin wits given (14 pg./0.2 ml.) a t 0, 3, 6, 8, 11, 14, and 17 hours, and a total of 1.666 mg. of puroniycin was given in 10 doses a t 0, 1.5, 3, 4.5, 5.5, 7.5, 9, 11.5, 13, and 14.5 hours. I n experiment 3, DMBA was applied a t 2 hours and actinoinycin D (5 pg./0.2 ml.) a t 0, 5, and 8 hours. All groups received croton oil once weekly beginning one week after the start of the experiment. Experiments 1, 2, and 3 were terminated a t the end of 22, 24, and 17 weeks, respectively (Gelboin el al., 1965).
intervals before and after a sinall dose of DMBA, on subsequent tumor formation. This technique permitted examination of the dependence of the initiation process on simultaneous gene activity as well as the period of time required for the completion of initiation. Table XV shows the effect of actinoinycin D and puromycin on DhlBA-initiated, croton oilpromoted skin tuniorigenesis. The inhibitor was applied on the same
74
H. V. GELBOIN
day as the single dose of carcinogen. In three experiments actinomycin inhibited tumor formation of 79, 69, and 65%. Puromycin had no effect. In the same study puromycin was not found to inhibit protein synthesis in the skin. This, of course, clouds the relevance of the biological data with puromycin. A n altered rate of carcinogen removal was not the reason for the actinomycin D inhibition, since the rate of disappearance of HJ from DMBH-H3-treated skin was not affected by actinomycin D. This study also reported the inhibitory effect of actinomycin D on skin tumorigenesis induced by a single large dose of DMBA (200 pg.) or skin tumors induced by urethane. Gelboin e t al. (1965) also studied the effect of actinornycin D on “initiation” when it was giveii a t varying times relative to the time thc, carcinogen was given. hlice treated with actinomycin D on day zero, the day the carcinogen was applied, developed 5 to 33% as many tumors as the controls. Actinomyciii D given 1 day after the carcinogen was equally effective as an inhibitor as when it was given on day zero. When actinomycin D was given 7 days prior to the carcinogen, there was only a slight inhibition which was probably due t o the presence of residual actinomycin D a t the time the carcinogen was applied. When actinomycin D was given a t day 4,the inhibition observed was considerably less than that observed at day zero, and when it was given a t day 7, there was essentially no inhibition. These studies show that the stage of transformation sensitive to actinomycin is largely completed 4 days after the carcinogen is administered; the fact that actinomycin D applied a t day 1 inhibited tumor formation as effectively as when it was applied a t day zero indicates that the process of initiation takes a t least one day for completion. The most reasonablc explanation for the actinomycin inhibition of tumorigenesis is that the initiation of skin tumorigenesis requires thc simultaneous presence of genetic activity, and that a block of this activity prevents initiation. Several other reports suggest that viral-induced malignant transformation requires the simultaneous participation of gene activity. Todaro (1965) reported that low, nontoxic levels of actinomycin D inhibit polyoma-induced transformation of cclls in culture, and Bader (1965) found that exposure of chick embryo cells to cytosine arabinoside at the time they are exposed to Rous sarcoma virus prevents viralinduced transformation. I n the latter study, when the inhibitor was added 16 hours prior to or 16 hours after the virus, it was ineffective. This inhibitor blocks DNA synthesis but not RNA synthesis. These results suggest that i t is DNA-dependent DNA synthesis that is required for transformation. Todaro and Green (1966) have shown that the transformation of cells in culture by SV 40 depends on their undergoing
CARCINOGESS, E NZ YME INDUCTION, A K D GENE ACTION
75
a mitotic cycle. Thus there are several lines of evidence suggesting a dependence on DNA activity for tumor initiation or transforma t'ion. The woi-k of many investigators has demonstrated an alteration in the enzyme profile of tissues during carcinogenesis and in the resulting tumor. Further, by electrophoric analysis, Sorof et al. (1963) have shown marked changes in the distribution of soluble proteins during the carcinogenic process. Since enzymes are the end products of gene-action systems, the altered enzyme profile of the tumor cell must reflect an alteration in the metabolism and profile of specific messenger RNA molecules. The studies of Kidson and Kirby (1964a, 1965) are consistent with this conclusion. They have shown that the pattern of messenger RNA synthesis during carcinogenesis and tlic resulting tumor is different from the pattern of messenger RNA synthesis in normal livcr. JlcCartliy and Hoyer (1964) have shown that the RNA from mouse tumor cclls, grown in culture, liydridizes with DNA diffcrcntly than tlie RNA obtained from normal mouse tissue. Furthcrrnore, Abelcv et al. (1963) has shown that liver tumor cells contain an antigen which is identical to an antigen present in embryonic tissue but not in normal adult liver. This suggests that genetic information which was one expressed in the embryo and subsequently repressed after differclntiation is again being exprcsscd in the liver tumor. T h a t carcinogenic agents may induce the expression of repressed genes is also suggested by tlic appcarancc of new antigens in tumors induced by X-rays or chemicals. These antigens are unique for each tumor, and there is ail absence of cross-reactivity between individual tumors. The antigens in tumors from virnlly induced neoplasms, however, do cross-react. It is clear that antigens in chemically induced tumors are derived from host DNA, since this is the only place from which the information can bc derived. Viruses, bringing infinitely more information to bear on the host cell, may de-repress identical gcnomic areas and thereby produce cross-reacting antigens. It is also well established that a number of endocrine tumors develop from disturbances of endocrine balance. Thus, tumors of the ovary, testes, adrenal, thyroid, and pituitary arc all associated with the abnormal manipulation of tlie endocrine systcm. A number of recent studies (Litwack and Kritchevsky, 1964; Kidson and Kirby, 196411) on the mcchanisni of horinonc action suggest that hormones exert their effects by altering gene activity. If hormones are acting on the same basic system as chemical and viral carcinogens, then one would expect certain types of hormone disturbance to result in neoplasia. The hypothesis of altered gene expression in carcinogenesis also accommodates thc known modulatory effects of ho:moncs on chemical carcinogenesis.
76
€1. V. GELBOIN
This hypothesis requires confrontation with the following qucry : Are structural changes in the DNA a requisite for carcinogenesis or can cnrcinogenesis proceed with intact gene structure but with an altered expression of gene information? I n order to answer this question there must be a n understanding of the mechanisms that control gene activity and of factors controlling their heritability. This includes a n understanding of those environmental factors, both of intracellular and cxtracellulnr origin, which are required to maintain a tissue in a differentiated state. Second, information is needed as to the nature of the gene activities which cliaractcrize the tunior state. Knowledge of the nature of tlicsc genes and their regulations will lead to a better Understanding of the nature of carcinogenesis.
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ACI~SOWLEDGNEST I would like to thank my friends and colleagues at the National Cancrr Institute for their many helpful suggestions and criticisms.
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In Vitro STUDIES ON PROTEIN SYNTHESIS BY MALIGNANT CELLS A. Clark Griffin' The University of Texas M.
D.
Anderson Hospital ond Tumor Institute, Houston, Texas
I. Introduction . . . . . . . . . . . . . 11. Current Conccpts of Protein Biosynthrsis . . . . . . A. Activating Mechanism . . . . . . . . . . B. Ribosomal-Polysomal Complex . . . . . . . . C. Transfer Reaction . . . . . . . . . . . D. Code . . . . . . . . . . . . . . E. Release Mechanisms . . . . . . . . . . 111. Protcin Syntlirsis by in Vilro Systems Dcrived from Tumor Cells IV. Comparison of Protein Synthrsis in Tumor with Microbial and Normal Mammalian Systems . . . . . . . . . A. Specificity of Aminoncyl Transfer Ribonucleic Acids and Transfer Enzymes . . . . . . . . . . . R. Fiirthrr St,udirs on the Aminoacpl Transfer Rihonuclric Arid Ribosomal System from Ascites Tumor Cells . . . . C. Ribosomal-Polysomal Complex . . . . . . . . D. Coding Characteristics . . . . . . . . . . V. Summary and Conclusions . . . . . . . . . . References . . . . . . . . . . . . .
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I. Introduction
The present knowledge of protein synthesis has been obtained largely froin in vitro systems derived from microbial cells, reticulocytes, and to a lehser extent, liver tissuc. Gcncrally, it would appear that the over-all mechanism for the polymerization of amino acids is the same in these widely divergent systems. The activating mechanism resulting in the formation of aminoacyl-ribonuclcic acids, the subsequent transfer of the amino acid to thc messenger rihonucleic acid-ribosomal or polysomal complex, followed by pcplide bond foriiiation, have been established for thcsc cellular systems, as well as for many othcr species and organs. Within this generalized sequence there are components and reactions wherein specificity may play a major rolc. In othcr words, many of the components of in vitro systems that are present in a given species would not function in systems derived from other species. From limited observations there is already preliminary indication that protein synthesis in the indignant cell proceeds by the same over-
' American
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A. CLARK GRIFFIN
all mechanism that has been proposed for microbial and normal animal cells. Within this framework it still remains to be established if there are differences in individual components, i.e., aininoacyl synthetases, ribonucleic acid (RNA) mcthylases, soluble or transfer ribonucleic acids (t,RNA), transfer enzymes, or ribosomes between normal arid cancer cells. An attempt will bc inarle to review the procedures that have been developed for the isolation of in vitro systems from cancer cells and the characteristics and preparations of these systems. These findings will he compared and contrasted with the results obtained from systems from microbial cells and animal tissues. The major objective will be to ascertain whether tlicre are diffcrences a t any level in the over-all process that may have a bearing or relationship upon malignant transformation or upon the behavior of malignant cells. Admittedly, this approach may be premature in view of the few coniponents of protein biosynthesis that have been purified or characterized and the limited studies that have been reported on the specificity or interchangeability of these coinponents. With the great emphasis on all aspects of the nucleic aciddirected biosynthesis of proteins we may reasonably expect to ascertain in the not too distant future whether any event or component in this sequence may hc unique for the malignant process. With a definitive answer a t this level we inay be in a better position to decide the emphasis that is to he placed a t different levels of thc transcriptional process or the regulatory mechanism that may have a bearing on the cancer problem. II. Current Concepts of Protein Biosynthesis
It is beyond the liniitations of this chapter to cover the extensive literature that provides the basis for our present concepts of the biosynthesis of proteins. This review will be restricted largely t o t,he niore recent in z)itro studies of protein biosynthesis. Attention is called to the thorough coverage of this subject by Campbell (1958). It is of interest that this review coincided with some of the early studies indicating the important role of the nucleic acids in the direction and regulation of protein synthesis. Many excellent recent reviews are available and reference may be made to the following: Zamecnik (1962), Watson (1963), Nathans e t al. (1963), Lipmann e t al. (1964) , Arnstein (1965), Ingrarn (1965), and Schweet e t al. (1965). These have been selected from the literature to provide a background for the role of rihonucleic acids in protein synthesis and for the generally accepted mechanism for the assembly of amino acids into peptide units.
PROTEIN SYNTHESIS BY MALIGNANT CELLS
85
A. ACTIVATING MECHANISM Thcre are specific activating enzymes (aiiiinoacyl synthctascs) for each amino acid, the function of which is to catalyzc the reaction between the carboxyl group of the aniino acid and adenosine triphosphate (ATP) to yield aniinoacyl-adenosine nionophosphatc (AbJP) and pyrophosphate. The aminoacyl adenylatc is united on this same cnzyrne with a transfer RNA specific for the amino acid (Berg, 1961). There is indication that more than one enzyme for each amino acid is present in the cell (Imamoto et al., 1965; Barnctt, 1965). Allende and Allende (1964) purified arginyl synthctasc from r a t livcr and studied its substratc specificity. Of the several amino acid analogues tested, only canavanine could be incorporated into both tRNA and protein. Highly purified tyrosyl synthetases from Escherichia coli and Bacillus subtilis were assayed for activation of a number of tyrosine analogs in the amino acid-dependent ATP-pyrophosphatc exchange system. Only 3-fluorotyrosine, tlihydroxyplienylalaiiine (dopa), and 5-hydroxy-2-pyridinc alaninc were activated (Calendar and Berg, 1965). The transfer or soluble RNA’s are composed of approximatcly 77 nucleotides in a siiigle chain. Separation of the RNA’s may be achieved by countercurrent distrilntion techniques (Goldstein et al., 1964) (Agpar e t al., 1962) and by other procedures (see Kelmers et al., 1965). The nuclcotidc sequence on the end of the chain whcrc the aiiiino acid is attached is always CCA, whereas a guunylic acid rcsiduc is found on thc other end of the molecule. The completc sequcncc of alanine-tRNA, isolated from yeast, was reported recently by Holley et al. (1965). This structure contains 77 nuclcotidc residues which give a calculated niolccular wcight of 26,000 for thc sodium salt. The multifunctionnl charactcr of tRNA has been considcrcd by Zamccriik (1962), Lamborg et al. (1965), and Ingram (1965). The nucleotide arrangement and secondary structure must provide for specific sites for recognition of the amino acid, the amiiioacyl adciiylatc-synthetase complex, coding, and ribosome recognition. For many of the amino acids more than one specific tRNA has been discovered (Weisblurn et al., 1962) (Bergquist and Robertson, 1965) (Connelly and Doctor, 1966) (Agpar and Holley, 1964). It is interesting to spcculate whether there are separate tRNA’s to correspond to the different triplet code designations. I n addition, there is considerable indication that tRNA’s for an amino acid may posscss species specificity (Berg, 1961; Imamoto et al., 1965; Yamane et al., 1963; Brody and Yanofsky, 1963). Another interesting aspect of tRNA metabolism is the presence of
86
A. CLARK GRIFFIN
methylated purines and pyrimidines in these structures. Approximately 2% of the bases in transfer RNA are methylated; however, there are some deviations depending upon the source and preparation. Borek (1963) has described some of the characteristics of the RNA methylase system. This system is present in liver, brain, spleen, kidney, heart, plants, and microorganisms. There appears to be a high degree of species specificity for both the enzyme and substrate (tRNA). However, there is no direct evidence to indicate that methylation is involved in amino acid receptor activity of tRNA (Gold and Hurwitz, 1963; Littauer et at., 1963). Bergquist and Matthcws (1962) have described a procedure for the isolation and estimation of ribonucleotidcs from subcellular fractions. The tRNA fraction bad the highest proportion of methylated bases. High levels of methylated purines were found in fractions from a mammary adenocarcinoma of C3H mice. Relatively high levels of the methylated purines, especially 6-amino-2-methyl purine and 6-methylaminopurine wcre also found in fractions obtained from the Sarcoma 180 ascites tumor. The significance of thcse findings, in terms of the carcinogenic sequence or the characteristics of malignant cells, is not immediately evident. It is interesting to speculate that the methyl groups present in many of the chemical carcinogens may have some involvement in the methylation of the bases of tissue ribonucleic acids (Cowdry, 1953; Miller and Miller, 1953; Magee and Farber, 1962; Craddock and Magee, 1965). Borek (1963) has suggested that some methylating enzyme complex may function as a naturally occurring carcinogen. The findings of Norris and Berg (1964) provide a clear explanation of the mechanism of aminoacyl synthesis. Synthetase-aminoacyl adenylates function as interiiiecliatcs in the biosynthesis of aminoacyl-RNA’s as shown in the equations:
+ ATP + Ail C synthetase AA - AMP + PPi - A M P + tRNA synthetase + AA4tRNA + AMP
Synthetnse Synthetnse .[A
These workers isolated the enzyme-amino acid adenylate complexes from reaction m i x t u m by gel filtration on Sephadex G-75 columns. Utilizing highly purified isoleucyl synthetase from E . coli they demonstrated the formation of both enzyme-isoleucyl adenylate and enzymevalyl adenylate complexes. Both complexes reacted with PPi32 to form ATP32. However, only the enzyme-isoleucyl adenylate complex would transfer the aininoacyl group to tRNA. It is therefore possible for the purified synthetase, specific for isoleucine, to form a complex with vnline. As this reacts with tRNA there is a breakdown of the complex rather than the formation of the aminoacyl-RNA. Several explanations
PROTEIN SYNTHESIS BY MALIGNANT CELLS
87
were given to account for the failure of the enzyme-valyl adenylate complex to transfer its aminoacyl group to tRNA: ( a ) failure of the complex to bind the isoleucyl-tRNA; ( b ) inability of isoleucyl tRNA to bind a valyl group to its terminal ntienylate; and (c) a n inability of the enzyme-valyl adenylate to cffect normal transfer of the aminoacyl group to the bound tRNA. The terininal structure of the amino:icyl-RNA has b c ~ nestablished, as sliowii, in Formula I: Cytosine
The amino acid is attached through its carboxyl group t o form a n ester linkage to either the 2’- or 3’-hydroxyl group of ribose in the termin:tl adenosine of the tRNA (Z:tchau et al., 1958; Prciss et al., 1959). Recently, Feldiiiaii and Znchau (1964) have obtained chemical evidencc for the 3’-liiikage of aniino acids to the tRNA. The above structure (I) of the aniinoacyl-ribonueleic acids would appear to be a universal one in nature with the terminal adenosine-cytosine-cytosine being coiiimon to all of the tRNA’s, regardless of species or tissue typcs. There is a high tlcgrce of sl’ecificity of the synthctases for aminoacyl adenylate and aininoacpl-RKh formation (Bcrg e t al., 1961). Enzymes specific for an amino acid will not ordinarily catalyze the formation of the aniinoaeyl-AMP complex for any other amino acid. An exception, Iio~vever,is the ex:iniplc noted abovc for the isoleucyl synthetase-valyl ntlcnylatc. Species specificity does exist a t the synthetase and transfer ribonucleic acid levels. These differences and the possibility of differences in synthetases and transfer ribonucleic acids between normal and malignant tissues or cells will be considercd later in this review. 1%~ I B O S O n l . 4 L - P O L Y S O ~ l . ~COMPLEX L
Watson (1965) and Ingrnm (1965) have reviewed the structural and functional characteristics of ribosomes. The existence of polysomal aggregates has been demonstrated in mammalian cells (Warner et al., 1963; Wettstein et al., 1963). The most common number of single ribo-
88
A. CLARK GRIFFIN
somes per polysornal unit was fivc. However, polysomes of much greater length have been found in r a t skeletal muscle (Breuer et al., 1964). Chains wwc consistcntly fourld of 60-100 monomers which could account for proteins with molecular weight of 350,000 or greater. Peninan et al. (1963) demonstrated that there arc, in the cytoplasm of HeLa cells, polysorncs of variable sizes which appear to be associated with a messengcr-liltc RNA. An additional observation of interest was that in poliovirus-infected cclls, whcrc the messenger R N A may have h e n the virus RNA (mol. wt. 2 x lo(,), there was indication of polysomal units of considerable size. Rich and associates (1963) have proposed that single ribosomes move along the lcngth of the rncssenger RNA forming a polypeptide chain during the movement. As the growing peptide reaches the end of the messcriger RNA-polysonial complex it is releascd, as is tlie ribosomc to whicli it is attached. As a ribosome leaves one end of the messenger RNA, another ribosome attaclies itself to the opposite end and will start its morcincnt along tlic template. The initiation of a new chain must involve the addition or attachment of an aminoacyl-tRNA (fo. mylmethionyl-tRNA may be the initiator of protein synthesis for ccrtain species) to tlie ribosome ant1 the messenger RNA. The oncoming aininoacyl-tRNA attaches to the next position on the messenger RNA and a peptide hond is forincd between the amino group of this oncoming amino ncid and the carboxyl group of the initial OT precetl!ng aniinoacyl-tRNA. The initial and now deacylated tRNA is ready to be rclcased and the polypeptidc expands as this procedure is repeated with continued movement of the ribo~oiiie-polypeptidealong the template. From the rccent observation of Thach et al. (1965) and Salas et al. (1965) there is reason to believe that messenger translation piocectls in a 5' to 3' direction along the polyribonuclcotide ch:tin.
C. TRANSFER REACTION The generally accepted mechanism of transfer of the amino acid from the aminoacyl-tRNA to the messenger-polysomal complex has developed from the investigations of Nathans and Lipniann (1961), Allende e t al. (19641, Rendi and Ochoa (1962), Fessenden and Moldave (1961), Bishop and Schwect (1961), and Arlingliaus e t al. (1964). There is general agreement t h a t the binding, transfer, and polynierization require two separate enzymes, monovalent cation, guanosine triphosphate ( G T P ) , and a sulfhydryl source, although there is some lack of agreement as to the precise niechanism that is involved. Allende e t al. (1964) have separated from E . coli two complcincntary factors (A and B) required for phenylalanine polymerization from
PROTEIN SYNTHESIS BY MALIGNANT CELLS
89
phenylalanine-tRNA on E. coli ribosomes. The starting material was prepared from the supernatant solution after sedimentation of the ribosomal fraction. Nucleic acids were renioved by precipitation with streptomycin sulfate. Anunoniuiii sulfate (28.0 g./100 nd. of supernatant fraction) was added and the resulting precipitate watb discarded ; 10 g. of ammonium sulfate/lOO ml. of the oi-iginal volume was added and the precipitate collected, resuspended in buffer, and dialyzed. Aliquots were applied to a DEAE-Sephadex A-50 column. The column was eluted btepwise with 0.2 M and 0.3 A 1 phosphate buffers, p H 7.4, and 0.0005 M with respect to 2-rnercaptoetlianol. Two definite‘ peaks (designated A and B) were detected and the complementary nature of the two factors was established. Froin these and related data, the authors tentatively identified the A fraction witli the polymerization of the amino acids. The B fraction, a more unstable fraction, appeared to be linked t o involvenient of G T P and guanosinetriphosphatase (GTPasc) in the incorporation reaction. A further correlation of the GTPase effect with amino acid polymerization was reported by Conway and Lipniann (1964). Evidence is prescntcd in support of the concept of an interrelationship between G T P split and amino acid polymerization. T h e two factors responsible for polypeptide synthesis were finally separated from GTPase (Nishizuka and Lipmann, 1965). The GTPase-frec enzymes catalyzed polymerization of phenylalanyl-RNA. Another enzynie was separated from the supernatant fraction which catalyzed a specific inorganic phosphatcG T P exchange. With this highly resolvable system there is reasonable expectation that the role and function of G T P in amino acid polymerization will be elucidated completely. Conway (1964) reported the involvement of ammonium or potassium ion in the transfer and amino acid-incorporating mechanism. I n their previous studies they had routinely used G T P with phosphoenolpyruvate and pyruvate kinase as the regenerating system. I n a later study they found that the ammonium sulfate present in the kinase preparations was stiinulatory in the incorporating reaction and could actually replace thc rcgenerating components. Potassium was almost as active a s arnmonium ion in this respect while sodium ions were inactive. It was conclutletl that aiiiiiionium or potassium ions arc essential in the binding or fixing of aniinoacyl-tRNA to the ribosoincs (Spyrides, 1964). Preincubation of E . coli ribosomes with phenylalanine-RNA, poly U, and ammonium or potassium ions eliminated the lag in the onset of polymerization that is observed in the presence of transfer enzyme (A or B) and GTP. The binding, a slow reaction, is thus rate determining in the polymerization reaction. Nakamoto and Lipmann (1964) have shown
90
A. CLARK GRIFFIN
further that the template-bound aminoacyl-RNA initiates peptide chain formation. Arlinghaus e t wl. (1964) prepared transfer enzymes from rabbit reticulocytes. The starting material was obtained by protamine treatnicnt of the high-speed supernatant fractioil followed by ammonium sulfate precipitation (between 40 and 70% saturation). The resuspended pm3pitnte was absorbed onto calcium phosphate gel and the peptide syritlictasc (TF-2) was eluted with 0.1 M phosphate buffer while the binding enzyme designated TF-1 was eluted with 0.3 d l phosphate huffcr. Further purification of both fractions was achieved by passage over DEAE-cellulose columns. The binding of phenylalanyl-RNA to the reticulocyte ribosome required poly U, MgC1, and TF-1. Preincubation of the ribosomes was required to destroy the endogenous hemoglobin synthesis. Leucine-RNA, valine-RNA, and lysine-RNA were not bound under the same conditions as reported above. The binding reactions required addition of G T P (1.3 x 10-5M ) . I-Iowever, reaction components, including the C14aminoacyl-RNA, had t o be purified carefully in order t o demonstrate a G T P requirement for the binding step. The peptide synthetase (TF-2) catalyzed nonspecific G T P breakdown. The authors suggcsted that during the binding reaction a highenergy intermediate is formed which breaks down rapidly, causing GTP hydrolysis. It was concluded t h a t the synthesis of a polypeptide must involve the alternating action of the binding enzyme which requires G T P and tlic peptide synthetase reaction (Arlingliaus e t al., 1964). This proposed mechanism differs in several major aspects from that proposed for tlic E . coli system. Kurland (1966) has determined the minimum requiremcnts for the binding of a specific tRNA by ribosomes. These include a messenger R N A to direct tRNA positioning and suitable ionic conditions for the formation of the complex. There is no requirement for transfer enzymes or G T P and the tRNA can be bound equally well in the aininoacylnted form or in the deacylated form. A resolution and partial purification of liver enzymes involved i n the transfer of aminoacyl groups have been reported by Gasior and Moldave (1965). These investigators used the pII 5 supernatant fraction obtained from the high-speed centrifugation of homogenized rat liver. The supcrnatant solution was passed through a Sephadex G-25 column. Calcium phosphate gel was added to the eluate, the suspension centrifuged, and the resulting residue was resuspended in phosphate buffer. Ammonium sulfate was added to 25% saturation, the precipitate discarded, and the supernatant fraction was adjusted to 65% of saturation with further addition of ammonium sulfate. The resulting pre-
PROTEIN SYNTHESIS BY MALIGNAXT CELLS
91
cipitate was resuspended in tris buffer, p H 7.2, KCl, and glutathione, and placed on a Sephadex G-200 column. Transferase I emerged with the lead fractions, traiisferase I1 appeared later, and RNA emerged after the bulk of the protein had passed through the column. When the individual enzyme fractions were assayed with purified ribosoiiics, prepared by thc proccdurc of Tak:an:imi (1960), no activity was observed. When combined, they catalyzed the transfer of aiiiino acids from aininoacyl-RNA to ribosonial peptides (as indicated by insolubility in hot trichloroacetic acid). Fi om the column characteristics it WVLS estimated that aminoacyl trurisfcusc I lias :I nio!cculnr weight of 300,000 or greater, and transferase I1 has a molecular weight of less t1i:rn 100,000. Since these investigators d i d not study the binding reaction per sc, no direct comparisons may be made with the transfer enzyiiie studies made on the E . coli and reticulocyte syctems. Keller and Ferger (1965) have reported recently the clironiatographic separation and purification of an enzyme fraction from E. colt responsible for the polymerization of phcnylalanine-RNA in a system containing washed ribosomes, poly U, GTP, and regenerating components. The molecular weight of the soluhle protein factor was estimated to be 80,000. A partial purification of the transfer enzyme from yeast has been achieved also by Heredia and Halvorson (1965).
D. CODE The excellent reviews of Nirenberg et u1. (1963), Ochoa (1963), and Watson (1965) on the gcnctic code provide a l~nckgroundfor the subsequent section on the coding characteristics of malignant cells. From the agreement between the code letters derived froin the E . coli system with amino acid replacements in mutants of tobacco mosaic virus, hemoglobins, and other proteins, Ochoa proposes that tlicre is but one genetic code for all living systems (sce Yanofsky, 1963; Wittnianii and Wittmann-Liebold, 1963; Tsugita and Fraenkel-Conrat, 1962). A rapid method for measuring C'4-aminoacyl-tRNA iiiteractioii with ribosomes prior to peptide-bond for mation was described by Nirenberg and Leder (1964). The trinucleotides, UUU, AAA, and CCC, directed the binding t o ribosomes of phenylalanine, lysine, and proline tItNA's, respectively. Dinucleotides were ineffective in this respect. I n a subsequent study Leder and Nirenberg (1964) cstablishcd the nucleotide sequence GpUpUp for a valine RNA codewortl. Nishimura e t al. (1964) have utilized synthetic deoxyribopolynuclcotides as templates for ribonucleic acid polymerase. With these techniques they have prep a r d ribopolynucleotides containing repeating di- and triiiucleotide sequences complementary to those in the short chain deoxyribonucleo-
92
A. CLARK GRIFFIN
tides. The known polyrihonucleotides may then be used in studies of binding of specific aminoacyl-tRNA's or in the direction of the incorporation of specific amino acids (Nishimura et al., 1965; Khorana, 1965).
E. RELEASE MECHANISMS From studies reported on in vitro incorporating systems there are some doubts as to the exact nature of the release of polypeptides from the messenger-polysonial complex. According to Lamborg (1962) the in vitro release of protein from ribosomes requires Mg++and a relatively high concentration of ATP. Puromycin and deoxyribonuclease (DNase) stimulated the release of labeled protein. Further studies on the release mechanism have been reported by Hultin et al. (1961) and Morris (1964). Stretton and Brenncr ( 1965) have suggested t h a t the triplets with the sequences of UAG and UAA have the function of terminating polypeptides in protein synthesis. Only polynucleotides containing UA produced the release of polypeptide chains from ribosomes in ccll-free, amino acid-incorporating systems of microbial origin (Takanami and Yan, 1965). The release of completed polypeptide units may depend upon the prior treatment of ribosomes. Hardesty et al. (1963) demonstrated that reticulocyte ribosomes which were once pelleted by centrifugation were more effective in apparent hemoglobin synthesis than were twice pelleted ribosomes. A 40-minute incubation period of the in vitro system resulted also in a greater proportion of the labeled polypeptides in the supernatant fraction [&23 vs. 4800 counts in the ribosomes and supernatant fractions, respectively, based on counts per iiiinute (c.p.ni.) per nig. ribosomes added to system]. Partially completed polypeptide chains could be released from the ribosomes by the addition of puromycin. I n the Novikoff ascites tumor system the incorporated amino acids are not readily released from the ribosomes (Griffin e t al., 1965a). I l l . Protein Synthesis by in Vitro Systems Derived from Tumor Cells
One of the initial studies of the incorporation of labeled amino acids by a n in vitro system of tumor origin was reported by Littlefield and Keller (1957). These investigators, utilizing the Ehrlich mouse ascites tumor, prepared p H 5-insoluble and microsornal fractions. Dcoxycholatc-insoluble particles and an NaC1-insoluble fraction were obtaiiictl from the crude microsoinal pellets. Ultracentrifugal analysis showed prominent peaks corresponding t o 50 and 43 S for the tumor rnicrosomal preparation, in agreement with the earlier observations of Peterman (1954) for this and other tumors. The dcoxycholate-insoluble particles
PROTEIN SYNTHESIS BY MALIGNAKT CELLS
93
showed three major peaks with sedimentation rates of 57, 54, and 50s. The NaC1-insoluble particles showed considel ably smaller sedixiicntation v a l u c ~ indicating , soiiic nltei at-ons in s1i:ipc and sLzeby tliis prep.irative procedure. Incorporation of C13-amino acids into p: oteins was detcrinriied by the addition of glucobe, buffer, and labeled amino acid to the whole ascites fluid. Approximately 33 pnioles of leuciiie was incorporated per gram of protein per hour (based on initial 10-min. incorporation in tumor cells obtained 7 days after transplantation). Tlie authors concluded that their findings were consistent with the concept that most of the amino acids incorporated into whole cell proteins pass through the ribonucleoproteins. Incorporation of C14-amino acids (as indicated by insolubility in hot perchloric acid) was observed in the tumor cell-free system developed by Littlefield and Kellcr. The system appeared to be similar to that of rat liver developed earlier in the same laboratory. Tlie niicarosonial and pH 5 enzyme fractions of the tumor and liver systems were interchangeable, indicating that a t this level the two tissues havc similar mechanisms for amino acid activation and incorporation. The tumor microsonial system required phosphoenolpyruvate and ATP. Cond e r i t b l e incorporation occurred in the absence of added GTP. When tlie deoxycholate or NaCI-inboluble ribonucleoprotein particles were substituted for the microsonies in the in vatro system, active incorporation was maintained, indicating that the membranous component of the microsome is not required for incorporation. The tumor ribonucleoprotein particle-pH 5 enzyme systein, treated with Dowex 1-X8 to reduce the inherent nucleotides, exhibited a complete dependence upon added A T P and to a lesser exteiit, GTP. The system was haturated by as little as 0.05 pmole of G T P per nil. I n general, it was concluded that the in vitro system was less active than corresponding liver cellfrce systems. However, minor variations in the cell fractionation procedures, as well as the stability of the isolated components, may account for the small differences that were observed between the liver and tumor systems. Another investigation to compare the mechanism of protein synthesis of tumor cells with that of other mammalian cells was reported by Oclioa and Weinstein (1964s). Strain DNA X Swiss hybrid mice were inocculatcd intraperitonially with L-1210 mouse ascites leukemia cells. Six days later the ascites fluid containing 2 to 4 X 10’ cells per nil. was collected, the cells washed and finally homogenized in a standard buffer (tris-HC1, 0.01 M ph 7.8; magnesium acetate, 0.005 M ; KCl, 0.06 M ; 2-mercaptoethanol, 0.006 M ; arid sucrose, 0.25 M ) . The suspen-
94
A . CLARK GRIFFIN
sion was subjected to low-gravity centrifugation in order t o sediment unbroken cells, nuclei, and mitochondria. The supernatant fraction was centrifuged a t 122,000 g to obtain the supernatant and the microsomal pellet fractions. A pH 5-insoluble fraction was obtained by established procedures. Further refinement of the microsomal pellet was achieved by homogenization in a 1% sodium deoxycholate standard buffer solution and recentrifuged to sediment the ribosomal pellet. Following usual assay procedures the ribosomal and pH 5 fractions incorporated approximately 30 ,+moles of C'" phenylalanine per mg. ribosomal protein. Addition of polyuridylic acid resulted in an approximate tenfold increase in the incorporation by this in vitro system. The microsomes were also fractionated through sucrose gradients and pooled into several fractions differing by their sedimentation coefficients. Phenylalanine incorporation was greatest in the heavy microsome peak (150-350 s) and least in the pool containing microsomes with sedimentation coefficients of 80-90 S. Poly U-dependent incorporation, in contrast, was associated with all classes of microsomes with the greatest specific activity in the region of the lighter particles. These findings would suggest that the poly U reacts with the 80s ribosome to produce polysomal formation. An active amino acid-incorporating system was developed in this laboratory (Griffin and O'Neal, 1962; O'Neal and Griffin, 1963) utilizing the Novikoff rat ascites tumor (Novikoff, 1957; Weber, 1958). This hepatoma, initially induced by the administration of diets containing carcinogenic azo dyes, possesses many advantages for the investigation of in vitro protein biosynthesis. It grows rapidly, either in a solid or free state as single cells, has a reasonably homogeneous cell population, and may be obtained in the quantities required for the isolation and purification of the various components involved in the biosynthetic sequence. This tumor possesses certain disadvantages in that i t is highly anaplastic and has lost many of the properties of liver, the tissue of origin. There is some doubt as to whether this tumor arose from the liver parenchymal cells or from elements of the bile duct. Ascitic fluid was withdrawn from the intraperitonial cavity of albino rats injected 7 days previously with the tumor. Approximately 40-60 ml. of fluid was obtained per rat, this fluid containing 40-60 million cells per ml. (equivalent to 0.2 g. of solid cell suspension per ml.). The ascitic fluid was diluted with 2 volumes of cell wash (NaCl, 0.14 M; glucose, 0.02 M ; tris, 0.04 M ; p H 8.5) and centrifuged a t 600 r.p.m. for 10 minutes. The supernatant, fraction, which contained most of the red blood cells, was decanted off and discarded. Cell wash, 4 to 6 volumes, was added to the lightly packed cells, mixed, and centrifuged a t
PROTEIN SYNTHESIS BT MALIGNANT CELLS
95
1000 r.p.m. for 10 inmutes. The supernatant fraction was discarded and the washing procedure repeated one or two additional times. At this stage the packed cells were combined and centrifuged a t approximately 1500 r.p.m. for 5 minutes in order to concentrate the tumor cell mass. (All centrifugations to this stage were carried out in a n International Centrifuge, Head #253. An attempt was made in these and all subsequent steps to maintain the temperature of the preparation below 4OC.) Six volumes of cold deionized water were added to the packed tumor cells, the mixture stirred, and a few minutes allowed for the cells to undergo hypotonic shock. The suspension was ground in an ElvehjemPotter Teflon-glass homogenizer. Approximately 12-15 strokes were required to disrupt the tumor cells, as judged by phase microscopy. One tcnth volume of a concentrated solution (0.25M KCl, 0.05 M MgCl,, 2 . 5 M sucrose) was added to the disrupted cells and the suspension centrifuged a t 12,500 r.p.m. for 30 minutes (Spinco Model pR2 Centrifuge, #21 rotor). The light amber-colored, clear supernatant fluid was centrifuged a t 150,OOOg for 80 minutes (No. 50 rotor, 50,000 r.p.111.) resulting in the sedimentation of the tumor microsomal fraction. The niicrosomal pellets were homogenized in a deoxycholate-glycyl-glycine buffer, p H 8.0 (approximately 6 ml. added to pellets obtained from 40 nil. of packed cells). This was diluted 20-fold with standard buffer and recentrifuged a t 150,000 g for 40 minutes. Ribosomal pellets were collected and the same deoxycholnte washing procedure was repeated. At this stage the pellets were homogenized in the standard buffer in the approximate concentration of 10 ing. ribosomal protein per ml. T h e ribosomal fraction, thus obtained, was frozen in small aliquots and stored a t -10°C. or lower. Under these conditions they retained almost full activity for periods of a t least 2 weeks. The clear supernatant fluid resulting from the original high-speed centrifugation was adjusted to p H 5.0 by the careful addition of 1N acetic acid. Following centrifugation the precipitate was redissolved in standard buffer (bucrose, 0.25 ill; KCI, 0.025 111; RIgCl,, 0.005 ill; tris, 0 . 0 5 M ; p H 7.6) reprecipitated a t p H 5.0, centrifuged, and the prccipitatr taken u p in standard buffer, in order to provide a final conccntration of 10-12 nig. protein per ml. This fraction (designated S 100, p1-I 5 . 2 X ) rctained a high degree of activity for several weeks when stored at -10°C. Treatment of the original p H 5.0 supernatant fraction with Scplindrx G-25 (0.2 g. per nil.) provided a concentrated source of transfcr cnzyrneb. This fraction also retained a high activity for many weeks when stored a t -10°C. Reconibination of the tumor ribosomal and activating fractions resulted in an active ainino acid incorporating system as shown in Table
96
A . CLARK GRIFFIN
TABLE I SPECIFICITIES OF ACTIVATINQAND RIBOSOMAL FRACTIONS FROM ASCITESTUMOR AND NORMAL LIVERa
CI4-arnino acid
Tumor system
Liver system
114 145 105 ti5 165 51
53 70 18 8 ti0 22
Tumor S 100, Liver S 100, pH 5 liver p H 5 tumor ribosome ribosome
+
+
90 72 45 8 56 20
35 65
~~
Alanine Arginine Glutamic acid Glycine Lericine Tyrosine
1ti
13 56 24
5 The assay system included 0.1 ml. ribosomes (1 mg. protein); 0.2 ml. activating fraction, deBignated as S 100, pH 5 . 2 X, (approx. 2 mg. protein); 0.005 pmole C14-amino acids (Nuclear-Chicago) and 0.005 pmoles of each of 19 cold amino acids; 0.25 pmoles ATP; 7.5 X lop3 pmoles GTP; 2.5 pmoles phosphoenolpyruvate; 5.0 pg. phosphoenolpyruvate lrinase. Buffer was added to a total volume of 0.5 ml. The system was incubated a t 37°C. for 40 min. Samples of 0.05 ml. were pipetted onto Whatman # 3hIR.I paper discs. These were twice extracted with cold 5% TCA, heated 15 min. in 5% TCA a t 90°C., reextracted with cold TCA, and washed twice with cold ethanol. After drying, the discs were counted in a Packard Tri-Carb Scintillation Counter, with a counting efficiency of 70%. The specific activities were based upon the original ribosomal protein added to the assay tubes. Valiies are expressed in micromicromoles amino acid incorporated per milligram ribosomal protein (Griffin el al., 1965b).
I. For most of the C"-labeled amino acids that were assayed in this system the extent of incorporation, as measured by insolubility in hot trichloroacetic acid, was approximately 100 ppnioles per mg. ribosomal protein. This reprcscnts about 10% of the incorporation obtained with corrcsponding microbial and rabbit reticulocytc ribosomal systems (Arlinghaus and Schweet, 1962). For comparative purposes, the incorporation of amino acids by rat liver ribosomal system (O'Neal and Griffin, 1963) are included in Table I. It may be observed that the incorporation for most amino acids was less than half of that observed for the tumor system. For glutamic acid, glycine, and certain other amino acids not included in this table the incorporation was considerably lower in the liver system than in the tumor system. Substitution of the activating and ribosomal fractions provided indication that the two systems were qualitatively similar (Table I ) , Of interest was the observation that the tumor-activating fraction did enhance incorporation for certain amino acids by the liver ribosomes, while the combination of liveractivating fraction and tumor ribosomes resulted in a generally lower incorporation than was attained with the complete tumor system. However, i t would appear that a t this level of comparison there are no
PROTEIN SYNTHESIS BY MALIGNANT CELLS
97
qualitative differences between the components involved in protein biosynthesis in the noriiial rat liver and thc ascites tumor in vitro systems. IV. Comparison of Protein Synthesis in Tumor with Microbial and Normal Mammalian Systems
From the initial observations of Littlefield and Keller (1957), of Oclioa and Weinstein (1964~1.)~ and the findings fiom our laboratory it would appear that the activating and ribosomal fractions from tumor cells and normal nianimalian tissues are completcly interchangeable. I t has also been observed (Dunn and Griffin, 1965) that the activating and ribosomal fractions obtained from beef pituitaries could be exchanged with the comparable components from the ascites tumor cells. ,Many other investigators (i.e., Breuer et al., 1964) have reported that major coiriponents involvcd in protein synthesis are interchangeable with coniponcnts from other nmnmalian tissues and organs. From these many findings it may be assuined that the same general meclianism for amino acid activation, aminoacyl tranbfer, and amino acid polymerization is operative in iiianmialian cells, and t h a t malignant cells do not differ radically in this respect. The aminoacyl RNA-ribosornal systems provide more opportunity to study specificity a t various I C V C ~ S in protein synthesis. Some species specificity does appear to exist a t thc aminoacyl syntlietase and transfcr RNA level. Rlonicr e t nl. (1960) found that liver synthetases would catalyze the formation of yeast valyl-tRNA. Synthetases from mammalian sources or ycast formed the mammalian or yeast tyrosyl-tRNA, but would not react with tyrosyl-tRNA from E. coli (Clark and EyzagUirre, 1962). Connelly and Doctor (1966) purified two ycast scrinctRNA’s by countercurrcnt distribution and obuxved that each was equally active toward either yeast or liver aminoacyl synthetases, whercas both werc inactive toward E . coli synthctases. Rendi and Ochoa (1962) demonstrated species specificity in amino acid activation, as shown in Table 11. It may be observed t h a t mainmalian synthetases will esterify CI4-leucine to tRNA from liver or yeast but not E . coli or other microbial species tested. Conversely, the E. coli synthetases would not catalyze the formation of aminoacyl-tRNA in the presence of yeast or mammalian tRNA’s. Loftfield and Eigner (1963) observed t h a t a single amino acid syntlietase catalyzed the aminoacylation of tRNA’s from different species, but a t varying rates. They pointed out the importance of the rate of reaction in asscssmcnt of specks specificity in terms of this reaction. Yamane e t al. (1963) and Iniainoto e t al. (1965) have provided
98
A. CLARK GRIFFIN
TABLE I1
TRANSFER RNA W I T H AMINOACYL-sRNA SYNTHETASES FROM VAR~OUS SOURCES~~~ ESTERIFIC.4TION O F C’4-LEUCINE TO
Specific radioactivity of R N h Source of supernatant (aminoacylsRNA synthetases)
Rat liver (c.p.m./mg.)
Yeast Bscherichia coli (c.p.m./nig.) (c.p.m./mg.)
Experiment 1 Itat liver Yeast E. coli R a t liver E. coli
1340 1510 18 1270
450 370 20 420
16 36 1780 1770
Experiment 2 Rat kidney Ox liver Pigeon liver Frog liver Lactobacillus arabirwsus Streptococcus jaecalis Azolobacter vi,rdaiidii Propiottibacteriicm shermanii
1680 990 1440 1200 0 0 2 2
1240
23 41 120 33 770 125 152 127
+
0
b
870 1120 940 0 0 0 0
Assay conditions aye as descrihed under “Preparations and Methods Reprinted with permission (Rendi and Ochoa, 1962).
”
additional evidence t h a t species specificity does exist for the aminoacyl synthetases and tRNA’s. The activating fraction (pH 5 insoluble fraction) obtained from ascites tumor cells, rat liver, and E. coli were treated with DEAEcellulose (0.2g. per 4.0 ml. activating fraction). This resulted in almost complete removal of the tRNA’s. Other aliquots of the activating fractions were treated by the usual phenol procedure in order to obtain crude prcparations of the tRNA’s. Preliminary studies were then carried out to determine if the aminoacyl synthetases of tumor and liver would rntalyze the formation of aminoacyl-RNA’s when incubated in the presence of specific C**-labeled amino acids, ATP, and tRNA’s from either tumor or liver. As would be predicted from these related cells, the tumor or liver synthetases catalyzed the formation of seventeen labeled aminoacyl-RNA’s in the presence of tRNA from either source (Griffin et al., 1965b). When tRNA’s from E . coli or yeast were added, the enzymes of mammalian origin failed to catalyze the formation of the aminoacyl complex for many of the amino acids. An interesting preliminary observation was noted in that the tumor synthetase preparation did result in the formation of yeast arginyl-tRNA while liver synthetase was inactive in this respect. Other possible differences be-
PROTEIN S YNT HE S IS BY hL4LIGNANT CELLS
99
tween tumor and liver werc detected ; liowwer, them findings require confirmation enip!oying inore refined techniques. A synthetase fraction from E. coli was active in the estcrificntion of all tltNA’s of the same source. However, only a limited number of arninoacyl-tRNA’s wcre formcd when the microbial syiithctax preparation was assayed in the presence of yeast or manimalian tKNA fractions. The specificity of the nrninoncylation reaction may warrant further investigation in tcnns of changes that may occur during malignant transformation. Employing tlic tc~lniiqucsof Sueoka and Kano-Sueoka (1965) and others, studies arc now in progress in our laboratories to ascertain if there are differences bctwecn thc synthetases and tRNA’s of normal liver and the Novilioft’ nscites tuinor cells. This same approach will be extendcd to thc livcrs of rats fed diets containing azo carcinogens and also to a widcr variety of tumors. Sueoka and Kano-Sueoka (1964), using niethylated albumin kiescJ1guhr colurnn chromatography, compared aminoacyl-tRNA’s of E . coli with and without phage T2 infection. There was a difference in only one aniinoacyl tRNA, leucyl-tRNA. The normal leucyl-tRNA of E . coli showed two major peaks, Leu I and Leu 11. I n infected cclls the chromatogram showed that Leu I was greatly decreased. From the data obtained it appeared that the change was in the tRNA and not in the synthcta>c. Norton and Rogers (1965) described a thin-layer chromntography pi orcr1ui.e for diff ercritiation of aminoacyltRNA’s. Specific livcr and P388 lympliocytic leukemia aminoacyltRNA’s were detected by autoradiogrnphy. Tlicy indicated that several spots were found for most aniinoncyl-tRNL4’s and that reproducible differences in chroinntographic pnttcrns were found bctwecn the liver and leukemia cclls. The synthesis of protein was studied in a mutant of E . coli possessing a plicnyl:zlaiiyl-RN~4 synthctase with a decreased capacity to utilize p-fluoropl~cnylnlaiiinc(Fangman and Neidhardt, 1964). It was concluded that the activat on of p-fluorophcnylalaninc is requisite for incorporation into pi.oteiiis :ind for pet miss’on of RNA synthesis. Stent (1964) has suggested that protein biosynthesis may be regulated by special tRNA specics and Sucoka and Kano-Sucoka (1965) have investigated the possibility of the role of tRNA in biosynthetic control. Brody and Yanofdiy (19F3) have intlic:itcd that alterations in the tRNA’s or tlic nminoncyl syntlietases might rcsult in translation errors in protein syntheqiq.
A. SPECIFICITY OF AUINOACYL TRAXSFER RIBOSUCLEIC ACIDS AKD TRANSFER E:Nznim Nathans and Lipniann ( 1960, 1961) ohscrved that aniinoacyl-tRNA’s derived from mammalian or microbial sources were interchangeable in
100
A. CLARK GRIFFIN
ribosomal-incorporating systems of various species. Similar findings were reported by Rendi 2nd Ochoa (1962) in that rat liver or E. coli ribosomal systems would accept leucine from rat liver tRNA yeast tRNA, or E . coli tRNA. Canning and Griffin (1965) also showed that CI4glutamic acid, Cl4-arginine and CI4-leucine were transferred from either the tRNA of tumor or E . coli to ribosomes of tumor, liver, or E . coli (Table 111). Bloemendal et al. (1964) have found that labeled polynucleotides from normal liver, hepatomas, and mammary tumors were transferred to niicrosoines or ribosomes of normal and cancerous tissues. In addition, thcy demonstrated that tumor rnicrosomal protein incorporated label from C14-aminoacyl-tRNA. While the source of the aminoacyl-tRNA is not an important factor in the transfer of amino acid to the ribosome, there is a certain degree of specificity of the enzyme or enzymes that catalyze this transfer reaction. Nathans and Lipinann (1960, 1961) and Rendi and Ochoa (1962) reported that partially purified supernatant fractions, used as a source of transfer enzymcs, could be cxchanged with certain limitations between species. Supernatant fractions from rabbit, pigeon, or calf liver could replace rat liver supernatant in a rat liver ribosomal system. It mas observcd, however, that partially purified bacterial transfer factor prepTABLE I11 ACIDS FROM SRNA TO TUMOR, LIVER, Escherichiu coli RIBOSOMES~
TR.ZNSFER O F cI4-;\MINO AND
CI4-glritamicacid C14-arginine (70) transferred (%) transferred from from Source of ribosomes
Cl4-leucine
(Yu)transferred from
Source of transfer factor
Tumor sRNA
E . coli sRNA
Tumor sRNh
E. coli sRNA
Tumor G-25 Liver G-25 E. coli transfer factor
27 32 18
22 23 12
12 10 6
41 60 4
50 51 11
37 40 5
Liver G-25 Tumor G-25 E. coli transfer factor
37 31 0
30 26 2
11 31 0
6 32 0
48 40
0
32 35 0
E . coli transfer factor Liver G-25 Tumor G-25
14
18
22
23
22
11
0 0
0 0
-
1
Tumor sRNA
E. coli sRNA
__________
Tumor
Liver
E. coli
1
Reprinted with permission from Canning and Griffin (1965).
0 0.5
0 0
PROTEIN SYICTHESIS BY MALIGNANT CELLS
101
arations were incapable of catalyzing the incorporation of amino acids from aminoacyl-tRNA by niammalian ribosomes and vice versa. Specificity studies a t the transfer enzyme level carried out in this laboratory arc in essential agreement with the observations of the Lipmann and Ochoa groups. From Table I11 it is apparent t h a t the transfer factor preparations from tumor and liver are completely interchangeable in C14-aminoacyl-tRNA ribosomal systems of the tumor or liver. The mammalian enzyme preparations were incffective in the transfer of amino acid to the E . coli ribosonics. \Trhile the E . coli transfer fraction was completely ineffective in the liver ribosomal system, there was an indication that the microbial enzyme (s) were a t least partially effective in catalyzing the transfer of amino acids to the tumor ribosome. This study was extended over a wider range of amino acids (Table IV). It may be observed that the E . coli transfer factor fraction did result in a significant amino acid incorporation for almost all of the aminoacyltRNA's that were tested. There is no explanation a t this time for this apparent difference in transfer specificity between the tumor and liver ribosomal systems. Further exchange studies are in progress employing other tumors and also in tho further resolution and purification of the transfer factor fractions. It would be of major interest if it could be firmly established that tumors differ from normal tissues in terms of the specificity of the enzymes involved in the amino acid transfer and polymerization reactions.
B. FURTHER STUDIES ON THE AMINOACYL TRANSFER RIBONUCLEIC ACID SYSTEM FROM ASCITESTUMOR CELLS RIBOSOMAL Development of a tumor aminoacyl-tRNA ribosomal system was essential for study of the later stages of incorporation of amino acids into polypeptides and for comparison with the events occurring in microbial and mammalian systems. Aminoacyl-tRNA's were prepared by charging of tumor or liver S 100, p H 5 fractions with C'"-labeled amino acids. The usual phenol procedurc (see Canning and Griffin, 1965) was employed and the preparations were subjected to dialysis and passage over Sephadex G-25 columns to lower the background of the system. The labeled aminoacyl-tRNA preparations could be stored for several weeks in a dry ice chest with no apparent breakdown. Transfer enzyme preparations were prepared from the p H 5-soluble fractions of liver and ascites tumor cells. These fractions, containing approximately 80% of the transfer enzyme(s) of the cells were treated with Scphadex G-25 (0.2 g. per ml.) which provided a concentrated and highly active source of transfer enzymes. A further concentration of the transfer activity was achieved by collection of the precipitate between
102
A. CLARK G R I F F I N
TABLE IV
AMINOACIDS TuMoR, LIVER,A N D Iikcherichia coli RIBOSOMAL 8YSTEMSatbZC
S P E C I F I C I T Y O F ’rR \ N S F E R FACTORS I N THE I N C O R P O R A T I O N O F BY
Ribosomal system Tumor ribosomes transfer factor from s R N h source Tumor Valine A rgin in e Proline Isoleucine Phenylalanine Glutamic acid Leucine Lysine Serine Tyrosine
Liver
87 100 110 80 103 174 86 89
Liver ribosomes transfer factor from
B. coli
Tuinor
lj. coli
3 51 32 3 11 17 22 36 26 26
100 36 138 87 76 118 95 147 102 103
E. coli ribosomes transfer factor from Tumor
Liver
0 5c
0 3 0 0 0
E. coli Valine Arginiiie Proline Isoleiicine Phenylalanine Glritarnic acid Leucine Lysine Yerine Tyrosine a
147 108 8G
92 105 80 98
30 2 42 17
77 G 89 54 00
0 1 151 79 116 110 li2 50 10::
0 0 2 0 <1 0 0
0 0 0 0 0 0 0 1 0 0
0 0
Reprinted with permission from Canning and Grifhn (1965).
* All values expressed as per cent of incorporation obtained when the transfer fraction
was from the same source as the ribosomes. 6 I t is doubtful that this value represents incorporation by the E coli ribosomes since it has been demonstrated in previous studies that some “apparent” incorporation does occur when C14-argininewas added to activating and other nonribosomal fractions obtained from the ascites tumor.
50 and 70% saturation with ammonium sulfate. Chromatography of the G-25 concentrate or the ammonium sulfate fraction on Sepliadex G-200 columns provided a further purification of transfer activity, as indicated in Fig. 1. It may be observed that the transfer activity appeared in the leading front of thc elution pattern. The tubes containing the greatest transfer
PROTEIN SYNTHESIS BY MALIGNANT CELLS
103
activity were usually pooled, the proteins precipitated by the addition of aininonium sulfate ( 4 0 ~ ;per . 100 nil., retaining the pII a t 7.0 during the addition of tlie salt). The precipitate was redissolved in the tris-KC1 buffer and exhaustively dialyzed against tlie same buffer, This preparation, termed G-200 TF-,4, retained almost full activity for several weeks of the G-200 T F A on a Sephadex if stored a t -40°C. Recliro~nntogi~apliy G-200 colunin resulted in a single elution peak (Fig. 2 ) , suggesting that the fraction is niade u p of high-mo!ecular weight proteins (200,000300,000) and that no further resolution could be achieved on this type of column.
il ;
0.5
\
\
\
I I
__-2 4 6-*8
I012
Tube
number
Fro. 1. Elution pattern of transfer factor from Sephadex (3-200 column. Sephadex G-200 column 3 x 30 cm. equilibrated 24 hours with 0.05 M tris, 0.15 M KCl, buffer pH 7.6; 10 ml. transfer fraction (G-25 concentrate of tumor S 100, pH &soluble fraction) placed on column. Eliition (3-4 ml. aliquots) with same buffer.
A further purification of the deoxycholate-washed ribosomes was achieved by passage over Sepliadex G-200 columns or by the procedure of Taltanami (1960). Recornbination of the system (C14-aminoacyltRNII, transfer enzymes, and purified ribosomes) resulted in incorporation values as shown in Table V. The data presented in Table V are representative of results obtained with several C1l-aminoacyl-tRNA's added to the assay system. It is apparent that this tumor in uitro system is almost completely dependent
104
A. CLARK GRIFFIN
upon transfer cnzymcs and an apparent energy source for maxiinurn ainino acid incorporation. There was little indication t h a t exogenous G T P is required for incorporation by this system-a finding difficult to cxplain in view of thc establislicd rcquircincnt for G T P in the biosynthesis of protein by many investigators (see Griffin e t al., 1965a). All of the components involved in this systcin have becn subject t o quitc rigid purification procedures and attcmpts have been inade to reduce the G T P that may bc present. Ncvcrtliclcss, addition of G T P O V C ~a wide con-
Tube
number
FIG.2. Recl~romatogra~~liy of partially purified transfer enzyme fraction on Sephadex G-200 column. Sephadex G-200 column 3 x 30 em. equilibrated 24 hours with 0.05 M tris, 0.15 M KCI, buffer pH 7.6; 5 ml. concentrated transfer fraction (leading fraction of preceding column) placed on column and eluted with same buffer.
centration range has not cnhanccd amino acid incorporation by this system. I n prcvious studies in this laboratory a inarkcd G T P effect was obtained when incorporation was studied in a less refincd system consisting of the pH 5-insoluble fraction and ribosomes or rnicrosomes (Table V I ) . There is no immediate answer to this apparent discrepancy in terms of G T P involvement in protein biosynthesis. It is possible that the tumor aminoacyl-tRNA ribosomal systcin has a low rcquireinent for G T P and that this requirement is provided by tracc amounts of G T P that still remain in one or more of the components. Clarification of the complex
105
PROTEIN SYNTHESIS BY MALIGNANT CELLS
TABLE V INCORPOR.ATION OF AMINO ACIDS BY ASCITES TUMOR AMINoacuL-tRNA RIBOSOM.AL SYSTEM Assay systeni Complete system" -P.E.P., lcinase, GTP -Transfer enzymes - GTP -P.E.P., GTP - GTP, kinase P.E.P., kinase, GTP heated '30" +Mercaptoethanol (0.6 pmoles) -P.E.P., kinase, GTE', NH4C1 (10 pmoles)
Counts per mg. ribosomal protein
1480 170 191 1440 1110 305 1x90
1450 lGl0
Complete systein includes 0.05 nil. tumor ribosomes (10 nig. per ml.), 0.01 ml. transfer enzyme fraction, 0.5 pmole phosphoenolpyruvate (P.E.P.),2.0 p g . P.E.P. kinase, 0.05 pniole GTP, 0.01nil. C14-isoleucine-tRNA(approx. 2500 coLmts) to a total volume of 0.1 ml. hlixture incubated for 30 minutes a t 37", 0.05-nil. aliquots removed arid adsorbed on paper discs. The discs were washed twice in cold 5(/h TCA, heated in 5y0 TCA, 15 miiiutes a t W, alcohol-washed, dried, and connt,ed in Paclcard Scintillation Counter (efficiency 70');). (1
role of G T P in the mechanism of protein biosynthesis may provide an explanation to account for these sceiiiingly contradictory data (see Hoagland et al., 1964). The apparent rcquireiiient for an energy sourcc in the aminoacyltRNA ribosomal system (Table V) was difficult to understand since phosphoenolpyruvate (P.E.P.) and kinasc were as effective as P.E.P., kinase, and GTP. It sceiiied possible t h a t the P.E.P. and kinasc were regenerating tlie guaiiosine p1iosl)liates as t h y were utilized in the biosynthetic reactions. HOWCVC~., 11c:tting of thc cncrgy coniponents a t ternpcratures that would destroy P.E.P. ant1 kinase did not niarkcdly affect amino acid incorporation. This problem was finally resolved when i t was observed that the systein required considerably inore monovalent cation than was present in the stxmdnrd buffer in which all of the components were dissolved. The high content of aiiiiiioniuiii ion in thc kinase preparation, arid to a lesser cxtcnt, the sodium ion of tlie P.E.P. enhanced the incorporation of the lalwlcd amino acids. Addition of NH,Cl to the system, in the absence of added energy components, resulted in riiaxinium incorporation, indicating the essentiality of monovalent cation in this system. Conway (1964) reported a high requirement for ammonium ion in the transfer and amino acid incorporation iiieclianism with E . coli systems. Potassium ion was lcss active and sodium ion was inactive in this
106
A. CLARK GRIFFIN
TABLE VI EFFECTOF GUANOSINE TRIPHOSPIIATE (GTP) O N AMINO ACID INCORPORATION BY TUMOR A N D LIVERin Vilro SYSTEMP* Tumor ribosomal system
Turnor microsomal system
C14-arninoacid
-GTP
+GTP
-GTP
+GTP
-GrP
SGTP
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Praline Serine Threonine Tyrosine Valine
1030 1290 270 1340 230 200 1770 2680 1330 220 3000 670 364 170 310 405
1480 1700 600 2950 950 510 3870 7650 3720 42 0 6450 1550 1230 330 490 980
740 1230
740 1130
670 690
780 650
650
690
210 TO
390 110
1140 3300
2100 3930
360 660
760 2440
180
183
190
170
300 650
250 740
180 180
260 280
~
~~
~
Liver ribosoinal system
~~
From Griffin et al. (198%). b Values are expressed in counts/min./mg. ribosomal or microsomal protein. The game procedure is used as outlined in Table I except that GTP was added to half of contents of assay tube (0.75 pmole/0.25 ml.). 5
respect. A further study in our laboratory indicated that potassium ion was almost as effective as ammonium ion and that sodium ion was also stimulatory for both the liver and tumor aminoacyl-tRNA ribosomal systems. The most effective concentration for ammonium ion was approximately 0.1 to 0.15M. A considerable effort has been extended in the further resolution of thc transfer enzyme fraction. I n view of the possibility of trace amounts of these enzymes being associated with the ribosomes, the deoxycholatewashed ribosomes were chromatographed on Sephadex G-200 columns or were purified according to the procedure of Takannmi (1960). Representative incorporation data employing the aminoacyl-tRNA purified ribosome systems are shown in Table VII. These data indicate that the system requires only transfer cnzymes, monovalent cation, and mercaptocthanol for maximum incorporation. There was still no indication of an exogenous GTP requirement a t this stage of purification of the components of the incorporation system. The above assay system was utilized t o ascertain if the transfer
107
PROTEIN SYNTHESIS BY MALIGNANT CELLS
TABLE VII INCORPORATION OF AMINOACIDSBY TEMOR A n m o , w y b t R N A PURIFIED I~IBOSOXAL SYSTEX Assay system Basic systema +P.E.P., kinase, G T P +NHaCI (10 rmoles) +Mercaptoethanol (0 6 pmoles) NH&l, mercnptoethanol +P.E.P., kinase, G T P , merca~~toetliniiol +NH,Cl, mercaptoethanol minus trarihfer eiizynic5
+
Counts/min./mg. ribosomal protein 92 190 302
150 b05
690 101
Basic system: 0.05 ml. purified ribosomes (10 nig. per ml.), 0.005 ml. transfer enzyme (G-200 TF-A), 0.01 ml. ~ ~ J - u m i i i o a c y l - t l (approx. ~ N ~ ~ 2000 counts). T h e amounts of P.E.P., P.E.P. kinase, and C T P were the same us shown in legend of Table V (total volume of the assay system ]+as0.1 nil.). 1ncul)ittioii and counting procedure its descrilwd 111 Table V.
cnzyine fractioii could be rcsolved into two or inore components. Thus far, it lias bceri impossible to fractionate the tumor transfer enzyiiie fraction on DEAE-Sephadcx coluiiiiis as rcported by Allciidc et al. (1964) for E . coli enzymes. There is little indication of a second transfer enzyme appearing later in the elution pattern froni Sephatlckx G-200 columns as employed by Gaaior and AIolclave (1965) for liver preparations. All fractions emerging from colunins have bccn assayed. However, only the leading fractions werc active in stimulating ainino acid incorporation (Fig. 1 ) . Combinations of the fractions were also assayed with little indication of complementary activity. Studies are now in progress to determine if calcium pho~phntegel or other adsorbing materials will separate the tunior transfer enzyiiic fraction into two or inore components. The activity of the ascites tuiiior transfer fraction (G-200 TF-A) is illustrated in Fig. 3, Approximatcly 10 pg. of the enzyme preparation was required for maxiinum transfer of the C1"-amino acid to 0.5 mg. ribosomal protein, as indicated by insolubility in hot TCA. As little as 2-3 p g . of this preparation catalyzed measurable transfer and incorporation of the ninino acid. From findings obtained thus far the ascites tumor aminoacyl-tRNA ribosomal systeni differs in a t lcast two aspects froni the E . coli and reticulocyte systciris, i.c., no exogenous GTP requirements and no indication of involvement of more than one transfer enzynie. A possible explariation for thew dilfcrenccs niay reside in the basic nature of the assay s y s t e m employed in various laboratories. I n the E . coli and
108
A. CLARK GRIFFIN
reticulocyte studies the investigators used ribosomes with greatly reduced endogenous messenger RNA and have takcn advantage of the excellent incorporation that occurs with the addition of poly U and phenylalanyl-tRNA preparations. I n the studies in this laboratory we have relied upon the “inherent)’ message associatcd with the polysomes and have determined the incorporation of several Cl4-amino acids (transfer from C14-an~inoacyl-tRNA).I n the E . coli and reticulocyte systenls the over-all incorporation mechanism may include the following sequence of events: polysome formation, initiation of new polypeptide chains, binding of aminoacyl-tRNA, polymerization, and release of peptide units
/
5001
u I
I
2
3 4 5 6 Transfer foclor
7 pg
8
9
10
Fra. 3. Activity of tumor transfer enzyme fraction. Assay system: 0.05 ml. purified ribosomes; 0.001-0.02 ml. transfer enzyme fraction, G-200 TF-A; 10 pmoles NH4Cl; 0.6 prnoles mercaptoctlinnol ; 0.01 ml. C14-isoleucine-tRNA (approx. 2000 counts). Incubation and counting procedures a s described in footnote to Table V.
(polyphenylalanine) . The tumor system we have developed may be limited essentially to the binding and polymerization steps. Only a small per cent of the acid-insoluble activity is released from the ribosome-polysome complex (Griffin et al., 1965a). The tumor polysomal complex undoubtedly contains many partially completed peptides which are extended, but not completed or released, during the incubation period. Perhaps this part of the biosynthetic mechanism may be catalyzed by a single enzyme and may not require GTP. The question may be askcd if G T P and another enzyme may be involved in events other than the binding and polymerization steps. Thus far all attempts have been unsuccessful to demonstrate a G T P and multienzyme requirement by the tumor system similar to that reported for other incorporating systems. A major obstacle has been
PROTEIN SYNTHESIS BY MALIGNANT CELLS
109
encountered in rcmoving tlie inherent messenger RNA from the liver and tumor ribosomes. Preincubation, addition of puromycin, and KC1 shock trratment (Arlinghaus and Schweet, 1962) reduce tlie incorporation of labeled amino acids; however, the ribosomes have a limited capacity to accept new messenger RNA (Griffin e t al., 1965a). It will be necessary to await further investigations in this area of protein biosyntlicisis in order to ascertain if there is a universal mechanism for all living things or if there are differences between widely varying species or between normal and tumor cells.
C. RIBOSOMAL-POLYSOMAL COMPLEX Specificity and the possible regulation of protein biosynthesis a t the ribosomal-polysomal level have been reviewed by No11 (1965). Webb ef 02. (1964) studied polysoriial patterns in rat liver and in several minimal deviation tumors, and some abberations were observed. However, they concluded that there was no evidence that these changes bore any relation to changes essential to the neoplastic process. Electron micrographs and sucrose density gradient patterns of tlie tumor polysonml preparations obtained in this laboratory (Griffin et (d., 1965:t) closely resembled patterns obtamed from normal liver. In a follow-up study Webb et nl. (1965) estimated the degree of association between the polysomes and the endoplasmic reticulum by comparing the yield of C-ribosomes obtained from the postmitochondrial supernatant fraction in the presence or absence of dcoxycholate. The fraction of bound polyribobomes in thr postmitocliondrial supernatant fractions in the iiiininial deviation tumors correlates with the degree of differentiation, and invcrsely, with their growth rate. The fraction of bound polyribosomes was very low in immature liver and in the Novikoff hepatorna but approached 60-7070 in normal and regenerating liver. These authors concluded that : “Free polyribosomes synthesize proteins concerned with cell growth and proliferation, whereas bound polyribosomes synthesize proteins charactcristic of the cell type (differentiated functions) whether or not they are for export.” Pitot and Peraino (1964) and Pitot (1965) have proposed t h a t the ericloplnsmic reticulum st:tbilizcs the messenger RNA of the polysomal complex and that defects in the membrane are responsible for the anomitlies in the enzyme induction found in neoplastic tissues. Of rclated interest is tlie cholesterol-negative feedback systeIn prcst.nt in nornial liver. Siperstein and Fagan (1964) found that the feedback system was retained in regenerating liver but was not present in most of the hepatomas that were studied.
110
A. CLARK G R I F F I N
D. CODINGCHARACTERISTICS Only a limited number of studies directed t o the coding problem have been carried out in in vitro systems derived from tumor cells. From these limited investigations it would appear that the coding characteristics of malignant cells do not differ from those of microbial or other mammalian cells. Griffin et aZ. (19654 added several synthetic polynucleotides, as well as various exogenous RNA preparations, to the tumor ribosomal system and determined the effect upon incorporation of several amino acids (Table VIII). It may be observcd that poly U, poly A, and poly C resulted in significant increases in the incorporation of phenylalanine, lysine, and prolinc, respectively. With other synthetic polynucleotides there were indications that the tumor system responded in the same manner as microbial systems for tyrosine, threonine, histidine, isoleucine, glycine, and aspartic acid. The tumor ribosomal system TABLE VIII
EFFECT OF EXOGENOUS RIBONUCLEIC ACID (RNA) UPON INCORPORATION OF AMINOACIDSBY TUMOR RIBOSOMAL SYSTEM"^^
(%) Increase following addition of RNA
Amino acid
TMVRNAc
Tumor nuclear RNA
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine
42 31 104 90 19 80 70 44 100 68 64 92 30 70 50 47
11 28 33 <10 50 31 38 28 24 13 43 42 75 40 42 43
ME-RNA
PolyU
PolyA
<10 <10 <10 11
<10 <10
<10 <10
< 10
<10
<10
125
< 10
1450 <10
<10 <10
<10
< 10 < 10
< 10 <10
<10 <10
<10
PolyC
Y2
10 12 10
From Griffin et al. (1965a). Assay conditions are the same as described in Table I. Contents of assay tubes (0.5 ml.) were divided and 20 pg. RNA was added to 1 portion before incubation. 0 Abbreviations used: TMV, tobacco mosaic virus; ME, mouse encephalitis virus; Poly U, polyuridylic acid; Poly A, polyadenylic acid; Poly C, polycytidylic acid. a
PROTEIN SYNTHESIS BY MALIGNANT CELLS
111
also responded in a limited manner to tobacco mosaic virus RNA and to a tumor nuclear RNA preparation. The inherently low activity of the tumor and liver preparations has presented a major problem in the study of coding characteristics as well as the effects of messenger RNA preparations. Attempts to remove the inherent messenger RNA, as reported for microbial and reticulocyte ribosomes, have met with limited success since the treated ribosomes gave even less response to synthetic polynucleotides or other messenger RNA preparations. It is indeed possible that an acceleration of the release of completed peptide chains would result in a greater response to synthetic polynucleotides and messenger RNA’s. Maxwell (1962) reported that phenylalanine, leucine, valine, glycine, tryptophan, and serine incorporation in a liver cell-free system was stimulated by synthetic polyribonucleotides of known base ratios. These findings were in complete agreement with results obtained from the E . coli system. Oclioa and Weinstein (1964b) arrived a t the conclusion that the genetic code of mouse ascites leukemia cells is qualitatively similar to that of bacterial, plant, and other animal cells. V. Summary a n d Conclusions
Space does not allow the inclusion of many studies of amino acid incorporation and protein biosynthesis that differ from the classical ribosomal system. Attention is directed to the nuclear incorporating system developed by Allfrey and Mirsky (1963). Nuclear biosynthesis of proteins may play an important role in tumor cells. However, relatively little has been reported in this area. Several investigators (i.e., Kaji et al., 1963, 1965) have described soluble amino acid-incorporating systems. The nature of this nonribosomal amino acid incorporation and the radioactive products that are formed have not been elucidated. End group addition of amino acids to existing peptides could account for a t least a portion of this apparent incorporation. It is of interest to note that significant amounts of labeled arginine and lesser quantities of glutamic acid, lysine, and valine are present in a hot TCA-insoluble form following incubation of the pH 5-insoluble fraction of the Novikoff ascites tumor system. This fraction is free of ribosomes; however, no further attempts have been made to characterize the acid-insoluble product. While many new advances have been made in the elucidation of the mechanism of protein synthesis there is still no definite answer as to whether there are differences between normal and cancer cells. It is obvious that further exhaustive studies will be required employing normal animal and also tumor systems. New tumor systems should bc
112
A. CLARK GRIFFIN
developed for comparative purposes. The minimal deviation hepatomas (see Pitot, 1965) offer certain advantages in view of the established gradations in morphology and function from normal liver. Plasma cell tumors that elaborate proteins related to immunoglobulins produced by normal plasma cells can be induced in inbred stains of mice. Several tumor lines are available that secrete relativcly large quantities of specific proteins. Kuff et al. (1964) studied the RPC-20 tumor previously converted to a n ascitic form. This tumor secretes a n immunologically distinctive protein with a sedimentation rate of 2.8 S and an approximate molecular weight of 24,000. This protein appears in the urine. With antisera prepared against the urinary protein it was shown that the ascitic tumor cells produced the protein during in vitro incubation. The virus-induced tumors may offer unusual opportunity t o determine if alterations in protein biosynthrsis may be associated with malignant processes. Watson (1965) has presented a convincing rstionale for utilization of the polyoma virus or the Rous sarcoma virus for studies related to causation of cancer. Employing in vitro conditions it has been established that these viruses will transform normal cells into malignant cells. Teniin (1965) infected several types of cells with different avian sarcoma viruses (Rous sarcoma virus, Schmidt-Ruppin Rous sarcoma virus, Fujinami virus, myeloblastosis virus, and leucosis virus) and studied many properties during cell conversion. Watson (1965) is of the opinion that this approach may be rewarding and has stated: “These viruses multiply in cells that we are only bcginning to study at the molecular level. Nonetheless, most important is thc fact that a t last the biochemistry of cancer can bc approached in a st:aightforward rational manncr.” It would appear also that studies of the effects of cancer-inducing chemical and physical agents on the enzymes and other components involved in protein synthesis may be rewarding. In viewing the total mechanism of protein biosynthesis there appears to be a striking similarity or universality in living things. The cancer cell presents no exception in this respect. There is incrc:ising evidence from many laboratories, however, that specificity is a n irnportant feature of many of the componcnts that are involved in the assembly of amino acids into proteins. This is especially evident a t the aminoacyl synthctase and tRNA level and in enzyme systems involved in mcthylation of nucleic acids. Further detailed investigations will be required to asccrtain whether cancer cells are unique in terms of the specificity of ally of these components. The possibility of control or regulation at this level, including malignant transformation or behavior, cannot be overlooked. Cancer cells may differ from normal cells in the enzymes
PROTEIN SYNTHESIS BY MALIGNANT CELLS
113
catalyzing the twtctions of amino acid transfer, polymerization, and of peptitle release. IIotvevcr, this remains still to be established. Control and rclgulation of protein synthesis or other cellular activities a t the ribosomal-polysoriial level lias been proposed by many investigators. It may be of interest t o restate the impressions of Campbell (1958) in his review of protein synthesis in cttnccr cells. “A coinparison between the synthesis of protein in tunior arid normal tissue has had to await developments in the field RS a whole. It is clcar t h a t in certain diseases the tumor cells syntlicsizc proteins which differ from those synthesizcd 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, a t present, whctlier the mechanism of protein synthesis in tumor cells differs froiii that in normal cells. The need now is for carcful cornparieons between normal and tumor tissue to be made employing the techniques wliirli have heen developed in recent years.” Since Cnmpbell’s 1 cvicw major atlv:tnrcs have been made in establishing the rolc of nuclcic ncidh in tlic tlirclction and control of protein biosynthesis. Nevertheless, we arc still unable, at present, t o provide a conclusive answcr as to wlicthc~rp~oteinsyntlicsis differs in any aspect between normal :ml cancer cells. \\7itli the availability of newer techniques for resolution and cl1:tractcrization of the transfer ribonucleie acids, enzymes, ribosoii~cs,etc., definitive answers as to whether there are differences that may have a hcwing on tlie problem of cancer should be available for tlie next rcvicwer of this subject.
Studirs reported in this rcvicw were supported by grants from the Robert A . Wclrh Forintlation and the Amrrican Cancer Society. I wish to ncknawledge the contributions of Barbara H . Holland, Dr. L. Canning, and Dr. T. F. Drinn in the investigations reported in this review.
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THE ENZYMIC PATTERN OF NEOPLASTIC TISSUE* W. Eugene Knox Deportment of Biological Chemistry, Harvard Medical School and the Cancer Research Institute, New England Deaconess Hospital, Boston, Massachusetts
I . General Ideas about Neoplasia . . . . . . . . A. Warburg’s Generalization . . . . . . . . B. Greenstein’s Generalization . . . . . . . . C. The Generalization Snggestccl by Enzyme Physiology . . 11. The Measurement of Enzymes in Tissues and the Prediction of Metabolic Brhavior . . . . . . . . . . . 111. Glycolyeis and the Enzymes of Glycolysis . . . . . IV. Glycerolphosphate Dehydrogenase Levels and Glycolysis . . V. The Enzymes of the Penlosc Pathway . . . . . . VI. Enzymes of Glrtconeogenesis and Glycogen Formation . . VII. Tentat,ive Formulation of the Pattern of l
117 118 120 122 123 125 133 134 137 143 144 152 155 158
I. General ideas about Neoplasia
The general ideas about ncoplasia arc cssentially coinparisonsRmong neoplastic tissues and between neoplastic and normal tissues. The diversities of the characteristics of both normal and neoplastic tissues are so great that few mcaningful goncralizations about neoplasia have stood the test of time. The most significant one, of course, is the morphological generalization by wliicli neoplastic tissues are rccognized as such. Normal tissues vary widely in their rates of cell growth, patterns of cc.llul:tr arrangemciit, a n d the structural peculiaritics of tlw cells. I n none of thcse charactcristics are a11 normal tissues qualitatively different from all tlie ncoplastic tissues. Yet neoplastic tissues as a group show unifoimity, a t Icast in rclatioii to the diversity of nornial *Aided, in part, by U. S. Public Hcalth Scrvicc Grant AM 00567, by Research Career Award AM-K6-2018 from the Kational Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, U. S. Public Health Service, and by U. S. Atomic Energy Commission Contract ilT(30-1)-001 with the New England Deaconess Hospital. 117
118
W. EUGENE KNOX
tissues, by converging in the direction of persistent cell growth (ncoplasia) , by the disordered cell arrangement (heterotopia) , and by the structural abiiormalities of cells (anaplasia). I n these ways they tend to resemble each other morc than normal tissues, and by use of this complex gcneralizntion tlie skilled and experienced pathologist can usually identify a fragment of tissue as neoplastic. Often he can classify it among the some 300 recognized types. Two other equally complex generalizations, both of them biochemical, arc those of Warburg (1930, 1956) and Greenstein (1954, 1956). These also point to a similar convergence toward uniformity in the propertics of the neoplastic cells relative to tlie diverse properties of normal cells. The properties concerned are the uniformly high lactate formation by neoplastic tissue slices (Warburg) and the similar patterns of enzymes in neoplastic tissucs (Grcenstein). Like the morphological generalization, neither of these generalizations can claim that there are established qualitative differences between the nornial and the neoplastic cells. Neither generalization, however, has been iiieaningfully extended or put to practical use, as has the morphological generalization. They have been affirmed, or denied. Apart from such exegeses, they have not yet led to a deepening of our understanding of the neoplastic process. The measurements of respiration and of thc enzymes present in neoplastic tissues that are now available are inore detailed than the data upon which these two generalizations were originally based. The new measurements provide extensive support for both generalizations. They also indicate the way in which Warburg’s generalization is a consequence of Greenstein’s generalization. The pattern of enzymes that is now being found in neoplastic tissues, when interpreted in the light of the developing knowledge of the function and regulation of enzymes in cells, may explain how neoplastic cells behave as they do. There is in prospect, a t least, a biochemical corollary to the morphological generalization, that neoplastic tissues have some similar appearances and metabolic behavior because they have siinilar enzymic compositions.
A. WARBURG’S GENERALIZATION The Coris discovered in 1925 that tumors in situ used more glucose and gave off more lactate to the circulation than did normal tissues (Cori and Cori, 1925a,b). The methods developed by Warburg for the measurement of the respiration and lactate formation in tissue slices made it possible to make similar observations more accurately in a great variety of tissucs. Although the tissues were removed from the animal, they were relatively intact during these “in vitro” measurements. As has become increasingly clear, the metabolism of carefully prepared
119
T H E ESZI'MIC PATTERN O F NEOPLASTIC TISSUE
tissue slices is inore like that of intact tissues than of cell-free preparations. The measurements are niade i n vitro, but the conditions approach those in vivo. This is an important restriction on thc comparison of LACTATE
Ti1BLE I SLICES (FROM GLUCOSE) ANI) HOMOGEN.ITES (FROM F D P ) NORMAL A N D NEOPLASTIC TISSUES O F RAT (UNLESS SPECIFIED)
FORMkTION IN
OF
Slices (with glucose)"
Homogenates (with FDP)b
Aerobic Anaerobic Anaerobic [pnioles/min./lOO mg. dry tissue (37"C.)]
Normal lissiies Thyroid Liver Intestinal niucosa Diaphragm musrle (Long, 1981) Kidney (Long, 1981) Spleen Testis Thymus Placenta Brain Embryo Retina Neoplasms Flexner-Jobling carcinoma Jensen sarcoma Walker 250 carcinosarcoma Sarcoma 37 (mouse) Spontaneous tumors (mouse) 'rar carrinoma (mouse) Melanoma Yale T tumor (mouse) Bladder carcinoma (man) Sarcoma (man) Laryngeal carcinoma (man)
0 0.05 0 1 0.16 0 0.2 0
0.05 0.7 0.2 0.4 3.4
1 9 1.3 0.9 0.6 1.1 0.4 0.5 1.8 1.2 1.1
0.2 0.24 0.3 0.37 0.38 0.6 0.6 0.6 1.0 1.4
1.7 6.0 2.3 2.5
-
2.1 1.9 1.9 1.2 1.2 2.7 2.1 1.4
5.8
-
12.1 7.3
-
5.7
-
5.5 6.0 5.0
-
-
-
-
From compilation of experiments by Warburg and others in Greenstein (1954, p. 451), modified as indicated by values i n Long (1961), A more catholic compilation of values for normal and neoplastic t,issries is that of Aisenberg (1981). The usual Q notation (pl./hr,/mg. dry weight) X O . O i 4 = pnioles/niin./IOO mg. dry weight. * From Le Page (1950, Table 11) for reinforced homogenates, with added glucose and fructose-1,G-diphosphate (FDP). c For later, higher values, see footnote, Table IV.
measurements in sliccs and homogenates. The rates of lactatc production from glucose in neoplastic tissuc slices were within the very wide range of rates found in slices of normal tissues, but the rates were less
120
W. EUGENE KNOX
divergent in the neoplastic tissues. They were comparable to the higher values found among normal tissues (Warburg, 1930). These observations by Warburg were promptly confirmed (Dickens and Simer, 1930, 1931). A selection of the classical values is shown in the first columns of Table I, which make it clear that anaerobic lactate production is relatively high and uniform in neoplastic tissues. Even in oxygen, which diminishes lactate formation (the Pasteur effect), the rates remain relatively high. It is unnecessary to consider now the altered control, or “defective” respiration, that may be indicated for neoplastic tissues by the latter observation. Much has been written about this; there is an authoritative compendium available (Aisenberg, 1961). It is clear that Warburg’s data supported his generalization that there was a convergence among the neoplastic tissues, in relation to the great diversity among normal tissues, in the ability of tissue slices to form lactate from glucose. This generalization encouraged the elucidation of the molecular mechanisms for glycolysis, the first coherent inetabolic pathway for which all enzyme steps were known. There was later elucidation of the other respiratory mechanisms, in both normal and neoplastic tissues. The result was clear: the molecular mechanisms involved in the respiration and the formation of lactate in neoplastic tissues were not qualitatively different from those in normal tissues (Weinhouse, 1955).
B. GREENSTEIN’S GENERALIZATION There was an almost random choice of the enzymes that were measured in neoplastic tissues until recently. The choice was dictated by the fragmentary knowledge of enzymes that was available, plus the interest and expertise of the particular investigator. Many measurements were the direct result of Potter’s popularization of the tissue homogenate, and of his search for enzymes that might be deleted in neoplasia (Potter, 1944). The methods of assay, the units and bases for expressing the results, and the type of controls chosen were as varied as the quality of the work. Furthermore, confusion existed about the measurement of enzymes in tissue slices versus cell-free systems : the former represents a functional measure in a relatively intact tissue, the latter a type of chemical analysis that seeks to measure the full amount of a substance which may be in cells. Comparisons between enzymes and between tissues, which are essential for the evaluation of these findings, were necessarily dubious. A most remarkable fact is that any generalization was possible on the basis of Greenstein’s collection in 1954 of the amounts of enzymes in cancers (Greenstein, 1954), which is itself a n emended and annotated
121
THE ENZYMIC PATTERN O F NEOPLASTIC TISSUE
compilation that faithfully reflected the disorder of the field. Only careful study will reveal that there is more than a casual basis for the generalization that: “NO matter how or from which tissues tumors arise, they more nearly resemble each other chemically than they do normal tissues or than normal tissues resemble each other” (Greenstein, 1954, 1). 589). Fortunately, the data for this generalization were later recast and epitomized in only eleven pages, under the title, “Some Biochemical Characteristics of Morphologically Separable Cancer” (Greenstein, 1956). The chart reproduced in Fig. 1 is taken from this summary to I
I
I
I
I
r
I
I
I
I
w I
I !
I Li
Hepatoma Lung tumor Mammary co Gas odenoco Int adenoca Sq corcin L rnphoma Myelanoma 37
s
Cr 00
Prim sorc Rhabdornso Osteogen
Arginase
FIG.1. The levels of various enzyme activities in cell-free extracts of different normal and ncoplastic tissues of mice. The relative activities for a given enzyme are indicated by the length of the horizontal bars in a column. The arrows denote a higher order of magnitude of activity. (From Greenstein, 1956.)
provide a basis for evaluating the generalization. The figure illustrates the largest of several blocks of data that could be compared: some enzymes in transplanted tumors of mice. Even here a certain license in the choice of relative activities was necessary, but the comparisons are convincing. With regard to these particular measurements, a t least, the tumors certainly resemble each other more than normal tissues do. Less certainly, one tumor may resemble other tumors more than it does the presumed tissue of origin (the identity of which is a weak point in all
122
W. EUGENE ICKOX
such coinparisons). However, the main weakness lies in the type of measurements. These are not of a kind to carry conviction t h a t this is an important phenomenon. At least three of the ten activities do not represcnt single enzyine reactions. Only three or four of the other activities can be assigned any possible physiological roles. As a result, the metabolic behavior cannot be predicted for a neoplastic tissue with this pattern of enzyme activities. However, additional measurements are now available to delineate the pattern more clearly and to test the validity of any such correlations between the metabolic behavior of whole cells and their enzyme patterns.
C. THEGENERALIZATION SUGGESTED BY ENZYME PHYSIOLOGY We have learned a great deal since the time of Warburg’s mcasurcnicnts of tissue slice respiration a t the beginning of the enzyniological explorations of the cell, and since the first quantitative ineasurements of enzymes in cells that were utilized by Greenstein. We often know which enzymes are present in cells, arid for many of them, how much is there. An enzyme physiology has grown u p that concerns itself with the relation between enzyme chemistry and physiological function, with what enzymes actually do and how they do it in vivo. An important element of this knowledge is the way enzyme levels change with alteration in physiological states (Knox and Grcengard, 1965). If the metabolic behavior of a tissue does reflect its enzyme pattern, then knowledge of the high and similar rates of lactate formation by neoplastic tissue slices should lead to the prediction that the amounts of the glycolytic enzymes would be more nearly similar and higher in the neoplastic than in most normal tissues. Since the enzymes involved in glycolysis made up the first coherent group to bc discovered, they offer a particularly attractive test of the significance of the Greenstein generalization. Greenstein himsclf did not overlook the fact that, given his generalization, the relatively uniform lactate production by neoplastic tissues was appropriate. His comment was simply that (italics added): “. . . thc range of activity of each of the enzymes is narrower among tumors than among normal tissues; the picture of a uniform respiratory and glycolytic pattern in tumors . . . is thus consistent with this phenomenon and, indeed, since it is based o n a n enzymatic pattern, could hardly be otherwise” (Greenstein, 1954, p. 456). T h e prediction that tissues with uniformly high lactate production will have unifornily high amounts of t h e glycolytic enzynies remains to be tested, and, if true, extended to other metabolic pathways and their individual enzyines so as to characterize the metabolism of tumors.
T H E E S Z Y X I C PATTERN O F NEOPLASTIC TISSVE
123
11. The Measurement of Enzymes in Tissues and the Prediction of Metabolic Behavior
The need for comparative “elementary analyses” of normal and neoplastic cells is generally acknowledged, but in the past if such analyses were directcd toward functional elements like the enzymes, they often abandoned the rigorous quantification applied to constituents such a s trace metals, amino acids, potassium, or deoxyribonucleic acid (DNA). Enzyme-catalyzed product formations were measured in homogcnates or tissue slices, for example, without knowledge of the limitations by permeability barriers, substrate concentration, or product catabolism. Such measurements reflect neither the amount of the enzyme nor the rate a t which the particular reaction may have proceeded in vivo before interruption of the circulation or homogenization. Only in a fully supplemented system, in which the enzyme concentration alone is limiting, is the rate of the reaction related to the amount of the enzymc. Inevitably, this measures the potential function of the enzyme. Ideally it should be correlated with other estimates of the rate of the reaction
in vivo. Numerous studies have now accumulated which show t h a t the measurement in vitro of the total potential amounts of specific enzymcs often does correlate with the function of the enzymes in vim. Differences in the amounts so detectcd betwccn tissues dcrivcd from animals in changed endocrine, nutritional, developmental, and disease states (Knox et al., 1956; Knox and Greengard, 1965; Weinhouse, 1959) are appropriate to the physiological change and to the new rnctabolic statc in vivo. Furthermore, this approach is often instrumental in identifying the physiological function of an enzyme. For example, the relative importance of certain enzymes for gluconeogenesis was more readily identified by observing whether the amount of the enzymc in question increased or decreased after a gluconeogenic stimulus (Lardy, 1964). I n the field of cancer research, Weher has admirably exemplified the application of these concepts (Wcbcr, 1961) and has edited a series of volumes reviewing the progress in this area (Weber, 196S1966). The identification of the physiological roles of more enzymcs in various metabolic states is now more prcssing than accurate enzyme measurcments, but the former depends on the latter, and the comparisons of the one with the other. To simplify the comparison of enzyme activities, which are rates of amount of substrate reacted per unit time, it has been recommended that they be expressed in international units, inicromoles per minute a t 25°C (I.U.B., 1961). The choice of the basis (units of tissue weight,
124
W. EUGENE KNOX
protein or cell number) to which the activity is referred may limit the comparisons that can be made. For example, many measurements in neoplastic tissues that were made of oxidative enzymes localized in the mitochondria but expressed only per unit of tissue or homogenate are omitted here. Since the amounts of mitochondria are low in neoplastic tissues (Price et wl., 1949; Allard e t al., 1952; Striebich et al., 1953; Laird, 1954), the proportionately low enzyme levels would only add new information if they were accompanied by measurements of the enzyme specific activities in the mitochondria of the neoplastic and control tissues. I n general, two kinds of reference bases are useful: a chcniiciil basis, sucli as the protein nitrogen of the sample for comparison of the enzyme specific activity in extracts, and a physiological basis, such as the unit of tissue weight or cell number for comparison of the physiological potentials of the enzyme in tissues. Conditions are now almost always chosen so that. the measured rates can be attributed to the amount of a particular enzyme species in the tissue. Because absolute rates can vary with the conditions of the assay, highly standardized ways of assaying and standard names and numbers of the enzyme are employed (I.U.B., 1965). Assays are also done comparatively, to sliow that there is more or less of a given activity in one tissue than anotlier, when both are measured a t the same time and under the same conditions. The mensured amounts of specific enzymes in normal tissues have by now firmly established the starting point of the Greenstein generalization: that all normal tissues do not contain the same enzymes, and that different tissues possess some enzymes in greatly differing amounts. The enzymes present and their relative activities constitute the pattern of enzymes of the tissue. While it is now common knowledge that the pattern of enzymes differs between tissues, the actual data on which this is based are scattered among the studies dealing with particular enzymes. Compilation of this information to provide the actual enzyme pattern of a particular tissue has been done for very few tissues: the gastrointestinal mucosa (Spencer and &ox, 1960), the placenta (Hagerman, 19641, the thymus gland (Long, 19611, and less completely for several other tissues. Such compilations could provide support for the common belief that the functional and morphological characteristics of a tissue reflect its particular enzyme pattern. The compilations would also supply the standard pattern of enzymes of a tissue with which, inevitably, comparisons must be made in studies such as the present one. I n view of the lack of enzyme patterns in normal tissues, if a common pattern of enzymes is approached by all neoplastic tissues, it may be the first tissue pattern to become known in great detail.
THE E N Z Y M I C PATTERN OF NEOPLASTIC TISSUE
125
Ill. Glycolysis and the Enzymes of Glycolysis
T h e systematic study of enzyme amounts in tissues began with the enzymes that carry out glycolysis. The over-all reaction was recognized to be uniformly high in ncoplastic tissues, and, appropriately, the enzymes that catalyze this reaction were the first series of enzymes to be known in tlctail. Eventually, by such a systematic appionch, the possible connection betwcen the Greenstein and W'arburg generalizations can be tested. The potential rates of glycolysis, represented by the coinbined enzyme activities freed in the hoinogcwites of tissues, werc much lower when first measured than the observed rates of glycolysis in the intact tissue slices. The discovery of tlie conditions and supplements that are necessary to obtain all thc different cnzymes free of cells, and a t tlie same time to allow them all to be optiinally active in the same solution, is still t o be made. Although it has becn closely approached, it 1s riot :I gcncrally realized cxpwiniental technique to obtain over-all glycolysis a t the same rate in cell-free systems as in the intact tissue slices. Such experiments, however, dion ed that only some of the enzymes were present in limiting amounts or activities, and attention could be focused on these. Thus the rates of glycolysis in slices of different tissues (Greenstein, 1954) that are listed in Table I can be compared with I,e Page's values for the partial glycolysis of fructosediphosphate in reinforced homogcnates of the same tissues (Le Page, 1950). The measurements are all recalculated in the same units. The rates of anaerobic glycolysis of glucose in normal tissue slices are variable and unusually low, and in neoplastic tissues arc more nearly uniform and high. On the other hand, glycolysis of fructosediphosphate is uniform and high in lioniogcnatcs of both normal and neoplastic tissues. The amounts of the enzymes for the conversion of fructosediphosphate t o lactate are clearly not limiting in lioiiiogcnates of thew tissues. Their amounts, or activities, in thc cclls piobably do not account for the difference in rates of glycolysis bctwecn normal and neoplastic tissue slices. It is unlikely that they account for the different rates between slices and homogenates. It is most likely that these differences reside in the amounts, or activities, of the enzymes not tested in the homogenate experiments, i.e., those converting glucose to fructosediphosphate (Fig. 2, through phospliofructokinase, P F K ) . At least, these enzyme systems must be examined first. The first nearly complete analyses of the glycolytic enzymes in tissues were of normal tissues characterized by relatively high rates of glycolysis when intact (Table 11). Detailed work was done by Beck for leukocytes (Beck, 1958a) and Racker and co-workers for brain and
I26
W. EUGENE KNOX
[GLYCOGEN]
()
Plase
GlySyn
G6Pase
1
PGM G6PDH A
HK PHI
7
PENTOSES
1
I
Aldolase T Pisom PGlyADH PGlyK PGlyM Enolase
LDH
1
[LACTATE[FIG.2. The enzyme sequences in glycolysis, gluconeogenesis, glycogen formation, and the pentose pathway. The abbreviations are translated in the text except for phosphohexose isomerase (PHI) (EC 5.3.1.9) and phosphoglyceraldehyde dehydrogenase (PGlyADH, elsewhere as GAPDH). Key enzymes which exert control are located a t sites a t which two different reactions function in the opposite directions (circular arrows). (Adapted from Weber el al., 196513.)
certain neoplastic cells (Racker e t al., 1960; Wu and Racker, 1959; Wu, 1959). The general methodology owes much to the studies of Bucher and his school (Delbriick et al., 1959). The later analyses by Shonk, Boxer, and co-workers are models for this kind of work (Shonk and Boxer, 1964; Shonk et al., 1965b). I n all the tissues the rates of
RATES O F GLYCOLYSIS 4ND LEVETS OF Rat h e r b (Shonk and Boxer. 1964) 25°C.
Glycolysk ( N ? inta t cells) Hexokinase (HK) tEC 2.7.l.l)c Glucose-&phosphate isomerasr (EC 5.3.1.9) Phosphofructokinase (PFK) (EC 2.7.1.11) Fruetosediphosphate aldolase (EC 4.1.2.13) Triosephospbate isomerase (EC 5.3.1.1) Glyeeraldehydephosphate dehydrogenase (NADP) (EC 1.2.1.13) 3-Phosphoglyeerate kinase (EC 2.7.2.3) 3-Phosphoglyceratr phaphomutase (EC 5.4.2.1) Phosphopyruvate hydratase (EC 4.2.1.11) ("enolase") Pyruvate kinase (EC 2.7.1.40) Lactate dehydrogenase (EC 1.1.1.27) Ratio
gl yrolq-sis
lllultllrg enz?.mr
(X2)
(XZ)
0 34 (J7"C.j 1 1 102.0
Leukocytes (chicken) (Racker ef al.. 1960) 26°C.
0 4 (26°C.) 9 4
-
TABLE I1 ENZYMES I N NORMALAXD
C:LYCOLYTIC
NEOPLASTIC
T~SSUES~
Leukemia (man) Rat kidney* Erlich ascites (Shonk and HeLa (Wuand Lymphocytesb Myelocytesh Boxer. 1964) (Wu.1959) Racker, 1959) (Beck, 1958aj (Beck, 1958,) 26°C. 37°C. 37°C. 25°C. 26°C. (rmoles triose equivalent/min./100 mg. cell protein)
0 72 (37°C.) 0 9 (37°C.) 4 4 5 5 106 0 234 0
1 4 (30°C.) 4 6 58 0
I 8 (37"r.) 2 l(37"C.j 3 3 3 8 469 0 138 0
Rat braid (Shonk and Boxer, 1964) 25°C.
2 6 (37°C.) 20 0 146 0
Leukocytcs (man) (Beck, 1958a)
37°C.
6 7 (3iY:.) 8.9 440.0
(X2)
1.8
28 0
3 2
6 5
57 0
4 2
4 6
27 0
10 2
(X2)
52
31 2
6 6
8 5
19 0
11 8
15 8
15 0
13.8
216.0
-
342 0
41 0
13 0
20 6
29 4
378 0
26 0
29 0
19 0
38 0
65 0
69 0
21 3
30 3
74 0
46.5
34.0
165 0
47 0
610.0
366 0
66 0
16.0
30 0
7 8
28.0
23 0
25 0
4.4
21 0
9 4
18.0
15 0
14.0
11 0 74 0
55 0 111 0
20 0 49 0
115.0
284 0
79 0 132 0
0.31
0.04
0.23
0.16
0.30
4 4 40 0
>0.55"
5.7 :34 0
> O .iP
116 0 58.0 0.71 (0 13)
14 4 53 1
> O i3d
0 All the values are expressed in thc same units to the same base. hut different temperatures were used. The temperatures of the enzyme assas-s are given in the heading of each column. Thr ttmperatures a t which glycolysis was measured are given in parentheses in the first line. Twice the actual units of the first four glycolytic enzyme artivities (" X2") are tabled. since one glucose forms t n o lactate molecules. The values for anaerobic glycolysis with glucose in intact cells are from the references cited in the column headings, except for rat liver. kidney and brain. These are calculated from the values given in Table I. To interconvert the various bases on which data were expressed in this and other tables, the cell protein and dry weight in mg./g. fresh tissue, respectively. were taken as 220 and 310 for liver, 180 and 340 for kidney. 110 and 204 for brain. 200 and 230 for heart, and 130 and 182 for rat tumors (Long, 1961). N X 6.25 = protein. c Enzyme Commission number (I.U.B., 1965). Published values are the means, with standard deviations, of a significant number of determinations. Enzymes measured a t the same instead of lower temperatures than glycolysis. tending to decrease these ratios relative to the other ratios.
m
128
W. EUGENE KNOX
anaerobic glycolysis in the whole cells (Table 11, first line) were less than the optimal activities (expressed in similar units) of the limiting enzymes found in the honiogenates (e.g., hexokiiiase, second line). The activities of enzymes in cells are apparently held in check, a t least to the extent of these discrepancies between their actual activities measured in whole cells and their potential activities measured in homogenates. They show about half or less of their full activity when in the cells (Table 11, last line). Thcre is no definite suggestion in these figures that the enzyirie activities are suppressed relatively more in normal than in neoplastic tissues. The chemical nature of this discrepancy of activity in and out of cells, and its variation within cells (e.g., the Pasteur effect), is the object of intensive current investigation. The goal is to account for the variable but lower activity in chemical terms (substrate and cofactor concentrations, etc.) ; this activity must be calculated for a n enzyme both inside the cells and when an equal amount is extracted from the cells and assayed under optimal conditions. I n the meantime, these analyses also suggest that the a m o u n t of an enzyme can sometimes be used as an initial approximation of its activity in vivo, especially if a limiting or “key” enzyme in a series is chosen (Fig. 2 ) . Table I1 shows that the enzymes most often limiting in amount are hexokinase ( H K ) and phospliofructokinase ( P F K ) . These two enzymes catalyze reactions between glucose and fructosediphosphate, where the differences in rates of glycolysis could reside between slices and homogenates or between normal and neoplastic tissues as discussed above (Table I ) . The tissues described from left to right in Table I1 range over a twentyfold difference froin low t o high glycolytic rates. This extends the possibilities of correlating the levels of the limiting enzymes with the rates of glycolyxis in the tissues. The ratios of the glycolytic rates to the amounts of the limiting enzyme (last line) do tend to be similar. With the exception of chiclien leukocytes, they cover less than a fivefold range. Additional allowances must be made for the fact that the data used are from different sources and were not collected for the purpose of this comparison. While the possible correlations between metabolic function and enzyme content in this type of data will repay additional studies, the correlation of cnzyine contents with the known glycolytic pattern among neoplastic tissues is of more immediate concern. The neoplastic tissues group theinselves together in the middle of Table I1 with somewhat similar but not irlcntical enzyme patterns. Patterns of the same enzymes have been determined for some solid tumors, in particular those reported by Shonk and co-workers (Shonk
THE EXZI’RIIC PATTERN O F NEOPLASTIC TISSUE
129
et al., 1964, 1965:1,b). Tlicsc pcrmit direct comparisons bctwccn iiorrnal arid neoplastic tissues studied hy the s:me nicthotls. The data have been expressed in international units per gram of fresh tissue, which has the advantage of siinplicity. Biir et al. (1963) also indicated in their extensive studies that the glyccr:tldchydc~~hosphate dchydrogcnase (GAPDH) activities in the difl’erent ti.ssucs tended to be similar, and to fall in the middle range of the 0 t h enzyme values. The samc tendency can be seen in the data of Table 11. Their suggestion of expressing other enzyme activities in relation to the G A P D H activity has been followed here. I n addition, tabulation of only those enzymes whose relationships are of the most interest reduces the array to a comprehensible pattern. The glycolytic rericb of enzymes can be rcpresentcd by the limiting or “key” steps (Wcber e t al., 1965a) that involve potentially large cnergy changes and that arc circumvented by other reactions when the general sequence of glycolysis is reversed during gluconeogenesis (Fig. 21. T h y are H K , PFK, and pyruvate kinasc (PI<).Aldolase, which appears to correlate rather well with glycolysis in Tahle 11, has been acldccl for comparison with the “key” enzymes, and GAPDH, n.hicli catalyzes the critical oxidation step in glycolysis, has beeii employed as tlic reference stand:trd. The relative levels of these enzymes for several nornial ti,wies :md for certain tumors are given in Tables I11 and IT’. Table I11 also shows the important point, made explicit by Smeency et a l . (1963), t h a t the rates of glycolysis in slices of hepatomas correlate with their rates of growth. Enzymes t h a t determine glycolysis therefore also will determine that most obvious property of a nc‘ophsni, its ccaselcss growth. I n normal rat tissues, the ahsolute amounts of G A P D H per gram of fresh tissue are similar, as are the aniounts of aldolase, while the relative amounts of the key cnzymcs of glycolysis can be seen to increase in proportion to the known :microbic glycolytic metabolism of these tissues (Table 111). This clear itlationship was obscured in Table 11 partly by the manner of presentation. The transplanted tumors with high glycolytic metabolism (per milligram dry weight) have lower absolute amounts of GAPDH per gram of tissue (neoplastic tissues have higher water content than normal tissues, see footnote, Table II), but the relntizx :tmounts of the glycolytic enzymes approach or exceed those in the patterns of the normal tissues with the highest glycolytic rates. Furthemore, the slowest growing hepatomas have rates of glycolysis in slices virtually identical to that of liver (Aisenberg and Morris, 1961), and their rclativc glycolytic ciizyme patterns depart only slightly froni that of liver toward the pattern of the tissues with the higher glycolytic rates. These slower-growing tumors have intermediate enzyme
130
\V. EUGENE KNOX
TABLE I11 I<EY
GLYCOLYTIC ENZYMES
IN
N ~ R M AA NLD NEOPLASTIC RAT TISSUES"
GAPDHb Rate of glycolysis ____ HK (pmoles/ (Aisenberg, 1961) rnin./g. Q c o , ~ ' ~ Qco2Nz tissue) Aornial tissues Liver Kidney Heart Brain Neoplasms Hepatomas, slow-growing (8 tumors)c Hepatomas, fast-growing (4 tumors)* Walker 256 carcinosarcoma
1.0 0 3.0
0.5d
3.0 3.0 19.0 1.Od
63.0 69.0 90.0 81.0 50.0
PFK
Ald
PK
GPDH
Relative activity (GAPDH = 100)
1 . 9 3.2 9 . 2 38.0 103.0 5 . 6 4.2 8.7 51.0 36.0 5.0 8 . 3 13.3 135.0 12.0 13.5 18.6 10.5 158.0 9.4 2.5
5.4
7.2
35.0
28.0
6.0
12.0
47.0
10.5 12.3
7.5 339.0
8.0
24.0
42.0
30.0
15.0 16.0
7.9 417.0
2.0
a Tissues are arranged in the order of increasing glycolysis (normal tissues) and increasing growth rate (neoplastic tissues). Enzyme levels are recalculated from the data of Shonk and Boxer (1964) for normal rat tissues and for neoplastic tissues (Shonk et al., 1965b). The standard deviations of the original values indicate that absolute differences greater than about 20% may be significant. * GAPDH, glyceraldehydepliosphate dehydrogenase (EC 1.2.1.13); HK, hexokinase; PIX, phosphofructokinase; Ald, fructosediphosphate aldolase; PK, pyruvate kinase; GPDH, glycerolphosphate dehydrogenase (EC 1.1.99.5). c The eight slow-growing hepatomas averaged here are 7787,7793, 7316$,7800, 5123 (A, C and D), and H35. The growth periods needed before transplantation ranged between 1.7 and 9.8 months. The incorporation in v i m of CL4-aminoacids into the protein of two of these (H35 and 7288C) was 90 and 130% of that in normal liver (Wagle et al., 1963), and the incorporation i n vivo of CI4-formateinto the DNA of five of these (7288C, 5123 [t.c. and C], H35 and H35 t.c.) averaged 290y0 of that in normal liver (Wheeler et al., 1964). dRespiratory quotients cited are for the Morris hepatoma 5123 (Aisenberg and Morris, 1961). Other values are in Rweeney el al. (1963). 5 The four fast-growing hepatomas averaged here are the Dunning, Novikoff and Morris 3924A and 3683. Azo-dye-induced tumors gave similar values, but are not included. The growth period needed before transplantation was between 0.25 and 0.6 months. The CL4-aminoacid incorporation in vivo into the protein of 3924A and 3683 was 300 and 275% of that in normal liver (Wagle el al., 1963). The C14-formateincorporation in vil!o into the DNA of the Novikoff, Morris 3683 and 8647 (another fast-growing hepatoma) averaged 780y" of that in normal liver (Wheeler et al., 1964).
131
THE ENZYMIC PATTERN O F NEOPLASTIC TISSUE
TABLE IV I N NORM.4L A N D NEOPLASTIC TISSUES OF MAN. KEY GLYCOLYTIC ENZYMES GAPDHb
HK PFK Ald Glycolysis Tissue
Qco,Oz
QCo,Nz
Liver Iiidnep Heart Rectum Rectal adenorarcinoma
-
-
1@
10"
-
14c 2oc
(pmoles/ niin ./g. tissiie) 65.0 63.0 61.0 25.0 67.0
PIC
GPDH
Relative activity (GAPDH = 100) 1.9 2.5 10.0 5.3 5.0
0.8 0.8 3.3 5.8 2.0
3.7 2 8 . 0 2 4 . 0 3 . 0 83.0 2 7 . 0 6 . 5 151.0 6 . 2 5 . 8 116.0 2 . 6 4 . 5 100.0 1.0
Calculated from the data of Shonk el al. (1964, 1965a), which give means and standard deviations for a significant number of samples of each tissue. Abbreviations: See Table 111. The respiratory quotients are for normal rat jejunun~(Dickens and Weil-Rlalherbe, 1941) and for the mean of nine human rectal adenocarcinomas (Rosmthal and Lasnitzki, 1928). The values indicate that some intestinal mucosae normally have higher rates of glycolysis t,han those cited in Table I .
patterns. The most significant change in the glycolytic enzymes with the increase in glycolytic capability in this series is the sevenfold increase in the relative amount of the first enzyme of glycolysis, the HK. There arc also similar increases in the relative amounts of the second key cnzynic, PFK. Weinhouse and liis co-workers had independently cstablishctl that glycolysis in tliese hcpatomas was limited by the amounts of total HK (Elwood et al., 1963), and that the increase in this total activity was correlated with increased growth rate (Sharma et al., 1965). Thus, the data in Table I11 illustrate three significant facts. There is a particular pattern of thc glycolytic enzymes toward which those in normal tissues progrcss as the rates of glycolysis in these tissuc slices increase. The neoplastic tissues share this same pattern to the cxtent that they also have siiiiilarly high glycolytic rates in slices. Finally, growth rates follow hot11 tflic rates of glycolysis and the amounts of the key glycolytic enzymes. As illustrated in Table IV, the same facts hold for normal and n.eoplastic human tissues. T h e nicasureiiients (Shonk et al., 1964, 1965a) were done in the same laboratory as those in Table 111 and arc presented in the saiiie manner. The absolute values are virtually identical to some similar measurements made earlier by Schmidt and Schmidt (1960) for normal huiiian liver, and by B a r et al. (1963) for norrrial and neoplastic human tissues. Tliere are small species differences between the patterns of enzymes in human and rat tissues. The absolute
132
W. EUGENE KNOX
amounts of the enzymes, indicated by the amounts of GAPDH, are very similar in the different human tissues and similar to those in the rat tissucs, cxccpt that they are lower in the rcctal mucosa. But again, the rclative amounts of the kcy glycolytic enzymes arc higher in the normal human tissues that are characterized by the higher glycolytic rates although this elevation is less striking than t h a t seen in tlic r a t tissues. The erizynic measurements are very similar for the normal and neoplastic rectum, and these wcre indistinguishable from those also givcn for the normal and neoplastic colon. These patterns would indicate a glycolytic potential somewhat higher than t h a t for kidney or heart, and such high viilucs are citcd in the table. The absolute enzyme amounts in normal rectal tissue were less than half of those measured in other normal human tissues. Thc major change with neoplasia of the rectum mas an increase in the absolute amount of GAPDH and other enzymes to the level found in tlic other tissues (Table I V ) . This occurrcd with maintenance of the type of enzyme pattern associatcd with the higher glycolytic rates. Although tlie amount of enzymes per niilligrain of soluble protein was not differcnt between normal and ncoplastic colon ( B k e t al., 1963), there was a real increase in enzymes, and presumably in glycolysis, per gram fresh weight. This illustrates the value of a second, physiological, base for expressing activities. Analyses of thirtythree different human tumors of sixteen types and some normal human tissues provided other illustrations that the glycolytic enzymes arc increased or a t least preserved in amount in human neoplastic tissues (Biir et al., 1963). Warburg’s generalization t h a t neoplastic tissues tend to have high glycolytic rates (Table I) and Greenstein’s generalization that neoplastic tissues tend to have the same pattern of enzyrncs (Fig. 1 ) arc both illustrated by the data from rats and man in Tables I11 and IV. Also, the similar pattern of glycolytic enzymes to which tlic neoplastic tissues tcnd is the particular pattern that can be associated with the high glycolytic rates in any tissues. This demonstration derives part of its strength from the occurrence of intermediate types. Normal tissues have a range of glycolytic rates, which generally corrclatcs with the incrcasing relative amounts of the key glycolytic cnzymes. The same is true for neoplastic tissues, and among these the slow-growing hepatomas represent the intermediate types. While the intermediate types strengthen the above correlation, their existence also reaffirms the fact that no qualitative difference between normal and neoplastic tissues has yet been demonstrated with regard to glycolytic rates or enzyme pntterns. The two generalizations about glycolysis and enzyme patterns are linked, yet the convergence toward a particular type of metabolism
THE ENZYMIC PATTERN O F NEOPLASTIC TISSUE
133
and ciizymc pattern in tumors is descriptive, not diagnostic. The same ib true for morphological criteria taken individually. IV. Glycerolphosphate Dehydrogenase levels and Glycolysis
The particular enzyme pattern toward which neoplastic tissues tend is neverthclcss of geneid interest. \\'lien extended to more than the glycolytic enzymes, the pattern might ultimately be recognized as unique. Advances in the untlcrstanding of the nature of neoplasia can tie expected from further studieb of the particular enzyme patterns s h a r d by such tisbucb. At the very least I t would help to know more about the pztrticular type of niet:ibolisiii that the pattern of enzymes in tumors will permit. In this senbe, a proper study of ncoplasia is the study of neoplastic tissues. For example, in addition to the increased relative amounts of the key glycolytic enzymes shown in Tables I11 and IV, the relative :mounts are given for another enzyme, glycerolphosphate dehydrogcnase (GPDI-I). Neoplastic tibsucs generally have none or greatly rcducctl amounts of this (~nzyiiic,:I\ nvis first pointed out by Bucher c t al. (Dclbriick e t al., 1959) and others (Boxer and Shonk, 1960; Angeletti et nl., 1960a). The enzyme was also very low or absent in the analyses of Ehrlich ascitcs cells (\\-uand Racker, 1959) and HeLa cells (Wu, 1959) cited in Table 11. The values assembled in Tables I11 and IV corroborate these fncts. The wlues alro show that the relative amount of this enzyme decreases in both normal and neoplastic tissues as their glycolytic potenti:il rises. This may help to predict the metabolic behavior of a tissue from its pittern of enzymes. Information that is still commonly lacking is required to relate the pattern of enzymes in a tiscue to its metabolic behavior. The actual reaction that is catalyzed tiy :I given enzyme zn vivo and the physiological role this reaction subserves must be known for a number of enzymes in older to clarify the rc.l:itionsliip. The reactions and the physiological role of the glyrolytic enzymes, a t least, RI’C clear. They convert glucose into Inctatc, or inore iiiimedintcly, into pyruvntc (Fig. 2 ) . Biicher has demonstrated that, in a variety of tissues, a role of the G P D H reaction is to t r w s f e r electrons, via reduced nicotinamide adenine dinucleotide (NA4DH),from their formation by the oxidative reaction of glycolysis (GAPDH) into the mitochondria for terminal oxidation (Delbruck e t nl., 1959; Schimassek e t al., 1963). One consequence of this reaction, then, is to regenerate nicotinaiiiide adenine dinucleotide (NAD) from NADH so that glycolysis can proceed without lactate forniation and $0 the pyruvate that is formed is available for irrimecliate oxidation (Fig. 2 ) . The measurements of GPDH in nor-
134
W. EUGENE KNOX
ma1 and neoplastic tissues provide further evidence of such a function of the enzyme because it should be less, as it is, in those tissues characterized by higher rates of lactate formation, and espccially in those with the higher rates of aerobic lactate formation that are typical of the neoplastic tissues. A corollary of the finding that low amounts of G P D H promote acrobic lactate formation is that the role of lactate dehydrogenase (LDH) would be expected to increase in a tissue as the amount of G P D H decreased. It has already been noted that the relationship between the amounts of these two enzymes may have a direct bearing on the rate of acrobic glycolysis (Boxer and Devlin, 1961). As expected from the previous discussion, the ratio of L D H to G P D H is high, or higher than that of tlic tissue of origin, in all of the neoplastic tissues examined so far (see references in Shonk e t ul., 1965a). Such a relationship would be prcdictcd from the combination and extension of the generalizations of Warburg and Grcenstein about the nietabolic and enzyme patterns of neoplastic tissues that is illustrated here. V. The Enzymes of the Pentose Pathway
The enzyme pattern of neoplastic tumors in addition to the increase of glycolytic capability and the associated decrease of the G P D H can be defined further by other functional groups of enzymes. One such group is responsible for the direct oxidative pathway of carbohydrate metabolism leading, inter a&, t o pentoses. This pentosephosphate or hexosenionophosphate pathway was recognized in part by studies of the persistent aerobic metabolism of glucose in neoplastic tissues. Dickens and Glock (1951) demonstrated the presence of the first enzymes of this sequence in rat hepatomas. Thc remaining enzymes in the sequence leading to scdoheptulose, pentosc, and hcxose formation have been demonstrated in four typcs of transplanted mouse tumors (Bosch e t (ul., 1956). The first enzymes of the sequence were also present in Ehrlich ascites cells (Williams-Ashman, 1953), mouse lymphosarconia (Villaviccncio and Barron, 1957), human myelocytic, and lymphocytic leukemia cells a t almost the level that is present in normal leukocytes (Beck, 1958a) and in cultured HeLa cells (Wu and Racker, 1959). Quantitative measurement in a variety of normal and neoplastic tissues showed that the first and second enzymes, glucose-6-phosphate dehydrogenase (G-6-PDH) and phosphogluconate dehydrogenase, were present a t comparable levels in ten different rat tumors examined. The levels in tumors were higher than those in normal muscle, and usually higher than in heart, brain, or liver of male rats. The levels in tumors were less than in adrenal cortex or in the richest normal tissuc, lactating
THE ENZYMIC PATTERN O F NEOPLASTIC TISSUE
135
rnaiiiniary gland (Glock and McLean, 1954). T h a t levels of a n enzyme in neoplastic tissues were often intcriiiediate between the extremc lcvcls found in different nornial tissues was repeatedly pointed out by Greenstein (1954). The significance of the presence in neo1)lastic tissues of G-6-PDH and the enzyrncs following it can be judged hy tlic relative levels of these enzymes found in the differcnt type5 of tumors aiid in particular normal tissues, possibly tlie tissues of origin. For exaniplc, R normal inuscle has a very low level of G-6-PDII. T h e level increases significantly in rhabdomyosarcomzi (Angeletti et nl., 1960a). On the other hand, high levels occur in inamiii:~i~y :~deiioc:ircinonias, but not the very high lcvcls seen in the lactating nianiniary gland. More thxn the inininiuni, but less than tlie niaxiiiiuiii, :mount of the eiizyiiic appears. One of the most valuable comparisons is Letween the relatively low level of G-6-PDH in n o m i d Iivcr :ind the higher levels in the series of tranhplaiitcd rat hepatomas tliat grow a t different rates. Glucose-6phosphate dehydrogenase w:is clcvated in the fast-growing Novikoff hcpatoinas (Weber, 1961) :tnd in the slow-growing Morris 5123 hepatoinas (Potter et al., 1960). The later coiupnrieons between seven differcnt hepatomas rcvealcd levels of tlic crizynic in all of thein that werc equal to or higher tli:tn tliobc in nornial liver. The levels in sonic of the slowcr-growing tumors were 15 to 20 times the level in iiornial liver (Weber and Morris, 1963). T h e averaged levels arc given in Table V for an even larger scrics of thcsc hepatoinas and for some normal tissues taken from tlic stutlics of Slionk and Boxer (1964) and Shonli et nl. (1965b). The t1:it:i :trc again exprcssctl relative to GAPDH and :ire separated for the slow- and fnst-growing hepatoinas. The relative I c v c l ~of G-6-PDII in t l i c l i c ~ ~ a t o i i niitl i a ~ in tlic \TTitlker 256 carcinoina, included for coiiip:~i~isori,arc a t I(,: twicc that of nornial rat liver. Tlic al)solutc valucs iii the hepatoiiias :ire albo d o u l ~ l cthat of liver, and they are about equal to that of liver in the \\7alker 256, which has less of the reference enzyme. The measurements of tlic G-6-PD1-I in human tissues that are also listed in Table V show that tlic relative and absolute levels found in liver are :tlso present in rectal iiiucosa and pcrslst in rectal adenocarcinoma (and in colon and colon atienocarcinomas) (Shonk et al., 1964, 1965a). Ca!culation in tlie smie way of the data of B i r et al. (1963) also givcs an average level of G-6-PDH equal to 2.4% (range, 0.9-Fi.4) of the reference enzynic (GAPDI-Ij in twenty-one analyses on nine types of Iiunian tumors. Nine additional single analyses on other tuniors were soincwhat higher or lower than this range. The :tnalyses of eight normal tissues, including liver, were in this same range (one
TABLE V GLCCOSE-~-PHOSPHATE DEHYDROGEKASE (G-6-PDH) I N ~ \ T o R ~ ~ A LA N D I\ITEOPLASTIC
TISSUES OF RAT A N D
MANa
Man
Rat GAPDH [pmoles/min./g. tissue (25'C.)]
G-6-PDH Relative activity (GAPDH = 100)
63.0 69.0 90.0 81 . O
4.0 2.5 0.8 2 .o
GAPDH [pmoles/min./g. tissue (25OC.)]
G-6-PDH Relative activity (GAPDH = 100)
65.0 63.0 61.0 25.0 -
1.4 2.1 0.6 1.5 (14)
Normal tissues Liver Kidney Heart Brain
Liver Kidney Heart Rectum Assorted normal tissues (8)
w
2
Neoplasms Hepatomas, slow-growing (8) Hepatomas, fast-growing (5) Walker 256 carcinosarcoma
50.0 (28-86) 47.0 (37-57) 30.0
11.2 (3.S 2 3 ) 10.5 (7.2-15) 8.1
0
Rectal adenocarcinomas Assorted tumors (9)
67.0
1.5
-
2.4 (0.9-5.4)
0 The values are calculated relative to glyceraldehydephosphate dehydrogenase (GAPDH) activities in the same tissues, from the data of Shonk and Boxer (1964) for normal, and Shonk el al. (1965b) for neoplastic rat tissues, and Shonk et al. (1964) for human tissues, and from that of Bar et al. (1963) for the assorted human normal tissues and tumors. The numbers of tumor types and the ranges of values are given in parentheses. The same rat hepatomas are described in Table 111.
x
T H E ENZYMIC PATTERN OF NEOPLASTIC TISSUE
137
analysis in testis was higher, 15%). Thus a relative or absolute level of G-G-PDII coiiiparable to, or higher than, that found in noriiial liver seeiiis to be characteristic of most aninial and human neoplastic tissues. It is possible that this level results froiii an increase wlicn the levcl in the tissue of origin is low, or from a dccrcase when the level in the tissue of origin is high. Coinparisons have not yet been inade that convincingly illustrate bucli qu:mtit:ttive changes during carcinogenesis. There are, of course, ample studies showing that intact neoplastic tissues carry out the over-all reactions of the pentose pathway, utilizing such techniques as the preferential oxidation to C"0, of glucosel-C14 over glucose-G-C14 (Kit and Griffin, 1958). Correlations, such as have been compiled here for glycolysis, have not been reported between other fuiictional measurements and the amounts of the neccssary enzymes present in the tissues. Nor has the physiological purpose of these reactions in the neoplastic tissue bccn so precisely defined as has that of the cnergy-producing functions of the glycolytic enzyiiics. But tuinors tend to have a cliaractcristic levcl of the cnzyrncs of the pentosc pathway, and a characteristic function of these enzymes in tumor nietabolim can be anticipated. VI. Enzymes of Gluconeogenesis a n d Glycogen Formation
It is usual to consider the next two functions together: gluconeogeiiesis, defined as glucose formation from noncarboliydratc precursors through rcactions, in part, the reverbe of glycolysis ( Krebs, 1963) ; arid glycogen formation and degradation, rcspectively f i 0111 and to glucosc-G-phosphnte (Fig. 2 ) . Thew functions are separatctl here because tlicy arc sepsrnted i n iiiobt tissurs, ouly liver and kidney having both functions to a significant degree. Glucoiicogcnesis occui's mainly in liver and kidney. It is apparently diipens:ible for neoplastic tissues. On the other hand, it is possiblc that a retluccd capability for glycogen formation, or for part of this bystein, iiiay persist in neoplastic tissues. Gluconeogencsis has been studied in relation to neoplasms only in liver. Biochemists have made little usc of the few experimental renal tumors that are available. A number of precursors, such as the amino acids, can be converted in liver by diffcrcnt reactions to the pi*oducts of glycolysis, and then by reversal of glycolysis, to glucose. A few enzymes, diffcrcnt froin those participating in glycolysis, act to circuinvent the energctically unfavorable reactions in reversed glycolysis, and these are the sites of the control of gluconeogcnesis: pyruvate carboxy1:lsc ( P c ; P y carboxylasc in Fig. 3 ) , phosphopyruvate cai%oxylitse ( P E P C K ) , FDP phosphatase (FDPase) , and glucose-G-phosphatasc (GGPase or G-
6-Pase) (Figs. 2 and 3 ) . I n his review, Weber described the complete loss of glucose-6-phosphatase a n d f1~uctose-l-6-depliosphate(FDP) phosphatase from the Novikoff hepatomas, as well as loss of their ability to forin glucose. Thcrc was also loss of tlic glucosc-6-phosphatase activity during azo dye (DAB) 1ieI)titocRrcinogencsis (Webcr, 1961). H e later examined all the key enxyiiies of gluconeogenesis in the iiitermeciiate series GLUCOSE-
G-6-Pase
FD Pas
pr
GK
P FK
Aldolase
I
TP Isomerase P Gly A DH P Gly K
I
P Gly M Enolase
Pyruvate
kinase
A f FIG. 3. Th e enzymes of glycolysis and gluconeogenesis shown in Fig. 2 are separated into the controlling Bey enzymes for each of these functions and the remaining bifunctional cnzymcs. (GK, glucokinasc.) (From Wcber et al., 1965a.)
of slower-growing rat hepatomas; the results are shown in Table V I (Weber et al., 1965b). The enzymes are present but decreased in amount in the slow-growing hepatomas, and they substantially disappear in the fast-growing hepatomas (Fig. 4) . The more rapidly growing hepatomas lose the necessary enzymes and lose the normal liver function of making glucose for the rest of the body (the Cori cycle). I n this way they conie to resemble most other normal and neoplastic tissues.
TABLE VI BEHAVIOR OF KEY GLGCONEOGEXIC ENZYMES IS HEPATOMAS OF DIFFERENTGROWTHRATE^^* Growth rates of hepatomas Slow Enzyme G lucose-6-phosphatase (EC 3.1.3.0)" Fructose-1 ,g-diphosphatase (EC 3.1.3.11) Phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32) Pyruvate carboxylase (EC 6.4.1.1)
Normal liver
Medium
Itapid
5123D
7800
H35
5123t.c.
7288C
i288B
3924A
3683
Novikoff
100
5gd
18d
50d
4Zd
<2d
<2d
100
30d
5id
lgd
22d
lld
27d
5d
3d
100
96
63d
-
-
35d
-
9d
5d
-
100
99
-
-
-
-
-
14d
9d
-
Adapted from Weber el al. (1965b).
* Enz>me activities were calculated as pmoles of substrate metabolized/hr./average
cell a t 37°C. The activities were eqressed in percentages of values found in normal liver. The mean represents data from four or more livers or tumors. c Enzyme Commission number (I.U.B., 1965). d Statistically significantly different from corresponding normal liver values.
140
W. EUGENE KNOX
The capability for glycogen formation in neoplastic tissues is less clearly decided than that for gluconeogenesis. It is well known that the glycogen content of neoplastic tissues, even of the slow-growing hepatomas, is low (Ball e t al., 1957; Nigam et nl., 1962; Weber e t al., 1961; Weber, 1963). The enzymic reactions for the synthesis and degradation of glycogen are phosphoglucomutase (PGM), glycogen synthetase (glycogen-UDP-glucosyltransferase) (GlySyn) , and phospliorylase (Plasc)
60 40 20
$1
401 60
20 0
Glucose- 6 - Phosphatase
FIG.4. Glucose-6-phosphatase and fructosc-l,6-diphosphatase activities in normal and neoplastic livers. The hepatomas are arranged in the order of increasing growth rate. They show the progressive loss of these enzymes of gluconeogenesis with increased rate of growth. (From Weber, 1963.)
(Fig. 2 ) . At least the last two enzymes, however, are activated and deactivated by a sequence of othcr enzymes acting in cascades, and also depend upon additional enzymic products in order to function. This can cause confusion in measuring the amount of enzyme by its activity unless the necessary precautions are taken to obtain the full activity. The precautions may not be the same for neoplastic and normal tissues. Because of the uncertainty that such enzymes have been fully activated in a given assay, their activity is more convincing than their inactivity. The reported activities of these enzymes are often decreased in neoplastic
THE ENZY,MIC P A W E R N O F NEOPLASTIC TISSUE
141
tissues, but not to the same extent as are the key enzymes of gluconeogenesis. Phosphoglucomutase was low and glycogen formation insignificant in both normal and leukemic white cells of man (Beck, 195813). HOWever, cultured HeLa cel!s contained respectable amounts of phosphogluconiutase (Wu, 1959), glycogen synthetase, and phosphorylase (Racker et al., 1960). The last two were elevated when the cells were grown on a high glucose medium. The phosphoglucomutase in the graded series of rat hepatomas (Weber, 1963), even with activation by Mg++ and iniidazole (Weber et al., 1964), was lower than in normal liver (Fig. 5 ) , but this enzyme was not so low and its loss not so well corre-
*
69
FIG.5. Phosphoglucomutase activities in normal and neoplastic livers arranged in order of increasing growth rate. Loss of the enzyme is less complete and follows a different pattern than the gluconcogenic enzymes in the same tumors of Fig. 4. Asterisks indicate significant differences from liver. (From Weber, 1963.)
lated with the growth rates as were the key enzymes of gluconeogenesis (Fig. 4). Phosphoglucon~utasedecreases in the faster-growing rat hepatomas to the low rclative level characteristic of other tissues of rat but does not disappcnr (Table V I I ) . The same fraction, 10 to 15% of that in liver, persists in human intestinal adrenocarcinomas (Table VTI) . Glycogen synthetase, in addition to its presence in HeLa cells as
142
W. EUGENE KNOX
TABLE VII PHOSPHOQLUCOMUTASE IN NORMAL AND NEOPLASTIC TISSUES~~~
Tissue
Glyceraldehy dephosphate dehydrogenase (GSPDH) Phosphoglucomutase [pmoles/min./g. relative activity tissue (25"C.)] (GAPDH = 100)
Rat Liver Kidney Heart Brain Hepatomas, slow-growing (8)c Hepatomas, fast-growing (4)"
Human Liver Kidney Heart Colon Rectum Colon adenocarcinoma Rectum adenocarcinoma
63.0 69.0 90.0 81.0 50.0 47.0
30.0 6.0 8.6 4.1 31.0 (27.O-35.0) 3.9 (2.8-5.3)d
65.0 63.0 61.0 26.0 25.0 73.0 67.0
35.0 5.1 9.4 5.4 7.9 5.2 4.5
Data from Shonk and Boxer (1964);Shonk et al. (1965a,b). The absolute levels of GAPDH and the relative levels of phosphoglucomutase (EC2.7.5.1)are given for tissues described in Tables I11 and IV. The number of the rat hepatomas, described in Table 111, are given in parentheses. The range of the relative activities among these tumors is in parentheses.
mentioncd previously, was present in the Novikoff liepatoma a t one third of its normal activity in liver (Nigarn e t al., 1962; Nigam, 1962). Phosphorylase, in addition t o its presence in HeLa cells grown in high glucose (Racker e t al., 1960), was present in the Novikoff hepatoma in the same total amount as in normal liver (Goranson e t al., 1954), or at least in half this normal amount (Nigam, 1962). Interestingly enough, it was also present in the same amounts as in liver in a mouse mammary carcinoma (Goranson e t al., 1954). I n all of these tissues, the inactive form, phosphorylase b, predominated. Demonstration of the enzyme, therefore, depended upon activating it fully. Yet in a variety of ascites tumors, cultured tumor cells, and a solid tumor, in which negligible amounts of phosphorylase were present, the activating enzyme could be demonstrated (Nirenberg, 1958, 1959; Nirenberg and Hogg, 1958). Phosphorylase was also very low in DAB-induced primary hepatomas (Hadjiolov and Daucheva, 1958). The near absence of the enzyme in HeLa cells grown in low glucose concentration, and its pres-
THE E S Z Y X I C PATTERN OF NEOPLASTIC TISSUE
143
encc with higher gluco3c coiiccntration (Racker e t al., 1960), plus the unsolved problems for full activation emphasize t h a t greater significance must be attaclied to tlie positive rather than to the negative findings about this enzyme. I t persists, or even increases, in a sufficient number of tumors to suggest tliat it niuy be :t c1i:tractcristic enzyme of these t1ssues. The available measurements are thus inadequate to indicate whether neoplastic tissues tend to lose their glycogen-storing ability or to preserve some of it. There are sufficient instanccs in which some or all of tlie ciizynies are present, and adequate reasons why in certain instances the activities could he overlooked to reinvestigate the question. Since neoplasms arc voracious consumers of glucose, sonic glucose storage capability would not seem inappropriate. It is also possible that other pliysiological functions besides glycogen storage are subservecl by particular enzymes in this sequence. Phosphoglucomutase, for example, can forin C-l-phospliates of other sugars besides glucose (Najjar, 1962). Reactions analogous to that of thc uridine tliphosphoglucosc (UDPG) pyrophospliorylase (EC 2.7.7.9) in glycogen synthesis could use such sugar phosphates for otlier synthetic reactions in the growing cells. VII. Tentative Formulation of the Pattern of Enzymes in Neoplastic Tissues
The incomplete analysis given above of some of the enzymes of carbohydrate metabolism suggests that there is a iiiinimal pattern of enzymes tliat is characteristic of all neoplastic tissues. More of certain enzymes, or additional enzyiiies, may be added to the pattern in part(icu1:tr neoplastic tissues. These enzymes may indicate the origin of the tumoi-, and they can endow the tumor with their own peculiarities, but presumably they are not essential for tunior survival. The added enzymes give intermediate types of enzyme patterns in neoplasms. Study of tlie progrcssion of tlicsc iiiternicdiatc patterns toward the iiiiniirial pattern of neoplastic tissues could illuiiiinate several problems. Presuniably, enzyme patterns change with different growth rates, with the classification of tumors into grades, with the “progression” of a single neoplasni from early to late in its clinical course (Foulds, 1954), and with its evolution during rcpcatcd transplttntations, e.g., from depend~ n c cto autonomy (Greene, 1951 ; Furth, 1953), to dedifferentiation, polyploidy, and ininiunological tolerance. The minimal enzyme pattern, if known, would predict the nietabolic behavior of tumors beyond their high glycolysis. The graded series of rat hepatomas now available offers an opportunity to distinguish such intcrinediate patterns of enzymes in addition
144
W. EUGENE KNOX
to the minimal pattern of enzymes that must be present in all the tumors. An arbitrary choice of some of the enzymcs that have been measured in neoplastic tissues will illustrate this approach. Additional listings of enzymes in tumors are those by Greenstein (1954, 1956), Weinhouse (1959), Weber (1961), Aisenberg (1961), and Reid (1962). I n general, those enzymes associated with nucleic acid metabolism and with electron transport systems have not been included here.
A. DISPENSABLE AND PERSISTENT ENZYMES IN NEOPLASTIC TISSUES There are a number of enzymes that are limited to a few tissues, not necessarily liver, or that occur a t relatively high concentrations in only some tissues, and that are absent or greatly reduced in amount in neoplasms of those tissues. Table VIII lists some enzymes such as these collected by Greenstein (1954), with a few additional examples. It illustrates the well-known fact that all enzymes of a tissue need not persist in a neoplasm arising from that tissue, but that even an enzyme with a highly specialized function may sometimes survive the neoplastic transformation. Intestinal mucosa has the highest level of alkaline phosphatase in the body, and only the stomach niucosa has pepsin, but both of these enzymes disappear from adenocarcinornas of these tissues. The function of the intestinal alkaline phosphatase that is lost by the neoplasm remains unknown. Equally unique are the alkaline phosphatase in bone, or the tyrosinase in the pigment cell, which have wellknown and very specialized functions in these tissues. They may persist in neoplasms, along with their functions of forming bone and pigment, and then disappear, with thcir functions, a t a latcr stage of neoplasia. The presence or absence of enzymes such as these in Table VIII is obviously unrelated to the neoplastic process itself, although the enzymes can contribute to the character of the neoplasm. The behavior of these enzymes shows that they are not essential for neoplastic tissues. They also show that the nature, and the changing nature, of a neoplasm can be correlated with its enzyme content. The simultaneous loss of bone formation and alkaline phosphatase in an osteogenic sarcoma, for example, helps to reveal the functional role of this enzyme in living cells. Conversely, if the physiological role of an enzyme is clear, its presencc or absence in a tumor should reveal somcthing of the tumor’s functional behavior. For example, lacking the key gluconeogenic enzymes, thc hepatoinas arc not glucosc-forming organs. An enzyme of highly unique occurrence in tissues is almost autoniatically eliminated as an essential component of the wide variety of neoplasms. Commonly occurring enzymes deserve more attention. It is not possible, or even desirable, to extend the list in Table VIII so as
TABLE VIII ENZYMES OF L I ~ ~ I T E T~ssr D E DISTRIBL-TIOX (SPECIALIZED FVXCTION) THATARE DISPESSABLE TO NEOPLASMS Enzyme Alkaline phoephatase (EC 3.1.3.1)“
Dehydropeptidase I1 (aminoacylase, EC 3.5.1.14) D-Amino acid oxidase I (EC 1.4.3.3) Gluconeogenic enzymes Pepsin and rennin Tyrosinase (EC 1.10.3.1) (“DOPA oxidase”)
Reference tissue Mouse intestinal mucosa Mouse osteogeriic sarcoma Human osteogenic sarcoma Rat and mouse liver, etc. Rat liver Rat liver Mouse gastric mucosa Human and mouse melanomas
Enzyme Commission number (I.U.B., 1965).
* Osseous formation ceases when the enzyme disappears.
A1)sent (less than 5y0of referenre tissue) in: Int,est,inal adenocarcinoma
E References
Chondro~arcoma
Greenstein (1942), Greenstein (1954, Tables 88 and 89) Greenstein (1954, Tables 88 and 89), Woodard (1956) Woodard (1956)
Hepatomas
Greenstein (1954, p. 380)
Hepatoma, Jensen sarcoma
Shack (1943), Lan (1944)
Hepatomas Gast’ricadenocarcinoma
(See Text,, Table VI, Fig. 4) Greenstein and Stewart (1942)
Amelanotic melanomas
Greenstein and Algire (1944), Greenstein et al. (1944), du Buy et d. (1949)
Repeated transplantations*
m
xN
*
5 *
+d
3
E i P Z
H
%r +u, cj
+I
0
2
i/,
TABLE IX LIVEREXZYMES APPARENTLY DISPEXSABLE FOR NEOPLASTIC TISSUES* Per cent of reference liver activity
Enzyme
Reference tissue: Normal (L) or tumorSlowbearing growingr (TB) liver6 Species hepatomas
Gluconeogenic “key” enzymes
L
Rat
Glycerolphosphate dehydrogenase (EC 1.1.99.5)‘
L
Rat
22
-
Man
L
Rat
-
Mouse/
Alanine-&etoglutarate minotransferase (EC 2.6.1.2) (“GI”’)
L
Rat
L
Mouse
L(-)
Rat
L
Mouse
Glycine acyltransferase (EC 2.3.1.13) (”1;-amino hipmrate synthetase”)
L
Rat
Histidase (EC 4.3.1.3)
L
Rat
Phenylalanine 4-hydroxylase (EC 1.14.3.1)
TB
Rat
Cysteine desulphhydraseo (EC 4.4.1.1)
Glutamiue synthetase (EC6.3.1.2)
References
Fastgrowingd (and all mouse) hepatomas
References
(See Table VI)
6
(See Table 111)
(See Table 111)
Other nonhepatic t UmOTs
1
<5 <10
(0)
135
Wu el al. (1965)
0
Auerbach and Waisman (1958) Greenstein (1954. Tables 88-90)
<14
Wu ef al. (1966). Auerbach and Waisman (19581, Williams and Manson (1953)
12
Masayama ef al. (1938). Viollier (1950)
Cohen and Hekhuis (1941)
<5
2-1500
<5
Rosen (1963) et ol. (1961). Rosen
Levintow (1954) Tung and Cohen (1950)
<3
Auerbach and Waisman (1958). Pitot ef al. (1963)
<8
Auerbach and Waisman (1958)
References
(See Table 111). Delbrfick ef d. (1959), Boxer and Shonk (1960), Angeletti et al. (1960a) (See Table IV)
Rosen (1963) el al. (1961). Rosen
(0) Cohen and Hekhuis (1941)
<14
Levintow (1954)
2
Levintow (1954)
Tyrosine-o-ketoglutarateaminotransferase (EC 2.6.1.5)
TB(+) Rat
p-Hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2)
TB
Rat
Fumarylacetoacetase (EC 3.7.1.2) [‘p-diketonase.” rtc., Edwards end Kcox (1956)l
L
Rat
L
Mouse
Threonine dehydrataseh (EC 4.2.1.16)
L(TB(-)) Rat
SHydroxytryptophn decarboxykse (EC 4.1.1.28)
L
Rat
L
Mouse
Tryptophan p)i-rolase (EC 1.13.1.12)
L(TB(+)) Rat
L
Mouse
1RS9fiO
0-1700’
140
Pitot and Morris (1961). Pitot et al. (1963)
Pitot et al. (1961). Pitot (1960). Pitot et al. (1963). Bottomley et al. (1963) Kizer and Chan (1961)
Pitot
ef al. (19591, Pitot (1962). Pitot el al. (1961). Pitot and Morris (1961). Pitot (1960). Cho et d. (1964). Pitot el al. (1963), Dyer el al. (1964)
110
Auerhach and Waisman (1958)
<10
Auerbach and Waisman (1958)
5
Meister (1948. 1949)
30
Meister (1948. 1949)
0
2
m m
zN
Auerhach and Waisman (1958)
c:
z
12
Kizer and Chan (1961)
<7
Kizer and Chan (1961)
<5
Aucrbach and Waismau (1958). Dyer el al. (1964, see Table HI). Ichii (1958)
z ‘
d P
3 E!
M
0
CJ
0
Claudatus and Ginori (19.57). Chan el al. (1960)
The activities in hepatomas and other, nonhepatic tumors are given as the ‘70of the activity found in livers of normal (L) or tumor-bearing (TB) animals. this is known, the levels in livers of tumor-bearing animals that are higher (+) or lower (-) than in normal livers are so indicated. c TI e slow-growing rat hepatomas include the Reuber H35 and most of the numbered Morris hepatomas not cited in footnote d. See partial listing in Table 111. d The fast-growing rat hepatomas are the Dunning, Novikoff, Morris 3687 and 39248, and DAB-induced primary and transplanted tumors. The various mouse hepatomas are included in this classification, although there are some that might be analogous to the slow-growing rat hepatomas. Enzyme Commission number (I.U.B., 1965). I These early assays of glutamic-pyruvic transaminase were done before its coenzyme was known. D The soluble enzyme (Auerbach and Waisman, 1958) is probably identical with cystathionase (Jollk’s-Bergeretel d..1963). and the reaction in homogenates (Greenstein, 1954) is a compasite one also involving transamination. The same enzyme acts on serine (serine dehydratase). and appears to be identical with cystathionine synthetase (EC 4.2.1.13) (Goldstein et al.. 1962). * A l l these assays of threonine dehydratase in slow-growing hepatomas are highly variable. They appear to have been done with suboptimal concentrations of substrate and coenzyme (Goldstein el ol.. 1962). 6 When
+
2 * m
%r 2 2 m 0
u,
3
P I&
4
148
W. EUGENE KNOX
to eliminate more of the enzymes of limited tissue distribution from the essential minimal pattern of enzymes in tumors. The preference of biochemists for liver is so great that more extensive comparative enzyme studies of neoplasia must use liver as the reference tissue despite its numerous disadvantages. One disadvantage is tlic rich cornplcmcnt of enzymes liver has. Many liver enzymes are uniquely in liver and not in other normal tissues or in neoplastic tissues. Table IX lists some of these enzymes of liver that are apparently dispensable for neoplastic tissues. Among them are a few which persist or are even elevated in slow-growing hepatomas, but all of them decrease in fast-growing hepatomas to about 10% or less of the lcvel in normal (or tumor-bcaring) liver. For a very few, the apparent dispensability of the enzyme has been confirmed by its absence in other, nonhepatic neoplasms. The actual dispensability of an enzyme will be difficult to prove. An enzyme could be presumed to be dispensable if less were present in several neoplastic tissues than in normal tissues containing the lcast amount of the enzyme in question. No investigations have been sufficiently dctailcd t o establish this. It is not certain that relatively low values are actually negligible. Even so, there are over two dozen enzymes in the (incomplete) Tables V I I I and IX that are apparently not essential for tlic neoplastic cell. This alone represents very creditable progress in the search for enzymes “deleted” in neoplasia. Table IX consists largely of enzymes that catalyze steps in the degradation of amino acids, a n important function of the liver. The carbon skclctons of many of these amino acids are diverted to gluconeogenesis in the liver, the enzymes for which are also included in thc table as dispensable. The loss of amino acid-degrading enzymes is apparently associated with the loss of the “anabolic” gluconeogenic cnzymcs in neoplastic tissues. Some other enzymes having possible synthetic functions are included, however, and on further study, these may turn out to be essential. The alanine-a-ketoglutarate aniinotransferase, for example, normally has such a low activity that the persistence of any of its activity in tumors may be physiologically significant. There is insufficient information about the selection of enzymes presented in Table X to decide whether or not they persist in neoplastic tissues. Respectable levels have been reported for most of these in certain hepatomas, but too few have been measured in nonhepatic tumors to decide whether their presence in hepatomas represents an intermediate type of enzyme pattern or whcther they are part of the minimal pattern of neoplasia. Since the actual physiological functions of most of the enzymes are uncertain, it could be argued with equal merit t h a t these are or are not essential for growing cells. Additional measure-
T WHICH3 I . i ~OR MAYSOT PERSIST 13 YEOPLASTIC TISSUFP LIVEREXZYMES Per rent of liver activity
Enzyme Glyrogeii-folming em) mes Asparagitie-ketoacid aminot ransferase (EC 2.6.1.14)* (“aspardgi nase I I ’ ’) Glutamine-ketoacid aminotransferase (EC 2.6.1.15) (“glutaminase 11.’) Glutammyl-sRXA sgnthetase
Fastgrowing rat Slow(and all growing rat mouse) Species hepatomas References hepatomas
Rat Rat Mouse Rat Mouse Rat
References
>30
8’2
Other nonhepatic tumors
Reference?
(t-100
(See text)
0 7
TTu el al.
m
Y.
el al. (1949) el al. (1949)
36 33 58 83 10
Greenstein Greenstein Greenstein Greenstein
50
Tung and Cohen (19.50)
16
Auerbach and Waisman (1958)
< 17
IGzer and Chan (1961) Kizer and Chan (1961)
et al. (1949) et al. (1949) (1965)
Ornithirie carbamoyltransferasec Rat (EC 2.1.3.3) Phenylalanine-pgruvate aminotransferase Rat “Proline oxidase”
Rat
25
Pyrroline-5-carboxylate reductased (EC 1.5.1.2) hlonoamine (serotonin) osidase (EC 1.4.3.4)
Rat
31-310
Rat Mouse
83
Pitot et al. (1963) Pitot et al. (1963)
30-70
a The data are presented as in Table IX, with the activities in hepatomas and other tumors, when available. given as the % of the activity in liver. b Enzyme Commission number (I.U.B., 1965). c Carbamate kinase (EC 2.7.2.2) was not controlled, but was probably not limiting in the assays cited. Reversibly forms L-proline.
Y
A
W
TABLE XI LIVERENZYMES WHICHPERSIST IN NEOPLASTIC TISSUES~
cn #-A
Per cent of liver activity
Enzyme Glwose metabolism G l u e o d p h o s p h a t e debydrogenaee CEC 1.1.1.49)b
Glyeolytic enzymes PhosDhodueomutase (EC 2.7.5.11 Amino acid metabolism Arginsse
Fssegrowinn Slowrat (and all growing rat mouse) Species hepatomas References hepatomas Rat, man
200
200
Rat, mouse. man
>SO‘
>75’
Rat. man Rat
80 Present
(EC 3.5.3.1)
Pitot (1962)
Rat
1-14
>20
Greenstein (1954,Tables 66 and 90).Auerbach and Waisman (1958)
Rat
Dyer et al. (1961),Sheid and Roth (1965), Sheid et al. (1965), Otani and Morris (1965)
Bremick (1964)
2CCI-500
Rat
I5d
Schimke (1964)
0-30
Pitot (1960.1962), Allard el al. (1957), Birns et al. (1959)
3 3 H
20
880 120
3 0
Greenstein (1954.Tables 88 and 89) Dyer et el. (1961),Cohen et el. (1942)
Cohen and Hekhuia (1941), Angeletti et al. (1960b), Angeletti et al. (1960s)
Auerbach and Waisman (1958).Bresnick (1964)
Mouse Glutamate dehydrogenaae (EC 1.4.1.2)
(See Table VII)
12-20
100-150
(See Table V)
13
Increase’
Mouse
Aepartate carbamoyltransferase (EC 1.4.1.2)
References
(See Tables 11. 111. IV)
14 200
Other nonhepatic tumors
100
10
Mouse Aspartate aminotransferase (EC 2.6.1.1) (“GOT”)
References
0
Bresnick and Hitchings (1961). Calm et al. (1959)
w
Mouse
Waravdekar et al. (1955)
5-11
32 ~
Increase.'
Man Hydrolases Carboxylesterase (EC 3.1.1.1)
Rat
33
Grwnstein (1954, Table 90). Allard and de Lamirande (1959)
25
Greenstein (1954, Table 90)
~
Ivlouse
>25
3
~~~
Greenstein (19-14. 1954, Tables 88 and 89)
Aminopeptidase (EC 3.4.1.2) Subst rate: ply-gl y-gly
Rat
300
200
100-300
Wu and Bauer (1963)
Dipeptide hydrolasesp (EC 3.4.3) Substrates: alr-als
Rat
150
100-150
50-200
Wu and Bauer (1983)
Rat
50
10-50
Wu and Bauer (1983)
250
Wn and Bauer (19W)
leu-gly
Wu and Bauer
10-30
Wu and Bauer (1963), Greenstein rt al. (1949)
Mouse
450
Greenstein et al.(1949)
Rat
240
Greenstein et al. (1949)
Mouse
170
Greenstein et al. (1949)
(1963)
gly-leu
gly-n&ala
Rat Mouse
prc-gly Glutaminase (“I”)
(EC 3.5.1.2) D-Glutamyltransferase (EC 2.3.2.1 )
Rat
14
Rat Mouse Rat
60-120
60
Greenstein et al. (1949)
450
Greenstein e2 al. (1949)
so GOO
Greenstein el al. (1949)
350
Greenstein et al. (1949)
220
Wu et al. (1965)
Present
-
Waisman et al. (1956)
Roberts et al. (1958)
100 (IIeLa) Williams and Manaon (1958)
0 The data are presented as in Tables I X and X. with the activities found in hepatomas and nonhepatic tumors given as the % ’ of the activity in liver of animals of c Limiting key enzymes are relatively higher. *Enzyme Commission number (1.U.B.. 1965). the same species. d Arginase was induced in HeLa cells by &In++and arginine. Arginase in mouse mammary carcinoma was 20 times higher than in estrogen-stimulated nonFourfold increase over white cells in human leukemic cells. cancerous mammary gland. a These may not all be activities of specific enzymes. though there are several different patterns of changes among the group.
152
W. EUGENE KNOX
ments of the appropriate kind should provide more decisive inforrimtion. Some enzymes found in normal liver which appear t o persist in a wide variety of neoplastic tissues are listed in Table XI. With due allowancc for the need of more weighty evidence in almost every case, this list represents a beginning of the enumeration of the minimal enzyme pattern of ncoplastic tissues. As would be expected, the tablc lists groups of enzymes that catalyze essential reactions, supply energy, alter sonic amino acids, and initiate pyrimidine synthesis (aspartatc carbanioy1transfcr:~se). The inclusion of arginase may cause surprise. Tumors contain only a siiiall fraction of the very high arginase activity of normal liver, but still show an activity t h a t is comparable to somc other normal tissues (Knox and Greengard, 1965). It is significant that the cnzyiiic caii be induccd by its cofactor and substrate, Mn++and arginine, in cultured HeLa cells (Schimke, 1964). The physiological role of arginase in nonliepatic tissues is not known. The hydrolases and peptidases, whose physiological roles are most uncertain, are among those that most clearly persist in all types of neoplastic tissues. CRITERIA FOR DETERMINING T H E b j I N I M A L ENZYME PATTERN OF NEOPLASMS The tentative tabulations of enzymes as dispensable or persistent in neoplastic tissucs in Tables V I I I to XI provide some criteria for dctermining the minimal enzyme pattern, but they do not deal with the reliability of the expcriiiiental procedures upon which the measurements are based. The condition of the animals used, whether livers of normal or tumor-bearing animals scrved as the rcference standard, the typc of assays, and the basis for expressing the activities can all affect thc comparisons. Thcse all-important considerations have been minimized in order to stress the nccd for a strategy to deal with the rapidly accumulating information about crizymes in cancer. The tables suggest a way of comparing the results of enzyme analyses in normal and neoplastic tissues for the purpose of classifying which enzymes are most probably essential for the neoplastic cells. The operation consists merely of a series of comparisons of the amount of each enzyme in normal and in neoplastic tissues. Certain kinds of comparisons are more practical and more efficient than others. I n comparison with normal liver, which must be recognized as the most available standard, an enzyme activity in a neoplastic tissue may be (1) equal or higher (aspartate carbanioyltransferase, Table XI), (2) somewhat lower (aspartate aminotransferase, Table XI), or (3) much lower (arginase, Table X I ) . As indicated by the examples given, enzymes in all of these categories may be judged essential for neoplastic
B.
THE E S Z Y l I I C PATTERN O F NEOPLASTIC TISSUE
153
tissue. But the initial ~~roiiability that a n enzyme is essential in neoplastic tissue is higher if it is in category 1 ancl the probability decreases in categories 2 and 3. Tlic relatively high ;activity of an enzyme in category 1 could rcsult from the invasion of the tumor inass by ubiquitous normal cells containing the enzyme in question, or from a peculiarity of the particular tumor exaniincd. Examples in Table V I I I and the slow-growing 1iepatoni:ts rc>prcsent tumors with such peculiwrities, i.e., those having intermediate types of enzyme patterns. The lower but still significant level of enzymes in category 2 may also represent such a n intermediate pattern. On the other hand, category 2 could represent the loss of that fraction of tlie activity in the standard normal tissue that is associated with sonic unique function of that tissue, with preservation of the lower level requircd by all growing cells. The very low levels of enzymes in category 3 would appear not to represent significant activities; however, objective criteria are iieedcd for the decision that even very low activities are negligible. Additional coinparisons of the enzyme level in other noriiial tissues and in other neoplasms can increase the certainty of these preliminary classifications. It must be recognized that whichever norinal tissue serves as the norrnal standard of coniparison, it has a more or less unique enzyme pat1,ern. Ideally, a series of norinal tissues representing the range of different activities of one enzyme should serve as t,hc standard. An enzyme activity in neoplastic tissue within the range found in normal tissues could then be presumed to be physiologically functional in the neoplasm. The inclusion of arginase as persistent in neoplastic tissues (Table XI) was based in part on such a comparison. Liver is rich in arginase, but neoplastic tissue and many nornial tissues are poor in this activity. However, a second comparison between normal or estrogenstimulated breast tissue and mammary carcinoma showed that the arginase is higher in this neoplastic tissue than tlie still significant activity in the nonhepatic normal tissues. Reiteration of the comparisons with a third nornial tissue, and so on, can increase to any desired degree the significance of the low but definite level of an enzyme activity found in a neoplastic tissue. The other side of the comparisons with norinal tissues, the neoplastic tissues themselves, vary considerably in their enzyme patterns. I n regard to this variation, the t,wo separate purposes of comparisons of this type must be clearly distinguished. The enzyme pattern of any tumor is of interest as a n aid to knowing its particular biological properties and potentials. Enzyme patterns that are intermediate between the “normal” and the niiniinal necessary for neoplastic tissue are particularly instructive in this regard, when correlated with the biological behavior of a
154
W. EUGENE KNOX
particular tumor. The progressive loss of some enzymes and gain of others in the Morris rat hepatonias that show increasing growth rates have been cited in the various tables. These are examples which may have practical as well as theoretical value for cell biology. The second purpose is to identify thc lcast common complement of enzymes that a neoplastic tissue must have. Tumors with intermediate type enzyme patterns are of less interest in this connection. However, it is conscrvative and therefore advantageous to work with a spectrum of neoplasms, rather than to choose some particular neoplasms as “typical.” A spectrum of neoplastic tissues shades from significant to negligible amounts of an unessential enzyme, and concomitantly, has relatively constant amounts of another esscntial enzyme. An enzyme that decreases succcssively in normal liver, slow-growing hepatoma, fast-growing hepatoma, and nonhepatic tumors may probably not be essential for neoplastic tissues-if it ultimately decreases to negligible amounts in some of the tumors. The relatively mechanical comparisons and reiterated comparisons between a series of normal and neoplastic tissues that have been described will serve a t lcast to define the question of the essentiality of any enzyme in neoplastic tissuc. The selection of the enzymes to be so investigated, and the final decision about their classification, can be greatly helped by the mutual interaction that operates between the metabolic function of a tissue and its particular enzymic machinery. Working hypotheses based on what is known about the biological behavior of neoplastic tissues can suggest that certain enzymes, or whole groups of enzymes, will be dispensable or will be preserved in neoplastic tissues. The enzymes that persist should have a role in the economy of the neoplasm, and this should be demonstrable from the study of the metabolic behavior of the tissue under physiological conditions. Presuniably the minimal enzyme pattcrn of neoplastic tissues represents a stripped down version of a living cell, every component of which can be demonstrated to play a metabolic role in its growth and survival. Components for which this can be shown are probably esscntial. I n general, to determine the minimal enzyme pattern of neoplastic tissues from the type of information that has accumulated to date, it will be necessary to take comparable analyses for a large number of enzymes in a number of different kinds of normal and neoplastic tissues. The individual enzymes can then be identified as part of the minimal pattern, or eliminated from this pattern, with increasing certainty as more comparisons between the normal and neoplastic tissues are made. Confirmation of these identifications, as well as their significance, will dcpend upon the dcmonstration of the physiological roles of the enzymes judged to be essential.
THE ENZYMIC PATTERS O F NEOPLASTIC TISSUE
155
VIII. Discussion
Insights derived from studies in enzyme physiology suggested that there was a meaningful link between the generalization of Warburg and that of Greenstein which can be extended in a further generalization about the nature of neoplastic tissues. The metabolic behavior (and possibly even the morphology) of neoplastic tissues are similar because they tend to have similar enzyme patterns. The principles of enzyme physiology would predict that the metabolic behavior and the enzyme pattern of a tissue are different aspects of the same thing: to the extent that one is understood, predictions can be made and tested about the other. In the first test of these ideas, the high glycolytic function of tumors led to the prediction that the glycolytic enzymes must persist in tumors, as they do. If the persistence of the enzymes had been known first, the high glycolytic function of these tissues could have been predicted. Some other metabolic functions of neoplastic tissues and the enzyme systems responsible for them apparently follow the same rule although correlative data are less complete than for glycolysis. The interrelation between the enzyme pattern and the metabolic behavior of a tissue amplifies the ways by which meaningful correlative information needed in cell biology can be obtained. The correlation of the intermediate and minimal types of enzyme patterns in tumors with the metabolic behavior of these tumors provides a means for identifying the physiological role of individual enzymes in living processes. Neoplastic tissue represents a particularly favorable type of biological material for such inquiries: it is derivable from most kinds of normal tissues; it grades toward a simplicity of enzyme pattern and behavior (growth) ; and its study is socially desirable. While the description of the ultimate minimal enzyme pattern and behavior that is common to neoplastic tissues will not constitute in itself knowledge about the cause or cure of cancer, information about the nature of any phenomenon is a prerequisite for studies of its genesis and control. Comparisons of enzyme levels in tissues of the sort exemplified here should be able to approximate the pattern of enzymes that is essential for neoplastic tissues. The pattern should be known in greater detail than for the few kinds of enzymes of carbohydrate and protein metabolism considered here. Many studies have been made about other enzymes whose classification as dispensable or persistent in tumors was not attempted here. Unfortunately, there are not even relatively complete listings of which measurements have already been made. Even the few enzymes that have been provisionally listed here are subject to change in their classification, perhaps simply upon reinterpretation
156
W. EUGENE KNOX
of the same data. Nevertheless, there is a n orderly way to go about the evaluation of both old and new experimental results that can sharpen our biochemical definition of neoplasia. A large number of differences have already been shown in the enzyme patterns between normal and neoplastic tissues, whethcr or not these differences are essential ones. The large number in itself suggests that these cannot all bc primary changes, with their basis in the nature of neoplasia. Many must be secondary conscquences of the neoplastic transformation. The variability between different tumors reinforces this view. The biological mechanisms that can produce such widespread variability in the metabolic machinery of cells are of general interest. This variability of enzymes in neoplastic tissues was recognized a t the time when the enzymatic and metabolic adaptations that occur so frequently in normal tissues were compiled: “There are in addition, a large number of enzyme analyses of tumors, originally undertaken t o detect a difference between normal and neoplastic tissues. Instead of a single tliff ercncc, these studies revealed surprisingly numcrous differences between tumors and the normal tissues of origin, and also diffcrences between different tumors. It will be necessary to reexamine these differences, identifying the oncs referable to adaptations t o the altered metabolic state of tissue or of the animal, before the original purpose of detecting a fundamental abnormality of tumors can be realized.” (Knox et al., 1956, p. 240). More recently there has been awakened interest in such metabolic control niechanisins, sonic of which have focused upon the problems of regulation in ncoplastic tissues (Pitot and Hcidelberger, 1963 ; Pitot, 1964-1965; Henderson, 1965). The particularly relevant question in connection with the enzymc pattern of ncoplastic tissucs is whether the large number of enzyme differences that exist bctween normal and neoplastic tissues are the cause or the effect of the neoplasia. It is now realized that the autonomy of neoplastic tissues is limitcd. Some response of tumors to their envirorimcnt is therefore to be expected. Are they sufficiently responsive so that most of their enzyme diffcrcnces could be caused by the altered metabolic state in neoplasia? The classic example of responsivencss of one enzyme in a neoplastic tissue to an extraneous influence is the dramatic decrease of the acid phosphatasc activity in prostatic carcinoma by estrogens. The control of the level of other enzymes has now been studied extensively in normal liver. Table XI1 illustratcs thc levels of such a n enzyme, tryptophan pyrrolase, and the control of its level in hepatomas studied by Dycr et al. (1964). Table XI1 shows in greatcr detail than the entry in Table IX t h a t the tryptophan pyrrolase in slow-growing hepatomas is comparable to that
157
THE EXZTSIICI PATTERN O F NEOPLASTIC TISSITE
TABLE XI1 I N D U C T I O N O F TRYPTOPHAN PYRROLhSE I N ANI)
n.4~HEP.\TOhZAS
Hosr LIVERSB Y T R Y P T O P H A N ~ ~ ~ Hepatomas
Host livers
No No tryptophan Tryptophan tryptophan Tryptophan Type of Hepatoma G i m ~ pI (7316B, 7800, 5123A, B & D) Group I1 (H35, 5123C, 73168, 7794A) Group Ill (7793, 7794B, 7795)
[pmoles/hr./g. dry weight (37"C.)]
(19)
<0.6 (19)
1 3 . 5 (19)
64.5 (19)
1 . 4 (21)
5 . 2 5 (23)
10.0 (21)
62.0 (23)
5.7 (10)
40.5 (8)
11.9 (10)
48.0 (8)
0
Derived from Dyer el al. (1964). bearing variow transplaiited liepatomas were untreated or received 51 mg. L-tryptophan per 100 g. body weight 4 to 5 hours before death. The average activity of the tryptophan pyrrolase in hepatomas and host livers is tabled, with the number of animals in parentheses. The hepatornas are grouped into those with absent, low or iiorrnal tryptophan pyrrolase basal levels. ,.U1 are relittively slow-growing hepatomas. a
* Rats
in normal liver and decreases to undetectable levels in the fast-growing hepatomas. The interest in the present discussion is t h a t the adaptive response of thc tryptophan pyrrolase is also present in thc hepatomas after tryptophan administration. This response is proportional to the basal level of the enzyme, entirely similar t o the response of the host liver in slow-growing hepatomas with normal levels of the cnzyiiie, and with no response in the fast-growing hepatomas that have no detcctable love1 of the enzyme. If the enzyme is present in these tumors, its control appears to be no different from that of the enzyiiie in normal liver. It would appear from other studies that the amounts of other enzymes in neoplastic cells can also be modified, within limits, by host or environmental factors (Rosen e t nl., 1961; Rosen, 1963; Dyer e t al., 1964; Pitot and Morris, 1961; Weber e t al., 1965b; and Weber, 1963), and even by specific chemical stimuli (Scliimke, 1964; Rackcr e t al., 1960). The particular controls of enzymes in tumors t h a t have been studied FO far are riot expected to have the pilactical significaiicc t h a t was realized froin the control of the acid phosphatase level in prostatic carcinoma. The examples do illustrate, however, that thcrc is R growing theoretical and experimental knowledge about the control of the enzyme patterns in normal and neoplastic tissues. It is in relation to these studies that the definition of the minimal pattern of enzymes in neoplastic tissues will be most useful. It is upon the base line of this minimal
158
W. EUGENE KNOX
pattern that the controls of enzymes are superimposed in neoplastic tissues. Since it appears that the enzyme patterns and the metabolic behaviors of tissues are related, the control of the enzyme pattcrn should lead to the control of the behavior of neoplastic tissues.
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Dickens, F., and Glock, G. E. 1951. Biochem. J . 5 4 81-95. Dickens, F., and Simer, F. 1930. Biochem. J. 24, 1301-1326. Dickens, F., and Simer, F. 1931. Biochem. J. 25, 985-993. Dickens, F., and Weil-Malherbe, H . 1941. Biochem. J. 35, 7-15. du Buy, H. G., Woods, M. W., Burk, D., and Lackey, M. D. 1949. J. Natl. Cancer Inst. 9, 325-336. Dyer, H. M., Gullino, P. M., Ensfield, B. S., and Morris, H. P. 1961. Cancer Res. 21, 1522-1531. Dyer, H. M., Gullino, P. M., and Morris, H. P. 1964. Cancer Res. 24, 97-104. Edwards, S. W., and Knox, W. E. 1956. J . Biol. Chem. 220, 7%91. Elwood, J. C., Lin, Y., Cristofalo, V. J., Weinhouse, S., and Morris, H. P. 1963. Cancer Res. 23, 906-913. Foulds, L. 1954. Cancer Res. 14, 327-339.
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Hadjiolov, A. A., and Dauclieva, I<. I. 1958. Nature 181, 547-548. Hagerman, D. D. 1964. Federation Proc. 23, 785-790. Henderson, J. F. 1965. Prog. Exptl. Tumor Res. 6, 84-125. Ichii, S. 1958. Gann 49, 125-136. International Union of Biochemistry. Commission on Enzymes. 1961. “Report of the Commission on Enzymes of the Internat,ionnl Union of Biochemistry, 1961.” Macmillan (Pergamon), New York. International Union of Biochemistry. Standing Committee on Enzymes. 1965. “Enzyme Nomenclature.” Elsevier, Amsterdam. Jollt5s-Bergerct, B., Brun, D., Lebourssc, J., and Chatagncr, F. 1963. Bull. SOC.Chim. Biol. Franc. 45, 397-412. Kit, S., and Griffin, A . C. 1958. Cnncer Res. 18, 621-656. Kizer, D. E., and Chan, S. 1961. Cnncer Rcs. 21, 489-495. Knox, W. E., and Greengard, 0. 1965. Advan. Enzyme Regulation 3, 247-313. Knox, W. E., Aucrbach, V. H., and Lin, E. C. C. 1956. Physiol. R e v . 36, 164-254. Iirebs, H . A. 1963. Advan. Enzyme Regulation 1, 385-400. Laird, A. K. 1954. Exptl. Cell Res. 6, 3 U 4 . Lan, T. H. 1944. Cancer Res. 4, 37-41. Lardy, H. A. 1964. Advan. Enzyme Regulation 2, 39-49. Le Page, G. A. 1950. Cancer Res. 10, 77-88. Levintow, L. 1954. J . Natl. Cancer Inst. 15, 347-352. Long, C., ed. 1961. “Biochemist’s Handbook.” Spon, London. Masayama, T., Iki, H., Yokoyama, T., and Hasimoto, M. 1938. Gann 32, 303-306. Meister, A. 1948. J . Natl. Cancer Inst. 9, 125-128. Meister, A. 1949. J . Natl. Cancer Inst. 10, 75-80. Najjar, V. A . 1962. I n “The Enzymes” (P. D. Boyer, H. A. Lardy, and K. Myrback, eds.), 2nd ed., Vol. 6, pp. 161-178. Academic Press, New York. Nigam, V. N. 1062. Nature 196, 478-480. Nigam, V. N., MacDonald, H. L., and Cantero, A. 1962. Cancer Res. 22, 131-138. Nirenberg, M. W. 1958. Biochim. Biophys. Acta 30, 203-204. Nirenberg, M. W. 1959. J . Biol. Chem. 234, 3088-3093. Nirenberg, M. W., and Hogg, J. I?. 1958. Cancer Res. 18, 518-521. Otani, T. T., and Morris, H. P. 1965. Advan. Enzyme Regulcltion 3, 32,5434.
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Shonk, C. E., Arieon, R. N., Koven, B. J., Majima, H., and Boxer, G. E. 1965a. Cancer Res. 25, 2W-213. Shonk, C. E., Morris, H. P., and Boxer, G. E. 1965b. Cancer Res. 25, 671-676. Spencer, R. P., and Knox, W. E . 1960. Federation Proc. 19, 886-897. Striebich, M. J., Shclton, E., and Schneider, W. C. 1953. Cancer Res. 13, 89-2M. Sweeney, M. J., Ashmore, J., Morris, H. P., and Weber, G. 1963. Cancer Res. 23, 995-1002.
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CARCINOGENIC NITROSO COMPOUNDS
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P N Magee and J . M. Barnes Toxicology Research Unit. Medical Research Council Laboratories. Carshalton. Surrey. England
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I Introduction . . . . . . . . I1. Chemistry . . . . . . . . . A . General Chemistry . . . . . . 13 . Preparative Methods . . . . . . C . Analytical Methods . . . . . . I11. Acute Toxic Effects . . . . . . . A . Dimethylnitrosamine . . . . . . B . Other Nitrosamines and Nitrosamides . . C. Pathological Changes in Acute Poisoning . IV . Neoplast.ic Changes . . . . . . . A . Liver . . . . . . . . . B. Kidney . . . . . . . . . C . Bladder . . . . . . . . . D . Nose and Nasal Sinuses . . . . . E . Lungs and Bronchi . . . . . . F . Alimentary Canal . . . . . . G . Nervous System . . . . . . . H. Skin . . . . . . . . . I . Teratogenic and Other Effects . . . V . Some Special Features of Nitroso Compounds as VI . Mutagenic Action . . . . . . . VII . Metabolism of Nitroso Carcinogens . . A . Metabolism in Vivo . . . . . . B . Metabolism in Vitro . . . . . . VIII . Biochemical Effects . . . . . . . A . Protein Synthesis . . . . . B . Other Biochemical Effects . . . . I X . Reactions with Cell Constituents . . . X . Possible Mechanisms of Action . . . . A . The Proximate Carcinogen . . . . B . The Cellular Target . . . . . . C . Alkylation of Nucleic Acids . . . . XI . Public Health Aspects . . . . . . A . Hazards t o Chemists and Industrial Worliers B . Hazards t o General Population . . . C . Concluding Comment . . . . . References . . . . . . . . . 163
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164 165 165 168 171 171 172 174 175 175 185 186 186 157 188 190 190 190 191 193 202 202 207 209 209 213 220 227 227 231 233 234 235 235 238 235
164 I. introduction
After discovering that dimetliylnitrosaniinc was acutely hepatotoxic to a number of laboratory animal species (Barnes arid Magee, 1954), tests for its possible carcinogenic action were carried out and it was shown that nearly all rats on a diet containing 50 p.p.ni. dimethylnitrosaminc developed malignant liver tumors within less than a year (Magee and Barnes, 1956). From these observations has grown a large literature on the carcinogenic activity not only of dimethylnitrosamine hut of a whole range of nitroso compounds, The diversity of the carcinogenic activity of the nitroso compounds is indicated in Table 111, which also reveals the importance of tlie contributions of Druckrey, Preussmann, Schmihl, Ivanltovic, and their colleagues, who have recently published an excellent review on their work on the carcinogenic action of 65 different nitroso compounds in tlie r a t (Druckery et al., 1967). The toxicity of cycad plants to man and animals has long been of interest (Whiting, 1963). The observation that rats when fed meal from cycad nuts in their diet might develop liver and kidney tumors (Laqueur et al., 1963) suggested that tlie toxic principle miglit behave like an alkyl nitroso compound. This work was discussed a t length a t a Conference reported in Federation Proceedings 23, 1337-1386 (1964), and cycasin and related compounds are considered in this review. Not only have the nitroso compounds shown great versatility a s carcinogenic agents but from tlie first have bccn shown t o lend themselves to interesting metabolic studies (Magee and Vandekar, 1958). Such opportunities have been amply exploited in trying to identify those intracellular reactions that follow tlie introduction of the carcinogen into the tissues and which precede the development of the malignant changes. This review starts with a brief account of some of the relevant chemistry of the nitroso carcinogenic compounds and goes on to consider the pathological lesions induced by these compounds and their mutagenic activity. Then follows an account of their metabolism in the whole animal and in tissue preparations in vitro. The biochemical disturbances to which they give rise will be described and also their reactions with tissue constituents. It is possible that the nitroso compounds may prove to be very useful tools in the study of chemical carcinogenesis and ideas for their mechanism of action are put forward. With the exception of some discussion on the alkyl-tert-butyl compounds, there is no consideration of other apparently noncarcinogenic nitrosamines. The possible value of nitroso compounds as chemotherapeutic agents in cancer is being investigated (Schabcl et al., 1963; Wheeler and Bowdon, 1965) but this topic is not discussed in this review.
CARCINOGENIC KITROSO COMPOUSDS
1 65
The great activity and considerable diversity of action as carcinopens shown by iiitroho compounds have led t o a n increasing interest ill them as possiblc carcinogenic hazards in the human cnvironnicnt. This role of the nitroso compounds, whether as natural products such as cycasin or artificially produced in tobacco smoke or in food processing, is also briefly considered. Carcinogenic nitroso compounds have been reviewed previously by Mngee (1963), Magee and Sclioental (1964), and Druckrey e t al. I 1963~). II. Chemistry
A. GENERALCHEMISTRY 1. Nitroso Compounds
The N-alkyl-N-nitroso compounds to be considered include aliphatic ring conipounds, such :is ,~-iiitrosopipcritline and N-nitroso-morpholine, :ts well as conipounds of the type:
R’ \ N-N=O
/ R” in which one group, R’, is an alkyl ratlical. The other group, R”, may be one of many others including ester groups, .CO.O.C,H, (carbethoxy) , aniitle groups, CO . NI-I, (primary amide) and aromatic groups, C,,H, (phenyl) . As only the nitroso group is coinnioii the physical properties of these coinpounds cover a wide range. S-Nitrosodimethylarnine (diniethylnitros:~niine)is an oily liquid, miscible with water in all proportions. Others are liquids or solids, some very sparingly soluble in water, but solublc in many organic solvcnts. The gencrd chcinistry of N-nitroso coni1)ounds is found in standard tcxtbooks (c.g., Fieser and Fieser, 1956; Hickinbottoni, 1957). Only cert:iin important properties are summarized here. a. Photosensitivity. The nitroso cornpounds are characteristically photosensitive and thc nitroso group is split by exposure to ultra-violet light (Section I1,C) . N-Alkyl-1V-nitrosourethanes undergo photodecxomposition in aqueous alcoholic solution to give crystalline compounds among the reaction products. N-Methyl-N-nitrosourethane (R” = .CO .0C2Hj) undergoes partial reduction t o yield the dicthyl ester of dimethyl tetrazodicarboxylic acid, CH,-N (COOC,H,) -N=N-
166
P. N . MAGEE AND J . M. BARNES
N(COOC,H,)-CH,, and the iV-ethyl derivative behaves in a similar manner (Schoental, 1963b). b. Reaction with Acid and Alkali. The dialkylnitrosamines are stable to strong alkali but the nitroso group is lost under acid conditions with reversion to the secondary amine. The alkyl nitrosamides, e.g., N nitroso-N-inethylurea (nu’ = -CONHr) are unstable a t alkaline pH and decompose to yield the corresponding diazoalkane. c. Reduction. The nitroso group can be reduced to give the corresponding unsymmetrical hydrazine derivative. d . Oxidative Decomposition and Alkylating Properties. The oxidative brcakdown of some dialkylnitrosamines has been demonstrated by Preussniann (1964a) using the hydroxylase model of Udenfriend et al. (1954). This system consists of ascorbic acid, the Fe++complex of ethylenedinmine tetraacetic acid, and molecular oxygen and was shown by Udenfriend et al. t o mediate the oxidative decomposition of drugs in a manner analogous t o the microsomal enzymes. Several reaction products of the nitrosamines were demonstrated by thin-layer chromatography but their chemical structures were not identified. Decomposition was minimal or absent when nitrogcn was substituted for oxygen. Formaldehyde is formed from dimcthylnitrosamine by the liver microsomal enzymc system (Brouwers and Emmelot, 1960) which represents oxidativc rcmoval of a methyl group. The primary product when one methyl group is removed from dimethylnitrosamine is expected to be monomethylnitrosamine (Heath and Dutton, 1958). This compound is unstable and breaks down very rapidly (Muller et al., 1960) the products depending on the physicochemical properties of the system. In nonaqueous solvents the main product is diazomethane. In aqueous solvents the nature of the products probably depends mainly on pH and may be either diazomcthane or carbonium ions. The possible deconiposition products of monoalkylnitrosamines have been discussed by Ridd (1961) and Austin (19GO). These authors suggest a reaction sequence in which the monoalkylnitrosamine undergoes a tautomeric shift to give the alkyl diazohydroxide. This rapidly ionizes to give the diazonium cation which loses nitrogen to give the electron-deficient carbonium ion which is the alkylating agent. It cannot be said which products are formed under physiological conditions. Possibly both the carbonium ion and the diazoalkane are formed in proportions depending on the particular nitroso compound. Both products act as alkylating agents. The alkylating properties of monomethylnitrosamine derivatives were illustrated by Kriek and Emmelot (1964), who demonstrated methylation of deoxyribonucleic acid (DNA) by methylamine and sodium nitrite a t pH 4 when monomethylnitrosamine is the expected product. Mechanisms of alkylation by nitroso compounds in relation to carcinogenesis, have
CARCIXOGENIC NITROSO COMPOUNDS
167
been discussed 1)y Ilruclircy c t al. (1963d) and by Emnielot e t al. (1962). The formation of a n alkylating agent from the monoalkylnitrosamine is not the only possible pathway of decomposition; the latter might also renct with water to give methylamine and nitrous acid. Since dialkylnitiwsaniines can be rct-luccd to the corresponding hydrazines and also possibly form hydroxylaniines, these compounds must also be considered :is possible iiietabolic products (Hcath :cnd Dutton, 1958 ; see Section VII,A) . e. Reactions with Szcljhydryl Compozsnds. Ar-Methylnitrosoui-ethane reacts with sulfhydryl groups at neutral p H (Schoental, 1961, 1966a). This reaction occurs at rooin teniperaturc :Ind has h e n studied in dettiil with cysteinc and sonic of its dcrivatives : ~ n dN-alkyl-nitrosouretlinries and dinzoniethanc~. Complicated mixtures of products were fornicd. The main products of reaction between cystciiie and iiiethylnitrosourethane arc S-iiictliylcystcine :~nd S-etlioxycarboiiylcy~teine. S-Ethoxycarbonyl derivatives isomerized to the respective N-ctlioxycarbonyl derivativcs nhcn the pH was rmsetl ahove 7, the migration being particularly c:i,iy with cysteinc (Schoentnl and Rive, 1963, 1965) . S-Methyl dcriv:itives and methyl casters were tlie coiiimon products formed from the ttiiol compounds with both methylnitrosourethane and diazometli:wc. Tlie esters were unhtnble and hydrolyzed easily. Tliesc observations arc tliscusscd by Helioental (1966a) in relation to their possible role in the carcinogenic. action of alkyhiitrosouretlianes (see Section IX) . Uiiiicthyliiit~os:~ininc docs not react under tliese conditions and other dialkyliiitrosaiiiiiics are riot known to react with sulfhydryl cornpouiids a t neutral pH. 2. M e t hylaxozgme thanol GI ycosides Cycasin is one of a number of glycosides which occur in different genera of cycad plants bclonging t o the botanical family Cycadaceae and which are widely distributed in the tropics and subtropics. It is a white crystalline solid, readily soluble in water (Riggs, 1956). These glycosides liave a coninion aglycone, methylazoxynicthanol
C H 3-N=N-C'HsO
I 0
H
but tlie sug:~r varies. I n cyctisni it IS glucose (Korsch and Riggs, 1964) and in nincrozaniin it is primeverose (Lythgoe and Kiggs, 1949). The structure of the aglycone has been determined after extensive studies by Riggs and his colleagues (Langley e t al., 1951) and by Nishida and his group (Nishida e t al., 1955). Although the aglycone is unstable it has been isolated (RiIatsunioto and Strong, 1963; Kobayashi and
168
P . N. MAGEE A S D J. M. BARNES
Matsumoto, 1964, 1965). It is a liquid that crystallizes a t 3°C. and slowly decomposes in aqucous solution. The acctatc, prepared from the isolated aglycone, is stable in aqueous solution (Matsurnoto et al., 1965). a. Alkylating Properties. Riggs (1965) showed that cycasin and rnacrozamin may be effective niethylating agents in vitro by production of anisolc on reaction with phenol. Methylaaoxyniethanol methylatcs nucleic acids and other compounds in aqueous solution buffered a t pH 7 a t 37°C. (Matsumoto and Niga, 1966). Thc possibility that the same methylating intermediate may be formed i n vivo from cycasin and from dimethylnitrosarnine has been suggested (Miller, 1964; hlatsumoto and Higa, 1966; see Section IX).
R. PREPARATIVE METHODS 1. .Nitroso Compounds
a. Unlabeled Material. AT-Nitroso compounds are usually prepared from the respective alkylamino compounds by the action of nitrous acid (Klobbie, 1890; Hartinann and Phillips, 1943; Hatt, 1943; Heath and Mattocks, 1961 ; Prcussmann, 1962). The papers by Druekrey, Preussniann, and their colleagues contain much valuable information on the preparation and physical charactcristics of many of the nitroso compounds which they have tested for carcinogenicity (Table 111).Most of the nitrosamines are liquids and have been purified by fractionation. It is not always certain, therefore, that impurities up to 1 or 2% may not be present. b. Isotopically Labeled Material. Methods for the preparation of nitroso compounds labeled with C", N15, and H3 have been described. These are listed in Table I. 2. Methylazoxymethanol Derivatives Details of the preparation of mcthylazoxymetliariol and some derivatives arc givcn by Kobayashi and Matsuiiioto (1964). Cycasin was hydrolyzcd by a commercially available 8-glucosidase, almond emulsin, a t pH 5.2, and thc crude mcthylazoxymcthanol fractionally distilled. Mcthylazoxymethanol acctatc was prepared by acctylation with acetic anhydride.
C. ANALYTICAL METHODS 1. Nitroso Compounds
a. Polarographic Method. Dimethyl and other nitrosamines can be determined in tissues and body fluids by the polarographic method of
TABLE I ISOTOPICALLY LABELEDPirmoso COMPOUNDS Labeled Nitroso Compound Dimethylnit.rosamine-C" Diethylnitrosamine-l-U4 n-B~tyl-C~~-rnet.hylnitrosamine n-l-C14-Butylmethyhii trosamine tert-But.y1-ClJ-methylnitrosamine tert-l-C~4-But~ylmethylriitrosamine tert-2-C~4-But.ylmethylnitrosamine Dimethylnitrosamine-H3 Dimethylnitrosamine-?;~~O (Me2N.Y3O) Dimethylnitro~arnine-r\"~C (Me2S15.XO)
3 P
z
Labeled Starting Material
Iiefereiice
Methyl iodide-Cl4 Ethyl iodide-GI4 hlethyl iodide-CL4 Sodium butyrate-l-C14 Sodium formate-Cl* Acetone-2-CI4 Acetone-[ 1,3-'C] Dimethylamine-H3 Potassium nit rate-W5 Ammonium nitrate-W
Duttoti atid Heath (195Ga) Heath and Mattocks (1961) Heath and Mattocks (1961) Heath a i d Mattocks (1961) Heath and Mattocks (1961) Heath and Mattocks (1961) Heath and Mattocks (1961) Lee et al. (1964) Heath (1957) Heath and Dutton (1958)
2 z 0
3
Q
z
r(
rl
z
$ 0
Q
0
z+c $ z
a
170
P. N. MAGEE AND J . M . BARNES
Heath and Jarvis (1955) which was modified for this purpose from that of English (1951). The nitrosamine is extracted from the tissue with sulfosalicylic acid, the extract made alkaline, and distilled. The nitrosamine in the distillate is determined polarographically. The minimum amount of dimethylnitrosamine that can be determined is 1 pg. to a n accuracy of +0.05 pg. The method has been adapted for estimation of several other nitrosamines with quantitative recovery from biological material (Heath and Jarvis, 1955; Heath, 1962). b. T h i n - L a y e r C h i ~ o m a t o g m p h yand Colorimetric M e t h o d s . Nethods for the separation of nitrosamines by thin-layer chromatography and their detection by color reactions after photolytic dissociation have been describcd (Prcussmann, 1964b; Preussmann e t al., 1964a,b). After separation of the nitrosamines they are split by irradiation by ultraviolet light, with release of nitrites which are revealed by sensitive color reactions. Thin-layer chromatography is carried out on silica gel plates, developed with mixtures of hcxane, ether, and methylene chloride. Two methods are described for revealing thc nitroso compound. I n one the plates are sprayed with an aqueous diphenylaniine-palladium chloride reagent and the moist plate is irradiated for a few minutes with ultraviolet light (A,,,,, 240 mp). Blue to violet spots corresponding to the nitroso compound appear on an otherwise almost colorless plate. I n the second method the plate is sprayed, after irradiation, with a sulfanilic acid-a-naphthylamine reagent in acetic acid which produces violet-red spots corresponding to the nitrosamine. About 0.5 p g . of nitrosairiine can be detected by both methods. The specificity ranges of the two reagents overlap in such a manner that the authors claim that positive results with both methods can be considered as specific for N-nitroso compounds (Preussmann et al., 1964b). A method for the quantitative determination of nitrosamines has been developed by Daiber and Preussmann (1964) using a colorimetric procedure based on the colorcd reaction product obtained by reaction of the sulfanilic acid-a-naphthylamine reagent with sodium nitrite photolytically released from the nitroso compound. The sensitivity is about 1-2 pg. per ml. A micro method for the separation and identification of nitrosamines as the 5-nitro-2-liydroxybenzal derivatives of the corresponding unsymmetrical dialkyl hydrazines has been described (Neurath et al., 1964a; Neurath and Dunger, 1964). The nitrosamines are first reduced with lithium aluminum hydride to the corresponding hydrazine, which is then allowed to react with 5-nitro-2-hydroxy-benzaldehyde. Melting points and spectral characteristics of a number of these derivatives are given. The derivatives appear as yellow spots on thin-layer chromatography, which become yellow-brown and more intensely colored on
CARCINOGENIC NITROSO COMPOUNDS
171
spraying with alkali so that 0.5 pg. can be detected. The sensitivity can be increased to allow detection of 0.05 pg. by treatment of the plates with potassium hexaferricyanide (111) solution in hydrochloric acid which produces a blue color, but this is not specific. Serfontein and Hurter (1966) have reported a procedure in which reduction by lithium aluminum hydride is followed by reaction with 4-nitroazobenzene-4carboxylic acid chloride. The resulting hydrazides are separated by thinlayer chromatography. Although the lowest detectable quantity of nitrosamine was not determined, 2.5 pg. was detected with ease. This method is claimed to be particularly suitable for the identification and estimation of nitrosamines in complex organic mixtures and to have been successfully applied to the analysis of nitrosamiries in cigarette smoke. 2. Methylazoxymethanol Derivatives Cycasin and methylazoxymcthanol have the same characteristic absorption spectrum a t 217 nip owing to the azoxy structure and this has been used in the spectrophotometric assay of the compounds (Kobayashi and Matsumoto, 1965). One mole of formaldehyde is produced when 1 mole of azoxyglycoside is hydrolyzed with acid (Langley et al., 1951) and this property was used for determination of cycasin by Matsumoto and Strong (1963), who measured the libcrated formaldehyde by the chromotropic acid method. The same procedure was developed by Kobayashi and Matsumoto (1965) for estimation of cycasin in biological material. The same authors (Matsurnoto and Strong, 1963; Kobnyashi and Matsumoto, 1965) give details of various chromatographic procedures for cycasin. Ill. Acute Toxic Effects
A. DIMETHYLNITROSAMINE
Dimethylnitrosamine was originally studied in laboratory animals because of reports that it had produced acute or subacute poisoning in men exposed to it in industrial laboratories (Barnes and Magee, 1954; Jacobsen et al., 1955). I n single doses around 25 mg./kg. whether given by mouth, or by intravenous, intrapcritoneal, or subcutaneous injection, diinethylnitrosamine produces a striking centrilobular liver necrosis in rats accompanied by hemorrhages into the liver and lungs and frequently a n associated hemorrhagic ascites and blood in the lumen of the gut. Death usually took place within 2-4 days or else was followed by complete recovery (see Section IV,B). The liver lesion is characterized by a sharp demarcation between cells that seemed to be totally destroyed and those that survive and begin to divide within 48 hours after
172
P. N . MAGEE A N D J . M . BARNES
dinictliylnitrosarnine administration. Apart from congestion, cellular damage visible with conventional histological technique could not be detected in the kidney of acutely poisoned rats. Rabbits, mice, guinea pigs, and dogs all develop a severe liver necrosis after diiiicthylnitrosaniine ; the dogs show a greater tendency to hemorrhage, particularly into the lumen of the gut (Barnes and Magcc, 1954). When dogs were exposed for 4 hours to concentrations of dimetliylnitrosainine vapor that killed 11 out of 12 within 48 hours there was an immediate effect on sulfobrornophthalein retention, indicating liver damage and a sharp fall in circulating leucocytcs (Jacobscn et al., 1955). The effect on the white blood cells was attributed to a possible direct action of circulating dimcthylnitrosamine. The urine output of the dogs was altered and further studies of the kidney were made in rabbits acutely poisoned with diniethylnitrosamine. Loss of tubular function wab observed with only a slight dccrease in gloinerular filtration. The capsules of the kidneys were tense suggesting a back diffusion of water across the tubular epithelium. Histological examination showed vascular congestion only (O’Lcary e t al., 1957). It is interesting that despite the development of renal tumors in rats given single doscs of dimethylnitrosamine no structural changes in the kidneys of acutely poisoned animals have been reported.
B. OTHER NITROSAMINES A X D NITROSAMIDES The acute toxicity of other dialkyl and related nitrosamiiies has been rcvicwcd bricfly by Hcatli and Magec (1962). Liver damage, heinorrhagic lung lesions, and convulsions and coma are produced in varying degrees by the twelve compounds they discuss. The acute oral LD,, doscs for rats of tlic various nitroso compounds considered in this rcview are listed in Tablc 11. Some arc very reactive compounds and produce acute hemorrhagic destructive lesions a t thc site of entry into the body. N-Nitroso-N-mctliylurcthanc is so irritating to the lungs and skin that even with all precautions i t is difficult t o produce on an industrial scale (Watrous, 1947). Spills have led to sevcre signs of irritation of the eyes, lungs, and skin (Wrigley, 1948). Given in solution by mouth Nnitroso-N-methylurethane produces severe necrotic lesions in the stomach, congcstion of the lungs, and a periportal necrosis of the liver (Schocntal, 1960; Schm2ilil and Thomas, 1962). It is often used as a convenient source of diaaornethane. This compound produced “chest pain and air hunger” in the man who discovered it in 1894; during a fairly extensive use by chemists as a methylating agent it has led t o a t least one death and a few cases of poisoning in which the predominant symptoms have been in the respiratory tract (see Lewis, 1964). Rats and
173
CARCINOGENIC NITROSO COMPOUNDS
TABLE I1 ACUTE ToxIcrm OF NITROSOCoiMPouNDs" Compound
N-Nitrosodimethylamilie A'-Nitrosodiethyla~nine N-Nitrosodi-n-hut ylamine A'-Nitroso-n-butylmethylamine N-Ni troso-tert-butylmetliylaniirie N-Ni trosodi-n-amylamine N-Nitrosomethylphenylamiiie N-Ni trosobenzylmethylamine N-Nitrosoethylisopropylamine A*-Nitroso-n-butylethylamine A7-Nitroso-tert-hut,ylethylamine A'-Nitrosoethylvinylamine A'-N it,rosoethyl-2-hydroxyethylami11e 2L'-Nitroso-di-2-hydroxyethylamine h'-Nitrosobutyl-4-liydroxybutylamine X-Nitrosomorpholine N-Nitrososarcosine A'-Nitrososarcosine ethyl est,er A',"-Dinitroso-N,N'-dimethylethylenediamine A'-Methyl-N-nitrosourea Ar-Met~hyl-N-nitrosourethane A'-Xitrosobrimethylurea Azoxyethane Diazoethy1acet)ate a
Dose (mg./kg.)
l:cf.*
27-41 216 1200 130 700 1750 200 18 1100 380 1GOO
A A
88
C C D B F C
>7500 >5000 MOO 282 >4000 >5000 150 180 240 250 530 400
B A A F A
c c c c
c
C F G E II C
All figures are for single oral LDx dose for rats.
* Key to references:
A: Heath and Mftgee (1962). B: Druckrey et al. (1964~). C: Druckrey et al. (1963b). D : Schmilhl (1963). E: Ivankovic et al. (1965). F: Druckrey et al. (1961b). G: Druckrey et al. (1962a). €1 : Druckrey et al. (1965~).
mice exposed to diazonwthane develop acute lung edema and those t h a t survive show scarring of the lungs (Schocntal, 1960). N-Nitroso-A'methylurca a150 produces an infl:uiimatory hcniorrhagic lesion of the stomach, intestinc, and pancreas when given by mouth to rats (Druckrey e t nl., 1961b). Trinicthylnitrosourca has an anesthetic effcct for 2-3 hours after an oral dose, but dyspnea and cyanosis develop latcr and the animals dic with lung edcina (Ivankovic e t al., 1965). Some nitroso compounds have a very lorn acute toxicity so t h a t accurate LD,, data cannot be obtained. There is no correlation between the acute toxic
174
P. N. MAGEE AND J . M . BARNES
effects and carcinogenic activity. This is clearly shown by comparing dimethylnitrosamine and diethylnitrosamine, both of which produce lesions of acutc poisoning prctlominantly in the liver. While diethylnitrosamine has about one cighth tlie acutc toxicity of dinicthylnitrosamine, if given continuously to rats it is probably more active a s n liver carcinogen.
C. PATHOLOGICAL CHANGES IN ACUTEPOISOKING On the whole there is remarkably little published work on the acute lesions produced by these carcinogens but boine detailed studies of the liver in animals given diiricthyl- and diethylniti osaniinc have been made. 1. Light Microscope
I n rats after 20 mg./kg. dimethylnitrosarnine a pallor of the cells in the centrilobular and rnid-zone region of the liver develops and progresses so that by 18 hours tlie cytoplasm is miorphous and vacuolated and the nuclei pale and irregular. Necrosis of these cells is complete by 24 hours and confluent areas arc frequently hcniorrhagic. The hemorrhage is more pronounced by 48 hours and polymorph infiltration is prominent. By 72 hours-that is, in rats whose ultimate survival was probable-the repair processes are in full swing and the necrotic areas contracting, and within 3 weeks repair and restoration of tlie liver tissue is almost complete (Bar~icsand Magee, 1954). The acutc lesion in the liver of the clog, mouse, and rabbit shows a similar pattern. A venoocclusive lesion involving the hepatic veins may also be seen in rats 10 days after tin approximate JJDs0dose (AlcLean et al., 1965). 2. Electron Microscope
Under the electron microscope changes could be seen in the endoplasmic reticulum (ER) of some liver cells within 3 hours after a dose of 50 mg./kg. diniethyliiitrosamine intravenously. Tlie ER was swollen and ribonuclcoprotciii particles were detached from the membranes. The nuclei, mitochondria, rriicro bodies, and Golgi apparatus were unaffected. These changes progressed niarliedly within the next 13 hours (Emmelot and Bcnedetti, 1960, 1961). These changes in the ER induced by dimethylnitrosamine have been confirmed by Mukherjee et al. (19631, who correlated the electron microscopic changes with stimulation and depression of amino acid incorporation produced by different doses of dimethylnitrosamine (Section VII1,A). D e M a n (1964) was able to show t h a t cortisone could protect the ER t o some extent from tlie damage produced by dimethylnitrosamine.
CARCINOGENIC NITROSO COMPOUKDS
175
IV. Neoplastic Changes
I n Table 111 are listed those nitroso compounds which have been reportcd to bc carcinogcnic in various species. I n ninny cnses tlie reports are very brief and detailed accounts of the pathology of the lesions are not provided. I n soinc instances very detailed studies of the clevelopmcnt of thc malignant lesion have been made and these will be reviewed hcrc. Although the site of the tumors w r i e s with the nature of the nitroso coinpound or with tlic route of administration and dose schedule, there are no pathological cliaractcristics that would servc to distinguish whether a tumor in one particular tissue h:td been produced by one or another nitroso compound. The pathological changes in the various tissues affected by nitroso compounds will be considered seriatim. A. LIVER
1. Ruts
a. General Morphology. I n the original description of the liver tumors produced by diiiictliyliiitro~amiiit~( LIagee :mtl Ihriics, 1956) emphasis w a s p1:tced on the gloss distortion of the gcncral liver structure, tlic anaplastic nature of the nodulc>s of neoplastic Iicpntic tissue, and their tendency to hcinorrhagic disintegration. Rletastasch within the :ibdomen and to tlic lungs were frequent. Gross hypcrplasia of the bilr ducts with cyst formation and a single sarcoiiiatous lesion were drscribed. Iiicrcased and varicd sizc of the pai~enchyninlcells with enl:trgcd nuclei were noted. Thc same general picture was also rcportcd in tumors produced by cliincthylriitrosamiiic (Schmiihl and Prcussniaiin, 1959) and later with dietliyliiitrosamine (Schm5hl e t nl., 1960). Thomas (1961) studied rats given daily doses of tliethylnitrosnniinc ranging from 0.3 mg. to 19.2 mg. per rat pcr day. He distinguished the fatty and hyaline changes in tlie liver cclls leading to destruction nntl collapse of the liver architecturc from prolifcrativc c1i:inges affecting tlic bile ducts niid hcpatic cclls. With high doses the dcgcncrativc changcs predominatcd and with the lower do the climges were mainly proliferative. Each type of change eventually evolved into malignancy unless the animal died first finom liver dniiingc. Thcrc were no qualitative differences in thc lesions produccd by diethyliiitrosamiiic and other hcpatic carcinogens. A sarcomatous I i v ( ~tumor in a rat fed dictliylnitro~ai~iiiicwas described by Argus and I-Ioch-Ligeti (1961), who also noted the frequent invasion of blood vessels by the liver tumors.
TABLE I11 THECARCINOGENIC ACTIVITYO F I\'ITROSO Compound N-Nitrosodimethylamine (dimethylnitrosamine, DMN), Menh-.NO
Species
Organ
COMPOUNDSa*b
Treatment
Rat
Liver
Feeding L.S.
Rat
Kidney
Feeding 1-12 wk.
S.C. and oral 1-10 doses
N-Nitrosodiethylamine (DEN), EtzN*NO
Rat
Lung
Rat
Nasal sinus
Mouse
Liver Kidney, lung Liver
Feeding and Daily dosing P.O. Inhalation, Single and repeated Feeding L.S. Drinking L.S. and S.C. Drinking water L.S.
Liver Liver
Feeding Drinking L.S.
Kidney Esophagus
Per rectum 5 x weekly L.S. Single oral Single oral or I.V. Drinking L.S.
Hamster (Syrian) Trout Rat,
References hlagee and Barnes (1956), Schmahl and Preussmann (1959) Magee and Barnes (1959, 1962), Zak et al. (1960), Argus and Hoch-Ligeti (1961) Riopelle and Jasmin (1963), Terracini and Magee (1964) Zak et al. (1960), Argus and Hoch-Ligeti (1961) Druckrey et al. (1963f, 1964d) Druckrey et al. (1964a) Takayama and Oota (1963) Toth et al. (1964) Tomatis et al. (1964) Halver et al. (1962) Schmahl et al. (1960), Argus and Hoch-Ligeti (1961) Schmihl et al. (1963a) Druckrey et al. (1963f) Druckrey et al. (1963f, 1964d) Schmihl (1963), Druckrey , e t a l . (1963d)
P 7,
tr
4
5
Liver SLonlach Esophagus Nose Liver
Drinking L.S. Drinking 30 wk.
Schmiihl et al. (1963b) Shvemberger (1965)
Percut. daily 6 wk. Drinking water 30-40 wk.
Lung
Drinking water 30-40 wk.
Rabbit
Liver
Dog Monkey
Liver Liver
Drinking water approx. SO wk. Food and drinking water Daily oral from birth
Fish Hamster (Syrian)
Liver Liver
Hoffmann and Graffi (1964a,b) Argus and Hoch-Ligeti (1963), Druckrey and Steiuhoff (1962) Argus and Hoch-Ligeti (1963) Schmlhl and Thomas (1965a), Rapp et al. (1965) Schmiihl et aZ. (1964b) O'Gara and Kelly (1965), Kelly et al. (1966) Stanton (1965) Herrold and Dunham (1963) Herrold (1964b)
Mouse
Mouse Guinea pig
Lung and bronchi
Nose
N-N itrosodi-n-propylamine (Pr2N.NO) h'-Nitrosodi-n-but ylamine (dibutylnitrosamine), Bu2N.No
Rat Rat
Liver Liver Bladder, esophagus Bladder
Water Oral 2 X weekly, 7 months I.P., Percut. intradermal 1 X wk. Oral 2 X weekly I.P., Percut. intradermal 1 Xwk. Transplacental in pregnant hamster S.C., I.P., intradermal Percut. Feeding L.S. Feeding L.S. 75 mg./kg./day Feeding L.S. 37 mg./kg./day S.C. 200 mg./kg. 1 X wk.
Dontenwill and Mohr (1961a,b), Dontenwill et al. (1962) Herrold (1964b) Mohr and Althoff (1964), Mohr et al. (1965) Herrold (1964a,b,c) Druckrey et d. (1961a) Druckrey et al. (1961a) Druckrey et al. (1962b) Druckrey el al. (1964~)
w
2
TABLE I11 (Continued) Compound
Species
N-r\-itrosobutyl-4-hydroxybutylamine, Rat HO.[CH~]~.KBU.KO h'-Nitrosodi-n-amylamine, Rat (CjHii)?N.NO
Organ
Treat,ment
References
Bladder
Drinking water L.S.
Druckrey et al. (1964~)
Liver Lung
Drinking water L.S. S.C. 1 X n k . 25 wks.
N-Ntrosoallylmethylamine, CH?:CH.CHrNMe.NO Ar-Nitroso-n-butylmethylamine, BuMeN.NO AT-iYitrosomethylvinylamine, CH2: CH.NMe.NO
Rat
iYose, kidney
I.V. 1 X n-k.
Druckrey et al. (1961a) Druckrey and Preussmann (1962~) Druckrey et al. (1964a)
Rat
Liver
Oral 30 doses
Heath and Magee (1962)
Rat
Nose
Druckrey et al. (1963f) Druckrey et al. (1964a)
N-Nitrosomethylphenylamine (N-methyl-N-nitrosoaniliie) , C6HrNMeNO N-Nitrosobenz ylmethylamine , C6HjCHrNMe.N0 N-Nitrosoethylisopropylamine, EtPriN.NO N-Nitroso-n-but ylethylamine, BuEtN.NO
Rat
Esophagus
Single inhalation Weekly inhalation for 35 hour Feeding L.S. Drinking water L.S.
Druckrey et al. (1961~) Boyland et al. (1964)
Rat
Esophagus
Feeding L.S.
Druckrey et al. (196313)
m
Feeding L.S.
Druckrey et al. (1963b)
zi
Rat Rat
Mouse Rat Rat
Esophagus, liver Esophagus, liver IGdney Forestomach Esophagus
Feeding L.S.
Druckrey et al. (196313)
Single dose Drinking water L.S. Feeding L.S.
Druckrey et al. (1964d) Schmahl et al. (1963~) Druckrey et al. (1963b)
Liver Kidney
Drinking water L.S. Oral, 10 doses
Druckrey et al. (196313) Thomas and Schmahl (1964); Druckrey et al. (1964d)
cd
z
F 0 M
M
P
K-Xi trosomorpholine CHz.CHz
/
\
\
/
0
Liver
Drinking water L.S.
Hamster
Kidney Nose Liver Lung Nose Esophagus Liver Lung
Oral, 6 doses I.V. 1 x wk. Drinking water L.S. S.C. 2 X wk. S.C. 2 x wk. Drinking water L.S. Drinking water L.S. 8.C. 2 x Wk.
Druckrev el d.(1961a), Banriasch and &fuller (1964) Tlionias and Schmahl (1964) Drurkrey el al. (1964a) Banns-ch and Muller (1964) Dontenwill arid Mohr (196La) Druckrey d ul. (1964a) Druckrey et 01. (1961a) Boyland at nl. (1964) Dontenwill and JIohr (1962a)
Ilat
Esophagus
Drinking water L.S.
Boyland et al. (1964)
Rat
N.NO
CHz.CH, S-Xitrosopiperidine CHz-CH,
/
\
CHz
\
N.NO
PvIouse Hamster Rat
/
CHZ-CHz A’-Ni trosoanabasirie CH,-CH,
0
N.YO
CH?
/
\ CH?--CH
hT-~itrososarcosine, HOzC.CH2.SMe.NO N-Xitrososarcosine ethyl ester, EtOCOCH,NMe.NO h’,N’-Dinitrosopiperazine CHz-CHZ
Rat
Esophagus
Drinking water L.S.
Rat
Forestomach, Drinking water L.S. tongue Nose, esophagus S.G. 1-2 weekly S.C. 1-2 weekly Liver, lung
Druckrey
at
al. (19GBb)
P
\
H
2 ci Q
\
/
0
Rat hlouse
Druckrey et al. (1963h) Druckrey el al. (1964a) Schmihl and Thomas (196%)
e
00
0
TABLE I11 (Continued) Compound
Species
N,~~’-Dinitroso-hr,N’-dimethylethyleneRat diamine, ON~X’MeCH&H~Kh‘IeSO N-Methy1-N-nitroso-N’Rat nitroguanidine, MeN(NO)C(:NH).NH.NO? N-Xitrosomethylnrethane, Rat EtO.CO.NMe.KO
Organ
N-Nitrosotrimethylurea, Me2NC0.NMeN0
Mouse Rat
Mouse Hamster Rrtt
References
Esophagus
Drinking water L.S.
Druckrey et ul. (1963b)
Forestomach
Oral, 2-4 doses
Schoental (1966b)
Stomach, esophagus
Oral single dose
Schoental (1960), Schoental and Magee (1962) Druckrey et al. (1961b) Druckrey et al. (1962a) Thomas and Schmahl (1963a, 1964) Schoental (1963~) Druckrey et d. (1961b) Druckrey et d.(1963f) Leaver, Magee, and Swann (unpublished) Druckrey el al. (1965a) von Kreybig (1965) Graffi and Hoffmann (1966)
z
Lung Kidney N-Nitroso-N-methylurea, H2N.C0.NMe.N0
Treatment
Drinking water I.V. 1 X wk.
Stomach, lung Forestomach, intestine, kidney
A few oral doses Drinking water I.V. single injection Oral single dose
Brain Teratogenesis Skin Skin Brain, nerves
I.V. 1 x wk. I.V. single dose Local applications Local applications Drinking water L.S.
Ivankovic et d. (1965)
;P
n n m
W
;P
m
3UJ
Mouse Rat
Intestine Stomach Stomach Liver, kidney
Guinea pig
Colon Liver
Mouse
Liver, kidney
Diazomethane, CHy:?;?
Rat Rat
Duodenum Lung
Azoethane. E t S :K E t
Nouse Rat
Lung Liver, esophagus, stomach, iiose. brain, leukemia Skin, esophagus, tongue, stomach
iV-Nit row-N-et hyluretliane, EtO.CO.n'Et.PI'O Cycasin, MeXO: T\'.CH?.O-glycosyl
Met hylazoxymethanol, MeNO :P\'CH,.OH
Ethyl diazoacetate,
CH.CO.OEt,
S2:
Rat
Rat
I.P. 2-3 doses Oral 4 doses
Schoental (1965) Schoental (1963~)
Feeding L.S. S.C. single dose (newborn) Feeding 13-21 days Feeding 5 days/mk. 2-3 times Topical application to skin ulcers I.P. 12 injections Inhalation
Laqueur ef al. (1963) Magee (unpublished) Laqueur (1964, 1965) Spatz (1964)
Inhalation S.C. 1 X ~ k 20-30 .
I.V. sereral doses Drinking water L.S.
TV~.
O'Gara et al. (1964) Laqueur (1965) Schoental (1960), Schoental and Rlagee (1962) Druckrey et al. (19650)
Druckrey et nl. (196513) Druckrey et al. (196311)
A survey of the species tested, the organ in which tumors developed, and an outline of the treatment, administ,ered. L.S. = life span; S.C. = subcutaneous injection; I.V. = int,ravenous injection; I.P. = intraperitoneal injection, Percut. = percut,aneous; P.O. = orally. a
6
I82
P. N . MAGEE AND J . M . BARNES
b. Detailed Cytology. Grundmann and his colleagues h a w made a detailed study of the 1ivc.r parenchymal cells in ruts given diethylnitrosaiiiinc in their drinking water. I n rats given a daily dope of 2.5 mg. diethylnitrosaniinc carcinoiiia with iiietastnses appe:ircd within 140-150 days and involved exclusively the hepatic rather than the biliary cells. Within a few days of first giving diethylnitrosamine the liver nuclei showcd an increased variability in size and D N A content. There was ail increasing ancuploidy and hypcrploidy with some very largc nurlei. Shortly before fr:inlr tuinors :tppenretl snmll nests of cells were found in the ccntrilobular region from which the carcinomata appear to develop. These cells are diploid with a dark cytoplasm rich in ribonucleic acid (RNA) but contain little glycogen. (Buchner et al., 1961). The liver ccll cliniigcs 111 rats on lower doses of dicthylnitrosaniine (0.75 mg./rat/d:iy ) could be tlividcd roughly into three stages. During the first 45 days the cells a t the periphery of the lobule showed an increascci cytoplasmic basophilia, vesicular swelling of the cytoplasm, and variation in nuclear size. During the second phase, lasting about 50 days, the cells a t the ccntcr of the lobule unclcrwent similar but rather inore striking changes. During the next 50 days siiiall ncsts of basophilic cells began to appear as grayish graiiules to the naked eye. They coalesced t o form tumors during the next 50 days. The biliary tissue and connective tissue are very little involved in these changes (Grundmann and Sieburg, 1962). A more detailed analysis of the changes in nuclear size and DNA4 content a t various stages in the development of the liver cancer is given by Christie and Le Page (1961) and Hobik and Grundmann (1962). Grunclmann docs not consider that dictliylnitrosarnine differs in any essential wiy from other livcr c:wcinogcns such as hitter yellow in its action on the liver. H e believes the first point of attack is the ccll cytoplasm with loss of protein and RNA and suggests t h a t one way in which the ccll can compens:ttc for this is hy :in inci*eased tioiynthetic activity of the nucleus which might result in c1iar:actcristic polyploid changes. When the neoplastic cells appear their nuclear size is smaller and more uniform. The apparent damage to cytoplasm seen microscopically was confirmed hy Oehlert and Hartje ( I 963) using Icucine-H’ and autoradiographic tccliniqiics to study Iwotein synthesis (see Section VIII) . Synthesis was dcpressctl in the ccntrilobular liver cells up until tlie time carciriomata appeared. RNA synthcsis studied by cytidine-H I and autoradiography was raised in the ccntrilohulnr cells, particularly those with the very large nuclci. At the sninc time the RNA level in the cytoplasm of these cells was reduced. Once the cancer cells appeared RNA synthesis in nuclei and tlie RNA level in the cytoplasm were above normal. Elcc-
CARCINOGENIC NITROSO COMPOUNDS
183
tronniicroscopy of liver cells in those rats given diethylnitrosamine confirms the darnage to the cytoplasm in the early stages with increased vcsiculation of thc ciit1opl:imic reticulum. This is intcrpretcd as likcly to lead t o a loss of specific structural protcin and possibly to the formation of new protein which is not ahle to excrcise any regulatory function over DNA production :mtl ccll niultip1ic:ition (Jlolbrrt ct ol., 1962). Tliesc authors could cictcct no tlminge to mitochondria1 structure. Bannasch and hliiller (1964) fed rats N-iiitrosoinorplioliiie, which produces liver tumors, aiid found c:trly cliangcs i n the ccntri1obul:ti- cell< c*h:~rnc~terized by a loss of glycogen aiid tlihivption of the endop1:miiic reticulum. On tlic other hand, a t tlic y r i p I i ( ~ yof the lobule the cells stored excessive qumtities of glycogen and :tlso sho~vedsonic loss of cytoplasmic basophilia. Thc cntloplasmic rcticulum of these cells tcndcd to hypertrophy, p:irtic~lni~lyin the large cclls. With the clcvclolment of the ninlignnnt ph:tv thcrc is a decitase i n glycogcri storage in these cclls :ind increase i n cytop1:tsmic 1iasophili:i. The tumors appcnr in many sitcls :itJ tlic m i i e tmic. Thcscb :iuthorh tli(l not conhidcr tliow nuclear c1i:triges that preccdc~lt Iic onset of tumor- to be iinpoitant. Giissncr ant1 Frictli3cli-Frek.a ( 1964) sturlictl g l u c o s e - 6 - ~ ~ h o ~ p h ~ ~ t a s c histochcniicnlly in rat liver during c.ni.cinogcncsis Iiy dictliylnitro~amine and i\r-nitrosomorpholinc. Enzyme activity w:is tlc>monstr:itcd in thc cytoplasm of nornxtl liver ccl1.i ant1 gc~nc~t~illy the activity w:~s g r w t e r in the peripheral zoncs of the liver Iobulcs. The activity of the cnzymc was increascd in fasting animals. There was no histochemirally clcnionstrnhlc glucose-6-plio~~~li:~ta~c activity in liver carcinomas induced by the nitrosamines. After 6 wccks of nitrobaniinc fccding (after about 200-300 nig. per kg. dic,tliylnitros:~ininc) first m a l l nests and latcr larger islands of liver cells were found in which 110 glucose-6-phosphntase could lie tlcmonstlatccl. In conil):irison, tlic. h u r i ouncting liver parcnchym t slion.cd n strong enzyinc re:ictioii. T h e ih1:iiids contninetl cells of varying size and were iiwgularly distributed in the section, frequently in the ccmtriloliular Z O I W . ~ , and sh:q)Iy distinct from the enzyme-coiit:rining cclls. There appears, therefore, to be aliscncc of the microsoinal enzyme gluro~.c~-6-plio~I,11:lt:is(~ i n CIrciirii~('rihC'1rcgions of p:ircnchyin:iI cclls of pi ccmiccrous liver. Tlic.c findings are c o n i p a i ~ t lwith the tlcnionc tlntion of similar cellular islands with loss of liver-specific microtonid antigcns (Weiler, 19591, the regions of incrcasctl RNA synthe& dciiionstratcd nuto~:~tliog~apllic:lly (O(~1ilert:t11(1 II:ii,t,i(,, 19G3), tlic glycogcn-ficr cell islands (Grunclmann arid Sieburg, 1962), and the changes i n tlic endoplasniic reticulum as shown in the electron microscope (Emmclot and Bcnctlctti, 1960; Rliilhrt et a/., 19621, :ill of wliich h : i r ~ h w n roportcd in nitrosnminc carcinoqenesis of the liver. Loss of glucose-6-phosphatase
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from the centrilobular zoncs of livers from rats acutely poisoned with dimethylnitrosamine was demonstrated by histochemical staining methods in the electron microscope by de Man (1964). Gross cellular damage is not a necessary precursor of liver cancer with nitroso compounds. Laqueur e t al. (1963) undcrline this in their account of liver tumors in rats fed cycad nuts containing the related carcinogen, cycasin. Although liver necrosis severe enough to cause the death of some animals was produced by a diet containing 27% or more of a toxic cycad meal and liver cancer ultimately appeared in the survivors, a less toxic meal led to the appearance of tumors in liver tissue that showed no evidence of damage to the rest of the parenchymal and connective tissues. 2. Other Species Adult mice have developcd malignant hemangioendothelioma when fed dimethylnitrosamine (Takayama and Oota, 1963; Toth et al., 1964) and dicthylnitrosaminc (Schmiihl et al., 1963b ; Schmahl and Thomas, 1965b). Newborn mice given single injections of dimethylnitrosamine developed liver cell tumors (Toth et al., 1964). A detailed account of cytological changes preceding the appearance of tumors in the livers of rats and mice fed N-nitrosomorpholine is given by Bannasch and Aiiiller (1964). Liver cell tumors are readily produced in guinea pigs by diethylnitrosnmine and are similar to those in the rat, showing a good deal of fibrosis with gross distortion of the liver architecture and bile duct hyperplasia (Druckrey and Steinhoff, 1962; Argus and Hoch-Ligcti, 1963; Thomas and Schmahl, 196313). A single animal developed an adenocarcinoma of the gall bladder (Argus and Hoch-Ligeti, 1963). The cytological changes in the guinea pig are similar to those seen in the rat. I n hamstcrs dimethylnitrosamine produced widespread liver cell damage and disorganization so that i t was often difficult to distinguish true neoplastic changes amid the other alterations in structure. The tumors tended to have a pronounced vascular element. Cholangiocarcinomas were common (Tomatis et al., 1964). Rabbits given diethylnitrosamine in their drinking watcr developed liver tumors of a mixed type similar to those seen in the rat (Rapp et al., 1965; Schmahl and Thomas, 1965a). Rapp et al. (1965) found metastases in the lung. Two dogs given diethylnitrosamine developed severe liver cirrhosis (Schmahl et al., 1964a). A portal-caval anastomosis was performed on one of these animals and diethylnitrosamine was given again for a few months after the operation. The dog became very ill and a t post-mortem was found to havc a large liver tumor which histologically was a leiomyosarcoma (Schmiihl et al., 1964b).
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185
O’Gara and Kelly (1965) and Kelly et al. (1966) have produced hepatomas in monkeys with diethylnitrosamine. Fish also develop liver tumors when exposed to dimethyl- or dietliylnitrosamine (Halver et al., 1962; Stanton, 1965).
B. KIDKEY The tumors produced in the kidneys of adult rats by dimethylnitrosamine fell into two general types. One type is well differentiated and varies in size from massive to scarccly visible macroscopically. I n rats killed serially after dosing with dimethylnitrosamine the early stages of epithelial proliferation within the lumen of a tubule can be seen (Magee and Barnes, 1962). These tumors are well circumscribed, whereas those of the other type, described as anaplastic, infiltrate the normal kidney tissue and are composed mainly of masses of oval or spindle-shaped cells showing many mitotic figures (Argus and Hoch-Ligeti, 1961 ; Magee and Barnes, 1959, 1962; Zak et al., 1960). Tliese tumors resemble nephroblastomas in showing a great variability in structure and are highly vascular and often disrupted by massive hemorrhages. hlagee and Barnes (1962) pointed out that these renal tumors in the rat have their human counterparts of renal carcinoma and nephroblastoma (Wilms tumor). When diinetliylnitrosamine was given to newborn rats all the renal tumors were of the nephroblastonia type (Terracini and hlagee, 1964). I n a paper which is an excellent source of references on experimental kidney tumors, Thomas and Schmahl (1964) discuss the renal tumors in rats produced by several nitroso compounds and again found the two types which they call epithelial and mcsenchynial. They compared these tumors with those produced by a variety of other agents, concluding that all the renal tumors fall into these two general types. In this they are in agreement with Zak et al. (1960), who pointed out that the rare spontaneous renal tumors in rats may also take either form. Rats fed toxic cycad meal develop renal adenomata which may occur together with undifferentiated proliferative tumors as seen in rats given dimethylnitrosainine (Laqueur et al., 1963). Renal carcinomas were not seen in the rats on cycad meal. I n a detailed study of the renal tumors produced in Sprague-Dawley rats by dimethylnitrosamine, Riopelle and ,Jasmin (1963) paid particular attention to the nephroblastoma type and found that some of these appeared to proliferate less rapidly than others and showed considerable formation of smooth muscle fibers. They concluded that the character of these tumors is determined by their origin from immature cells with considerable potentialities for differentiation. In further studies these authors found that the epithelial
186
P. N . MACEE A N D J . >I. BARNES
tumors (rarcinoma) occurred more frequently in male rats and the sarcomatous type in the females, suggesting soinc horrnonal influence on the developnicnt of thcsc tumors (Jnsniin : m l Riopelle, 1964). Following their obscrvation that a high p i oportion of r e n d tumors could be induced in rats by six successive daily doses of I .5 mg. dimcthylnitrosamine, these workers have studied the early clcvelopmcnt of renal tumors. The earliest tlctcctnble change in the kidney was a mononuclear cell infiltration appearing wound some tubulcs within 1 week :inti disappearing within a further 4 wccks leaving the tubules ntrophictl and fibroscd. A t about 20 \\rocks atlcnonintous tubules could be found and these nlight either iegichs to form sln:ill cysts or progrcss to solid tuniors. The lesions weit thought to originate in the ascending put of the loop of Henle.
C. BLADDER Carcinoma of the t)l:Ldclei* caii be produced in rats either by injccting dibutyl nitrosnniine or fccding 4-liytlros~l~utyl-)i-butylnitrosarnine. The tumors vary in ch:iractcr h t h:ivc the usu:il fct:itui r s of tquanious cell, transition cell, or atlcnocarcinoiii~ita.In only one n i i i n i a l was thcit a tumor of the ureter ( D ~ u c k i ~ cety nl., 1 9 6 4 ~ ) .
1). XOSEA N D NASALSr~uscu Nitrosamincs have pi otlucecl nin1ign:int tumors i n the n:isaI c:ivities of mice, rats, and hitnistcrs when given by inhaltition, by niouth, or by skin apphcntion ( x c ‘rable 111 for rc3fcrcnccs). In the mouse given diethyliiit~osamiiicby skin application the nasal epithcl:um showed necrosis followed by progressive changes of hypcrtrophy leading to the tlcw~lopmcntof s q n : ~ i i i ~ccll ~ s c:trcinomatn in the iwijoi-ity of c:isc>: a fcw tuniors hacl glanclulnr clcnicnts (Hoffniann ant1 Graffi, 1964a,b). In the r a t and hxnistcr t1ici.c wc;c not only s t p i n i o u s ccll carcinoma : ~ n dadcnocarcinomata hut a good pi oportion of ncuroepithelial tumors (Herrold, 196421; Tlioinas, 1965) . Thcsc tumors., firtt described in man and citllcd “esthesioneziro epitlieliome o l f n ~ f i f ”ai e of uncertain origin but 1): ob:ibly arisc from thc olfactoiy epithelium and are considered to be a foirn of ncuroblnstonia (Hcrroltl., 1964:~).In the hamster epithelial papillomas of the nasal cavity can :dso bc protlucc~tl by dietliylnitrosaminc (Hcrrold, 1964b) :is well as iriidiffeienti:itcd carcinoina of the ethnioid region (Her1old and Dunhani, 1963) . As in thc kic1nc.y thc nitrosamines have produced tumors of scvcr:~l types in the nose of nninials which have the p:ithologic:il c1i:iracteristics of malignant lesions seen in the human nose.
CARCISOGENIC NITROSO COMPOUNDS
187
E. LUNGSA K D BRONCHI I n mice, rats, aiitl guinea pigs nitrosaniiiies liaw pi otluced luiig tumors which arc iiiaiiily iiiultl1)lc aclwoiii:ita, often well circuniscribccl m d situated a t the peripliery of thci lung (Z:tlt et (il., 1960; IJIagce :iiitl Barnes, 1962; Argus aiid IIoch-Ligcti, 1963). M7iiIe imny have a benign sppearancc others arc iiivasivc :ind in soiiic’ C:IW\ break iiito the bronchial luiiien (Scliniiilil and Tliomxs, 196.5~). Sqii:iiiiou~iiietapliisia of the bronchial cpitliclium has been ol)acrvctl aiitl t h i h iiiay be continuous with a squaiiious-cell carcinoma (A41’g,~iSand IIoch-Ligcti, 1961). A single example of a squamous carcinoina of tlic lung was found in a sinall group of rats exposed to diazomcthnne wliicli also iiitluccd the chnractcristic lung sdeiioma in niicc ant1 similar lesions iii r:its (Sclioental and Magee, 1962). S C ~ W U ~carcinoiiin ~ O L I ~ was iiitlucrtl iii one iiiousc in a group given iV-iiiti.osoinoi.pIioline in their driiilting water. Thc others had lung adenonia m t l broiicliiitl papillonin (11. A. Rliillcr, 1965) . Siiiiilar adeiioiiin of tile lungs tlcvclopctl in iiiiccl boim to nlothcrs wliicli had reccivcd five claily doses of tlictIiyIniti,os:uniiic clui,iiig tlic fifteenth and twentieth day of prcg1i:incy (LIohr and Altlioff, 1965). The hamster seeins t o react soiiicvhat diffcrcntly (Dontcnwill et al., 1962). Doiitenn.ill :miN o h r ( 19Gla,b) g:ivc clictliyliiitrosaiiiiiie daily by stomach tube and within a few weelis the tiwheal arid bronchial epithclium was sliowiiig in:irltctl t~1i:tiigc~. A \ well :is n gcncml q u n i i i o u s nictnplasin, papilhry bronclii:tl tumors tlcvelopcti :iml within 2-3 irioiiths carcinoina infiltrating the lung 1i:itl appc:ti~ccl. 1Icrrold and I1unh:tm (1963), reported similar experiinciits but with 10s f i q u e n t dosing of clictliyliiitrosaiiiine, found only scjuniuou~-cc~ll p:ipillonin of the ti~tchca aiid bronchus with no inva4on of tlie lung. Thehe lesions were siniilnr to those induced by iiitr:itixclie:il iii4:tllation of bcnzopyrcne. Eotli papers referred to :ibove eiiipliasizc tlie t1iver.w rcllular ch:tiigcs in tlie bronchixi and tracheal epitheliuiii in the :ti eas untlvi going iiic.tapl:tsia. When dietliylnitrossniinc was given to piqyiant haiii>tcis in tlaily doses of 2 mg. for 1-7 chys duriiig the sccoiitl half of tlicw prcgii:iiicy inany of t h e youiig sliowcd niet:iplnei:t :inti sonic pnpil!oiiin involving the tracheal cpitheliuni. The motlicrs were hiiiiilarly affected (Alohr e t al., 1965). R4easurcnicnts of iiuclear c1iainctci.s in tlic trachc.al and broiichial epitlicliuni of liaiiistcrs receiving clic~tliylniti~o.:iiiiiiicslionctl :I pcnk a t 1220 p,? in norinal :itiinials conil)nrcd with a 1wak witli a I iingc. 24-36 p2 in nictaplastic or cai ciiioniatous lesions (Dontcnwill niitl Rlolir, 19621)). Autoradiogr:il)hic studies liavc been in:de on Iinnistors receiving tlictliylnitrosaiiiinc and injected with tliyiiiitlinc-11’. Cells bhowing DNA synthesis (niarkcd cells) were found in the bnsal layers of the epithcliuiii
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undergoing mctaplasia and an increased proportion of cells was marked as hyperplasia proceeded to mctaplasia. The proportion of such cells did not increase once frank neoplasms had appeared. It was interesting that the carcinomata appeared mostly in tlic smaller bronchi and not in the region where there is normally a higher rate of epithelial proliferation such as the bifurcation of the trachea (Dontenwill and Wiebecke. 1964). Dontenwill (1964) has provided a useful summary of his findings in the hamster givcn diethyliiitrosaiiiine and stressed the value of 01)servsttions on animals after administration of the carcinogen has stopped. He points out the difficulty of distinguishing areas of intense metaplttsia from carcinoma by any criterion except invasiveness. He believes that the respiratory tract, particularly the trachea and bronchi in the hanistcr, shows a predisposition to the carcinogenic action of diethylnitrosnniinc, which in this species affects the liver much later than the lung. This organ specificity is not affected by the route of administration whether hy mouth, intratraclicnlly, pcr rectum, or subcutaneously. Dontenwill sti'esscs that the ninlignant changes do not occur in areas of the most rapid cell multiplication cither within the trachea and bronchi or in the mouth and pharynx where cell division is much more rapid than in the respiratory tract. IIe alFo compares the lesions pro(luccd in tlie respiratory tract of different specics by different agents and shows that the pathological lesion depends upon the specics tested rather than upon the agent used.
F. ALIMENTARY CANAL 1. Esophagus
Carcinoma of the Lase of the tongue has been produced in rats with diazoethylacetate (Druckrcy e t al., 1963a). Numerous nitroso compounds h a w produced carcinoma of the esophagus (Druckrey e t al., 1963h). Tlicsc may be single large tumors or the whole length of the esophagus may be studded with papillonlatous growths. Histologically these tumors are typical squamous-cell carcinoma Schoental and Magee, 1962). Although many of the nitroso compounds that produce csophagcal tumors arc highly reactive and may be expected to produce a local lesion when given orally, Druckrey e t al. (1963b) point out that lesions elsewhere in tlic mouth and pharynx are not usually pi*odurcd and esophageal tumors may also develop after the intravenous injection of N-nitrosopipcridine. 2. Stomach
Tumors of the forcstomach in the rat and mouse have been produced by several nitroso compounds. Schoental and Magee (1962) give a n ac-
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189
count of the histological features of the tumors produccd by N-nitrosomcthylurethane and include some study of the acute dcstructive lesion ~)roducedby this compound and its subscqucnt healing. They emphasize the fact that malignant tumors may arise after a single dose of this compound. A much more dctailcd study of the progression of changes in the forcstoniach of rats is provided by Toledo (1965), who gave N-nitrosoii~ethyluretlianea t varying concentrations in the drinking water. I n rats receiving 1: 10,000 N-nitrosomethy lurethane as drinking water, the iiuclci of the bnsal cclls round up niid the nucleoli become darker within 24 hours. During the next 24 hours there is vasodilation, which progresses to edema; the cell nuclei swell, and the cells die. By 7 days the necrosis is complete and a leucocyte infiltration is evident. Between 11 and 31 days regeneration starts with islands of cells which grow to form small papillomas. These cells have large nuclei and thcrc are nests of foam cells. By 96 d n y s multiple papillomas are visible on the surface of the iiiucosa :ind the transition to carcinonia takes place. From 173-326 days umbilicatcd papillomatous lesions with keratin foimation grow and invade the subniucosa to become cvciitually frank squamous cell carcinomata. Limited observations on rats receiving 1:80,000 and 1:160,000 N ii,tro~oiiictliyluretliaiie s h o ~ the ~ d same cellular changes a t 60 dnys but there was no preceding nccrosis. Cytophotometric measurements of DNA xliowed that after 3 t h y s exposure to 1: 10,000 the peak had shifted from diploid nuclei t o tetra and between tetra- and octoploid nuclei. The peak gradually inovcd back so that by 178 days i t was again normal. Labeling with thymidinc-H3 from a normal 4 4 % of cells rosc to 24-32r/o in the first 48 hours but then the cells died and a t 7 dnys 33-3670 of the cells a t the edges of the necrotic areas were labeled. The raised percentage of labeled cells was confined to the basal layers even after invasion of the subjacent tissues had started but became morc general after frank tumors had developed. When thc N-nitrosomethylurethane was stopped after only 2 days the carly degenerative changes in the epithelium procecdcd as before but healing and restoration of the epithelium was more rapid. The glandular part of the rat or mouse stomach is involved in the early acutc Icsions that follow the introduction of N-nitrosomethylurethane and in occasional rats an adenocarcinonia involving this part of the stomach has developed (Schocntal, 1963a,c).
3. Small and Large Intestine Adcnocarcinoma of the small intestine, colon, and rcctum have been produced in rats after a single intravenous injection of nitrosomethylurea
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P. N . MAGEE AND J . M. BARNES
(Druckrey e t al., 1964d) and in the small intestine after the intraperitoneal injection of N-ethyl N-nitrosourethanc (Schoental, 1965). Tumors of the forestomach and small and large intestine as wcll a s the kidney have followed a single oral close of iV-nitrosomethylurea to rats (Leaver, Magee and Swann, unpublished). Mucous adenocarcinomas of the large intestine were found in rats on cycad meal (Laqueur e t ol., 1963; Laqueur, 1964). Tumors of the duodenum have arisen in rats given intrapcritoneal injections of niethylazoxymcthnnol (Laqueur, 1965).
G. NERVOUS SYSTEM
A series of tumors involving the brain, spinal cord, and peripheral nerves of rats have been described in animals inj ccted with nitrosomethylurea (Druckrey e t al., 1964b, 1965a) and trimethylnitrosourea (Ivankovic et al., 1965). No detailcd histological descriptions of these tumors are yet available but they are rcportcd as including glioma, glioblastoma, oligodcndroglioma, sarcoma, ncurinoma, and cpcnclyinoma (Thomas and Kersting, 1964). H. SKIN The daily application of diethylnitrosaminc to the skin of mice lor up to 10 months led to no p~ec:~ncerousor other pathological changes; when croton oil was inclurlcd, the inflamrnatory lesions were siniilar to those produccd by croton oLl alonc (IIoffmmn and Graffi, 1964a,b). On the other h:tnd, N-nitro~omethylureaapplied as a solution in acetone to the skin of mice appeared to be as active as the polycyclic hydrocarbons in producing skin cancer. IIamstcrs wcrc also :tffrctrcl (Graffi and Hoff mnnn, 1966). Multiple squamous cell carcinomas were produced in thc skin of rats by the intravenous injection of diazoethylacetate (Druckrey e t al., 196513).
I. TERATOCENIC
AND OTHER
EFFECTS
When rats were given a single intravenous injection of nitrosomethylurea on the thirteenth and fourteenth day of pregnancy many fetuses were killed and resorbed and those surviving had many cleformities. As in rats treated with cyclopliosphamide the deforniities mainly involvcd the brain and limbs (von Krcybig, 1965). Arff man (1964) injected diethylnitrosamine and nitrosomethylurcthane into newts and observed the typical liyperplasia of the epidermis
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191
wen with other carcinogens, particularly those of the polycyclic hydroc:trbon group. Dietliyliiitrosai~iinewas only effective when injected in oil solution. V. Some Special Features of Nitroso Compounds as Carcinogens
I n Table I11 are summarized the published data on the carcinogenic activity of the various nitroso and related compounds discussed in this review. I n ninny instances the reports are preliminary and there are few expc’rimcntal details or figures for tlie incidence of tlic tumors recorded. Horvcvcr, work SO far done with the carcinogenic nitroso compounds indicates L: nunihcr of points of interest in their bcliavior as carcinogens for experimental animals. With dimcthylnitrosamine it was shown that with continual feeding at a level that allowed the rats to survive 30 weeks or longer only liver tumors appeared, but i f the compound was given a t higher concentrations for short periods or even as a single dose kidney tumors developed. The livers of tliobe aninials which were damaged hy the high Icvels of dirnet1iylnitros:Liiiine recovered with slight scarring and no malignant changes (Magee and 13:zrnes, 1962). Subsequent observations (Argus and Hoch-Ligeti, 1961) sliowed that liver :ind kidney tumors coultl boiiietiiiics occur together in rats fed dimetliylnitro9ainine. However, the generwl point rem:tins true and Riopclle and .Jasmin (1963) have shown that an incidence of 90-100% kidney tumors can be produced in rats by a, limited number of do5cs of tliiiietliylnitrosainiiie given on successive clays. Dimethylnitrosaminc will also produce lung tumors in some rats (Zak et nl., 1960; Argus and IIoch-Ligeti, 1961) but the particular conditions under which the incidence of these tumors may be iiifluenced have not been discovered. As Argus and Hoch-Ligeti (1961) pointcd out diinctliyliiitrosainine was the first example of a compound that could oclucc squanious-cell caitiiioma in the lung of rats aftcr ingestion. Dictliylnitrosaniirle is cnpablc of producing tumors of thc liver in a great variety of bpecics (Thomas and Scliniiihl, 1965) and it is clear that the guinea pig, hitherto considered resistant to other liver carcinogens, is quite susceptible to tlie alkyl nitrosamines (Druckrey and Steinhoff, 1962). T h a t the liver is the most commonly affected organ is t G I x expected since the main metabolic hreskdown of the simple alkyl iiitros:mines takes pl:m in that organ (Section VII) . The fact that a single dose of diniethylnitrosamine could lead to kidncy tumors in rats (i\l:igce a n d Barnes, 1962) was of spcci:rl interest, i):wticularly as tlicre werc no markecl preceding renal changes (sce Section IV,B) . Druckrey and his colleagues have studied other nitroso com-
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P. N. MAGEE AND J . M . BARNES
pounds given as single doses and found that several can give kidney tumors and dimethylnitrosamine itself by a single inhalation will produce ethmoid carcinoma. A single intravenous dose of nitrosomethylurea produced 11 different types of tumors in a group of 16 rats (Druckrey et al., 1963f, 19Md). A single oral dose of the same compound will produce tumors in different parts of the gut and also in the kidney (Leaver, Magee and Swann, unpublished). Single doses of nitrosomethylurethane will produce malignant and N-methyl-N-nitroso-N'-nitroguanidine tumors in the stoniach of thc rat (Schoental, 1966b). The importance of a carcinogenic hazard from a single exposure to a poison cannot be over emphasized when considering risks to those exposcd to thesc compounds in industry, where an accident could lead to an acute but not necessarily fatal poisoning. If the method of administ>ration remains the same but the daily dose is altered the site of tumor production may change. Thus Druckrey et al. (1963d) describe cxpcriments in which rats received diethylnitrosamine in doses ranging from 14.2 down to 0.075 mg./kg. daily. At the highest dose the animals died early with liver cirrhosis and some early liver cancers. Betwecn 4.8 and 1.2 mg./kg. daily thc animals had liver cancer but a t 0.6 mg./kg. daily and below there was a n appreciable number of cancers of the esophagus as well a s liver tumors. These findings are also discussed by Schmlhl (1963). The dose-response relationships for carcinogens including nitrosamines have been discussed at length by Druckrey and Schmahl (1962). When N-nitroso-di-A'-butylamine was incorporated in the diet at two levels all rats on the higher level devclopcd liver cancer while of those on the lower level some had esophageal and the others bladder cancer (Druckrey ct al., 1962b). Under some conditions the liver cancer may develop so rapidly that the rats may die within 40 weeks, before tumors in other organs have time to make their appearance. The variations in the response of different organs to the carcinogenic nitroso compounds is of interest in rclation to the biochemical changes that may be essential for the initiation of a carcinogenic change. The alkylation of cellular components in different tissues of animals given dimethylnitrosamine is discussed below (Section IX) . There does seem to be some organ specificity which is independent of the route used for administering the nitroso compound. While many nitroso compounds will produce esophageal cancer whcn fed t o rats (Druckrey et al., 1963b), these authors point out that some, such as N-nitrosopiperidine, will produce a high yield of esophageal tumors after repeated intravenous injection. N-Nitroeo-di-N-butylamine when injected subcutaneously leads only t o tumors in the bladder, whereas given by mouth tlie
CARCINOGEXIC NITROSO COMPOUKDS
193
same compound produces tumors of the liver, esophagus, and bladder with equal frequency (Drucbrey e t al., 1 9 6 4 ~ ) .Clearly a great deal awaits t o be done on the distribution and metabolism of each of these nitroso compounds before the differences in the reactions each can produce will be understood. At the present time there would appear to be a strong case to be made out for a detailed study of one or two compounds so that their mode of action may be better understood. Many of the detailed pathological studies of the evolution of tumors produced by nitroso compounds have been in animals receiving repeated daily doses continued until such times as the tumors appeared. It is now known t h a t single doses or very short periods of exposure may be followed by the appearance of tumors. More serial pathological studies are needed in animals treated in this way so that the evolution of malignant changes may be observed in tissues not continuously exposed to the carcinogen. Dontenwill (1964) has emphasized the value of such studies in animals after the dosing of the carcinogen has stopped. VI. Mutagenic Action
Many carcinogenic nitroso compounds are mutagenic (Table IV) but no attempt a t a detailed survey of the literature has been made in this review. It is clear that the nitroso carcinogens, in contrast t o some others, are unequivocally mutagenic. This is of considerable importance in connection with theories of the mechanism of carcinogenesis, which are discussed in Section X. There are several other interesting features of the mutagenic action of the nitrosamines. For example, N-nitrosomethyl- and N-nitrosoethylurea are stated t o be the most powerful known mutagens in Drosophila (Rapoport, 1962b ; Alderson, 1965) and N-methyl-AT-nitroso-N’-nitroguanidine is claimed t o be the most potent chemical mutagen for bacteria yet discovered (Adclbcrg et al., 1965). Table IV also shows the contrasting behavior between the nitrosamines and the nitrosamides in their mutagenic activity in Drosophila ancl in microorganisms. The relatively unstable nitrosamides which decompose spontaneously are active in all organisms while the more stable compounds may be inactive. On the other hand, the nitrosamines, which are thought to require enzymatic decomposition before becoming active carcinogens (see Section VII) arc mutagenic only in Drosophila and inactive in microorganisms such as Escherichia coli, Neurospora, and Saccharomyces. This may possibly be related to the presence of enzymes capable of a-oxidation of the nitrosamine in Drosophila and their absence in the microorganisms, but experimental proof of this is lacking. The fact that Geissler (1963) could obtain T 4 phages which were not
194
P. N. MAGEE AND J . M. BARNES
inactivated by exposure to 25% solutions of dimethylnitrosamine (3.4111) suggests t h a t the unchanged conipound has little biological activity (Section X ) . The mechanism of mutagenesis by the nitroso compounds has been discussed by several authors. Pasternak (1964) concludes t h a t the nitrosamiiies, after breakdown in vivo, exert their mutagenic action through alkylation by decomposition products. She suggests that a n identical niolecular mechanism may account for both the Carcinogenic and niutagenic activity of the nitroso compounds, An interesting parallel between carcinogenic and mutagenic action is shown by the behavior of te& butylethylnitrosamine, which is not carcinogenic in the rat (Druckrey et al., 1963b) and is not mutagenic in Drosophila (Pasternak, 1963). The tertiary butyl group lacks an &-carbon atom and therefore cannot be oxidized to give the diazoalkanc. Marquardt et al. (1964) also conclude that the mutation induced in Saccharomyces cerevisiue by nitrosamides is likely to be due t o methylation resulting in the formation of 7-methylguanine. Zimmermann e t al. (1965) observed t h a t N-metliylnitrosamid~s were mutagenic in Saccharomyces a t pH 2 and demonstrated that a t this pH, the compounds decompose to yield nitrous acid. They fuyther demonstrated that deamination of adenine occurred when the base was exposed t o some nitrosamides at pH 2. These results led to the ronclusion that N-methylnitrosamides a t low pH possibly excrt their mutagenic action via deamination by nitrous acid besides the alkylation. which probably prevails a t higher pH. Mere incubation of yeast cells in buffers a t low pH was not mutagenic. A protective action of cystcine against mutagenesis by N-nitroso-N-methylurethanc was demonstrated by Loprieno (1964) and compared with its protection against dimcthylnitrosamine toxicity in the rat (Miarahi and Enmelot, 1962). Loprieno suggests that a similar mechanism of action, involving trapping of the alkylating agent, may occur in both situations. Muller (1964) found no clear-cut differences between mutant spectra induced in Ambidopsis by nitrosoniethylurea, other alkylating agents, and X-rays. I n later work (A. J. Rliiller, 1965) he found t h a t respiratory inhibitors affected the radiomimetic action of nitrosomethyl and nitrosoethylurea but not that of N-nitroso-AT-methyl-N'-nitroguanidinc.He concluded that neither of the urea derivatives can owe its radiomimetic activity entirely to decomposition with the formation of an alkylating intermediate. Gichner e t al. (1963) did observe effects of metabolic inhibitors on the radiomimetic action of N-nitroso-N-methyl-N'-nitroguanidine on Vicia faba and concluded that it acted differently from nitroeomethylurethane in that decomposition to diazomethane did not appear
CARCINOGENIC NITROSO COMPOUNDS
195
to be involvcd in the radiomimetic action. They suggested a similar mode of action for nitrosomethylnitroguanidine to that proposed by Iiihlman for phenylmethylnitrosamine. The work of Kihlman (1961 a,b,c) requires some coninicnt. I n his systems with Vicia faba diphenylnitrosamine, plienyliiicthylnitro~a~~iiiie, and thc related compound cupfcrron (N-nitrosophenylliytlroxylamine) wcre actively radioniinietic but dimethyl and dictliylnitrosnmine were inactive, as was dimcthylaniline. The radiomimetic effects observer1 were structural chromosome changes in root tips of Vicia f a b a pretreated with acridine orange as a photosensitizer. The chromosome changes werc also obtained with sodium nitrite. Kihlnian (1961b) studied the radiomimetic effect of phenylmethylnitrosamine in greater detail and concluded that the active agent is not thc nitrosaniine pcr sc hut some dccoinposition product formed in the prcsence of oxygcn and a hcavy mctal-containing enzyme. H e suggested that the actire products rcsponsiblc for the radiomimetic effccts are peroxides, :tnd/or free radicals. It is intcrcsting in relation t o this that phenylnictliyl :mI diplicnyliiiti~osaminc,as well as diethylnitrosaniine, had no mutngcnic action in iVeuro.spor.n crassa although chromosome aberrations in Bellavalia romana were produced (Marquardt et al., 1963:~).Tlie mechanism of mutagenesis by nitroso compounds is discussed by Fahmy et al. (1966), who compared the mutagenic activity of dicthylnitrosainine and iV-riitrosoethylurcthanc in Drosophila. Although thcre was a hroad similarity in the niutagcnic mode of action of the two compounds, more dctailcd analysis of their results revealed differences which n'ercb difficult to explain by simple ethylation of the genetic ni:iteri:d. They suggest that either the compounds themselves or products of their metabolism, othcr than diazoethane, must be playing a role in the initiation or subsequent stnhilization of certain mutations. They point out that the nictabolic production of aldehydes and reduction products such as the corresponding hydrazines or hydroxylamincs, all of which are known to hc niutagenic in some systems, may be important and should not be excluded. Similar considerations in relation to carcinogenesis by the nitroso carcinogens are discussed in Sections VII,A, and X. From the foregoing w r y bricf summary it is apparent t h a t it cannot be assumcd that the nitroso mutagens act only by alkylation of the genetic material, or indecd that alkylation necessnrily plays any role in thc mutagenic process. Tlicre is, however, quite substantial support for this hypothesis. A t thc risk of repetition it is finally emphasized that the nitroso carcinogens arc a t least as active in mutagencsis as they are in carcinogenesis.
TABLE IV THEMUTAGEXIC ACTIVITYOF SOME NITROSO COMPOUNDS Compound N-Ni trosodimethylamine (dimethylnitrosamine), Me2N.NO
Organism Drosophila melanogaster Saccharomyces cerezrisiae Eschsrichia coli K12W 3102 (A) Serratia marcescens HY (XI Phage T4v+ indicator bacteria E . coli B
Vicia fabo
N-Nitrosodiethylamine, Et2N.NO
Drosophila melanogaster Drosophila melanogaster Neurospora crassa Saccharomyces cereirisiae
Mutagenic or related action Itecessive sex-linked mutation? of X-chromosome (hI-5 test) Back mutation in mutant ad 6 4 5 Spontaneous product of A and x phages Frequency of colourless colonies (w-mutants) in S. ntarceszns T4 phage inactivated in concentrations of dimethylnitrosamine stronger than 1 yo.Propagation of lyzates swviving two nitrosamine treatments pave lyzates resistant to 25% nitrosamine Chromosome aberrations in the light-acridine orange systcm A s for dimethylnitrosamine Sex-linked recessive IethaLQ and other effects Strain K3/17. Back mutation in adenine3 lorus As for dimethylnitrosamine
Result
L
CD
0,
References
+
Pasternak (1962, 1964!
-
Marquardt et al. (1964)
-
Geissler (1962)
-
Geiss!er (1962) Geissler (1963)
-
+ +
Kihlman (1961a,b)
Pasternak (1963, 1964) Fahmg et al. (1966)
-
Marquardt et al. (1963a)
-
Marquardt el d.(1964)
N-Ni trosomet.hylvinylamine, CH,: CH.NMeh’O N-Nitrosomethylphenylamine (N-methy 1-N-nitrosoaniline), CGHrNMe.NO
E. coli K12W 3102 (A) Serratia marcescens HY ( x ) Phage T4v+ indicator bacteria E. coli B Vicia faba Drosophila melanogasler Neurospora crassa
Vicia faba
N-Nitrosobenzylmethylamine, CtjHvCHrNMeNO N-Nitrosomorpholine CHrCH,
\
/ 0
Vicia faba Drosophda melanogaster Drosophila melanogasf er E. coli B/r
As for dimethylnitrosamine
Geissler (1962)
As for dimethylnitrosamine
Geissler (19B)
As for dimethylnitrosamine
Geissler (1963)
As for dimethylnitrosamine Recessive sex-linked mutations of X-chiomosome (M-5 test) Strain K3/17. Back mutation in adenine-3 locus
Kihlman (1961a,b) Pasternak (1964) Marquardt. et al. (1963a)
c,
Chromosome aberrations in the light-acridine orange system Chromosome aberrations As for dimethylnitrosamine
Kihlman (1961a,b,c)
2:W 5
Recessive sex-linked lethals and II/III translocations Biochemical mutations
N.NO
c, b-
0
CHrCH? N-Nitrosopiperidine CHz-CH?
\
/ CH?
\
/ CH,-CH,
N.NO
E. coli B/r
Biochemical mutations
3 8
Kihlman and Eriksson (1962) Pasternak (1964)
td
Henke et al. (1964), IGinkel (1964)
8
Trams and Kunkel (1964): Kunkel (1964)
/
\
T
Trams and I
5
0
zd
U u,
TABLE IV (Continued) Compound
Organism
Mutagenic or related action
Result
~~
References ~~
N,iV’-Dinitrosopiperazine
E . coli B/r
Biochemical mutations
Trams and Kiinkel (1965)
CH?-CH? 0N.N
/
\
\
/
a 1:
N.NO
CHr-CH, N-Methyl-hT-itroso-N’-nitroguanidine, MeN(N0)-C(:NH) . NH.KO2
E . coli, strain S
E . coli K12, strain 9 B 1621 Vicia faba Arabidopsis thaliana
Saccharomy e s ceretisiae
Induced ValR mutants (auxotrophs) Radiomimetic effects Recessive lethal mutations in the emhryo test Radiomimetic effects (unaffected by metabolic inhibitors) Back mutations in mutant ad -5
Salmonella typhimurium Drosophila melanogaster
Multisite auxotrophic mutants Lethal mutations, C.I.B. test
Arabidopsis thaliana
N-nitrosomethy lurethane, EtO.CO.NMeNO
Mutation to resistance to the compound itself. Product.ion of arixotrophs
+
Mandell and Greenberg (1960)
F
g +a B
+ + + + + + +
e Adelberg et al. (1965) Gichner el al. (1963) Muller and Gichner (1964)
4
F W &-
0
Z
A. J. Muller (1965) Marquardt el al. (1964), Schwaier et al. (1965), Schwaier (1965), Zimmermann el al. (1965) Eisenstark et al. (1965) Rapoport (1945)
4t
Drosop hila melanogaster Drosophila melanogaster E . wli B/r
Recessive sex-linked mutations of X-chromosome (11-5 test) Receqsive sex-linked lethals and I I / I I I translocations Biochemical mutations
Saccharomyces
Back mutations in mutant ad 6-45
Saccharomyces Ophiosioma multiunndatzim
Reversion of ad?-mutants Fonvard mutations in wild type strain and increased frequenry of back mutations in a methionineless strain nlet,hionine-requirinp. strain hlet-4. D19,h+. Iteversion to methionine independence Auxotrophic mutants
Schistosaccharom?jces pombe Colletotrichzim coccodes Vicia faba Amoeba
Itadiomimetic effect ; chromosome aberrations in lightacridine orange system Delayed lethality, polyploidy, change in size distribution Some effects carried through many generations.
4-
+ + + + +
Pasternak (1964)
Henke et a?. (1964) Iiunkel (1964) Tram? and Kiinkel (19G4), Iiunkel (1964) Marquardt el 01. (19631)), Zimmermann ct a/. (1963), Schwaier (1965) Zimmermnnn et al. (196.5) Zimmermann and Schwaier (1063) Zetterberg (1960, 1061)
+
Loprieno (1964)
+
Loprieno el al. (1964)
+ +
Iiihlman (19G0, 1961a), Iiihlman and Eriksson (196%) Ord (1965)
h3
8 TABLE IV (Continued) Compound N-Ni trow-N-methylurea, H,N.CO.NhleKO
Organism Drosophila melanogaster Drosophila melanogaster Drosophila melanogaster Saccharomyces cerevisiae
Arabidopsis Wheat seeds Vicia faba A'-h-itroso-N-ethylurethane. EtO.CO.NEt.NO
Drosophila melanogaster Saccharomyces cerevisiae Saccharomyces cerevisiae Collectotrichum corodes
Mutagenic or related action Lethal, sublethal, and visible mutations Recessive sex-linked mutations of X-chromosome (M-5 test) Sex-linked, recessive, lethal mutation Back mutation in mutant ad 6-45 Lethal mutations: radiomimetic effects Chromosome aherrations in meristem. Delayed death effect Radiomimetic effect, chromosome aberrations Sex-linked recessive lethals and other effects Back mutation in mutant ad -5 Reversion of ads-mutants Auxotrophic mutants
Result
+ + + +
References Rapoport (1962a,b) Paxternak (1963, 1964)
Alderson (1965)
+
Marquardt e t a / . (1963b, 1964), Schwaier (1965), Schwaier et al. (1965), Zimmermann et al. (196.5) A. J. Mdler (1964, 1965)
+
Zoz and Makarova (1965)
Kihlman (1960) Fahmy el al. (1966) Marquardt et nl. (1963b), Zimmermnnn et al. (1963) Zimmermann and Schwaier (1963) Loprierlo ct al. (1964)
Cycasin, MeNO :iV.CH2.O-glycosyl Diazomethane, CH2:IV2
Alliiim crpa
Chromosome aberrations
Drosophda melanagaster Il’eurospora crassa Saccharomy e s
Lethal mutations, C.I.B. test Back mutation Back mutation in mutant ad G 4 5 Lethal mutations, C.I.B. test Back mutation in mutant ad 6-45 Radiomimetic effect, chromosome aberrations
ceretrisine
Ethyl diazoacetate, Nz: CH.CO.OEt
Drosophda melanagaster Saccharomvces cerevisiae Vicia faba
+ + + + +
Teas et al. (1965) Rapoport (1948) Jensen et al. (1949) Marquardt et al. (1964) Rapoport (1948) d
-
Marquardt et al. (1964)
-
Kihlman (1960)
+ a
z
3
M
50
202
P . N . MAGEE AND J. M . BARiXES
VII. Metabolism of Nitroso Carcinogens
A, METABOLISM in Vivo The metabolism, distribution in the body, and excretion of dimethylnitrosamine were studied in rats, mice, and rabbits by h/lagee (1956) using the polarographic niethod of Heath and Jarvis (1955) for the determination of the compound in tissues and body fluids. Recovery of injected diiiicthylnitrosaiiiiiie from the whole animal fell rapidly with incrcusing time after injection such that only 30% of tlie dose (50 mg./kg. body wt.) was still recoverable a t 6 hours and none a t a11 a t 24 hours. Disappearance of the compound from the whole bodies of m k e was cvcn faster. Total urinary and fccal excretion of unchangcd dirncthylnitrosaniine was very low in rats under similar conditions; even when the injectcd dose of dimcthylnitrosamine was raised to 500 riig. per kg. body wt. intravenously only about 5-10% was recovered unchanged from the urine. The fall in total body coiitcnt of diniethylnitrosainine could riot be explained by inacccss:ibility of the coinpound t o the extraction procedures used, and it was concluded t h a t dimethylnitrosamine must be rapidly metabolized with the metabolic process starting almost immediately after administration. Expcrimcnts with totally hepatectomizcd rats and in animals with damaged livers suggested that tlic liver was the main organ concerned in tlie metabolism of diiriethylnitrosaniinc, although lower ratcs of nictabolism in other organs could not be excluded. The distribution of diiiietliylnitrosamine in different organs ant1 tissues was studied a t intervals after injcction. The concentrations in most orgaris wcre remarkably similar with no obvious accumulation a t any one site. Studies on the rate of decline in concentration of dimcthylnitrosamine in tlic circulating blood of rabbits led to tlie conclusion that it is evenly distributed throughout the hotly water. The selective damaging action of dimethylnitrosamine on the liver could not be explained by preferential concentration of tlic compound therein and it sccmed probable that a mctabolite of dirnetliylnitrosat1iine, formed inside the liver cell, might be the true toxic substance. Metabolism of dimethylnitrosamine in the rat and the mouse was unequivocally demonstrated by Dutton and Heath ( I 956h) using dinictliylnitros:tn~ine-C14.From both species the main radioactive product was expired carbon dioxide. In tlic mouse, 6*5% of tlw injectcd CI4 was recovered in the expired carbon dioxide 6 hours after st subcutaneous injection of 50 mg. dimethylnitrosamine per kg. body wt. I n the rat, metabolism was slower, but about 40% of the radioactivity was recovered 8 hours after the injection. The remainder of the C" was fairly
CARCINOGENIC NITROSO COMPOUNDS
203
evenly distributed in the tissues a t the end of the experiment apart from about 770 excreted in the urine. The authors concluded that diuicthylnitrosamine is rapidly dcmethylatcd in vivo and that tlie biochemical lesion is probably produced by a metabolite and not by dimetliylnitrosaiiiine itself. The same workers (Heath and Dutton, 1958) extended their original work with dinietliylnitrosamine-C'* and included further obseivations using dimethylnitrosamine labeled with N"O and N"C. Following thc suggestion that the toxic action of diinethylnitrosamirle iiiay be exerted by its metabolites, they undertook a search for tlicse metabolites in rats anti to a lesser extent in mice. Attention wah directed to the liver, since this is tlie organ most severely and acutely affected, and to the urine which might contain a high proportion of metabolites. Chemical tests for suspected metabolites in liver homogenates, after precipitation of protein, and in urine were carried out. Having previously shown that dimethylnitrosamine must be dcmcthylated in vizlo (Dutton and Heath, 1956b), they assumed that this process might have been preceded or succeeded by reduction of the nitroso group to a hydroxylaniino or amino group. Since the deinetliylation product, iiionoriietliylnitrosaiiiine, is unstable, they tested for mcthylaniine, hytlrazinc, nitrite, and hydroxylamine and, since most of thew groups can conjugate with acids in bioc1ieniic:tl systems, they included further tests for mctliylaniidcs, liydroxamic acids, oxirnes, and hydrazides. I n most cases the available analytical methods proved inadequate and considerable modifications were therefore devised. Full details of these analytical procedures are given in tlie original paper. The results with diinetliylnitrosamine-C" confirmed those obtained previously and led to the conclusion t h a t the compound is demcthylated to one-carbon intermediates which are either further oxidized to carbon dioxide or used in the normal metabolic processes of the body. This early realization t h a t much of the radioactivity incorporated into cellular components after the injection of labeled nitrosamines may represent normal metabolic turnover rather than specific interaction between a toxic molccule and cell receptor has been of great irilportance in the interpretation of the rcsults of subsequent work (Section I X ) . Traces of methylanline i n the liver and some labeled niethylamine in the urine were found a few hours after treatment. A trace of hydroxylamine W H S found in the urine in one cxpcriincnt and traces of nitrite were detected in urine from control arid treated animals. Experiments wit11 diriietliyInitrosamine-N\;i' showed that much of the amino nitrogen was converted t o ammonia since the ammonia derived from urinary urea by the action of urease was relatively heavily labeled. It is interesting t h a t the distribution of N15 after injection of dimethyl-
204
P. N . MAGEE AND J . M. BARNES
nitrosamine-W50 was very similar to that with the amino N-labeled compound, indicating that some of the nitroso groups were also reduced to ammonia. The labeling was quite unspecific and consistent with the assumption that both the NC and the NO nitrogen atoms become distributed evenly throughout the nitrogenous constituents of the body. The only conclusion drawn by the authors about the biochemical lesion is that it cannot involve the attachment of more than minute quantities of any metabolite of dimethylnitrosamine t o liver constituents since the methods used would have revealed the attachment of metabolites equivalent to less than 1% of the injected dose. I n the light of later work on alkylation of cell components by nitrosamines in vivo (Section IX) the finding by Heath and Dutton of extensive incorporation of both nitrogen atoms by biosynthetic reactions becomes important. Since the postulated mechanisms of alkylation involve the formation of diazoalkancs with the subsequent release of free nitrogen, this metabolic pathway would not be expected to give rise to incorporation of nitrogen and it cannot, therefore, be the only one. T h e suggestion on theoretical grounds t h a t dimethylnitrosamine may owe some or all of its biological activity to metabolic conversion t o the powerful alkylating agent diazomethane was put forward by Regina Schoental (personal communication) and independently by Rose (1958). This idea has led to much subsequent experimental work which has, in general, supported it, although there are observations which are difficult to reconcile with this mechanism (Arcos and Amos, 1962) (see Section X). A large series of experiments on rates of metabolism of dialkylnitrosamines in the intact rat have been carried out by Heath (1961, 1962). These experiments were designed to test the hypothesis that the acute hepatotoxic action of dialkylnitrosamines is attributable t o toxic metabolites and t o elucidate, if possible, which of the possible metabolic products is responsible. I n the first communication (Heath, 1961) the hypothesis that diazoalkanes may be produced metabolically was tested by comparing the actions of the isomeric nitrosamines nbutylmethylnitrosamine and tert-butylmethylnitrosamine on rat liver in vivo. On the alkylation hypothesis n-butylmethylnitrosamine should cause typical centrilobular necrosis of the liver since a-oxidation can give either diazomcthanc or diazobutane, according to which alkyl group is attacked. The tert-butylmethylnitrosamine, on the other hand, should not induce typical liver necrosis because the tert-butyl group cannot be attacked in the position and oxidation of the methyl group leaves a cornpound with no a-hydrogen atom which cannot thereforc yield a dinzoalkane. Heath observed that the n-butyl compound did cause acute centrilobular necrosis of the liver in rats given doses of 100-120 mg. per (Y
205
CARCIKOGENIC NITROSO COMPOUNDS
kg. body wt. With the tert-butyl compound, however, even survivors of iicw lethal rcpe:tted doses (totttls of 1400-1600 rng./kg. body wt. over 4 days (did not show ccntrilobu1:tr nccrosi+. Thcw r('sults are in accord with the diazoalkaiie tlicory but, :is 1Ic:itli pointed out, thc possibility that even higher do,ws of the te,d-butyl compound iniglit induce typical livcr lesions could not be excludc(l. In tlie second and longer paper Heath (1962) studied thc mct:ibolisni of diiiiethyl-, diethyl-, n-butyliiicthyl-, and te,.t-butyliiictliylnitl.osnmines iii fciiialc rats using C"-labeled arid uiilabelcd compounds. His ni:iin results can bc suri-~niarizedas follows: All the nitrosnmincs wcrc tlecori-~posedin the rats. Labclctl carhon dioxide wzts expired by rats given nitros:imines labclcd in methyl, ethyl and n-butyl groups but with tert-butylmctliyl-l-C1' and the tert-butyl2,2-C," compound oiily 1.1 and 1.6%, rcspcctively, of the C" injected werc recovcrccl in the expired carbon dioxide, indicating that the tertiary butyl group is only very slightly oxidized iri v ~ v o .Relationships were established between rates of expiration of labeled carbon dioxide and rattes of decomposition of the nitrosamines as determined from polarographic nieasureiiieiits in thc circulating blood. These cnablcd the latter to be calculated from tlie former uftcr allowing for the amount of nitrosniiiine cxcretcd unchaiigcd in the expired air and urine. The diffcrciit nitrosaniincs inhibited each other's clccomposition suggesting that they were partly decomposed a t the smie sites. Dimcthylformamidc u"is an cff ective inhibitor of dimethylnitrosamine oxidation and diethylformaniidc of dietliylnitrosai~iineoxidation but inhibitors had little effect on thc toxic action of the nitrosaniines. The previous finding that tertbutylniethylnitrosaniine was without necrotic action on the liver even a t relatively high doses, wits ~onfi1mc(3.Hcatli (1962) discussed his results iii relation t o the inec1i:iiiisin of hcpatotoxic action of nitroszmiiiies and arrived a t the followiiig conclusion: The dialkylnitrosainincs thcnisclves cannot induce the typical livcr necrosis bccnusc, if this were true, any treatment which increased the pcrsistence of the nitrosaniine in vivo should incrcasc its toxicity. %his is not the case, and tlircct action by the nitrosainines tlieinsclves could only be postdated if, in evcry case, the inhibitors of nitrosnniinc iiictabolism pr0tectc.d the rats just sufficiently to compcns:itc for the incrcasc in 13ersistcncc that they brought about which is very unlikely. The production of R toxlc nictabolitc as :Lproduct of the ninin oxitlation reaction, however, is consistent with :ill the results, in particular thc inactivity of tert-butylmethylnitrosaniine. Aldchydcs produced by oxidation of the alkyl groups are unlikely to be involved in the meclianisrii of injury to the liver cells since much more fornialdehydc would be released from a nonhepatotoxic dose of tert-butylmetliylnitrosnmiiic than froin n toxic dose of dimcthylI
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nitrosamiiie. Also methanol, which is probably oxidized in the liver to formaldehyde, does not induce lesions similar to dinietliylnitrosamine. Oxidation of only 3% of a toxic dose of methanol in the liver would yield more formaldehyde than a lethal dose of climetliylnitl.osanline (see Scctioii X) . The possibility that the monoalkylnitrosaniinc itself might be the toxic agent was also discussed but this molecule is very unstable indeed (Muller e t al., 1960) and its life a t 37°C. is presumably a fraction of a second. Heath, therefore, discussed some of its possible decomposition products emphasizing that this is a spontaneous chemical breakdown without the necessity of enzyme catalysis. H e postulated that nitrites may be released by hydrolysis of the nionoalkylnitrosamines but these are unlikely to be the toxic intermediates because this would not explain the facts that dimethylnitrosamine produces detectable liver necrosis a t 15 mg. per kg. body wt., but tert-butylmethylnitrosamine is not necrogenic even a t 1000 mg. per kg.body wt. Heath concludes finally that the acute hepatotoxic action of simple dialkylnitrosamiries can be explained if the compounds are converted in vivo into monoalkyliiitrosaniiiies and these, or cnrbonium ions or diazoalkanes formed from them, react with some vital component of the liver, i.e., the biochemical lesion is an alkylation. The reader is referred to the original paper for details of the experimental findings and of the discussion. It should be noted that Heath drew no conclusions on the possible relation of alkylation to carcinogenesis. This will be discussed in later sections of this review (Sections IX and X). The effect of aminoacetonitrile on the metabolism of dimethylnitrosamine has been recently studied by Fiume and Roffia (1965). Following the earlier observation that this lathyrogenic compound can prevent the necrosis arid the inhibition of amino acid incorporation into liver protein by dimethylnitrosaniine in v i m (Fiume, 1964), they suggested that aminoacetonitrile might prevent these biochemical and pathological lesions by inhibiting the enzymatic demethylation of dimethylnitrosamine and thus hindering the formation of the alkylation intermediate. This hypothesis was tested by comparing the concentrations of dimethylnitrosamine in rats treated with protective doses of aminoncetonitrile with those in untreated control animals. At 15 and a t 90 minutes after the intraperitoneal injection of 50 mg. per kg. body wt. the concentrations of dirnethylnitrosamine were significantly higher in the livers of the rats treated with aminoacetonitrile. These findings should be compared with the conclusion (Heath, 1962) that persistence of the nitrosamine in vivo did not increase its toxicity. The metabolism of di-n-butylnitrosamine was studied by Druckrey et al. ( 1 9 6 4 ~ )As . mentioned in Section IV, this compound aiid N-butyl-
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N-~utnnoliiiti,os:iiiiineare the only nitroso carcinogens which i ~ r cknown to induce bladdei- tumors ill the rat. After injection of 1000 ing. dibu tylnitrosamine per kg. body wt. into rats no unchanged coiiipouiitl was found in tlie urine by thin-layer chroiiintography. However, sevci-a1 water-soluble inrtabolitcs with nitrosainine structure were fouiid and their concentration in the urine was 10 to 100 times higher than in the blood or serum. The authors suggest that the dibutylnitrosamine first undergoes terminal hydroxylstion of a n alkyl group which is followed by conjugation and excretion with concentration in the urine. Hydroxylation of the a-carbon atom with consequent production of a n alkylating intcrniccliatc is suggrstcd to occur in the bladder niucosa.
R. METABOLISM in 1’ztr.o Metabolism of dimcthylnitrosamine in vitro was first reported by Magee and Vandekar (1958) using disappearance of the compound as determined by the polarographic method (Heath and Jarvis, 1955) as a measure of its decomposition by tissue slices and homogenates. Incubation with liver slices caused disappearance of dimcthylnitrosaminc from the medium in the presence of oxygen and the rate of decomposition was much reduced when the gas phase was air. Incubation in an atmosphere of N, gave no detectable decomposition nor did incubation with boiled slices in the presence of 0,. About 450 mg. wet wt. of liver slices brought about the aerobic metabolism of 30 pg. of added dimethylnitrosamine but there was no detectable nietabolism by this method in the presence of kidney or brain slices. More recent work (hiagee, unpublished) with dimethylnitrosamine-C14 has shown that CO,-CI4 is liberated on incubation with rat kidney slices, although much less than with liver slices. The significance of these results will be discussed later in relation to distribution of lesions induced by dimethylnitrosamine and to alkylation of cellular components by this compound (Section I X ) . Metabolism also occurred with rat liver homogcnates but this was rather capricious and slow. Rabbit liver homogenates proved to be more effective. Metabolism was again absent when incubation was anaerobic. Homogenates of other organs tested included kidney, spleen, heart, and lung, all of which were inactive with the possible exception of kidney. Fractionation of the liver homogenate into a nuclear plus mitochondria1 and a microsomal plus cell-sap fraction showed that the metabolic activity was cntirely in the microsomes plus cell sap. Neither microsomes nor cell sap was appreciably active alone but activity was restored on recombination. Activity was removed from these preparations by dialysis and it could be partially restored by the addition of riicotinamide
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adenine dinuclcotide phosphate (NADP) or nicotiiiamide adenine dinucleotide (NAD), the former being the inore effective. These findings were confirmed and extended by Brouwers and Emmelot (1960), who showed that formaldehyde was produced from dimethylnitrosamine incubated with slices and microsome plus cell-sap preparations from normal rat liver but not from hcpatorna slices. Formaldehyde production was used as a measure of the amount of decomposition of the dimethylnitrosamine and hence of the activity of the deinethylating enzyme. Since nietabolism of diiiiethylnitrosamine occurs in the postmitochondrial fraction of the liver cell cytoplasm it seenicd reasonable to suggest that some active metabolite formed locally might be responsible for the inhibitory action of diinetliylnitros,znine on hepatic protein synthesis (Section VIII) . On the assumption that an alkylating intermediate is formed, Emmclot and Mizrahi (1961) pretreated rats with cystcine before they were given dimethylnitrosaminc. This greatly reduced the inhibitory effect on hepatic protein synthesis and also gave some protection against the toxic action. No protective action against diethylnitrosaniine was observed, however. Tliese experiments are discussed in Section V I I I and are mentioned here only because they led to the observation that cysteirie treatment reduced the activity of the enzyme system which clemethylatcs dirnethylnitrosamine but had no effect on the de-ethylation of dicthylnitrosamine (Mizrahi and Emmelot, 1962). The same authors (Mizrahi and Emmelot, 1963) made a inore detailed study of the in vivo and in vitro effects of various sulfhydryl compounds on the metabolic conversion of dimetliyl- and dicthylnitrosamine. Using the cotnbinctl niicrosome-soluble fraction from 600 ing. of rat liver fortified with cofactors as before, they observed that neither the soluble fraction nor the microsonies were active when incubated singly, thus confirming the earlier observation of Magee and Vandekar (1958). The soluble fraction could be replaced by the addition of crystalline glucose-6-phosphate dehydrogenase to the microsonies for the generation of reduced N A D P (NADPH). The inhibition of the enzyine-demethylating diniethylnitrosamine by subcutaneous injection of cysteine and the lack of inhibition of de-ethylation of diethylnitrosainine were confirmed. Addition of cystcine and cystearnine, but not of enzymes, in contrast to the in vivo effects of cysteine observed prevition caused inhibition of both the demethylating and the cle-ethylating enzymes, in contrast to the in vivo effects of cysteine observed previously. These results are also in contrast to the effects of cysteamine in vivo which prevented the inhibition of amino acid incorporation into liver proteins by dimethyl- and diethylnitrosaniine (Section V I I I ) but did not affect the dealkylating enzymes for either nitrosamine. The
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various interlocking effects of sulfhydryl compounds and nitrosamines in viuo and in vitro arc clearly rather complex and the reader is referred to the original papers of hlizrahi and Emmclot for detailed discussion of their possible significance in cellular injury and carcinogenesis. Their final conclusion is thnt a mutual elimination of sulfhydryl compounds and nitrosamine metabolites may occur in the livers of rats receiving dialkylnitrosamines and cysteamine or cysteine, thus explaining the protective action of the sulfhydryl compounds. VIII. Biochemical Effects
A. PROTEIN SYNTHESIS The inhibitory effect of dimethylnitrosamine on the incorporation of amino acids into proteins of rat liver zn vivo was reported by Magec (1958). One of tlie earliest changes observed in the hepatic parenchymal cell with the light microscope in rats treated with dimethylnitrosamine is an alteration of the morphological character of tlie cytoplasmic basophilc material. This observation has been abundantly confirmed in later work with the electron microscope (Section 111),and suggested that an early biochemical lesion in the induction of liver necrosis by dimethylnitrosamine might involve the mechanism of protein synthesis. Incorporation of Cl4-aniino acids into liver proteins was reduced by about 50% by 3 hours after R necrotizing dose of dinietliylnitrosamine, thc extent of the reduction being about the same in the different subcellular fractions of tlie liver. Incorporation of amino acids into kidney and spleen proteins was unimpaired. The concentration of free amino acids in the liver was not significantly altered 3 hours after dimethylnitrosamine administration, nor was the incorporation of amino acids into the free amino acid pool, indicating that the reduction in incorporation into liver proteins was not due to failure of the labeled amino acids to reach the liver. Incorporation of P32into partially purified RNA was also inhibited during this period (Aiagec, 1958). The possible significance of tlie involvement of the same subcellular fraction, i.e., niicrosomes plus cell sap in the metabolism of dimethylnitrosamine and in amino acid incorporation, was discussed and the suggestion made that the inhibition of protein synthesis might be explained by clamage to microsome structures by a toxic mctabolic product of diiiicthylnitrosaniine produced in high local concentration closc to the site of most active amino acid incorporation. The action of dimcthylnitrosamine on protein synthesis was analyzed further by Hultin e t al. (19601, using in vitro preparations. Incorporation of valine-C14 into proteins of rat liver slices in vitro was inhibited by preincuh:ition of the sliccx with diiiicthyliiitrosamine a t
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concentrations of about 0.1 m M but no inhibition was observed with kidney slices. The smallest dose of dimethylnitrosamine necessary to produce definite necrosis of the liver in the rats used was about 20 mg./kg. body wt. Concentrations of dimethylnitrosamine in the livers of rats given this dosc reached levels of the order of 0.1 mM, calculated in terms of liver water, which suggested that the observations with slices in vitro probably reflected the same events in vivo. Preincubation of the slices with dimcthylnitrosamine was necessary to produce the inhibition of amino acid incorporation, which supports the hypothesis of a toxic inetabolite since diniethylnitrosaminc is known to be metabolized under these conditions (Section VII) and the compound was shown to penetrate the slices very rapidly. Incorporation of adenine-C14 into RNA of rat liver slices in vitro was also inhibited on preincubation with dimethylnitrosamine. The incorporation was measured in vitro of 1 e ~ c i n e - Cinto ~~ proteins of niicrosornc plus cell-sap preparations from livers of rats killed a t 2 and 3 hours after they had received dimethylnitrosamine (50 mg./ kg. body wt.) and compared with that in similar preparations from untreated rats. Incorporation was considerably less in the preparations from the treated animals and the decrease was also present when microsomes from treated rats were combined with cell sap from control animals. Amino acid activation as measured by the amino acid-dependent pyrophosphate-adenosine triphosphate (ATP) exchange was unimpaired in the treated animals as wcre oxidative phosphorylation and the induction of adenosinetriphosphatase (ATPase). The addition of dimethylnitrosamine to isolated mitochondria respiring in vitro in concentrations up to 10 mM had no effect on the oxidation of several substrates. I n agreement with this, Nickel and Diener (1965) found unimpaired oxidative phosphorylation by rat liver mitochondria in the presence of added diethylnitrosamine (0.13M ) and Bailie and Christie (1959) found no effect of added dimcthylnitrosamine (0.15 M ) on oxidation of several substrates by rat liver homogenates in vitro (Section VII1,B). Thcse results were thought to support the idea of a toxic metabolite as the agent responsible for cellular injury by diiiicthylnitrosamine and an alkylating intermediate was favored by Hultin et al. (1960). The findings of Brouwers and Emmelot (1960), which are also discussed in relation to metabolism of dimcthylnitrosamine (Section VII) , are csscntially similar to tliosc of Hultin et al. (1960). They also observed niarkcd impairmcnt of thc amino acid incorporation systenl of the combined microsomal-soluble fraction of livers from rats treated with dimethylnitrosamine and thcy further found no significant impairment of the reaction betwccn leucine-C" and soluble RNA (sRNA) in the presence of the pH-5 enzyme system of the supernatant fraction.
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This indicated that the impairment of protein synthesis involved transfer and incorporation of the amino acid from the sRNA to the microsoma1 protein rather than a t an earlier stage. Inhibition of amino acid incorporation into liver slices preincubated with dimethylnitrosamine was also reported but no such inhibition was found with slices of primary and transplanted r a t hepatomas. Since these tumors were incapable of metabolic conversions of dimethylnitrosamine (Section VII) this observation gave further indication of the necessity of inetaholism of dimethylnitrosamine before it becomes inhibitory. I n agreement with Magee (1958), Bailie and Christie (1959), and Hultin et al. (1960), Brouwers and Emmelot concluded t h a t the inhibitory effect of dimethylnitrosamine on hepatic protein synthesis is rather specific sincc respiration, glycolysis and a number of other enzymatic activities were not impaired under their experimental conditions. Emmelot and Mizrahi (1961) reported t h a t subcutaneous injection of cysteine prolonged the lives of rats treated with dimethylnitrosamine and reduced the inhibition of protein synthesis and the loss of glycogen from the liver (see Section VIII,B). They concluded that this protective effect of cysteine might indicate t h a t the methylating agent diazomethane is thc actual toxic derivative of dimethylnitrosamine, probably reacting directly with the sulfhydryl groups. Alternatively there might be transitory blockage of tissue sulfhydryl groups which thus become unable t o react with the derivative of dimethylnitrosaniine. The same group (Emmelot et al., 1962 ; Mizrahi and Emmelot, 1962, 1963) showed that cysteine docs not protect against the toxicity or the inhibition of amino acid incorporation due to diethylnitrosamine, and rclated this to the inhibitory action of cysteine on the metabolic dealkylation of dimethylnitrosamine but not of diethylnitrosamine (Section VI1,B). Cysteamine exerted a protective effect against inhibition of protein synthesis by both dimethyl- and diethylnitrosamine, but this compound had no effect on the enzymes responsible for the metabolism of either compound. The general conclusions drawn by Emmelot and his colleagues from this work are that both the toxic and the carcinogcnic effects of the N-nitrosodialkylaniines are due to the in situ formation of diazoalkanes which are alkylating agents and appear to react primarily with the endoplasmic reticulum of the liver cells. The mechanism of inhibition of incorporation of amino acids has been further analyzed by Mizrahi and Einmelot (1964), who investigated the possibility t h a t there could be loss of messenger RNA (mRNA) from the polyribosomes in the livers of dimethylnitrosamine-treated rats. This would be consistent with the appearance in thc clectroii microscope of detached ribosomes lying in the cytoplasmic matrix and lack-
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ing the aggregate structure characteristic of polysomes (Section 111). The response of the postmitochondrial fraction (12,000 g supernatant) and of ribosomes from control and dimethylnitrosamine-treated rats t o a synthetic messenger, polyuridylic acid (poly U) , was therefore tested. The animals were given the large dose of 100 mg. dimethylnitrosamine per kg. body wt. intravenously and were killed after 2 and 5 hours. The incorporation of phenylalanine-CIJ was stimulated to a greater extent in both types of preparation from the treated rats than from the controls, suggesting that available sites for exogenous messenger were saturated a t lower concentrations of poly U in the control than in the treated preparations. The incorporation pattern of the ribosomes from the dimethylnitrosamine treated rats resembled that of normal ribosomes after preincubation or treatment with ribonuclease, conditions known to convert polyribosomes to smaller aggregates and single ribosomes through the loss of mcssenger RNA. Further evidence that mRNA is lost from the treated preparation was obtained by sucrose gradient centrifuga t’ion of the ribosomal components. The number and size of the ribosomal aggregates were decrcased and there was a corresponding increase in the number of smaller aggregates and ribosomal monomers (80 S) in the diinethylnitrosamine liver as compared with the control. I n later work Mizrahi and de Vries (1965) observed t h a t a further breakdown of polyribosomes from livers of rats treated 5 hours previously with dimethylnitrosamine occurred during incubation in experiments on amino acid incorporation into protein. No significant inhibition of incorporation of P3?into nuclear or cytoplasmic RNA was found in the livers of the treated rats. Villa-Trevino (1965) observed progressive breakdown of the ribosomal aggregates which was detectable 1 hour after administration of dimethylnitrosamine ; the extent of breakdown was proportional to the degree of inhibition of protein synthesis. This breakdown of microsomal aggregates was not accompanied or preceded by inhibition of incorporation of orotate into nuclear RNA; thus, 2 hours after administration of dimethylnitrosamine, when in vivo incorporation of leucine was decreased by 48%, no significant difference was observed in orotate incorporation. The greater stimulation by poly U of incorporation of phenylalanine-C14 by ribosomes from dimethylnitrosaminetreated livers was confirmed by Magcr e t aE. (1965a), who suggested that this reflects a n increased availability of the ribosomal surface for interaction with exogenously supplied coding agent (i.e., poly U) . They presumed that destruction of messenger RNA bound to ribosomes and consequent unmasking of normally screcned combining sites can account both for the exccssive affinity of the system for poly U and the concomitant decline of its intrinsic amino acid incorporation activity.
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These findings (Mizrahi and Emmelot, 1964; Mizrahi and de Vries, 1965; Villa-Trevino, 1965; Mager et al., 1965a) led to the suggestion that there may be accelerated breakdown of messenger RNA in the livcrs of treated animals. This might be a result of alkylation of mcssengcr RNA, a possibility that receives support from the demonstration by Villa-Trevino (1965) that purified liver nuclear RNA is metliylated in the dimethylnitrosaminc-treated rat. Recently aminoacetonitrilc has been reported t o prevent the inhibition of protein synthesis induced by tlimetliylnitrosamine (Fiume, 1964). This lathyrogenic agcnt partially protects tlie liver against tlie necrosis induced by the carcinogen (Fiumc, 1962). Injection of aminoacetonitrile daily for 2 days before and on tlic same day as dimethylnitrosamine (about 100 mg./kg. body wt.) abolished the inhibitory effect on protein synthesis. This antedotal effect of aminoacetonitrile was further studied by Mager et al. (1965b), who found that thc protective action could be obtained when thc aminoacetonitrile was injected 12-20 hours before the dimethylnitrosamine but not when injected 2 hours before or simultaneously with the nitrosaniinc. Addition of aminoacetonitrile t o the in vitro preparations did not reverse the inhibition. The behavior of the in vitro system was detcrmiiied by the origin of the microsomes and ’was independent of the source of the supernatant fraction, indicating t h a t the site of both tlic intracellular injury arid the protective effect are in the microsomal particles thus confirming tlie obscrvations of Hultin e t nl. (1960). Pretreatnient with aiiiinoacctonitrilc also reduced the rcsponsc of the microsomcs from the nitrosamine liver to tlic addition of poly U (see above). The authors interprct their results to imply a “stabilizing” effect of aminoacetonitrile on the ribosomal particle. More recently, however, Fiume and Roffia (1965) h a w reported that aminoacctonitrile inhibits metabolism of dimcthylnitrosaminc (see Section VII) . With lower doses of dimcthylnitrosamine hepatic protein synthesis is stimulated rather than depressed. Mukherjee et al. (1963), in a combined electron microscopic (Section 111) and biochemical study showed that incorporation of amino acids into liver proteins of rats was increased a t 30 hours after 20 nig. per kg. body wt. This stimulation did not occur in adrenalectomized rats (Section VII1,B).
B. OTHER BIOCHEMICAL EFFECTS 1 . Acute Effects
These include biochemical changes that have been reported to precede or accompany the acute liver necrosis induced by adequately large doses of the nitrosamine. Most of this work has been done with di-
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niethylnitrosamine in doses ranging from 20 t o 100 mg. per kg. body wt. and it has been discussed elsewhere (Stoner and Magee, 1957; Heath and Magee, 1962; Magee, 1964b, 1966). There is no basis for linking any of the changes to be described with the carcinogenic action of the nitrosamines. Six hours after a necrotizing dose of dimethylnitrosamine to rats, Magee (1958) observed a reduction in the level of liver RNA but not of DNA or total phospholipid phosphorus. As the liver lesion developed there was an increase in stainable and chemically dctermined lipid and marked loss of glycogen. Three hours after the necrotizing dose, however, the level of liver glycogen was only slightly reduced and the diffcrence from the control levels was not statistically significant (Hultin e t al., 1960). Emmelot and Benedetti (1960, 1961) reported progressive loss of glycogen from livers of rats treated with dimethylnitrosamine. However they found that the glycogen content appeared to be normal 3 hours after 50 mg. per kg. dimethylnitrosamine when incorporation of amino acids into hepatic microsomal proteins was greatly impaircd (Section VIII,A) and when the typical early electron microscopic changes in the endoplasmic reticulum were already present (Section 111).The loss of liver glycogen could be very largely prevented by prior treatment of the animals with cysteine (Emmelot and Mizrahi, 1961). On the other hand, cysteine gave no protection against the glycogenolysis induced by diethylnitrosamine (Mizrahi and Emmelot, 1962), but cysteamine treatment greatly reduced the loss of liver glycogen induced by both nitrosamines. The sulfhydryl compounds alone were found to reduce liver glycogen ; this effect was counteracted by the nitrosamines, suggesting a mutual elimination of sulfhydryl compounds and nitrosamine metabolites (Mizrahi and Emmelot, 1963) (see Section VII1,A). After lower doses of dimethylnitrosamine (20 mg./kg. body wt.) and a longer interval of time, 20 hours, the level of liver glycogen is higher than that of fasted control rats and there is a corresponding increase in the spongy endoplasmic reticulum and its contained glycogen (Gustafsson and Afzelius, 1963) as well as increased protein synthesis (Mukherjee et nl., 1963; scc Section VII1,A) ; both these actions of the nitrosamine could be prevented by adrenalectomy. The authors postulate that these are secondary effects on the liver cell which are induced by potcntiation of the action of glucocorticoids. Changes occur in the livers of rats treated with cycasin which are similar to those with dimethylnitrosamine. Loss of cytoplasmic basophilia occurs early with feeding either cycad meal or cycasin and the light microscopic changes correlate with the apparent loss of ribosomes observed in the electron microscope. Chemical analysis revealed loss
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of RNA and phospholipid from the livers with increase in neutral glycerides and cholesterol but no change in the level of DNA. Tliese observations were carried out over a period of feeding for 5 days a t a 0.2% level of cycasin in the diet (Williams and Laqueur, 1965). The mechanism of production of fatty liver by dimethylnitrosamine in the rat was investigated by Rees and Shotlander (1963). They found no significant change in total lipid, triglyceride, cholesterol, or phospholipid in the livers of rats 5 hours after dimcthylnitrosamine (100 mg./kg. body wt.) but a t 22 hours the total lipid was about twice the level of the controls. The increase was due t o a &fold rise in triglyceride without significant changc in the lcvcls of cholesterol or phospholipid. The authors conclude that inhibition of protein synthesis by dimethylnitrosamine precedes inhibition of secretion of triglyceride from the liver by several hours but that the accumulation of fat is not solely due to reduction in lipoprotein synthesis (cf. Recknagcl and Lombardi, 1961; Robinson and Seakins, 1961). The acute effects of dimethylnitrosamine on NAD-linked and other mitochondrial enzymes have been studied by several groups. Bailic arid Christie (1959) studied the hcpatotoxic action of dimethylnitrosaiiiine in homogenate and mitochondrial preparations from rats treated with dimethylnitrosamine a t the high dose of 100 mg. per kg. body wt., which induces zonal necrosis involving nearly all the parenchymal cells. Aerobic oxidation of pyruvate, octanoate, L-malate, citrate, L-glutamate, a-oxoglutarate, and P-hydroxybutyrate progressively decreased after the twelfth hour following injection but were unchanged up to that time. Succinoxidase activity and anaerobic glycolysis, however, were unaffected even when necrosis was extensive. Oxygen uptake of the NAD-linked enzymes could be greatly increased by the addition of the coenzyme to the mitochondrial incubation medium but no other cofactors were effective. Dimethylnitrosamine added u p t o 7 hours bcfore incubation a t a final concentration of 0.15M to normal r a t liver homogenate had no effect on oxidation of several substrates (see Section VII1,A). Addition of ethylenediaminetetraacetic acid (EDTA) to mitochondrial preparations did not reverse the inhibition of aerobic oxidation, suggesting that intracellular accumulation of calcium ions did not play a part in the action of dimethylnit,rosamine. Levels of NAD were measured in liver preparations from dimethylnitrosamine poisoned rats (100 mg./kg. body wt.) by Gallagher and Rces (1960), who found no change in whole homogenate or mitochondria a t 554 hours but a fall in level a t 1036 hours. I n contrast t o this T. F. Slater (personal communication) has shown t h a t NADP concentration falls significantly as early a s 2 hours after administration of dimethylnitrosamine and h:ts confiriiiccl the later fall
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in NAD. These results are interesting in relation to the work of Stirpe and Aldridge (1960) and Christie et al. (1962). Both groups found little change in the activity of the enzyme diphosphopyridine nucleotide phosphorylase (ATP: NhlN adeiiyltransferase) in nuclei isolated from livers of rats poisoned with dimethylnitrosamine in necrotizing doses even as late as 24 and 19 hours, respectively. The latter authors discuss problems of relating enzyme activity to nuclear counts in view of the difference in frequency distribution of the nuclear ploidy groups in rat liver. These results indicate t h a t synthesis of NAD is unaffected even when the liver is severely damaged by dimethylnitrosamine. This is consistent with unpublished observations (C. J. Threlfall, personal communication) that the NAD content of the liver rises by the same absolute amount in the livers of treated rats as in untreated controls after injection of nicotinaniide. I n contrast to the stimulatory action of carbon tetrachloride, Cleveland and Smuckler (1965) found t h a t dinicthylnitrosainine (50 mg./kg. body wt.) had no effect on rat liver N A D H or NADPH cytochroine c reductase 2 hours after administration. Rees et al. (1962) studied the leakage of liver enzymes into the serum of rats poisoned with dimethylnitrosamine (100 mg./kg. body wt.) . Serum levels of isocitric dehydrogenase and malic dehydrogenase were raised a t 6 hours and continued to rise up to 24 hours. Glutamic dehydrogenase showed little change until 24 hours, when it also rose. These changes in serum enzyme content were reflected by losses of isocitric and malic dehydrogcnase activity of the liver liomogenate and they were mainly due to losses from the extramitochondrial cytoplasmic fractions with little mitochondria1 loss. The increase in liver lipid induced by dimethylnitrosamine was confirmed. Neither previous adrenalectomy nor treatment with the antihistamine drug Phenergan prevented the leakage of the cnzynies into the serum or the developmcnt of liver necrosis. Release of lysosoinal enzymes was not observed iii the early prenecrotic stage of liver injury by dimethylnitrosamine and it was concluded t h a t the lysosornes probably play no role in the early development of the lesions but that they may be involved in the later scavenging processes (Slater et al., 1963; Slater and Greenbaum, 1965). 2. Subacute and Chronic Effects
Enimelot et al. (1960) studied the effects of single acutely hepatotoxic doses and repeated lower doses of dimethylnitrosamine over scveral months on the swelling and enzymatic properties of rat liver mitochondria. They concluded that the mitochondria from the acutely poisoned rats showed an increased tendency t o swell and resemble previously aged normal liver mitochondria. The fine structure of the major
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part of these mitochondria, however, was unaltered whereas, after prolonged administration of dimethylnitrosainine a t lower levels, there was some loss of the internal structurc of the niitocliondria but tlieir in vitro pattern of swelling was normal. Fritz-Niggli e t n l . (1965) found no change in oxidative phosphorylation by mitochondria isolated from precancerous and cancerous liver induccd in the r a t by feeding diethylnitrosaniinc for up to 30 days followcd by return to normal diet. I n a histologically controlled study Nickel and Dicncr (1965) studied oxidative phosphorylation in isolated liver niitocliondria during 70 days feeding (daily dose 9 mg./kg. body wt.) and for varying periods after return to normal diet. On this regimc periportal fibrosis developed first, followed by cirrhosis and finally primary liver carcinoma. The P/O ratios did not change during administration of diethylnitrosamine or after withdrawal and were not significantly different from those in thc control animals. Heise and Gorlich (1964) studied changes in metabolic rate and activity of glycolytic enzymcs in rat liver during carcinogenesis by feeding diethylnitrosaniine for 120 days. Enzyme activities were expressed per livcr and also in terms of unit liver wcight and milligrains of protein in the homogenate. The liver weight increased during feeding but there was little change in respiration of slices or hoinogenates of liver prepared a t intervals of 2 weeks. Aerobic glycolysis increased in liver slices after the rats had received about GOO nig. diethylnitrosaniine and then remained on a steady level, but in homogenates there was little change during the period. Anaerobic glycolysis only became definitely raised in slices toward the end of the feeding period while the homogenates showed an early fall in level followed by a slight rise during the last weeks. There was an early fall in fructose diphosphatase activity in the livers followed a t about day 70 by it sharp fall to a lower level which then remained constant. Glucose-6-phosphatase activity showed little change until day 50 when it rose only to fall again on about day 70. The authors concluded that the transition to tumor metabolism did not occur gradually but by jumps. The activity of fructose diphosphatase fell to a third and that of glucose-6-phosphata~eto 6570 of tlie initial levels. The changes in metabolic rate were regarded a s the result of changes in activity of tlie cnzyines and possible correlations between the activity of fructose dipliosphatasc and glucose-6-phospliatase and the malignancy of the hepatomas were discussed. Sydow (1964) found increased hexokinase activity as compared with normal livers during induction of cancer in the rat with diethylnitrosamine. There was a marked difference in hexokinase activity between primary and transp1:tnted hepatonias induced by the nitrosaniine which was attributed to
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their different degrees of malignancy. Pro!onged feeding of diethyliiitrosamine to mice produced a decrease in liver glucokiriase with a slight increase in hcxokinase activity before the onset of cancer (Sydow and Korn, 1965). I-Eoch-Ligeti e t al. (1964) measured 8-glucuronidase and lactic dehydrogenase in tlie liver, kidney, and lung of rats during tumor induction by feeding diethylnitrosarnine or diinethylnitrosainine. Tumors in the liver were first observed on day 113 of treatment with dimethylnitrosamine and on day 122 with diethylnitrosamine. I n the livers of the treated rats the concentration of p-glucuronidase increased significantly over the controls between days 50 and 70 and it also increased in kidneys but more gradually and to a smaller extent. The level was increased in the lungs after fccding for 130 days. The increase in p glucuronidase was related to the time of exposure to tlie carcinogens but not, to the amount ingested. At the time of the increase, large cells with hyperchromic nuclei and vacuolated cytoplasm appeared in the central areas of the liver lobules. The lactic dehydrogenase concentration was not increased in the livers of rats during the induction of tumors by either riitrosamine and the concentration in tlie tumors was lower than in tlie surrounding tissue. The p-glucuronidase activity of the liver thus increases quite suddcnly some weeks before the tumor can be detected and the authors suggest that this increase may indicate the onset of irrevcrsiblc changes in the liver. Since certain toxic substances which are excreted as glucuronides increase tlic P-glucuronidase activity of liver and kidney, the authors suggest that damage to the liver by the carcinogen might cause an increase in the concentration of P-ghcuronidase in the liver. Stiniulation of glucuronide formation in the livers of rats fed diethylnitrosarnine has, in fact, been demonstrated by Greenwood and Stevenson (1965). The activity of uridine diphosphate ( U D P ) glucuronyltransferase was determined using o-aminophenol as acceptor and found to be increased up to 6-fold after feeding diethylnitrosnmine for 4 months; it was increased 2-fold within 6 days following a single intraperitoneal injection. It is very interesting that stimulation of glucuronide formation also occurred when diethylnitrosamine was added to homogenates and microsonial preparations of liver i n vitro, giving increases of up to 4-fold. No effect was obtained by adding tlic nitrosamine to liver slices, howcver. Since the concentrations of diethylnitrosamine needed to stimulate in vitro were greater than those in vivo the authors suggest that the in vivo effect may be mediated by increased synthesis of the enzyme UDP-glucuronyltransferase. Changes in activity of dihydroxyphenylalanine, 5-hydroxytryptophan, and histidine decarboxylascs were followed during the induction period of liver
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cancer by diethylnitrosainine and correlated with tlie corresponding 1iistologic:tl changes (Reid et al., 1962, 1963). Tlicse experiments were designed to test the hypothesis that a functional relationship exists between histidine decarboxylase activity and tissue growth and also to attempt to find out whether the specific or nonspecific histidine decarboxylases arc involved. Dietliylnitrosamine was added to the drinking water of rats over a period of 5 nionths. Two main histological changes in the liver dcvelopcd in sequence. First, there was gradual formation of hepatomas and later cholangionias (defined as progressive overgrowth of bile-duct epithelium). No histidine dccarboxylase activity could be dctectcd a t pH 5 in the livers of control rats but activity appeared in the livers of rats which had received diethylnitrosamine from 3 months onward. Activity was much higher in hepatoma than in surrounding liver arid declined as tlie livers became overgrown by cholangiomatous proliferation. The nonspecific, pH-8 enzyme, however, was present in both control and carcinogen-treated livers but the levels were lower after feeding diethylnitrosaniine for 3 months. A positive correlation was found between the activities of histicline decarboxylase measured a t pH 8, dihydroxyphenylaniline decarboxylase, and 5-hydroxytryptophan decarboxylase, but no correlation between the pH-6.5 histidine decarboxylase and the other cnzymes. The authors conclude that the specific histidine decarboxylase is associated with the growth of a particular cell, the hepatoma, but not with growth in general. The induction of enzymes in the liver during carcinogenesis by feeding N-nitrosomorpholine was studied by Kroger and Greuer (196*5), who also made a parallel histological study. The animals received 10 mg. per kg. body wt. of the carcinogen daily in the drinking water. Substrate induction of tyrosine-2-oxoglutarate transaminase was reduced after about 20 days on the Carcinogenic regime and that of tryptophan oxygenase (tryptophan pyrrolase) after about 70 days. Induction of hoth enzymes by cortisone, however, was not affected. Experiments using actinomycin D suggested that synthesis of messcnger-RNA during substrate induction of these enzynics was reduced in the animals fed diethylnitrosamine. Changes in patterns of mRNA synthesis during rat liver carcinogenesis by diethylnitrosamine have recently been reported and compared with carcinogcnesis by thioacetainidc (Parish and Kirby, 1966). An niRNA-DNA complex was isolatcd from the livers of rats on the carcinogenic diets and its countercurrcnt distribution was observed, the animals having been injected with H7-orotic acid. The patterns of distribution were different with the two carcinogens, possibly to some extent because dicthylnitrosamine inhibitcd incorporation of orotic acid under these conditions while t1iioacet:irnitlc did not.
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IX. Reactions with Cell Constituents
The first evidence of reaction between a nitroso carcinogen and a cellular constituent was presented by Magee and Hultin (1962). The classical demonstration of binding of carcinogenic azo dyes to rat liver proteins in vivo by Miller and Miller (1947) suggested the possibility that some form of reaction between dimethylnitrosamine and liver proteins might also occur since it appeared that both compounds were enzymatically dealkylated in the liver by similar microsomal enzyme systcms. This possibility was tested by incubation of rat liver slices with dimethylnitrosamine-C14 in vitro. Previous work (Magee and Vandekar, 1958) had indicated that dimethylnitrosamine was metabolized under these conditions and the metabolic formation of formaldehyde (Brouwers and Emmelot, 1960) was confirmed in cxperiments with dimethylnitrosamine-C14 by addition of carrier formaldehyde and precipitation as the dimedone derivative, which was found to be radioactive. The presencc of radioactive formaldehyde after the addition of carrier serine and subsequent periodic acid oxidation was also demonstrated, indicating incorporation of label from the dimethylnitrosamine-C14 into serine of the acid-soluble fraction of the liver slices. This demonstration of labcling of the one-carbon pool by C14 from dimethylnitrosamine-C14 is relevant to the interpretation of all subsequent work with methyl-labeled nitroso carcinogens, since i t indicates that the finding of radioactivity in any cellular component, whether in vivo or in vitro, nced not mean anything more than metabolism or breakdown of thc carcinogen with release of a normal 1-carbon prccursor and subsequent incorporation by the normal metabolic pathways (Section VII). R a t liver slices were incubated with dimethylnitrosamine-C14 and small but definite radioactivity was found in the slice proteins after aerobic incubation but negligible activity after anaerobic incubation. Kidney slices incubated aerobically showed a slightly higher protein radioactivity than the anaerobic liver preparation, which was considered to be significant and has since been confirmed (P. N. Magee, unpublished results). The nature of the binding of the radioactivity to the liver slice protein was investigated in samples with considerably higher specific activity, The proteins were hydrolyzed with acid and their compound amino acids separated by ion-exchange chromatography. Elution of the hydrolyzate from the colums yielded three main peaks of radioactivity in the regions of serine, methionine, and the basic amino acids, respectively. Much of the radioactivity in the first two peaks could probably be explained by normal incorporation via formaldehyde-C14. The third peak was the largest and appeared in the region of lysine and histidine. A
CAHCIKOGEKIC NITROSO COMPOUNDS
22 I
large part of this radioactivity was shown probably to be composed of 1-methyl and 3-methyl histidine. This provided the first direct experimental evidence for tlie alkalation of cellular components by dimethylnitrosamine. Cratlrlock (1965) treated rats with dimcthylnitrosamine-C" and demonstrntcd the presence of very small amounts of S-methylcysteine and of methylated histidines in acid hydrolyzates of liver proteins. Alkylation of cysteine in the proteins amounted to about 0.02% of the cystinc and was less extensive than that of histidinc where the corresponding figure was 0.08%. hiiriute amounts of free S-methylcysteine were found in the acid-soluble fraction of the livers from the treated animals and also in the urine. The methylated histidines did not persist in the livers of tlie trcated rats and had largely disappeared 6 days after administration of diniethylnitrosamine. The possible significance of alkylation of proteins in carcinogenesis is discussed in Section X. I n their experiments on rat liver slices incubated with dimethylnit rosamine-C" Magee and Hultin ( 1962) observed that the radioactivity of the protein-coiitaining precipitate, after extraction of acid-soluble and lipid components, was higher than t h a t of the final product after extraction of thc nucleic acids with hot trichloroacetic acid. Extraction of the combined sodium salts of the nuclcic acids from the slices by hot 10% NaCl showed them t o have considerably higher specific activity than the proteins. Ion-exchange chromatography of the RNA nuclcotides after alkaline hydrolysis and precipitation of the DNA showed radioactivity exactly coincident with aclenylic acid and also a larger radioactive peak which appeared close to, but was definitely distinct from, guanylic acid. Subsequent work (Farber and hiagee, 1960; Magee and Farber, 1962) suggests that this material was probably a derivative of 2,4-diamino-6-liydroxy-5-~~-iiietliylforiii~midopyrimidine which is known to be produced by ring opening following exposure of 7-methylguanosine to mild alkaline conditions (Haines et al., 1962). This indicated that reaction had occurred between dimethylnitrosamine or one of its products of decomposition and one or both of the liver nucleic acids. The nature of this reaction was further investigated in the intact rat by Magce and Farber (1962). In these experiments the animals were injected intraperitoneally with diinetliylnitrosaniine-CI4 in doses of 30 mg. per kg. body wt. (about the median lethal level), which was sufficient to cause acute centrilobular necrosis of the liver in all the injected rats and to induce tumors of the kidney in about 20% of the survivors, about, a year later. The aninials were killed 5 hours after injection when the liver injury was not far advanced. Incorporation of radioactivity into RNA and protein of liver, kidney, spleen, and pancreas was observed and compared with that following similar intra-
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peritoneal injections of C14-labeled methionine, leucine, valine, and formate. With dimethylnitrosamine the specific radioactivity of the liver RNA was higher than that of the proteins by a factor of about 5 while the ratio was less than 1 with the other compounds. Incorporation from dimethylnitrosamine-C14 also occurred into DNA of liver and probably of kidney, and the lipid fraction also became labeled. Alkaline hydrolysis of the liver RNA and ion-exchange chromatography gave very similar results to those obtained in vitro (see above). Acid hydrolysis of the labeled liver RNA released most of its radioactivity as 7-methylguanine, identified from its behavior on ion-exchange and paper chromatography and from its ultraviolet spectrum. Treatment of this material with nitrous acid gave a product which was identified as 7-methylxanthine by the same criteria. Radioactive material in the expected position of 7-methylguanine was found on ion-exchange chromatography of acid hydrolyzates of kidney RNA and liver DNA but insufficient material was present for ultraviolet absorption spectra. Traces only of radioactivity in the position of 7-methylguanine were found in hydrolyzates of pancreas and spleen RNA. The latter showed considerable incorporation into guanine and adenine which was considered to reflect the rapid turnover of splenic RNA and incorporation of l-carbon precursors derived from oxidation of the dimethylnitrosamine methyl into the purines. The chromatographic profile of splenic RNA from rats treated with radioactive formate was very similar to that with dimethylnitrosamine but there was no measurable radioactivity in the position of 7-methylguanine. No evidence of 7-methylguanine was found in acid hydrolyzates of the acid-soluble fraction of liver from rats treated with dimethylnitrosamine. The identification of 7-methylguanine in the nucleic acids of rats treated with dimethylnitrosamine was the first demonstration of a defined reaction between a carcinogen and the genetic material of a target somatic cell; and its significance will be discussed in Section X. Evidence for methylation of DNA of kidney was presented by Craddock and Magee (1963) based on probable identification of 7-methylguanine in acid hydrolyzates of the nucleic acid prepared from this organ from rats injected with dimethylnitrosamine-C14 as before. The time-sequence of methylation of liver and kidney RNA and DNA was then investigated after administration of diniethylnitrosamine-C14 to rats. Methylation of RNA reached a maximum in both organs Fj-12 hours after injection and then decreased reaching very low levels a t 3 weeks. In the liver, where methylation is considerably more extensive than in other organs there was a very rapid initial loss of methyl groups from RNA during the first two days during which there is concomitant necrosis. In the case
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of DNA the methyl groups were lost even more rapidly since maximal methylation occurred 5 hours after injection and had already fallen substantially a t 12 hours. Experiments were not continued beyond 48 hours when the degree of methylation was only a small fraction of the maximum but still definitely detectable. The level of methylation of kidney DNA, as with RNA, did not attain more than about 10% of that in the liver a t the time of maximuin methylation, but the 7-methylguanine was lost much more slowly from the kidney, so that it could still be detected in the DNA 48 hours after injection. With both nucleic acids, the rate of loss of methyl group was much greater than the normal turnover rate. Rats fed dimethylnitrosamine in the diet a t a hepatocareinogenic level for 23 weeks still showed methylation of liver DNA when treated with single doses of dimethylnitrosamine-C". Methylation and subsequent loss of the methyl groups occurred a t about the same rate as that following treatment of normal animals (Craddock and Magee, 1965). Using dimethylnitrosaniine-H3 prepared by the Wilzbach exchange reaction Lee et al. (1964) observed that methylation of nucleic acids occurred in rats and mice as with the O4compound and that H3 was also incorporated into nucleic acids via normal metabolic pathways. The normal incorporation, however, was considerably less relative to the methylation. Using dimethylnitrosamine labeled with each isotope a comparison was made of the degree of methylation of RNA in eight organs in the rat and in the mouse. I n both species the liver showed much the highest level of methylation. In rat, kidney RNA was the next highest after liver with about lO-lSPr, of the liver level, and lung was next with about half that in kidney. The spleen and esophagus plus squamous stomach showed very low but detectable levels, appreciably greater than those in the small intestine and pancreas, which were virtually zero. The pattern of methylation in the mouse organs showed certain differences since the kidney was very low in this species while the lung, relative to the liver, was higher than in the rat. These different levels of methylation of RNA in the different organs are interesting in relation to the distribution of lesions induced by dimethylnitrosamine (Section V) . I n both species, the liver is much the most severely affected acutely but tumors only appeared in the liver and kidney of the strain used with very occasional tumors of the lung (Magee and Barnes, 1962). Other strains of rat, however, do develop lung tumors in response to dimethylnitrosamine (Zak e t al., 1960; Argus and Hoch-Ligeti, 1961). I n the BALB/C mice used, however, the very low incidence of pulmonary adenoma in the control animals was raised to 85-890/0 by diniethylnitrosamine feeding and there was a high incidence of liver tumors but only very occasional renal adenomas (Toth e t al., 1964).
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Possible reasons for the quantitative differences in degree of methylation of the RNA of different organs were also discussed. The simplest explanation is that each organ is capable of metabolizing the nitrosamine to a greater or less extent. The different degrees of methylation would then reflect the different metabolic activities of the organs. The observation of low but dctcctable capacity to metabolize dimethylnitrosamine by in vitro preparations of other organs as well as the liver is consistent with this view (Magee and Vandekar, 1958; hlizrahi and Emmelot, 1962; P. N. Magee, unpublished). The possibility that a methylating agent in formed metabolically in the liver and then released into the circulation is unlikely since such an agent would be expected to damage organs with rapid cellular turnover such as intestine, bone marrow, and lymphoid tissue, and this is not the case. Another possibility would bc release of 7-methylguanine or a derivative from the liver and its subscquent biosynthetic incorporation into the nucleic acids of other organs. This is also unlikely because organs such as the small intestine which incorporate nucleic acid precursors very rapidly would be expected t o show high levels of methylation but, in the rat, 7-methylguanine was virtually undetectable in the RNA of this organ. These results strengthen the hypothesis that dimethylnitrosamine must undergo metabolism before it can produce cellular injury and cancer but do not necessarily indicate that alkylation is the essential reaction since other metabolites must also be formed which may be active intermediates (see Sections VII, VIII, and X ) . It is possible that the injury and carcinogenic changes will only take place in those cells that can metabolize the nitrosamines. This may explain why the nitrosamines are more selective carcinogens than the nitrosamides which do not require enzymes for their decomposition. Lee and Spencer (1964) reported methylation of liver and kidney RNA in rats injected with dimethylnitrosamine in the neonatal period. A very slight methylation of fetal liver RNA was observed after the mothers were treated with the carcinogen. The methylation increased rapidly soon after birth in the treatcd babies, reaching adult levels after injection a t 3 days of age. Methylation of kidney RNA was relatively higher in the newborn rats than in the adult. These observations are of interest in relation t o the induction of kidney and liver tumors by single doses of dimethylnitrosamine (Terracini and Magee, 1964; see Section 111). Since the biological alkylating agents vary very considerably in their capacity to induce cancer, some explanation must be forthcoming for the extraordinarily high carcinogenic potency of the nitroso compounds if their mode of action is t o be explained by alkylation. With this end in view Swann e t al. (1965) undertook a comparison of the degrees of alkylation of RNA and DNA in the organs of rats treatcd with a series
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of nitroso and non-nitroso alkylating agents. The compounds compared were diiiiethyliiitrobainine, nitrosomethylurea, iiiethylnietliane sulfonate, climethy1 sulfate, and iiiethyl iodide. Diiiietliylnitrosainiiie was by far the most active iiicthylator of DNA arid RNA in liver, but considerably less so in kidney and only very weakly active or inactive in the other organs. N-nitro some thy lure:^, after intravenous or oral administration, did not show as high a level of methylating activity in liver as dimcthylnitrosainine but methylated nucleic acids in all the organs studied a t about the level observed with diniethyliiitrosaniine in the kidney. Dimethyl sulfate was less active, even in the high doses used, than the iiitroxo carcinogens. Illethylmethane sulfonate, on the other hand, WLS iibout ccpilly as active as iV-nitrosomethylurea in the metliylation of the nucleic a(~ids. hZetliyl iodide gave no detectable metliylation of the liver RNA (DNA was not studied) a t a time after injection when the level of glutathione in the liver was drastically reduced by the formation of nicthylglutathione (M. K. Johnson, personal communication). These results allowed certain predictions to be made. Since the level of methylation of nucleic acids in kidney was slightly higher with nitrosomethylurea than with diiiicthylnitrosainine the former was anticipated to be an effective renal carcinogen after single oral administration. This proved to be the case (Table 111) and the incidence of liidncy tumors was higher than with dimethylnitrosamine. The other prediction is t h a t methylmethane sulfonate should also induce renal tumors if methylation of nucleic acids per se is sufficient to induce the neoplastic change. This possibility is being tested (P. F. Swann and P. N. RIagee, unpublished) . Alkylation of nucleir acids in rut l i l w also occurs in rats treated with diethylnitrosaiiiine and n-hutylinethylnitrosaiiiine but not with tert-butylmethylnitrosaiiiine (Magee and Lee, 1963, 1964). With diethylnitrosaiiiine-C1', even a t approximately the median lethal dose, the incorporation of radioactivity into liver RNA is relatively low compared with the dimethyl compound. Acid hydrolysis and ion-exchange chromatography revealed a peak of radioactivity emerging from the colunin immediately before adenine. This material was identified as 7-ethylguanine. Incorporation of radioactivity into RNA and protein from n-b~tyl-C~~-inethylnitrosaiiiine was more active than from the diethyl compound and, in fact, approached t h a t of dimcthylnitrosamine. \'l'hen, however, the compound labeled in the n-butyl group was injected the incorporation into RNA and protein was very much lower and the ratio of the specific activity of the RNA to that of protein was 0.24 in contrast to the ratio of 4.5 found with the n-butyl-C1'methyl compound. Hydrolysis and chromatography of the RNA from
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rats treated with the compound labeled in the methyl group revealed that virtually all of the radioactivity was preseiit as 7-mcthyl-guanine. 'I'he metabolic behavior of tert-butylinetliyliiitt~o~aiiiincwas greatly ciifferent. With the C14 label in thc tert-hutyl iiioicty of tlie molecule there was virtually no detectable incorporation into nucleic acid howand very little into protein. With tert-b~tyl-C~~-methylnitrosamine, ever, there was active incorporation with more into proteins than RNA, the ratio being 0.53. Hydrolysis and chromatography of the labeled RNA showed that all of the radioactivity was present in the pyrimidines and purines presuiiiably by normal metabolic incorporation from the one-carbon pool. This result was in striking contrast with that obtained with the n-butyl-C14-niethyl compound and supports the hypothesis that alkylation of cellular components may be important in the biological action of the nitrosamines. Unfortunately tert-butylmethylnitrosamine appears not to have been tested for carcinogenicity but the tert-butyl ethyl compound is inactive both as a carcinogen and a mutagen (Section VI). The n-butyl cornpound is actively carcinogenic (Heath and Magee, 1962) and, furthermore, produces acute centrilobular nccrosis of the liver while tert-butylmcthylnitrosamine does not induce this typical acute lesion of the carcinogenic dialkylnitrosamincs (see Section VII) . As emphasized in Sections VII and X, dimethylnitrosainine and other nitroso carcinogens are potentially capable of yielding other metabolites as well as alkylating intermediates. Among such possible decomposition products are nitrous acid and derivatives of hydroxylamine and hydrazine (Heath and Dutton, 1958; Heath, 1962; Suss, 1965). Although Heath and Dutton (1958) could not detect any of these compounds in rats treated with dimethylnitrosamine the possibility exists that trace amounts might have been formed which reacted with available chemical groupings in the cell and destroyed themselves in the process. Similar reasoning applies to the fate of alkylating and, in fact, any chemically reactive intermediates formed in small amounts inside cells. An attempt was made to test for the transient presence of nitrous acid or a hydroxylamine derivative in the livers of rats treated with dimethylnitrosamine by Craddock and Magee (1966). I n these experiments it was assumed that nitrous acid would deatninate the nucleic acids with conversion of guaninc and adenine to xanthine and hypoxanthine, respectively, and that hydroxylamine or a derivative would convert cytosine to NGhydroxycytosine. The sensitivity of the analytical procedures was increased by using rats whose nucleic acids had been prelabeled with C14 and H3 by injections of nucleic acid precursors, and account was taken of nrtifactual deamination which is known to occur (luring isoIa-
CARCINOGENIC NITROSO COMPOUNDS
22 7
tion and hydrolysis of nucleic acids. The presence of 7-metliylguaninc in DKA and RNA of rats trcated with dimethylnitrosaniine was confilmed but no evidence of xanthine, hypoxttnthine, N',-liydl.oxyniethylcytosine or of any other :~bnornial base was found. These results do not exclude the formation of tibiiormal nucleic acid bases but tlicy suggest that any such formation must be quantitatively minute and much smaller in amount than that of 7-methylguanine. X. Possible Mechanisms of Action
Mechanisms of action of the nitroso carcinogens have been discussed by several authors (Druckrey et al., 1961a, 1963c; Arcos and A ~ C O S1962; , Magee, 1962, 1963, 1964a,b; Magee and Schoental, 1964; Weisburger and Weisburger, 1963; Miller and Miller, 1966; Schoentul, 196613). The induction of cancers by single doses of rapidly eliminated nitroso carcinogens implies an interaction between the carcinogen and/or a product of its decomposition with some component or components of the cells which must occur within a short time after administration. In this section the nature of the proximate carcinogen (Miller and Miller, 1966) and some of its possible interactions with cellular components will be discussed.
A. THEPROXIMATE CARCINOGEN 1. T h e Unchanged iMolecule The possibility that some unchanged nitrosamines may be actively carcinogenic themselves without the necessity of any form of metabolic or other decomposition has been discussed by Arcos and Arcos (1962). Argus et al. (1961) showed that dimethylnitrosamine and some other carcinogens are agents of protein denaturation and considered this in relation to the hypothesis of Rondoni (1955), which suggests that denaturation may play a part in carcinogenesis. However, diniethylnitrosamine is uniformly distributed in the body water (Magee, 1956) but tumors have only been reported in liver, kidney, and lung in rats after oral administration, which is difficult t o reconcile with simple protein denaturation as a causal factor in carcinogenesis unless the unaffected organs are more resistant than those affected. Argus et al. (1964) showed that several nitrosamines, including N-nitrosopiperidine, and other compounds, including diethylformamide, diethylacetamide, and dioxane, had similar surface-active properties.
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The same group (Argus e t al., 1965) tested tlie last three compounds for carcinogenic activity by prolonged oral administration t o the rat. Dioxane was found to be a liepatic carcinogen, producing liepatornas in 6 out of 26 rats. Diethylacetalnide and diethylformalllide, however, did not induce livcr tumors but showed only possibly very wcak activity to kidney and lymphoid organs, respectively. Thcse authors maintain that the carcinogenic activity of the cyclic dioxane and of the cyclic alkyl nitrosamines, such as iv-iiitrosoinorpholine, cannot be accounted for on the alkylation hypothcsis suggested for the dialkylnitrosamines (see below). They suggest that it is liiglily unlikely, on chemical grounds, that dioxane could yield metabolic products resembling diazoalkanes and they also throw doubt on the possibility of ring opening in tlie cyclic nitrosamines.* They conclude that two coricurrent and mutually not exclusive mechanisms may be operating in tumor induction by the nitrosamines and related surface-active compounds, namely, denaturation of cellular macron~oleculesinvolved in metabolic control and alkylation of nucleic acid bases after mctabolisin to reactive diazoalkanes. The first mcchanisin is based on their earlier observations that all the nitrosainines tested by them, a s well as dioxane, show potent hydrogen honding and protein-denaturing ability. 2. Possible Active Decomposition Products
a. T h e Corresponding Aldehyde. Formaldcliyde is unlikely to be an active intermediate in the induction of thc acute livcr injury by dimethylnitrosaiiiine (Heath, 1962 ; see Section VI1,A). Many drugs containing N-alkyl groups are metabolized predominantly in the liver to give tlie aldehyde (Brodie et al., 1958) but they are not known to be Carcinogenic. Formaldehyde has been tested for carcinogenicity with negative results (Ilartwell, 1951 ; Sliubik and Hartwcll, 1957). A special action of tlie aldehyde after intraccllular release from the nitroso compound cannot be excluded but it appears improbable that the induction of cancer by thcsc agents is mediated by the aldehyde. b. Nitrous Acid. Nitrous acid is a very effective mutagen which is thought to act by deamination of nuclcic acid bases (Schuster and Schramm, 1958). Sodium nitrite, however, was not carcinogenic on prolonged feeding to rats (Druckrey et al., 1963e) but it is possible t h a t release of nitrous acid from a nitroso compound inside the cell close to a critical receptor site might be carcinogenic. Craddock and Magee (1966) were unable to detect deamination of liver nucleic acids of rats *Lce and Lijinsky (1966) have recently reportcd thc alkylation of rat liver
RNA by cyclic N-nitrosamines in uiuo.
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229
treated with dirnethylriitrosamine (Section IX) and very little deamination would be expected a t p H 7 (Lochmann and Stein, 1963). There seenis to be no experimental evidence in favor of nitrous acid as an inteix~ediatein carcinogenesis by tlie nitroso coinpounds, but deamination of nucleic acids sufficient to induce gene mutations would certainly not have been detected since this is thought to require reaction with only one base (Gierer and Mundry, 1958). c. H ydroxylnmine Derivatives. Hydroxylamine is mutagenic (Freese et al., 1961) and induces chro~nosoiiiedaniage (Somers and Hsu, 1962; Borenfreund e t al., 1964), but it has not been reported as a carcinogen. Hydroxylamine reacts with nucleic acids, predominantly on cytosine, to give a product which yields N"-hydroxycytosine on acid hydrolysis (Brown and Schell, 1965). This reaction has been suggestcd to be its mechanisin of mutagcncsis. Craddock and Magee ( 1966) could not detect N"-hydroxycytosiiie in liver nucleic acids of diniethylnitrosaminetreated rats but again the presence of minute amounts could not be excluded (Section I X ) . ti. H y d r a z i n e Derivatives. Them are possible active intermediates in riitrosmiine carcinogenesis sirice hydraeine is carcinogenic in the mouse (Biaricifiori et al., 1964) although unsyminetrical dimethylhydrazine was reported inactive in the rat (Druckrey et al., 1961a; Argus and Hocli-Ligcti, 1961). Hydrazine and some of its derivatives are mutagenic (Lingens, 1964). Reduction of iV-nitrosoniorpholine to the hydrazine derivative by rat liver preparations in vitro has been reported (Suss, 1965; see Section VI1,B). e. Alkylating Intermediates. The evidence for the formation of these has been presented in Sections VII and I X and the possible nature of the active molecular species discussed in Section 11. For the underbtsnding of the molecular mec1i:znism of carcinogenesis tllc fact of alkylatioii of nucleic acids, proteins, and probably other cellular components inay lie of greater significance than the actual nature of the alkylating agent. T h e idea that carcinogenesis by nitroso compounds rnay be related to alkylation receives considerable support from the extensive studies on structure-activity relationships by Druckrey, Preussniann, Schinahl, and their colleagues (Druckrey et nl., 1961a, 1963c, 1967; see Scction I V and Table 111). I n gcncral, there is good correlation between tlie possession by the nitrosainine of a chemical structure which could yield a diazoalkane on decomposition and its ability to induce cancer. This is well shown in the comparison of n-butylethylnitrosamine which is active and the tert-butylethyl compound which is not. Difficulties arise, however, with the heterocyclic iiitrosamincs such as ~r-nitrosomorl,holirie,which is
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highly active, because, as pointed out by Argus, Arcos, Hoch-Ligeti, and their colleagues (see above) there is no experimental proof that the rings can be opened metabolically. If this is not the case therc is no possibility of the formation of a diazoalkane or other alkylating intermediate. On the other hand, in the absence of evidence, i t may be premature to assume that no ring opening can occur (see footnote p. 228). The induction of esophageal cancer in the rat by phenylmethylnitrosamine is also difficult to reconcile with the alkylation hypothesis, assuming that metabolism is predominantly via demethylation. If alkylation is related to carcinogenesis some explanation for the relatively low carcinogenic action of some alkylating agents in comparison with the nitroso compounds must be provided. Although several alkylating agents are undoubtedly carcinogenic (Walpole e t al., 1954; Haddow, 1958; Dickens and Jones, 1961) some have only weak activity or activity confined to the site of injection. The behavior of dimethyl sulfate is interesting in this context. It is an effective local carcinogen after repeated subcutaneous injection in the rat, producing local sarcomas with metastases (Druckrey e t al., 1966), but was only very weakly active or possibly inactive when tested by oral and parenteral administration (B. Terracini and P. N. Magee, unpublished). Since diinetliyl sulfate, even in nearly lethal intravenous doses, induced levels of methylation in the nucleic acids of all organs tested which were considerably lower than those obtained with dimethylnitrosamine and nitrosomethylurea (Swann e t al., 1965; see Section IX), i t seems that the difference in carcinogenic potency might be explained by the quantitatively lower methylation obtainable with dimethyl sulfate which would presumably apply to other cellular components as well as the nucleic acids. Quite small quantitative differences in reaction between dirnethylnitrosaniine or a decomposition product and some cellular component may be crucial for the induction of cancer; Riopelle and Jasmin (1963) observed that rats given 0.5 mg. diinethylnitrosamine twice daily for 6 days had 100% incidence of kidney tumors a t 7 months while animals receiving 0.2 mg. twice daily for 1 month had none. Although it is possible that some of the rats on the lower dose level might have developed renal tumors later there is no doubt that there was a marked difference in carcinogenic response to this very small difference in dose. There is some correlation between the organs in which cancer is induced by dimethylnitrosamine and nitrosomethylurea and the extent of methylation of their nucleic acids, which presumably reflects the extent of methylation of proteins and other cell components as well (Section IX) . There are also discrepancies, notably in the liver, where methyla-
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tion is relatively very extensive but tumors have not been observed after single doses althougli kidney tumors may be induced in the same animals with considerably lower degrees of methylation in that organ. This may reflect the great capacity of the liver for regeneration and repair. The methyl groups disappear rapidly from the liver DNA in the intact rat treated with dimethylnitrosaniine ( Craddock and Magee, 1963) and i t is possiblc that, some form of rcpair enzyme may be involved. Such enzymes wliich remove methyl groups from methylated DNA in microorganisms have been reported by Strauss (1963). It seems, therefore, that if alkylation is causally related to carcinogenesis by the nitroso compounds reactions with a cellular receptor or receptors must occur to a critical extent and for a critical duration of time.
B. THECELLULAR TARGET 1. Proteins
Emphasis has been placed on reaction with nucleic acids in this review partly because there is more evidence for this than for reaction with other cell components. There is no experimental proof, however, that reaction with these macromolecules rather than other cellular components is related to carcinogenesis. Since the discovery of protein binding by azo dyes (Miller and Miller, 1947, 1953) and by polycyclic hydrocarbons (see Heidelberger, 1959) the well-known “protein deletion hypothesis” of carcinogenesis has been developed by Potter and others (see Potter, 1957), in which reaction of the carcinogen with cellular protein is postulated as the primary event. Alkylation of cellular proteins cannot be excluded as such a possible primary factor. Schoental ( 1966a) has recently eniphasized the possible importance of alkylation of protein-bound sulfhydryl groups. I n particular she suggests that there may be “sensitive centers” where a protein thiol is linked or close to a nucleic acid irnidazole possibly in links between nucleic acids and residual proteins. She suggests that these centers may be involved in the carcinogenic action of alkylnitrosourethanes and of some other chemical carcinogens. 2. Lipids and Other Cell Components
The lipids have not received much attention as primary cellular targets for carcinogens but there seems no good reason for this. Recent studies (P. F. Swann, P . N. Magee, and K. Lee, unpublished) of liver
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lipids from rats treated with dimethylnitrosamine-C14 have not provided any evidence of abnormal 1nethylat:on of lipid components but they do not exclude the formation of unstable mctliylated products. Although there is no experimental evidence of alkylation of other cellular components in animals treated with nitroso carcinogens it is unlikely that this does not occur. I n view of the present ignorance of fundamental mechanisms of carcinogenesis this possibility should not be ignored. 3. Nucelic Acids
The idea that the induction of cancer might follow mutation in a somatic cell was put forward by Boveri (1914), who suggested that an abnormal chromosome constitution might be the primary cause of malignant change. Other theories have bcen advanced which postulate altered activity of extrachromosomal genetic factors (Haddow, 1944, 1947; Darlington, 1948). Aspects of the somatic niutation hypotliesis have been 1959; Kaplan, widely discussed (Burdete, 1955; Burnet, 1957; ROL~S, 1959) and the conclusions have been widely different (Pullman, 1964). Whatever the nature of the initial event in carcinogenesis it seems that there must be soiiie change in the heritable propertics of the transformed cells. The simplest explanation of a hereditary cellular change is that there is some change in the genetic material which is now generally accepted to be composed of nucleic acids. The change might involve a direct action on the chromosomal DNA by the carcinogen or nn indirect action on nuclear histones or on some extrachromosomal cytoplasmic component. The latter possibility is consistent with tlic cxtrschromosomal mutational hypotheses of Haddow and of Darlington and also more recent suggestions of Jacob and Monod (1961a,b) which have been developed by Pitot and Heidclberger (1963). These latter author5 have proposed t h a t carcinogenesis does not necessarily involve primary modification of D N A but might result from changes in repxssor or othcr regulatory molecules which could be either protein or RNA. The demonstration of alkylation of DNA, RNA, and proteins in organs of animals treated with nitroso earcinogcns (Section IX) is consistent with all the above hypotheses and there is no experimental evidence for distinguishing any one type of rnacroniolecule as the primary target in carcinogenesis. However, the evidence for alkylation of nucleic acids by the nitroso Carcinogens (Section IX) docs give support to the hypothesis enunciated by Haddow (1958) in which he stated: (‘Whatever the precise chemical mechanism of the action of carcinogens, there can be little doubt of the importance of their combination with genetical
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material or its precursors, if (as seems likely) this is essential t o those a t least which function through biological alkylation.”
C. ALKYLATION OF NUCLEICACIDS 1. Possable Mechavisnzs of Mutagenesis and Carcinogenesis Although it is not established that there is any relationship betwcen tlie mechanism of mutagenesis and cnrcinogcnesis, it may he worthwhile to compare possible iiicclianisriis of the two processes by the nitroso compounds which are equally effective in both. Since DNA is accepted as the genetic material in most orgariibrns all mechanisms of mutation must, directly or indirectly, involve some change in it. It is widely believed that mutation, a t the molecular level, involves a change in the sequence of the bases in the DNA nioleculc, that is to say, a change in the genetic code (see Freese, 1963; Krieg, 1963). Possible consequences of alkylation of DN-4 have been discussed by Brookcs and Lawlcy (1964a,b) and by Pullman (1964). Brookes and Lawley suggest t h a t two types of molecular change might be expected t o result in mutation. First, the amount of genetic material could remain constant but the base sequence could he changed by substitution of one or more bases by different ones; second, :t loss of one or more base pairs might occur in which the loss of genetic material is not such as t o inactivate the descendant cells. The principal site of alkylation is a t N-7 of the guanine moieties in DNA, and could be predicted on theoretical grounds (see Pullman, 1964). This increases the acidity a t the N-1 atom of the purine ring so that the amount of the ionized form present a t neutral pH will be much increased in comparison to the normal base. I n this ionized state the alkylated guanine could form a pair with thymine instead of cytosine, which normally pairs with guanine. This anomalous base pairing would le:d to replacement of the affected guanine-cytosine pair by an adenine-thymine pair in subsequent replication of the DNA. Alkylation of both guanine and adenine in DNA iesults in slow depurination with rclease of 7-alkylguanine and 3-alkyladenine from the macromolecule. It is possible that this depurination could result in mutation by causing a base-pair deletion. Grosser deletions of genetic material might also be produced by chemical degradation of the alkylsted DNA or by the action of specific tieoxyribonucleases (Brookes and Lawley, 1964a,l)). The experiniental results outlined in Sectiorl IX are consistent with the occurrence of both these mutational mechanisms in tlie cells of somatic organs in which cancer has been induced by a single dose of a nitroso carcinogen.
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2. Possible Significance of Enzymatic Methylation of Nucleic Acids An interesting recent development has come from the discovery of enzymes which activate methylation of nucleic acids a t the polymer level by methyl transfer from S-adenosylmethionine. The first obccervation by Fleissner and Borek (1962) showed the presence of enzymes that methylate RNA (RNA methylases) in E . coli and later work revealed that they also occur in mammalian systems (Srinivasan and Borek, 1963). Enzymes of similar typc which activate methylation of DNA a t the polymer level have been reported in E. coli by Gold et ul. (1963). The possibility that these normally occurring enzymatic methylation reactions might be related to carcinogenesis has been discussed by Borek (1963) and Srinivasan and Borek (1964). They suggest that if imposed alkylations of nucleic acids by chemical carcinogens have a causal relation to the induction of tumors, then such chemical alkylations may find a counterpart in aberrant or excessive methylations of RNA or DNA by the naturally occurring methylating enzymes. It is therefore very interesting that Bergquist and Mathews (1962) have shown that some tumor tissues contain the highest levels of methylatcd bases in RNA ever reported; very recently Mittleman et al. (1965) have reported that RNA methylase activity in SV 40 virus-induced hamster tumors is higher than that in normal liver by a factor of about 14. The carcinogenic action of ethionine, the ethyl analog of methionine, on rat liver is relevant to hypotheses relating carcinogenesis to disordered cellular alkylation reactions. There is evidence that cthionine can replace methionine to form S-adenosylethionine and can then participate in some alkyl group transfers, resulting in ethylation instead of the normal methylation of acceptor groups in the cell (Farber, 1963). Rat liver RNA becomes cthylated in animals treated with ethionine and the levels of ethylation are markedly higher in the sRNA, as would be expected (Farber and Magee, 1960; Stekol et al., 1960; Farber et al., 1966). Incorporation of the ethyl group of ethionine into DNA has also been dcmonstrated (Stekol et al., 1960) and 7-ethylguanine is formed in both DNA and sRNA (Stekol et al., 1964). These results support the hypothesis that aberrant alkylation of nucleic acids may be important in carcinogenesis and emphasize that changes in RNA may be significant. XI. Public Health Aspects
There can be little doubt that the nitroso compounds discussed in this review might offer a serious carcinogenic hazard to those people who might be exposed t o them.
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A. HAZARDS TO CHEMISTS .4ND INDUSTRIAL WORKERS Serious attention must be given to the use of diazomethane and some of its precursors under laboratory conditions so t h a t every chemist is presented with information that will enable him to take appropriate precaut,ions. Druclircy and Preuvsniann ( 1962a) suggest that nitrosomethylurethane should not be used as a source of diazomethane but t h a t the noncarcinogenic prccursor p-tosylmet hylnitrosamine be used instead. The same point is made by Arndt et al. (1963), who suggest a number of other precursors whose biological activity should be tested before they can be recommended a s Fources of diazomethane. There is good experimental evidence for having the question of the industrial production of certain nitroso compounds considered in the same way as t h a t for 2-naphthylaniine even though an undoubted association of exposure to nitrosaniine and tumor production in man has not been establishetl. Since a wide range of animal species are affected and tumors of many different types resembling those seen in man can be produced by nitroso compounds it seems very improbable that man will not be sensitive to their carcinogenic action. What is disturbing from the animal experiments is that a single dose of a nitroso compound may produce tumors. If a latent interval is 20 to 30 years in man it is unlikely that a chemist would remember a previous episode of acute poisoning if a tumor were diagnosed a t a time of life when cancer becomes more coinnion. Clearly a good deal of publicity about the possible hazards of handling such compounds and the long-term effects of exposure to thcm is needed for the guidance of chemists.
B. HAZARDS TO GENERAL POPULATION It may be worth considering whether some of the human population could be exposed to compounds of this type, albeit quite unwittingly. 1. Naturally Occurring Compounds
The story of cycasin indicates one potential hazard and underlines the need to scrutinize more carefully other natural products for constituents of this type. A toxic azoxy compound known to poison sheep and cattle was first shown to be present in a plant of the cycad family in Australia by Langley et al. (1951) but no toxicological studies seem t o have been made with the pure compound. It differs only in the nature of the sugar from cycasin, which has been the object of much detailed study referred t o earlier in this review. It is interesting t o reflect that had the biologist tested the crystalline material studied by Riggs and his colleagues in 1950 the story of the toxicity of the nitroso compounds
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might have begun a t least 5 years earlier. One hopes t h a t natural products will continue to excite as much interest as possible agents of disease as they have as potential cures. At least one fungus is known to contain a nitroso benzaldehyde (Herrmann, 1960). It might be worth looking a t the products of metabolism of othcr fungi and microorganisms. The ability of microorganisms to produce compounds likc cycasin is illustrated by elaiomycin, a possible antibiotic active against Mycobacteriuin tuberculosis, reported by Ehrlicli et al. (1954). These workers had reported that elaiomycin could produce liver damage in mice and guinea pigs. Dr. R. Schocntal has recently shown that, given as a single dose to rats, it is a t least as acutely toxic as dimethylnitrosamine and produces an identical type of acute liver damage. Its carcinogenic action awaits further investigation. The possibility that under sonic conditions the normal body microbial flora could produce nitroso compounds might be worthy of consideration. 2. Nitrosanzines in Food
Although Druckrey et nl. ( 1 9 6 3 ~ )were unable to produce tumors with sodium nitrite itself and concluded that nitrite niay be safely used as a food preservative, experience with a fish meal product indicates that this may not he the casc. An outbreak of illness among sheep led to the discovery that this was associated with a severe toxic liver necrosis and apparently followed the eating of a fish meal as a food supplement. Nitrite had been added to the fish meal as a preservative and a suggestion was made that nitrosamines might bc formed by a reaction of the nitrite with secondary amines in the fish meal. Dimcthylnitrosaniine was then actually detected in the treated fish mcal and it seems very probable t h a t this was the cause of the liver necrosis scen in the poisoned sheep (Ender et al., 1964; Sakshaug e t al., 1965). Nitritc is widely used in the treatment of meat products for human consumption and the possibility that nitroso compounds may be formed in the treated food should be investigated by the appropriate analytical methods. 3. Nitrates in Plants
Under some conditions plants and crops may accumulate large quantities of nitrate and this, after rcduction to nitrite in the rumen, may be absorbed and cause acute poisoning with methemoglobin formation in cattle t h a t have fed on the plants. In some cases of poisoning nitrosohemoglobin is formed and the cattle have no cyanosis (Case, 1957). Thc possibility that nitroso compounds may be formed in plants, particularly those t h a t can accumulate nitrate, might be worth investigating. Domestic animals are not often kept long enough to enter
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the cancer-bearing agc but an outbreak of esophageal cancer in cattle in Africa was never explained (Plowright, 1955). The possibility that there was a nitroso compound in one of the plants consumed must remain only an interesting speculation. 4. Nitrosuntines in Tobacco That N-nitroso compounds might be present in tobacco smoke and be responsible for tlic carcinogenic activity of cigarette smoke has aroused a good deal of attention. Druckrey and Preussiiian (196210) had suggested thcir possible forniation froin the nitrates and bases present in tobacco. Robertson (1964) claimed that nitrosarnines had been detected in cigarette smoke. Neurath e t al. (1964a,b) developed a sensitive method for detecting nitrosamines based on their conversion to unsymmetrical hydrazines. M’liile they could find no nitroso compounds in the smoke from norrnal cigarcttes they did find traces of AT-nitrosoniethyl-n-butylaniine in smoke from cigarettes with a high content of nitrate and volatile bases. Further work, while establishing the formation of iV-nitrosopyrrolidine aiid diiiiethylnitrosaiiiine in the smoke of these tobaccos, also indicated that the Ar-nitrosomcthylbutylamine was an artifact formed during the collection of the smoke (Neurath et al., 1965). The possibility that nitrous fumes in tobacco smoke could form nitrosaniines in tlie tisbues has been considered (Henxhler and Ross, 1963, 1966; Ross aiid Henschler, 1963). Thcse authors exposed mice to nitrogen dioxide for periods of 48 hours spaced twice weekly, every 10 days and every 30 days. The increase in lung adenonia was greatest in tlie last group who received the smallest dose of NO,. This was possibly accounted for by the fact that one dose of NO2 protects the lung to some extent against the next exposure. This protective mechanism was least effective when the intervals between exposure were greatest and the cellular reaction in the lungs of these mice was more marked than in those exposed more frequently. Henschler and Ross calculated that in 24 hours a t 100 respirations/minute of 0.3 inl. each a niousc would inhale 3.5 mg. NO, or 140 mg./kg. If 50% of this was converted to nitrosaniine in the tissues it would correspond to a close of 150 mg./kg.-yet with diethyliiitrosamine only 0.1 mg./kg. is enough to produce cancer. They concluded that nitrosamines are not formed in tissues exposed t o NO,. However, these calculations are not valid since they did not take into account tlic content of secondary amines in the tissues of the mice. Species may differ in the amine content of their various tissues. Investigations on the effects of nitroso compounds-real or suspected-on thv puliiionary and bronchial tissues are probably best carried out on the hamster, since the early changes have been well worked out in this
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species by Dontenwill and his colleagues. Herrold and Dunham (1963), in discussing the production of tumors in the respiratory tract of hamsters by diethylnitrosamine, emphasize that the nitrosamine need not necessarily be applied directly via inhalation. Rather the respiratory tract is sensitive to diethylnitrosamine by whatever route it is given. The carcinogenic activity of tobacco smoke may therefore result from materials swallowed as well as those inhaled in the smoke. The carcinogenic factors in polluted air may also include compounds of nitrogen, which have tended to be ignored while interest centers on either the oxides of sulfur or hydrocarbon materials.
C. CONCLUDING COMMENT It is too early to be able t o suggest with any confidence the part nitrosamines might play in the etiology of human cancer. I n a whole range of experimental animals tumors can be produced in a number of different sites which bear in some cases a striking pathological resemblance to cancers seen in man. However, the species, the nature of the nitroso compound, and the dose and route by which it is administered can all play a part in determining the nature of the malignant lesion produced. One may hope that in the years to come a further study of the carcinogenic activity of the nitroso compounds may not only reveal more about the chemical changes within the cell or tissues that precede the onset of malignant growth but also information on dose-response relations for chemical carcinogens that may make it possible to carry out better investigations into the role of environmental hazards in the etiology of human cancer.
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THE SULFHYDRYL GROUP AND CARCINOGENESIS J . S . Harington* Chester Beatty Research Institute. Institute of Cancer Research. Royal Cancer Hospital. London. England
I . Introduction . . . . . . . . . . . . . . I1. The SH Group and Normal Cell Division and Growth . . . . I11. The Stimulation of Cell Division by SH Groups . . . . . IV . SH-SS Cycles in Cell Division . . . . . . . . . . V . Growth Inhibition and Stimulation by Carcinogenic Substances . . A . Growth Inhibition . . . . . . . . . . . . B. Growth Stimulation . . . . . . . . . . . . C . Alternating Growth Inhibition and Stimulation . . . . . VI . The Interaction of Carcinogens with SH Groups . . . . . . A . Polycylic Aromatic Hydrocarbons . . . . . . . . B . Hormones . . . . . . . . . . . . . . C . Hepatocarcinogenic Substances . . . . . . . . . D . Alkylating Agents . . . . . . . . . . . . E . N-Alkyl-N-Nitroso Compounds and N-Nitrosodialliylaiiilin~.~ . . F. 4-Nitroquinoline N-Oxide . . . . . . . . . . G . Lactones, Quinones, and Related Substances . . . . . . H . Metals and Metallic Derivatives . . . . . . . . . I . Arsenic . . . . . . . . . . . . . . . J . Polymer Cancers . . . . . . . . . . . . . I<. Radiation . . . . . . . . . . . . . . 1.11. The Interaction of SH-Iteactivc Substancc)s of Unknown Carcinogenic Activity or without Carcinogenic Activity . . . . . . . A . SH Reagents . . . . . . . . . . . . . B. Lachrymators and Vesicants . . . . . . . . . . C. Alloxan . . . . . . . . . . . . . . . D . Maleimides and Related Compounds . . . . . . . . E . Maleic Hydrazide . . . . . . . . . . . . VIII . The SH Group in Carcinogrncsis . . . . . . . . . . A . Skin Carcinogenesis . . . . . . . . . . . . B . Liver Carcinogenesis . . . . . . . . . . . I X . SH Metabolic Systems Possibly Involved in Carcinogenrsis . . . A . The Glutathione Reductase System . . . . . . . . B . Inhibit,ion of Systems Specifically Requiring GSH . . . . . C . The Synthesis of GSH . . . . . . . . . . . D . Possible Participation of Hormones in GSH Metabolism . . . X . Other SH Systems and Carcinogens . . . . . . . . .
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*Present address: Cancer Research Unit of the National Cancer Association of South Africa. South African Institute for Medical Research, P.O. Box 1038. Johannesburg, South Africa . 247
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I. Introduction’
The importance of certain aspects of sulfhydryl metabolism in cell division suggests that the sulfhydryl group (SH) might be a poirlt of attack in carcinogenesis, one of the consequences of which is uncontrolled proliferation. The possible reactions of Carcinogens with SI-I groups in vivo and in vitro have therefore been examined. I n view of the considerable reactivity of this group, it is not surprising t h a t multiple rcaction with many carcinogens takes place. This in turn makes it more difficult to prove the significance of spccific reactions involving SH groups and to define a primary lesion probably obscured by “irrelevant” reactions. I n order t o overcome this difficulty in interpretation, an attempt has been made to define hypotlietic:dly more specific systems involving SH groups which could conceivably be modified by carcinogens a t a primary stage in carcinogenesis. Normal cell division and growth have been found to be associated in some organisms with a SH-SS cycle which can be modified by SH compounds and cert:tin SI-I-reacting substanccs. These aspects are discussed and the de rmvo synthesis of glutathione (GSH) and certain SHcontaining, acid-soluble materials during the cycle in some organisms is pointed out. Since growth inhibition is a characteristic property of certain carcinogens, both it and the interaction tictween carcinogens and SH groups are considered in relation to spccific systems associated with cell division and growth. Finally, it is suggested that a t least one form of the carcinogenic process may occur in three stages: inhibition of cell division through interaction of carcinogen with critical SH groups, an overcompensatory response to this, and a subsequent and permanent loss of control of division. This sequence of events could occur by fccdback adjustment
’Abbreviations uscd : SH, sulfhydryl; SS, oxidized sulfhydryl (disulfide) ; GSH, rrduced glutathione ; GSSG, oxidized glutathione ; CJ SH, cysteine ; S, sulfur; BAL, 2,3-dimrrrapto propan-1-01 ; ADP, adcnosinc diphosplinte ; ATP, adenosine triphospliate ; DNA, dcoxyribonucleic acid; RNA, ribonucleic acid ; NAD(P), oxidized nicotinamide adrninr dinucleotide phosphate ; NAD(P) H,, reduced NAD(P), (formerly triphosphopyridine nucleotidc) ; Pi, inorganic phosphate.
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after the initial reaction of carcinogens with particular S H compounds. I n view of the evidence presented, it is envisagcd that tlic pertinent SH groups may be found within one or more of the systems in which glutathionc is rcduccd, oxidized, or synthesized. This study is not intended to be a critical review of the enormous literature concerning sulfur and SI-I metabolism in normal and malignant tissues, or of thc chemical reactivity of tlie SH group. I t s main feature is to inquire whether reaction hctn.ecn a wide variety of carcinogens and SH compounds occurs, and if it docs, to suggest certain specific systems in SI-I metabolism which may be worth further investigation. Wherever possible, rcferencc is made to cell populations or organisms which apparently adapt tlicmsclves in various ways to rarcinogcns, especially where an inhibition of their growth and division precedes a stimul a t’1011. The dangers inherent in singling out any one system from the many others involved in the control aiitl execiit’on of cell division is recognized. Stern (1959, 1960, 19621, i n p:irticul:ir, 1i:th consistently warned against the belief that any one component in the process of cell division is more important than another. This seems doubly significant in view of the prcscnt state of knowlvtlgc of tylicb of mitotic control in mammalian arid indeed, in other tissues. A second aspect which is recognized is t h a t emphasis is placed in this paper on one feature only of mnlignant ncoplasia, namely, uncontrolled proliferation or “excesive mitosis” (sce Biescle, 1962), and in this sense what follows must necessarily be a n oversimplification of the carcinogenic process. Other facets, for example, invasiveness outside the normal stroma and ability to metastasize, are considered to be out of the scope of the present review. II. The
SH
Group and Normal Cell Division and Growth
Shortly after the isolation of GSH, tlie proposal was made (Hammett, 1932) that the SH group “is the naturally occurring, essential and specific, chemical stimulus to cell division.” The SH-SS redox cquilibrium was considered of utmost importance: shifts to the left coulcl lead to division, to the right to retardation of growth. It is now widely accepted, liowcvcr, that thc existence of a single, causal, mitotic substance is unlikely, although rclativcly high intraccllular concentrations of SI-I arc rcquircd in plants, animals and possibly, in microorganisms for the operation of mitosis and division (see Mazia, 1954, 1959, 1961a,b; Stern, 1956, 1959, 1960, 1962; Swann, 1957, 1958). Swann (1957, 1958) considers that several general mechanisms are important in the control of division and must develop in parallel for the process t o take place. I n particular, a hypothetical “energy reservoir”
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and the GSH cycle could be involved as principal mechanisms in the carrying through of mitosis. There is reason to think that the GSH cycle represents some mitosis-initiating and coordinating mechanism; according t o Swam the cycle might itself be involved in the “energy reservoir” niechanism or might act by regulating enzyme activity through effects on the intracellular redox potential. I n eggs it seems (Swann, 1957) that the DNA cycle is not a limiting factor in the control of cell division; rather the supply of energy from the (‘energy reservoir” or the GSI-I cycle (both probably found in cytoplasm rather than nucleus) may be the essential features. Stern’s vicw (1959, 1960, 1962) is that the SH group is simply one factor of many in thc “mitotic pool” of metabolism. SH groups, for example, “can behave in mitosis as specific inducers only in the sense that as a pool they are rate limiting.” While this cautionary assessment is of great importance in any understanding of the part played by SH groups in cell division, several careful studies have shown that SH compounds can, in certain circumstances, stimulate division in conditions where ordinary nutritional effccts can be excluded. Ill. The Stimulation of Cell Division by
SH
Groups
The process underlying the entry of Amoeba into mitosis depends upon the fulfillment of a complex set of conditions which are quite labile and easily disturbed. Nuclear growth and division are increased on exposure of the organism to GSH, the effect being apparently a direct one on cell division (Voegtlin and Chalkley, 1930; Chalkley, 1942). The very marked increasc in the rate of entry into mitosis in a short time was produced a t such low concentrations of GSH that an ordinary nutritional cffect can be ruled out. Copper in high dilution inhibited nuclear growth and division whereas GSH increased it and could overcome to some extent the effect produced by the metal (Chalkley and Voegtlin, 1932). More recent studies (Jamcs, 1959; Padilla and James, 1960) have shown that the fission time, but not the generation time, of synchronously dividing Amoeba longa and the flagellate, Astasia longa, is reduced to one half or more by SH compounds. (The fission time is the time that the cells spend in the terminal portion of division.) It was considered that the S H materials, apart from controlling the redox potential of the medium, might have provided the cell with an overabundance of a compound normally synthesized from the culture medium. Cysteinc (CySH) and mcthionine proved t o be key factors in the reduction of thc fission time of Astasia, the effects being ascribed (Padilla and James, 1960) t o (a) both substances possibly being precursors of compounds
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such a s coenzyme ,4 or GSH, ( b ) as protein SH residues, CySH and methionine could be directly involved in the composition of the mitotic apparatus, and (c) their primary role could be one dealing with the oxidation and reduction of the cellular environment. CySH and GSII, applied externally to growing but nondividing yeast cells, induce division in such cells, in keeping with the known data of S H metabolism in the yeast (Nickerson, 1948; Nickerson and Falcone, 1959; Nickerson and van Rij, 1949). The effect is inhibited by penicillin and heavy metals, the influence of which is in turn antagonized by CySH (Nickerson and van Rij, 1949). Cell division in yeast cells appears t o be under the control of a “unit enzymatic mechanism,” probably a protein disulfide reductase system, in which SH groups play a n important part. A stimulatory effect of SH compounds on cell division has also been reported in studies of Tubifex, Chilomonas, fibroblasts, and of rat and mouse skin (see R‘loment, 1952) but these early investigations require reassessment. I n a larger context, the important contribution made by SH groups to growth and cell division has been emphasized many times in substantial contributions (see Needham, 1942; Brachet, 1950; Mazia, 1954, 1959, 1961a,b; Stern, 1959, 1960, 1962; Swann, 1957, 1958). I n one of these, Rlazia (1954) concluded “there is certainly no reason to discount the results of investigations showing a stimulating effect of glutathione and other SH compounds on either total growth or on biological, recognizable components of the growth process. Indeed, the regulatory function of SH compounds for so many enzymes of all classes . . . would make it inevitablc that a widespread relation would be found, and that in most eases addition of reduced glutathione and comparable solublc thiols would operate i n the direction of stimulation.” Work on radiation protection and tumor development suggests a stimulatory role for sonic SH compounds. The incidence of tumors in rats protected against X-irradiation by cysteaminc and cystamine was found t o be significantly higher than in unprotected animals surviving a n equal dose of radiation (see Pihl and Eldjarn, 1958). This suggests that these substances not only offered little protection against tumor development but may have actually augmented the number of tumors obtained. According to Pihl and Eldjarn (1958) the result might be partly ascribed to a co-carcinogenic action of cysteamine. Acceleration of the growth of Ehrlich ascites tumors by cysteamine, but not by cystamiiie or CySH, has been reported (Koch, 1955). GSH, although protecting rats from otherwise lethal X-radiation, had no effect on the ultimate incidence of tumors although this was remarkably high after protection by p-aminopropiophenone, parabiosis (Brecher et al., 1953), and cysteamine (see Pihl and Eldjarn, 1958). a-Methylcystcine has been
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reported (Connors and Ross, 1958) a s a growth-promoter of Walker carcinoma cells. Cystine or methionine, given in the diet or parenterally, significantly increase the rate of healing of experimental wounds (see Kinnamon and Sutter, 1965). However, stimulation of division by SH compounds is not a uniform phenomenon. For example, injection of CySH into a rabbit inhibited cell division in the lens epithelium, the effect beginning about 18 hours after injection and lasting 4 days (Pirie and Lajtha, 1959). Anoxia also inhibited cell division but in a different way to CySH (Pirie and van Heyningen, 1960) ; when the animals were returned to air the rate of mitosis rapidly returned to normal. With CySH however, the rate was still depressed after 48 hours. It seems clear from the above experiments that the reactions between CySH (or cystine) and cell constituents probably take place shortly after injection of the S compounds because there is then sufficient of the injected materials present in the aqueous humor. Depression of the synthesis of D N A by CySH was suggested as a possible explanation of the mitotic arrest; the latter amino acid has been shown to be toxic t,o different stages of Drosophila (Wilson, 1946; Plaine, 1955). Finally, it may be mentioned here that both lipoic acid and mercaptoethanol arc active inhibitors of morphogenesis (Brachet, 1962). Oxidized lipoic acid markedly inhibits normal development in sand dollar and sea urchin embryos but has only a slight effect on early cleavage. The abnormalities are probably the result of the oxidation of SH groups by oxidized lipoate (Wolfson and Fry, 1965). IV. SH-SS Cycles in Cell Division
This subject appears as part of many excellent articles and reviews on the biochemical changes taking place in cell division. Of particular relevance are the following references (Rapkine, 1931 ; Swann, 1957; Mazia, 1954, 1961a,b; Nasatir and Stern, 1959; Stern, 1956, 1959, 1960; Wilson and Morrison, 1961 ; Morris, 1965). The history of thiol participation in cell division goes back to Rapkine, who in 1931 suggested t h a t a cyclic oxidation-reduction process involving SS and SH took place during cleavage of the sea urchin egg. H e believed that a low concentration of soluble SH (as GSH) corresponded with a n increascd concentration of fixed SH, these effects being produced by the reversible denaturation of proteins during spindle formation. As a next step, GSH (derived from GSSG) would increase in level before division, activate glycolysis, and so initiate the metabolic events which would lead the cell into division. Neither Neufeld alld Mazia (1957) nor Sakai and D a n (1959) could confirm t h a t the princi-
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pal S H conipountl involved in Rapkine’s cycle of cell division i n tllc sea urchin egg was GSH. The cyclic fluctuation of a trirhloroacetic acidsoluble SH compound was, however, found to be fundamentally correct (see Mazia, 1959) except that the suhstance involvctl is not GSH hut a SH-containing protein or polypeptide. hlorc details of the nature of tliis SH substance which may play an essential part in the regulation of the dividing cell in inorc organisnis than one would be most valu:ihlc (Llazia, 1961a; Morris, 1965). I n the sea urchin egg free and hound SH groups follow the course suggested by Rapkine, that is, the levels of the two alternate reciprocally, so that low free SH reflects high bound SH and vice versa. Mazia developed tliis relationship into one which showed t h a t a decreasc of free SH occurred when acid-soluhle SH was oxidized during the reduction of the intramolecular SS bonds of the spindle protein to fixed SH bonds. Conversely, the rihe in solublc SH accompanying thc final stages of gelation of the spindle was produced hy the reduction of soluble SS associated with the oxidation of bound SII of spindle protein to form SS linkages. [Of special interest are observations t h a t cell division in sca urchin eggs can take place without the mitotic apparatus (Hiramoto, 1965) or without spindle or asters (Hiranioto, 1956).] It is now known that GSI-I does not represent the only form of free SH involvcd in ccll division; the manncr in which the presumed requirement for a high SH content is met varies with cell types (Stern, 1960). I n the lily microspore GSII plays an cssential part in division. There is an abaolute increase in the concentration of this substance before mitosis, and this high concentration persists until wcll after mitosis is coinpletcd (Stern, 1956, 1960; Nnsatir and Stern, 1959). I n the pea xcdling GSH also p:irticipatcs in rcartions specific to cells preparing t o divide (Hughes and Spragg, 1958). On the other hand, the critical division tliiol in the sea urchin egg, Chlorelln, and the yeast cell is a SH-containing protein or polypeptide (see hlazia, 1959) , though not necessarily the same substance. One species of bacteria has about 5 times as much SII in the rapid growth phase as thcre is in the corresponding spores Ihlortenson and Beinert, 1953), though the nature of the tliiol is unknown. There are strong indications (see Morris, 1965) that in some organisnis the onset of cell division is controlled by the synthesis of various spccific compounds, in particular, spccific “division” proteins and various sulfur-containing macromolecules. I n Chlorelln, coenzyme A and a sulfur roniponcnt, or componcnts soluhle in tricliloroacetic acid, play an essential part in cell division and increase considerably in quantity a t the time of nuclear division (Hase e t al., 1959; Tamiya, 1963). T h e acid-
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J . S. HARINGTON
soluble material appears to be a peptide-nucleotide (polynucleotide) with the sulfur in the peptide moicty in some form other than cyst (e)ine, methionine, and sulfate, and with adenine and uracil as the major bases in the nucleotide part (Tamiya, 1963). A substance with almost identical properties has been found in the trichloroacetic acid extract of yeast cells (Hase et al., 1959; EIase and hlihara, 1960). This sulfur-containing substance may turn out to be an important link between sulfur metabolism and mitosis (Mazia, 1959, 1961a), possibly being analogous t o the acidsoluble matcrial which fluctuates in the dividing sea urchin egg. Increases in content of the sulfur-containing nucleotide may also be associated with DNA synthesis. A related finding of some interest concerns the identification by Chang and Wilken (1965) of a liver nucleotide-peptide which is the unsymmetrical disulfide composed of COenzyme A and GSH. The compound was found in trichloroacetic and perchloric acid filtrates from guinea pig, rat, and bovine liver. Its function is a t present unknown, but may bear some relation t o the polynucleotide found in lower organisms (Chang and Wilken, 1965). Work on thc lily microspore by Stern (1956) and Nasatir and Stern (1959) supports Rapkine’s thesis that soluble SH compounds increase prior to cell division. In this particular species, GSH plays an essential metabolic role in the cell division process. Stern (1960) has found, too, that once a population of Escherichia coli recovers from poisoning by the SH reagent, N-ethylmaleimide ( a t a concentration which produces selective and reversible effects on cell division and growth), the onset of the second division follows after a much smaller interval of time than that found in the controls. I n some cases, there was no detectable interval between the two divisions. Stern (1960) ascribed this effect to the inactivation (by an agent reasonably specific for SH groups) of some factor necessary for cell division. The substance in question, possibly a protein, seemed to be formed soon after division had taken place, so that if the other necessary premitotic processes continued unhindered during the prolonged first interphase of the maleimide-treated cells, a second division quickly followed. This suggests an adaptive response by the cell to maleimide by way of an increased de novo synthesis of some SH protein, possibly an enzyme. Bacteria differ from the lily anther in their response t o maleimide (Stern, 1960). There is no drop in content of GSH, which may even increase, suggesting a blockage in the utilization of the tripeptide, a state analogous perhaps to that found in pea seedlings treated with maleic hydrazide (Hughes and Spragg, 1958). In anthers, the drop in level of GSH is associated with the arrest of mitosis by N-ethylmaleimide. Coincident with the onset of mitosis there is a rise in GSH concentration.
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It seems therefore (Stern, 1960) that in the lily anther a lowering of the soluble S H in the environment is sufficient to delay the onset of the mitotic cycle. The biochemical evidence a t present points to an induction of SH synthesis during interphase when the existing concentration is inadequate. This could be expected to be a de novo synthesis and not a GSSG-GSH conversion, although the latter does occur in certain plant cells preparing to divide (Hughes and Spragg, 1958). De nouo synthesis of GSH has been suggested elsewhere: in radiation damage in the rabbit lens (Gray, 1954), in radioresistance (REv6sz e t al., 1963), and after stress reactions associated with the release of adrenocortical hormones (Beck et al., 1961; Kalser and Rcck, 1963). It is unfortunate that very little is known about fluctuations of SH or SII-SS levels in mammalian cells under various conditions. I n the rabbit lens there exists a spatial corrclntion between concentration of GSH and growth rate (Waley, 1959). Regenerating rat liver shows a peak of mitotic activity coinciding with one in the nonprotein SH level (we Neish and Rylett, 1963). Also, the diurnal periodicity of mitosis in regenerating livers shows high and low levels of mitosis a t times when the GSH levels (of normal rat liver) are itko high and low, respectively. Cyclic variations in distribution of SH and SS occur during the estrous cycle of the mouse, and are probably concerned with SH-SS conversions in keratinization (ilsscher and Turner, 1955). Studies of experimental carcinogenesis of skin and liver, t o be discussed in more detail later, report increases of acid-soluble SH material; these may be related, perhaps specifically, with carcinogencsis, although the exact nature of the compound or system involved has not been elucidated. I n recent studies of liver carcinogenesis induced by aminoazo dyes, there is evidence that GSH is involved (Calcutt, 1960; Calcutt e t al., 1961; Dijkstra, 1964), though this is not yet beyond question (Fiala and Fiala, 1959). Two aspects of the above section are of interest: ( 1 ) the correlations found between SH levels and cell division, in particular, the increased levels of free SH in cells ready to divide; and (2) the apparent de nozlo synthesis of GSH or SH to provide for this increase, as indicated in certain studies. V. Growth Inhibition and Stimulation by Carcinogenic Substances
A. GROWTHINHIBITION Physical and chemical carcinogens under certain conditions markedly inhibit the growth of animals, tumors, and cells. It is possible that these effects may bc due to interference with SH functions in the control of cell division. Brachet (1950, p. 1 1 ) has suggested that reaction between
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carcinogenic hydrocarbons and SH groups could slow down division and cause mitotic abnormalities of the SH proteins vital for this process. One of the most striking characteristics of carcinogenic polycyclic hydrocarbons as a class is their unusual and strong growth-inhibitory property first observed by Haddow and his associates (Haddow, 1938; Haddow and Robinson, 1937; Haddow et al., 1937). Normal animals, when injected or fed with carcinogenic hydrocarbons, suffered a rapid inhibition of growth ; this also applied t o tumors treated with carcinogenic hydrocarbons. The action is not a specific one but affects growth of the body a s a whole. No inhibitory effects were produced by a number of related, but noncarcinogenic, hydrocarbons, and a striking degree of correspondence was found between the inhibitory and carcinogenic activity of closely related compounds. Haddow and Robinson (1937) drew attention t o thc paradoxical situation in which the continued exposure of normal cells to strong growth-inhibiting agents could apparently rcsult in the appearance of rapidly growing, malignant tumors. An analogy between the growth-inhibitory effect in tumor production and the seeoridary colony development in bacteria was later made (Haddow, 1937) : cancer could rcsult from the cell freeing itself from the inhibitory effects of carcinogens to produce a new cell race without control of division. Among suggestions made t o explain the growth retardation caused by polycyclic hydrocarbons was that these substances caused a spccific deficiency in S-containing amino acids by using them in detoxification reactions (White and White, 1939). Others found this explanation unlikely and concluded that while their results supported the idea that combination with SH-containing substances was an early stage in the metabolism of carcinogens or of their biological action, the growth-inhibitory effect was not a direct result of this (Elson, 1958; Elson e t al., 1945). As an alternative, the proposal was made that carcinogens might interfere with the availability or the actual synthesis of proteins ncccssary for cellular growth. Another suggestion made somewhat earlier was that growth inhibition might be caused by carcinogens interfering with the normal functioning of the pituitary gland, perhaps suppressing the secretion of growth hormone (Haddow, 1938). It is now known that this substance contains four SS linkages (Lehninger and Neubert, 1961), and that it can act biologically through reaction with thiol groups; for example, it causes mitochondria1 swelling, by an SS-SH interchange reaction (Melhuish and Greenbaum, 1961; Lehninger and Neubert, 1961). The participation of growth hormone in possible SH reactions a t some primary stage of carcinogenesis is worthy of further attention for not only is there a connection between the action of the hormone and GSH levels, but also one between GSH levels and body size. As long
THE SULFHYDRYL GROUP AND CARCINOGENESIS
257
ago as 1942 Needham considered the possibility that GSH levels might be controlled by pituitary growth hormone because injection of this material into adult rabbits, which had stopped growing, caused a rise in the GSH content (but not in ascorbic acid) of their muscles; this could obviously not apply to stages of development before the appearance of thr pituitary, a s Needham points out. The levels of free SH in livers of rapidly growing rats are higher than those in tlie livers of normal mature rat,s, but stimulatioii of the lattcr with growth hormone increases the liver GSH level by 16% and by 19% in liypophysectomized animals (Bartlett et al., 1956; see Jocelyn, 1958). Growth hormone has been shown (in experiments on tumor growth and its effect on liver GSH levels) t o be associated with an increased synthesis of GSH (Shacter and Law, 1956; see Jocelyn, 1959). Thc apparent effectiveness of the hormone in restoring the response of hypophysectomized and thyroidcctornized animals to carcinogens has also been demonstrated (Bielschowsky, 1958, 1961), and it is of considerable interest t h a t the prolonged administration of growth hormone to rats (by the intraperitoneal route) results in the induction of a variety of neoplasms (Moon e t al., 1950a,b,c). As might have been expected, the rats injected with the hormone showed a very marked increase in body weight over the control animals (Moon e t al., 1950a). Support for the belief that growth hormone may be implicated in the growth-inhibitory effect of the carcinogenic polycyclic hydrocarbons by its action on SH levels may be found in Needham’s proposal (1942, p. 425) that the SH group could be a n “intermediary between the genotype mid hereditary size limitation in the phenotype.” This was based on earlier evidence (Gregory and GOSS, 1933) showing that there was a higher concentration of GSH in tlie embryos of larger rabbits than there was in races of smaller rabbits. This applied even after a small race of rabbits had been crossed with a large, when intermediate values for adult weight and GSH concentration in the embryos a t birth were found. The method of estimation of GSH t h a t was used did not exclude the presence of other reducing compounds such as ascorbic acid, which accounted for about 30% of the total values found. In another study (Painter, 1928) it was found that in races of both large and small rabbits, the ova of which were of identical size, the larger embryos possessed more cells than the smaller ones, and had a higher rate of cell division. Such a relationship was found in birds as well, though was not as clearly defined and could be established only after the eggs had hatched (scc Needham, 1942). The final conclusion drawn from these studies (see Gregory and Cross, 1933) was that size inheritance is associated with SH concentration, since (1) SH concentration is rclated to adult racial size, (2) birth
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J . S. HARINGTON
weight is associated with adult racial size, and (3) SH concentration is related to the rate of cell proliferation of the zygotes for the respective races. From this evidence emerges the possibility t h a t the growth-inhibitory action of the carcinogenic polycyclic aromatic hydrocarbons may result ( a ) from a general systemic suppression of GSH synthesis by a direct action of the carcinogens on GSH synthetases or on some other point in the synthesis of GSH, or ( b ) from an interference in the hormonal control of GSIl synthesis for the whole body, in which process growth hormone may play some key part.
B. GROWTHSTIMULATION Carcinogenic hydrocarbons have growth-stimulatory effects which, in general, arc not found in noncarcinogenic substances, although possible “over-sl1ooting” phenomena-such as are scen in certain reported growthstimulatory effects by radiation (Upton, 1963)-should be considered in any interpretation of results of work on the stimulation of growth. “Over-shooting’’ has been defined by Upton (1963) as “the relatively synchronous resumption of mitosis by cells accumulated in interphase through mitotic arrest.” Carcinogenic hydrocarbons significantly enhanced the growth of a nuiiiber of organisms including Paramecium, Obelia, and yeast cells, and GSH itself resembles certain carcinogenic hydrocarbons in stimulating the regeneration and multiplication of planaria (see Cook and Kennaway, 1940). C. ALTERNATING GROWTHINHIBITION A N D STIMULATION Growth-inhibitory and growth-stimulatory properties may also vary according to tlic concentration at which the carcinogens are applied, or may be so interrelated that a sequence of inhibition and stimulation of mitotic activity could lead t o malignant changes in cells. I n their study of the changes induced by methylcholanthrene on the mouse skin, Cramer and Stowell (1942) concluded t h a t a sequence of inhibition and stimulation of mitotic activity could lead t o malignant changes in the treated cells. The immediate effect of the first application of methylcholanthrene was injury t o the skin epithelium; after subsequent application the toxic effect was marked by an increasingly active stimulation of cellular proliferation. The authors concluded t h a t “the carcinogenic property of a hydrocarbon for the skin depends on its ability to inflict on the tissue an injury of a specific kind which leads to the formation by the skin of a substance inducing epithelial proliferation.” Wolbach (1937) had earlier come to rather similar conclusions. Following the ad-
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259
ministration of hydrocarbon carcinogens t o the mouse skin, a period of hyperplasia occurred after some degree of “resistance” to the carcinogen had been acquired by the affected cells. The hyperplasia seemed to be a reparative process which was niaintained and which preceded the developnient of the tumor. The concept of “resistance” is evident in studies of rat fibroblasts treated with 7,12-dimethylbenz [ a]anthracene (Starikova and Vasiliev, 1962). At all concentrations tested, this hydrocarbon caused severe inhibition of mitosis and growth in cultures of normal cells. I n the ease of explants of induced sarcomas, however, only the highest concentration of hydrocarbon had a n inhibitory effect and in some cases the lower concentrations appeared to enhance the growth of the tumor cells. Normal fibroblasts proved to be much more sensitive to the growth inhibitory action of dimethylbenzanthraccne than cells of rat sarcomas. Increased resistance was scen not only in sarcomas induced by the same hydrocarbon but also in those induced by subcutaneously implanted ciellophane films. The rcsults wcre taken to be in ‘lagreement with the suggestion t h a t formation of a new cell type rcsistant t o the toxic action of carcinogen occurs in the course of chemical carcinogenesis.” Another study (Hradec, 1959) showed t h a t although both carcinogenic and noncarcinogenic substances enhanccd and reduced growth a t different concentrations, only the carcinogens showed an alternating inhibiting and enhancing effect. VI. The Interaction of Carcinogens with SH Groups
This section is based in part on the contention of Brachet (1950) who, in discussing the possible niotle of action of carcinogens through SH groups, concluded (p. 182) that the “facts are understandable if the primary action of carcinogenic agents is concerned with the -SH groups of proteins.”
A. POLYCYCLIC AROMATIC HYDROCARBONS Apart from certain detoxification reactions which are not specific for carcinogenic hydrocarbons (Boyland, 1963, 1964a; see Furst, 1965, p. 472), there is little evidence of direct reaction of SH compounds with polycyclic aromatic 1iydrocarl)ons. Combination of benzo [ a]pyrene with t,he SH groups of CySH and other SH compounds has been occasionally rcported (Calcutt, 1949) and there is evidence (see Rondoni, 1955) that SH levels in vivo and SH enzymes in tissue preparations are lowered and inhibited respectively, by carcinogenic hydrocarbons. Wood and Fieser (1940) and Fieser (1941) suggcsted the possibility of the formation of
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SH derivatives of polycyclic aromatic hydrocarbons which could be intermediates in detoxification, carcinogenesis, or both. Several condensation products of polycyclic hydrocarbons with SH compounds were prepared, among them benzo [ a ]pyrenyl-5-mercaptan, benz [ a ]anthracenyl-7-methylmercaptan, benzo I n ] pyrenyl-6-S-cysteine, and benz [ a ]antlirocenyl-7-metliyl-S-~-cysteine (Wood and Fieser, 1940; see Cason aiicl Ficscr, 1940). It was later concluded (Fieser, 1941) t h a t reactions between such groups as OH, NH,, and SH seemed unlikely to be a primary step in carcinogenesis because noncarcinogens could also bind through such groups. This view does not, however, exclude the possibility of a reaction of the carcinogen with specific SH groups. Other studies (Elson, 1958; Calcutt, 1960, 1961; Calcutt e t al., 1961; Calcutt and Coates, 1961; Calcutt and Doxey, 1962b) support the concept that combination with SH-containing substances may be an early stage in the metabolism of carcinogens or in their biological action. 1. Anticarcinogenesis
Useful rcfcrences on this subject are to be found in Calcutt (1961) and Falk e t al. (1964). The early and fundamental work in this field is undoubtedly that of Crabtree (1941, 1944, 1945, 1946, 1947, 1948). The possibility of a carcinogen-SH reaction led him to believe, like Wood and Fiescr (1940) a t one timc, that two concurrent reactions could l x envisaged: (1) the detoxification of carcinogens by processes involving reactions with sulfur, and (2) the combination of carcinogens or their derivatives through S linkages as a primary phase of the carcinogenic process. These views were based on the antagonism of hydrocarbon carcinogenesis by inhibitors of S metabolism as a result of the prefercntial or conipetitivc combination of the inhibitors with SH-containing cell constituents. The inactivation of SH groups by carcinogen could be antagonized in three ways: 1. B y the use of inhibitors of S metabolism, for example, bromobenzene. 2. B y the use of competitive reactions between carcinogen and noncarcinogen for the active center of the SH enzymes believed to play a role in tumor induction. 3. B y the presentation to the cell of a suitable SH compound which might fix the carcinogen preferentially by a mechanism similar t o that iiormally occurring between a carcinogen and a specific cell substrate. Crabtree’s work showed that the presentation of any SH-containing molecule was not in itself sufficient to produce a biological effect ascribable to the SH group. The total molecule, with the SH groups ap-
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propriately disposed, was of prime importance. All the nionot1i:ols tested were without effect on hydrocarbon carcinogenesis, whereas BAL (2,3cliiiierca~~topro~~:tn-l-ol), a dithiol, had a retarding effect. This wa5 later confirmed by others (see Lusky et al., 1947). Heidelberger and Moldenhauer (1956) found that cantliaridin, malcic anhydride, and hcptaldehyde, all inhibitors of carcinogenesis, also inhibited the protein binding of benzo [ a ]pyrene and dibenz [a$]anthracene, whereas BAI,, which they found noninhibitory, did not affect binding. This work, except for tlic results with BAL, confirmed the earlier work of Crabtree (1941, 1944, 1945, 1946, 1947, 1948) and was itself supported by the findings of Calcutt (1961) and Falk e t al. (1964). The evaluations of tumor-inhibitory effects rnade by Heidclberger and Moldcnhauer were based on thc production of papillomas and carcinomas which were both inhibitccl I)y nialcic anhydride, cantharidin, and heptaldchyde. Examination of thoir results shows however t h a t BAL, while having no effect on papilloma production by the carcinogens used, reduced the number of carcinomas from 4 to 1 for bcnzo[a]pyrene and from 14 to 5 for dibenz[a,h]antliracene. So it can be taken that BAL does have a retarding or anticarcinogenic effect. Taken in consideration with tlic rcsults of the earlier work on anticarcinogenesis, these findings suggest that certain hydrocarbon carcinogens bind or react with SH groups in the cell, and t h a t this proccss can he prevented or diminished, apparently competitively, by certain anticarcinogciis. Fall;, Kotin, and Thompson, however, in their recent study ( 1 964) of aiitic:irciiiogciiesis, do not consider that a single mechanism is sufficient to explain all aspects of the process. Although competition for receptors, for instance, SH groups, might be important, such a concept seems inadequate to explain thc inhibition of bcnzo [ a ]pyreiie carcinogenesis by related polycyclic liydrocnrbons a t a molar ratio of antic:wcinogen t o carcinogen of less t1i:in one. The pioneering work of Crabtree indicates that if a n SH system is involvcd in tlie primary stage of carcinogenesis, it could possibly possess a t least two SH groups arranged in a suitable manner for “caicinogeiiic” interaction, in a n analogous nitinner, perhaps, to tlie presentation by lipoic acid of two SH groups to :menicals (see Peters, 1963, p. 45). Crabtree concluded that his rcsults could only be explained in terms of a concept of reversi hility imicle possible hy a n occnsional interference with SI-I nictabolisni in which the carcinogcn would form primarily a dissociable complex with cell constituents through S linkages. Wood and Fieser (1940) had suggested earlier that such :i reaction coul(l possibly Lw rc1:itcd to tlic first st:ige of hydr~carhoncarcinogenesis. Crabtree was one of tlie first to introduce the concept of competitioli
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between carcinogen and SH reagents for a common primary site, a process which he believed might be an early and fundamental phenomenon in carcinogenesis. He also drew attention to the possibility of competition between detoxification and carcinogcnesis; this idca has been subsequently supported in later papers (Calcutt, 1961 ; Schoental, 1961; Boyland, 1963). In this context, Schoental (1961) has pointed out that the binding of carcinogens with SH groups i n vivo (if this occurs) is likely to differ fundamentally from that of noncarcinogens, many of which are excreted as mercapturates. The action of carcinogens, she writes, is irreversible and compatible with the survival of the affected cells. The interaction products may possibly remain irrcvcrsibly i n a t u , so preventing the physiological replaccment of sonic specific SII compounds the action of which they block. Or alternatively (aiid the presetit hypothesis is concerned principally with this possibility), such an inhibition of some specific SH compound or enzyme may, by suitable feedback adjustment, eventually result in a compensatory response by which the compound in question is oversynthesized and maintninetl a t a new and permanently high level in specific parts of thc cell conccrncti wlth the control of cell division (Harington, 1967). 2. Cigarette Smoke
Tobacco smoke absorbed in buffer irreversibly inhibits SH-containing enzymcs; the effccts-apparently due to peroxides formed from free radicals-arc abolished by tliiols a t high concentrations (Lange, 1961). Preinhaled smoke has a negligible effect (Lange, 1961 ; Tongc, 1962). The catalyzed aerial oxidation of CySH by tobacco smoke has been ascribed to reaction between intermediate thiopcrosy radicals ant1 SH groups (Tonge, 1962). The watcr-insoluble fraction of cigat ettc smolte condensate hemolyzcs red blood cells, while smolte trapped in I)ufl'er contains several SH-binding substances and SH-enzyme inhibitors (Sat0 et al., 1962). Insofar as hemolysis is concerned, GSII is known to play an important part in maintaining the viability and stability of crythrocytes (Harley, 1965; Sass et al., 1965) and GSSG reductase is possibly associated with this function (Valeri and 1LIcCalluni, 1965). Erytlirocyte aging may be due to defective working of glycolytic enzymes PO that SH levels become diminished. Lower levels of GSII and GSSG h a w bcen found in older erythrocytes than in younger. This diffcrerice has not been substantiated in other reports, however, though SII cnzynics do npp a r t o decay in activity during aging of tissues (sce €I:wl~y, 1965 for references).
THE SULFHYDRI’L GROUP AND CARCINOGENESIS
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B. HORMONES
A biological relationship has beeti shown to exist between ctircinogenic and estrogenic activity : csti-ogcns are both growth-promoting and growth-inhibiting agents, producing cffccts like those of certain polycyclic hydrocarbons (Badger e t nl., 1942). I n this the estrogens are it second example of the apparently paradoxical situation in wlnch coinpounds possessing growth-inhibitory properties may also ultimately stimulate growth to a carcinogenic state. C1icmic:il and biological conncctions between certain carcinogciiic su1)~tancesand sex lioi moms have also been cnip1i:isized (see Nccdham, 1942, p. 400; Brachct, 1950, p. 243; Sexton, 1953, p. 340). Interaction of hornioncs with SH groups appears to bc extensive, and changes in SII levels tire influcwxd hy xtli.en:il, thyroid, pituitary, panciwitic, and pnrathyroid hormones ( ~ C C Iazarow, 1954; Jocelyn, 1958, 1959). I n the case of certain hornioncs not :ipparcntly rcl:ttcd to carcinogenrsis (for cxarnplc, the antidiuretic l~ornione, v:t~opressiii, and insulin), there is evidence that a covalent bond involving S linkage is formed in tlic interaction of the hoimonc and its rcccptor (hce Black, 1963). The tlisulfide bond thus fornied is the result of a SI-I-SS exchange ieaction (Fong e t al., 1960; Rasnius,icn et al., 1960). The concentration of both GSH and glutathione rcductase in some tissues is a function of tlic cndocrinc state (Lehninger antl Neubert, 1961). The enzyme is very strongly inhibited by the free phenylalanyl rliain of insulin in the reducccl state (T,angclon, 1960) and by thyroxine (hlize and Langtlon, 1962; sec .Jocelyn, 1959), while synthesis of GSH in certain situations is depcnclcnt upon carhohydrate metabolism aiid insulin ( I h h l , 1953). Hormonal control of SH inetabolisiii is strongly suggested in several studies (see Kasbeknr et nl., 1959). Stress of different kinds teniporarily depletes liver SH levels (Beck e t al., 1961; Knlser and Beck, 1963; Beck and Linkenhcimer, 1952; Beck e t nl., 1954; Bartlett and Register, 1954; Register antl Bartlctt, 1954b), mainly by inhibiting the synthesis of GSIl (Beck et nl., 1961). The levels of SH arc later restored, as shown for liver in one study (Beck and Linkenheimcr, 1952) and for kidney in another (Register and Bartlett, 1954b), by overcompensation or temporary d c 7 1 0 1 ~ 0 ovcrsyntlicsis of SH. The stress-SII connection seems rrasonablc since it is known that injcction of adrenaline (Register nncl B:wtlctt, 1954a) :tiid corticotropin (ACTH) (Goldzichcr e t al., 19531%)decreases tissue SH levels, perhaps, in the case of adrenaline, by iricrcasing the turnover rate of GSH in the liver (IIcnriques et nl.,
264
J . S. HARINGTON
1957). Adrenocortical function and control of atlrcnal GSII arc related, the adrenals playing a part in the synthesis aritl metabolism of liver GSH (Goldzieher e t al., 1953b; Wellers arid Aschkcnasy, 1957 ; Goldzieher et ul., 1958). The results of other studies (Coon e t al., 1949; Seneca et ul., 1950; Anderson e t al., 1951) testify to the appalt~ltly antagonistic effccts of adrenal steroids and SH-containing substances. Hemingway’s work (1960, 1961) implies t h a t active hormones such as cortisone and corticotropin act as growth-inhibitory hormones because their withdrawal from the rat allowed an approximate doubling of the normal rate of mitosis in the regenerating liver. The inhibition of mitosis by cortisone was overcome proportionately as the dose of growth hormone was increased. Withdrawal of the inhibitory state inclucecl by cortisone and corticotropin lcd t o a growth horniorie cffcct and the initiating of cell growth. This study is an interesting approach to the question of hormonal equilibria arid control in cell division and may have w r y wide implications. I n another approach, Alioduszcwska (1964) iound that corticotropin stimulated the growth of nw1:inonxi in vitro nnd hydrocortisone inhibited it. Repeated surgical traun1:i has n stimu1ntoiy effcct on tumor growth in transplanted and chcnnc:illy induced neoplasms, appearing to act as a co-carcinogen (Gottfrictl :~nclP\Iolomut, 1964). It is of interest t h a t the hormones used in the above studies are in some ways related to GSI-I metabolism. For example, the asbociation of growth hormone with GSH synthesis and the ability of thc hormone to induce neoplasms has already been discussed (see Section V,A) . In one study (Hess et al., 1951) it was shown t h a t although the lerels of total GSH and GSSG were unaffected by administration of corticotropiti and cortisonc, the level of GSH dropped, suggesting that the oxidatioiireduction state as a whole had been modificd. Such an action on the redox system is reminiscent of t h a t pertaining in rubhcr trees whclc growth-regulating hormoncs act by altcriiig the ratio of rcdurcd to oxidized, low-molecular weight SH substances (Barnes e t al., 1962), inost of which are present as GSH and CySH (McMullen, 1960). Finally, the idea has been mooted (Schocntal, 1963, 1964) that some specific, and possibly steroidal “mitotic hormone” may be inyolved in cell division. The biosynthesis of this niaterial could be associated with SH enzymes, vulnerable to attack by carcinogens. Alternately, some other stages in the biogcncsis of a steroidal cncl product m:iy bc afiected.
C. HEPATOCARCINOGENIC SUBSTANCES Certain aminoazo dyes and amincs, thioacetamide, ethionine, the Senecio alkaloids, carbon tctrachloridc, nitrosamines, and the aflatoxins,
THE SULFIIPDRTL GROUP AND CARCINOGEKESIS
265
are importaiit hcpatocarcinogens. Of them, the last two groups arc dealt with separately. Anticarcinogenic effects produced by other carcinogens against liver c:tiicer inducers have frequently been described, for instance, by niethylcliolaiitlirene (Richardson et al., I952), nitrogen niustard (Griffin et nl., 1951), Thorotrast and iron oxide (Spain and Clayton, 1958), antl copper (Iiiiig et al., 1957; Fare, 1964). Copper failed t o iuhibit carcinogcnesis 1)y 2-:iminofluorene, however (Goodall, 1964) . One of the enrlicht proposals that SH enzymes may be involvctl directly in licpntocai*ciiiogciic.sis was madc by Potter in 1942, when he suggested that the split products of p-diinethylaminoazobenzene, after oxidation, rniglit :act at- quinoncs and so inhibit essential SH enzymcs $is part of tlie carcinogciiic process (we also Burk and Winzlcr, 1944). The SH-eiizynic, umase, w:is rnarkedly inhibited by p-dimetliylaminoiizobeiizeiic split protlucts i n the osi(lizcc1, I)ut not in the reclucctl, state, and tlie inhibition w:is prcvcwtccl, hut not reversed, by CySH. Potter believed that the niecli:inisni might be R mutual oxidatioii-reduction lictwcen the enzyinc SII ant1 tlic oxidized split product, followcd by (BoInbination of the two end products. Inactivation of SH groups of )hosphntc cleliytlrogciiti~t~ hy split proclucts of p-dimethylaminoaxobenzene had been pobtulatetl :it about this time (Kensler et al., 1942). Kuhn and Beinert (1943) held that some azo dyes had a carcinogenic iictioii becauFc of tlicir mctal~olic conversion to fully oxidized forms which could possibly bc inhibitors of enzymes. GSH is rapidly increased following tlie injection of the strong hepatocarcinogen, 3’-methyl-4-dimcthylaminoazobenzene and other carcinogens (Ncish and Rylctt, 1963), hut decrcascs below the normal lcvel after the injection of a closely rc1:rtcd noncarcinogcn. Similar effects were found in regciier:tting livers of normal rats subjected to partial hepatectomy :tnd which c.ont:iinetl iiearly twice as much GSI-I as did control livers. Tlic import:incc of t h e in(’rcasc of lircr GSH in liepatocarcinogeiiesis has hecn correctly stressed (N‘cish a i i d Rylctt, 1963; Neish et al., 1964) 4ncr such a cliaiigc may be t l i c impctiis for liver cell proliferation. The results of other stii(lics of Iic.p:~toc~:ii~ciiiogciicsis (Fiala and Fiala, 1959 ; Dijkstrn, 1964; Dijkt-trn a r i d Peplcr, 1964) also show increases in the levels of solul)lc SII compouiitls :Iftei, tlic fcc(ling of niniiioazo dyes to animals; these studies are discussed in detail elsewhere in the present paper (bee Ycction \-III,B) . The cnrrinogciiic action of the clycstuff, light green FS, a substituted tiipliciiylinetliwne dyestuff structurally related to crystal antl geiitinn violet, probahly involves reaction with cellular SI1 groups (llillcr ant1 hlillcr, 1952). Crystal violet rcacts with protein SII gr011p+ to give a colorcvl dye protein-SH complcx (Braun, 1951); such formitlolit- iii:~y lie inpoiisiblc for the high toxicity of the triphenylmethane dyes (Hembcrg, 1949, 1950).
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J. S. HARINCTON
Reaction with SH groups has been suggested (Culvenor et al., 1962) a s a possible mode of action of the pyrrolizidine alkaloids, a group containing powerful hepatotoxins and carcinogens, and capable of acting as alkylating agents in the cell. Chromosome breakage in Allium roots by pyrrolizidine alkaloids can be prevented by CySH (Avanzi, 1961) although the way in which this is done is not yet known. Studies of ethionine hepatocarcinogenesis have thrown u p a few possibilities where defects in SH metabolism could occur. For example, the ATP-trapping propensity of ethionine (see Farber, 1963) could disturb GSH synthesis, possibly cutting it down. Rats injected with ethionine show a very striking decrease in the level of hepatic ATP and the latter substance (or certain of its derivatives) protects animals completely against the inhibition of protein synthesis and the induction of fatty liver by ethionine (Farber, 1963). A more direct biochemical reaction between SH groups and ethionine has been described (Brcwer and Greenberg, 1961) but is probably irrelevant insofar as ethionine carcinogenesis is concerned. R a t liver microsonies havc a n enzyme able to methylate foreign SH compounds such as 2,3-dimcrcaptopropan-l-ol (BAL) and mercaptoethanol. The donor for this transinethylation reaction is S-adenosylmethionine. I n two transethylations studied, S-adenosylethionine proved to be about as efficient an alkyl donor as the methionine derivative. “Physiological” SH compounds (homocysteinc, CySH, and GSH) were inactive as methyl acceptors; from the above evidence it may be inferred that X-adenosylethionine would in turn fail to donate ethyl groups to “physiological” thiol compounds.
D. ALKYLATING AGENTS A number of different properties have been reported for alkylating agents: they may be growth-inhibitory, carcinogenic, cytotoxic, mutagenic, arid may break chromosomes (see Ross, 1962; Wheeler, 1962; Elson, 1963; Warwick, 1963). As far as their mode of action as carcinogens is concerned, it is worth reiterating the warning given by Alexander and Stacey (1960) t h a t since the radioinimetic alkylating agents can react with almost all cell constituents, the detection of one particular reaction in a biological system need not constitute evidence that this process plays a part in the initiating lesion; this warning is of course timely when reactions between SH groups and carcinogens are being considered. Much has been made of the alkylation of nucleoproteins as the principal mode of action (see Wheeler’s review, 1962), but although DNA seems to be the most sensitive material involved in the process,
T I I E SIILb’HTDRYL GROIJP AND CARCINOGENESIS
267
reactions with other constituents may be equally, or even more, important. Nitrogrn niust:iid<, cthylcneimines, sulfonic acid esters, ant1 epoxides icict mitli n n u n i b c ~of different chemical groupings, including SH (see \l’lieclcr, 1962) . h “dctliiolation” of essential cellular constituents by ceitnin :ilkyl:itiiig agents, for esaniple, Mylerari (1,4-dimethanesulfoiiyloxyl)iit:incs), has been shown, and derivatives of alliylating agents with CySH havc bwn p i q m w l (Roherts :tiid Warwick, 19.58, 1959a,b, 196la,l~). (Thc status of RIy1er:in a s a carcinogen requires further ct1ucicl:ition; while it 1i:is hccn reported as being inactive both as a cnrcinogcn (Shirnkin, 1954) :ind as an initiator in skin carcinogcnesis (Roc and Snlnnian, 1955), a third study (Roberts and Warwick, 1961a) describes it as carcinogenic.) Urethane and iV-hydroxyurethane react slowly with the SH group of GSE-I (Boyland, 1962, 1963; Boylnnd et al., 1963). The N-hydroxy derivative, in acting as an alky1:iting agent, may perhaps be the active mctu1)olite re+ponbil)le for protlucing the r:tdiomimetic effect observcrl after tlic ncliniiiisti,atioii of urctlinnc itself (Boyland e t al., 1963). There IS cvitlcnce (bee Uoylantl, 1963; T3oyl:ind et nl., 1963) that some aromatic niniiicL: are carcinogenic aftor nr-liytli,osyl:itioii to arylhyclroxylaminc dcrivativcs w1iit.h can react with thiols. I3oth the carcinogen, 2-naphthyl:uiiine :tntl its tlerivntir-c, 2-n:~~~lith~lliytlrosyl:imine, like arylhydroxyl:~iiiines in geiicral, react with SII compounds, for example, GSH (in neutral solution a t room tempcr:iture) , t o give S-aminophenyl derivat’ives. Tumor resistance nnd crabs resistance to certain alkylating agents have h e n developed, 1)cing coiwlntotl in one study with a 11& to 2-fold ineinise of noiiprotciii $11 gi oups (Hirono, 1960) ; this increase could protect thc cells from tlamttgc by the ngcnt (\T”eeler, 1963). No correlation has bcrn found 1)etwcen SII content and the degree of natural resishnce of ascites tumors to alkylating agents (Hirono, 1961 ; see Wheeler, 1963). Tumor sensitivity is correlated with the ratio of protein-hound SII t o soluble S H (Calcutt and Connors, 1963) which helps detcimiine n.hethci* an alliylating ngcnt is detoxified by frec SH 01’ whether it inflicts tlnmnge on bound SH (Connors and Elson, 1962; Calcutt et nl., 1963; Calcutt, 1964). CySH reduces the toxicity of a number of aromatic nitrogen mustards which react with the SH group (C:tlcutt e f nl., 1963; Connors and Elson, 1962), possibly by i n c i ~ ~ s i n g the amount of frcc PH substances in the cells, perhaps t h a t of CySH itself, as h:in heen recwitly suggcstcd (Connors e t al., 1965). The incrc:isetl SH bul)d:inces woultl then he available for reaction with the alky1:iting :igCiit, so prtwmting a toxic renction with cellular sites. At :my rate in thc :tI)ovc studie.; (Calcutt et al., 1963; Connors et d., 1965), the mrchanism of the protective action appears to he an intracellular
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J . S. HARINGTON
reaction between thiol and the nitrogen mustard. Another suggested mechanism of protection from alkylating agents by SH compounds could be represented by the combination of the protective agent with protcin SH groups to form SS bonds (see Whceler, 1963).
E. N-ALKYL-N-NITROSO COMPOUNDS A N D N-NITROSODIALKYLAMINES These materials (see Magee and Schoental, 1964) apparently become carcinogenic after conversion in vivo to the corresponding derivatives, diazomethane or diazoethane (formed from dimethylnitrosamine and diethylnitrosamine, respectively). Both derivatives have bcen shown to interact with SH groups in vivo and also with RNA (see Magee and Schoental, 1964; Boyland, 1963; Craddock, 1965). N-Alkyl-N-iiitrosourethanes are effective carcinogens which interact nonenzymatically with SH groups in tissue preparations. Thiols (CySH, GSH) react with them a t room temperature and a t neutral pH, evolving nitrogen in the process and forming a complicated mixture of products (Schoental and Rive, 1965). N-Methyl-N-nitrosourethane, in particular, is a strong carcinogen, effective after a single dose. Its ability to substitute concurrently at several reactive groups is exemplified by its nonenzymatic, in vitro reaction with SH, when it yields both an active ethoxycarbonyl group and an “active methylene” (Schoental, 1966). Such reactions, according to Schoental, could be exercised a t specific “sensitive centres” in the cell, with sulfur links which appear to exist between nucleic acids and residual proteinaceous material. It is suggested that, provided they are compatible with cell survival, such specific changes in SH situations may lead to later tunior development.
F. 4-NITROQUINOLINE
N-OXIDE
This powerful mutagen and carcinogen (see Endo, 1958; Nakahara and Fukuoka, 1959) occupies a central position in the present thesis because of its rapid reaction within the physiological pH range with SH groups but not with other important cell constituents. No evidence of any direct reaction has been found between 4-nitroquinoline N-oxide and DNA, RNA (Endo, 1958), thymine (Endo, 1958; Searle and Woodhouse, 1963), adenine (Searlc and Woodhouse, 1963), or guanylic acid (Searlc, 1964). On present evidence the major reactions of 4-nitroquinolinc N-oxide may be said to occur with tissue S H and in vitro S H (Endo, 1958; Searle and Woodhouse, 1963, 1964), both of which take place rapidly under various conditions. Reactivity of the SH group in vitro has also been found to be correlated with the carcinogenicity of various nitroquinoline derivatives (Endo, 1958). At the same time, it is
‘11lK SULFHYDRYL GROUP AND CARCINOCEKESIS
269
important to strcsq, its Eiido (1958) did earlier, that it is not yet possible to conclutic that reaction with important cell constitucnts other than SH gi oups might not occur in vivo; labeling experiments should be done to chcck this. 4-Nitroquinoline A‘-oxide lias strong anticarcinogenic properties, inliihtiiig carciiiogcncsih by both benzo [ a ] pyrene and rlibenz [ a , h ]anthracciie (Scnrle aiid Rootlhousc, 1964) if applied simultaneously with the carcinogens a t tlic saiiic, but not a t separate, sitcs on mouse skin. The inhibitory effect docs not scorn t o be due t o simple, direct reaction with SH gi o u p ~beciuise maximal inhibition T W ~ S found after 4-nitroquinoline i\-ositlc 1i:id 1)ccii :Ipplictl 3 days before the bciizo [nlpyrenc arid not e:wlicr (Scnrle, 1965). Rcaction bctwecii 4-iiitroquinoline AT-oxide and SH groups is rapid so the 3-day effect suggests that if SH groups are involved in the anticarcinogenesis process in this particular case, the S-o.;idr mny iicctl suficicnt time to reach some site in t h e cell where tlic ciitical reaction is to take place; or alternatively, to modify sufficicsntly soiiie specific SI-I system t Scarlc, 1965).
G. LACTONES, QUINOKES, AND RELATED SUBSTANCES The lactones (see Dickens, 1964) represent a newly discovered group of carcinogenic substances aiid include as members, patulin (clavacin) , paiicillic acid, penicillin G , liexcnolartoiic, P-propiolactoiie, ancl the aflatoxins B arid G, both ol which have been shown to be a,p-unsaturated l:tcto11cs. 1,actoncs react extensively with SH groups both in vitro and in vwo, and SH substances are ant:tgoiiistic t o a variety of lactone antibiotics (Kraybill and Sliimkin, 1964; Dickens and Jones, 1961 ; Veldstra and I-Iavinga, 1944; I-Ittyiies, 1948; Scxton, 1953), for example, t o penicillin G, patulin, and penicillic acid. Tlic effect of lactones on cellular proliferation appears to lie cscrtcd through iwction with essential SH groups (for instance, CySII) of amino acids and proteins (Dickens and Jones, 1961) and t l i c i ~are iiidicntioiih t h t such reactions may be ciizymiitic (-4l-K:~ssabet nl., 1963). Reccntly llickenh and Cookc (1965) found tliat hctones nnd related substances showing lower reactivity with CySH were niaiiily of lon.cr carcinogenic activity or were noncarcinogenic. Afl:Ltoxin (R, and G,) proved to be a significant exception. Although l;ictoiics, t h e reacted only slowly with tlie SH groups of CySH. Possihly otlicr chciiiical features of thc molecule may account for their cstrcmcly potent carcinogenicity (Dickens aiid Cooke, 1965). A number of other interesting biological effects of lactones h a r e been dcwrilml. Thc growtli-iiiliil)itory action on plants of tlie unsnturatcd lnctones, couniariii, protoanenionin, and hcxenolnctonc, is prevented by
270
J. S. I-IARINCTON
BAL. SH-tlependcnt enzymes appear to be involved in this process, probably one which is normally a limiting factor in growth (Thimann and Bonncr, 1949a,b) , /3-Yi.opiolactone, a strong carcinogen, reacts with CySH to form S-J3-carboxyethylcysteine, which is very weakly carcinogenic, showing as Kraybill and Shimkin (1964) point out, “the cfficieiicy of sulfliydryl compounds in reduction of carcinogenicity.” Quinones as a class both oxidize SI-I to SS and react directly with SH groups (see fIoffniniin-Osteiiliof, 1963) and in certain plants participate in oxidation-reduction processes in which they inhibit mitosis. Reed (1947, 1949) has shown that mitosis becomes blocked if the oxidationreduction potential in the mcristems of the olive tree shifts so that the naturally occurring dihydroxyphenols (chiefly eatechols) becomc oxidized to quinones. If suitable hydrogen donors, such as SH compounds, are added in sufficient amounts to restore a reducing potential, cell division will recommence. Growth-inhibitory, unsaturated ketonic substances participate in the dormancy processes of certain plants; here GSH itself may play an active part (see Sexton, 1953; Leopold and Price, 1956). The suppression of bud dcvclopment in dormancy by growth inhibitors can be naturally relieved by the biosynthesis of GSH (Hemberg, 1949, 1950), and the dormancy factors involved may bc lactonc-like materials (Leopold and Price, 1956). Of substances rclatcd to lactones, mnleic and succinic anhydrides both induce cancer and react vigorously with the SI-I group of CySH in neutral aqueous solution (Dickens and ,Jones, 1965). On thc other hand, Lu,j3-dimethyl maleic anhydride, in spite of being a carcinogen, reacts only vcry slowly with SH under similar conditions. Since this also npplics to nflatoxin (Dicltcns arid *Jones, 1965), no simple correlation bctwccn SJ-I reactivity and carcinogencsis seems evident in thcse two instances, unless some specific SH system is involved.
H. METALSAND METALLIC DERIVATIVES Evidence from animal experimentation and epidemiological studies of man show that the following mctnls and metallic derivatives are carcinogenic: nicltcl, nickcl cnrbonyl, chromium, chromates, chromitc ore, hematite, iron ore, beryllium, selcriium, cobalt, cadmium, zinc, silver, and lead (see review by Roe and Lancaster, 1964). Special forms, such as the metal complcx, iron clextran (ferric hydroxide-dextran) , are also carcinogenic uridcr certain conditions. Cancer of the thyroid is possibly aspocinted with cnviroiimrntal tleficicncy of iodine (Axelrad arid Leblond, 1955; see Wynder, 1952) which, though not a metal, may be considered here for convenience.
T H E STJLFHYDRI'L GROUP AND CARCINOGENESIS
271
The oxidizing power of metals, their catalysis of the oxidation by air of S H compounds, and their effcctivcncss as riierc:il)title-forming agents :LIT well-known pi operties (see Madsen, 1963). Triv:tlcnt arsenic :and antimony, hivnlcnt lend, nici'cury, coppcr, cadmium, antl zinc, and monovnlcnt mercury, copper, silver, and gold readily form slightly dissociated mcrcnptidcs, the :iffhities for SI-I being roughly in the following order: Hg++,Ag+, Cut', Pb", Ni", and Zn++. Several rnetal-SH interactions in vivo arc of interest. Vcry low concentrations of cohalt inhibit those re:wtions catalyzed by ketoglutarate dchydrogcnnse in which tlic coenzyme form of lipoic acid functions in tlie reduced (dithiol) state (I-Ic:tth et al., 1961). Other cationb, for cx:imple, cadmium and zinc, in11il)it to the same extent or more effectively than cohalt, and coniplcte reversal of the inhibition is obtained in the presencc of BAL. In contrast, BAL only prevents the inhibitory action of cobalt if it is added either with or immediately after the cation. This inhibition may be rclatetl to thc demonstrated ability of col)alt to foi m :L chclate with tlihyrlrolipoic acid, the characteristics of which differ to sonic cxtcnt from those of the chelates formed by other divalent metal ions. Cobalt (and penicillin) in1iil)it division in yeast cells where a protein retluctase seems t o be princip:tlly involvctl : penicillin by promoting the oxidation of S11 t o SS,:~nclcol)alt by apparently forming coordination complexes with Cy8I-I (Nick(~rsonand van Rij, 1949). Selenite is a very active catalyst in vitro and in vivo in the oxidation of CySH, GSH, dihytlrolipoic acid, and coenzyme A, more efficient even than copper (Tbcii and Tuppcl, 19581, though it is not a strong inhibitor of SI3 cneyines (Tscn and Collier, 1959). The last-named authors consider that selcnium toxicity is probably not due t o direct inhibition of crizyine but to seleniuin-catalyzcd oxidation of such cofactors a s GSH, cocnzymc A, and tlihydrolipoic acid. Sclciiium is incorporated into various proteins in ninmmalian orgnnisnis (McConnell and Dallam, 1962) and may intcrch:angc with sulfur in plants antl microorganisms (Shrift, 1959, 1961). Division of Chlorella is inhibited by selcnomethionine but the effect is a temporary one, and inhibited cultures are able t o adapt and give rise to progeny which cannot be distinguished from the unatlaptcrl cells in most respects (Shrift, 1961). This seems to be a mass adaptation of the entire population :ind not the selection of resistant mutants; it is a ht:tble, liereditable change in tlie entire population of cells. Shrift bclicves that selcnoniethioninc, in blocking the incorporation of methionine into proteins, also cause5 the various intermediates between sulfate arid inethioninc t o accuniulate and act as inducers for an increased synthesis
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J . S. IIARINGTON
of tlic cnzyincs responsible for their nictabolism. It is believed that the end result would then bc the establishment of a stcady-state levcl for this particular metabolic route (the sulfate-reducing pathway) and a faster rate of methioninc synthesis which could then compete with the absorbed ~clcnonictliioiiinc.Sulfate or its products would he the actual inducing agents rather than scleiioiiictllioliilIc itself. Under sulfur staI*vation, the cnzynics involt ctl arc rctui.nct1 to tlicir normal lcvcls, giving cells OIICC again scnsitivc to ~clc~io~iictli:oiiiiie, a “dc-,zdaptation’Jpheiioni(won of niorc than passing intcrcst. Iodine is a uceful oxidizing agent for SE-I groups. Likc iodoacctic acid, it btops the clcavage of sen urchin eggs (R:tpkinc, 1937), probably because it iiihibits glycolysis. The effect is reversed when the eggs are placccl in GSH (Rapkinc, 1937; scc Rapkine, 1938; I<ensler et nl., 1942). Iodine (as sodium iodide) inhibits tlic glutathionc reductase of gcrminatcd peas (Mapson and Ishcrwood, 1963). Copper in certain circumstances has pi ofound effects 011 XI-I groups, particularly in wool, where a dcficicncy of copper in hhecp may considcrably increase the SH lcvcl of the wool fiber (Rlarston, 1950, 1952; B L W ~ 1954; ~ J ~ , H:wingtoii, 1961). Thew arc good re:isons, as Richmond (19Gl) has discussed, why SII groups might he in\ olvctl as part of iron-dcxtran carcinogcncsis ; such consiclcrations coultl well apply to asbestos and silica carcinogenesis m1iei.c nxtnls participate in catalytic rc:ictions involving SH groups (Harington and Roc, 1965).
I. ARSENIC Thc status of arsenic as a carcinogen is uncertain, cxccpt in thc casc of the intluction of skin c:~ncer in man ( w e V:illcc e t nl., 1960). Combination of nrscnicxls with SII groups has been cxtcnsirely studied (Peters, 1963; Johnstone, 1963) . I n their illurninnting studies of arsenicals Peters and his collengucs showed (Peters, 1963, p. 40 e t seq.) that monothiols did not protect against Lcwisite toxicity, whcrcas dithiols, in particular BAL, did, indicating tlic importancc of critically placed SH groups (in tlic form of reduced lipoic acid) in the p y r u w t e oxidase systcm with which BAL could form relativcly strainless rings. Resistance to arsenic as an insecticide is related in one specics of cattle tick (Thompson and Johnston, 1958; Harington, 1959; Whitehead, 1958, 1959, 1961) but riot in another (Roulston and Schuntner, 1960) to significant incrcascs in acid-soluble S1-I groups, in particular, GSH and CySH (Harington, 1959) . Sanguinarine, an isoquinolinc alkaloid found in ccrtain tylws of mustard oil used in cooking, reacts biochemically in a similar way to arsenicals, and is involved in the pathogenesis
THE bCLFHYDRTL GROUP A S D CARCINOtiEKESJS
273
of epidemic dropsy, a serious nutritional disease associiited with tlie pro1ifer:ttion of tlie bloocl vcsscls of tlie skin (see Peters, 1963, 11. 82 e t seq.) . Sanguinarinc, liowe~cr, does iiot weiii to he c:ircinogenic (Sclioental and He:itI, 1954) . Tlie :ilk:~loicl combines witli S1I gi oups in tlic pyruvate osiclasc systcni and tlie effect is rcvcrsctl by EAL (Peters, 1963) and 111 wiii(1 c a b c b 1)y CySII (II:iliiiii, 1957). J. POLYMER CASCERS Tlie origin of this type of c;arcinogcncsis ( SCT Alcsaiidcr and Horning, 1959), whether physical or chcniic.al, or h t h , is not yet liiiown. AS 1)o.jsible c:~uscs,both anoxia :ind “disco~iiiectioii”of tissue cells have beeii suggcstctl. 81 I groups 1ia1.c iiot I)ecii iriil)licatctl Ilut ni:iy conceir:ibly be if anoxia i):irtici1):xtes in tlils forin of cai,ciiiogcwcsis. Noriiial cells grown in culturc in itnosic conditions mere reported to have undergone malignant tr:insform:ition (Goldblatt aiid C:iincroii, 1953) hit this could not be confirnictl in a subscqucnt study. S:uiforcl (1965) found t h t no influcncc of :uinerohic trentniciit 011 tlic time or inanifcstnt,ion of the malignnnt conversion could be dctcctcd in fibroblasts. Otlicr objections 1i:icl I)cc,n ixisetl cwlier (see S:illfoi~(l)and sonie of tlic deleterious cffccts seen by Golclblntt and Caiiieroii (1953) might have been associated with t,lit: typc of culture iiietliuni usctl. Oxygen pressure witliiii cells plays an important part in radiobiological effects, and anoxi:t and SH coiiipouiids, in protecting aiiim:tla and microorgnnisms :qpinet tlic 1i:irinful cffccts of S - i x y s , arc alniost certj:iiiily related in some way (Cr:iy, 1956; Pill1 and Eldjarn, 1958; Alexander, 19621. Changes in osygeii tension in maiiimalinii liver cligests result in the reversible osiclntioii of GSI-I; in one study, liiglicr lcvcls of SE-I coinpountls were found in nnaerol)ic t h n in aerobic digests of tissues. During osygenation of tlic tissue tlic SI-I lei.els dcc~‘e:~se as a result of osicl:ition to SS (Hopliiiis niicl Elliott, 1931). Scnifc (1964), howevcr, in work on ratlioscnsitivity in cells, iountl that :inosin protlucctl only a small inciwwe iii noiiprotc~inSI-I contcnt :ind n sindl :iii(I tloubtful iiicre:tse in protein SH. Untler reduced oxygen tension (which could bc cxpcc‘tcd t o have :111 important effect on oxitlation-rctluctioii systcnis in the wll) tlic miouiit of SI-I groups in A ? ~ o c reiiiainccl ~u high during tlic first two hours of the cxperiment, wliercas under atmosplieric oxygen pi c1ccre:tse of SH occurred during tlic sniiie length of tiinc iC11:~lklcy ant1 Voegtlin, 1940). In this orgnniwni SII scv’iiis to rwt, in coiijriiict,ioii Lvitli osygcn in the rcgul:ttioii of cnzynic :ictii.ity, cspcci:illy of tlint coilcci*nc~l with nucle:ir growtli.
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J . S. HARINGTON
K. RADIATION Although protection against radiation damage by many agents, including SI-I compounds, has bccii widcly studied, no dircct relationship between protcction and radiation carcinogencsis has yet cmcrged. Thiols may possibly protect against radiation damagc because they lower oxygcn tension (see hlaycr and Patt, 1953; Salerno and Friedell, 1953; Gray, 1956; Pihl and Eldj:trn, 1958; Bacq and Alexander, 1964). The involvement of SH groups as a primary evcnt in radiation carcinogcnesis is also still problematical (see Bacq and Alexander, 1961) and several mechanisms may be involved, including D N A synthesis and SH metabolism. Gray (1954) considers t h a t if the likelihood of SH compounds being attacked during radiation is acccpted, a dosc of a fcw hundred roentgens, which will kill many cells, should not significantly reduce the activity of any onc type of SH-containing molecule unless the majority of the molecules of this particular type are preferentially situatcd with respect to the path of the ionizing particles. This appears to be the case with yeast cells where X-rays can completcly inhibit cell division (very probably through SH groups associated with this proccss) , yet have no effect on othcr aspects of cell metabolism, as measured by glucose oxidation and fermentation (Gonzalez and Barron, 1956). It is of some significance t h a t such irradiated cells had not bccn killed but had only lost their powcr of ccll division. Invcstigations on the possible existcnce in this organism of a specific coiitrolling mechanism for cell division would be fruitful. Such a system may opcratc through SH interaction (sec Nickcrson, 1948; Nickerson and van Rij, 1949; Nickerson and Falcone, 1959) or may be found in a separate coinpartmcnt of thc cell, cspecially scnxitivc to radiation. In fact, the concept of preferential effects bcing excrtctl by radiation and othcr carcinogens on critical S H sites in the cell would be undcrstandablc if it were assumcd that different and specific SH and SS systems wcrc housed in scparatc and compact units in thc cell. Eldjarn (1965) has recently drawn attention to this important mattcr, and Scaifc’s work (1964) indicatcs that only a small fraction of SII, localized in specific parts of the ccll, is probably involvcd in the development of sensitivity to radiation. A few specific enzyme systems have bcen shown t o be affcctccl by radiation. The growth of the bean seedling is inhibitcd by a decrease in auxin content caused by the inhibition of the enzyme responsible for the last stagc of the transformation of tryptophan to auxin (Gordon, 1956) . The system conccrncd is a lipoic acid-depcndent one strongly resembling the pyruvatc oxidase systcm in E . coli and mammalian tissuc (see Helc, in discussion by Gordon, 1956, p. 44).
THE SULFI-ITDRTL GROUP A K D CARCINOGESESIS
275
The coiitciit of GSH and glutathione reductasc falls progressively t o very low levels tluriiig tlic dcvelopii~entof cataract induced in rabbit lens hy irradiatioii (Piric, 1956; Piric c t al., 1953; van IIeyiiiiigcii e t nl., 1954). Four other ciizyiiics, not depcnclent on S1I groups for activity, were uiinffectcd, even in coiiiplctely opaque lenses. Thc GSIE-I content, the protein SfI content, :mtl the activity of glutathione reductasc were still urichaiigctl as late as 20 hours after irradiation and thereafter started to fall (Pirie e t al., 1953) ; 85% of GSH had been lost 9 months after irradiation and 63% of glutathionc rcthictase activity after 6 months. At thc same time as the fall of GSII, tlic level of A T P also began to drop, as shown in a separate study, : ~ n dthere is evidence t h a t both the licxosc moiiopliosphatc sliuiit arid aiiaci,obic glycolysis were impaired studies Gray a t the time (Imiiian, 1962, 1965). In discussing €'irk's (1954) concluded that irrat1i:ttioii a p l x w s to interfere with tlic syiithcsis of fresh glutathione reductasc rather tliaii to iiiactivate that which is prcscnt a t the tiiiic of 1rrat1i:ition. Tlic loiig-term effects of radmtion 011 GSH metabolisni arc of intclic.st ant1 i m y he directly related to tlie iriduc t ion of rntlia t ion c anccr . Total body irradiation of rats caused an initial iiicrciise of GSH in erythrocytes, liver, spleen, : ~ i i d1ie:irt iiiu,sclc for a period of up to 48 hours. The values later leI.clcd out ant1 after 21 days were Imcli to normal (Wernze et nl., 1965). The iiicreaic of GSH was ascribcd to a stimulation of GSH syritlicsis by radiation, particularly since tlie latter is known to decrease the lcvels of tissuc-ATP (Maass and Timm, 1964). P u t another wuy, the tlecrensc of ATP could he due to tlie stiniulution of synthesizing processes 1~iion.nto utilize ATP. I n a related study, Zic>lia e t a / . (196.5) fount1 that 24 hours after whole body irradiation, the GSH content of the liver increased to 169% of its initial v:Llue, after n h i c l i tliv v:~lucstlroppccl. Each riinxirnuni of GSH correspondcd with a minimum of GSSG rc(1uctase. I n the maturation of senile cataract in man, GSI-I and CyS1-I levels decrease distinctly in the lens while the lcvcl of 85 iiicrcases (Ilische :md Zil, 19.51). Loss of CrSII-syiitlicsizii~gactivity nlso occurs in Xirradiated lenses, though this docs riot seciii to be n primaiy effect (Daisley, 1955 ; see Wnley, 1959). SH groups in thymus riuclci arc not only oxidized hy X-irradiation but are made more retictivc toward SH reagents (Denkiii e t al., 1963; (3rd and Stocken, 1963). The fall iii SI-I content is inaiiitainetl (and detected) only if g1ut:ttliioiic reduct:w, a no1 ma1 componeiit of the iso1:tted ~iuclci,is iiihibitcd hy zinc (Zii- +) , hecnuhc the enzyme moultl rcverbe any oxidation of SII to SS. This cnzynic is pro1)al)ly important in reversing the oxidntivc effects of X-irradiation. Ord and Stocken
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J . S . HARINGTON
(1963) believe that agents which alter the nuclear SH + SS equilibrium produce metabolic effccts paralleled by X-irradiation. Thc reduction in the level of SH gi'oups in nuclei after irradiation effects of an oxidative nature may bc responsible, niiiong other tliings, for the early biochemical lesion and may chiefly account for the damage caused by irr:idiation (Smit and Stocken, 1963). Scaife (1964) has recently reported that irradiation decreased the nonprotein SH c o n t e n t m a i n l y GSH-of thymus cells and thymocytes, but not of ascites cells. The SH groups involved arc probably to be found in critical sites in the cell, and only a small fraction of SH may be involved in the sensitivity t o radiation. Resistance to anoxic radiation damage in cells is correlated with increased free SH levels in tumor cells (RBvBsz e t al., 1963; Caspersson and REvBsz, 1963; R6v6sz and Littbrand, 1964), the free SI1 probsbly acting as a physiological protector as it seems to do in arsenic and alkylating agent resistance. Thc ratio of nonpl otcin SlI to protein SH in cells also contributes to the development of radiosensitivity (Ri'vksz et al., 1963; Caspersson and Rbvirsz, 1963; Bott and IJuiidgren, 1964) and t o the protcction afforded by added mcrcaptuns (Siirho, 19G2 ; Ilkv6sz and Bergstrand, 19G3) . Results of studies employing SH inhibitors also implicate SH groups in tlic tlcvclopment of r:i&oresistnncc (Howard-Flanders c t al., 1963; Lee et al., 1963). Ascites cells treated witli the radioprotective substance, cystcaminc, sliowcd a Iargc incrc:isc of nonprotein SH of which about 3776 was duc to GSH (Rkvksz and Rlodig, 1965). Whether this rapid incieasc is clue to greatly accelerated, de novo syntlicsis of GSH is not known. Retluction of GSSG could only have playcd a small part i n augmenting thc G811 and R6vEsz ancl Alodig (1965) consider tlic foirnatioii of mixed tlisulfitles betwcen GSH and protein tlisulfide (GSSP) the most likely explan a t'1011. Diurnal variations in SII levels occur in both normal (Beck et al., 1958) and tumor tissues (Beck e t al., 1958; Calcutt, 1964) and in the latter case can be related to the radiosensitivity of cells and their protection against alkylating agents by SI-I compounds. In some radioresistant sublines, the nonprotein SH content pcr cell was not found to be associated with any considerable change in D N A contcnt. As a result it was concluded (Rbvbsz et al., 1963) that variant cells with an increased nonprotein SII contcnt per unit D N A could be induced and/or selected by sublethal X-irradiation such as had been shown earlier in the development of radioresistant populations. Such elevated SH levels appear to be the result of a stable alteration, and since the nonprotein SS lcvel is much too low t o account for the increases, the formation of higher levels of noiiprotein SII is therefore probably due to de novo synthesis.
T H E SCLPIITDRTL GROUP A S D C I R C I N O G E K E S I S
277
C1t r n v iole t R a &a t ion
Rlany cases of iii1iil)ition :tiid stimulation of growth and division have h e n rcpoitctl (see EI.I,cI.:L, 1959) , though a large pcopoi,tion of the stimuliitoi’y cffcvts niay be due to ‘*over-sliooting”(Upton, 1963). According to this :iutlior, one iiiingly valid stimulation occurs in yeast cells, mliere the iiici c : t s r ~ l rate of cell division persists through a number of gtiierat,ions, k i n g transmitted to successive cells, but not by niutntioii (Jaiiies and Rluller, 1961). 111 :in e:irly study, tlie alga, Stichococcus bacillaris, w:is fount1 to divide iiiorc r:tpidly after treatment with subIctlial tlo..cs of ultr:iviolct iri.atli:atioii. Tlic stimulation persisted for HCVCI.:II yoitrs, :iiitl the cells were sliortcr and witlcr than those of the original cwltui.ci (Illcicr, 1939; i\Ieier-Cliasc, 1941). The S1I groups of GSI-I (I?ose :mI Rnjewsky, 1962) and CySH (Foi~l)c~s and S:i\.igc, 19G2 ) are iii:~i~kcclly :\ffected by ultraviolet light, plio t 0- oxida t ioii and plio t ol y sis Iieing a common fi iitling. Cystine d isruptioii i i i ciizynicts may also occur (hugcnstein aiid Riley, 1964). There have bccii suggestions (Cantarow anti Scliepartz, 1962) that iriflammatory skin lesions caused by sunliglit iiivolre tlie destructioii of SH groups which ill one i1iYestig:itioii (Flcscli and Rotlinian, 1948) have hccn reported to be associated fiuictionally with skin pigmentation. VII. The Interaction of SH-Reactive Substances of Unknown Carcinogenic Activity or without Carcinogenic Activity
Except for tlie case of nialeic hytlraziclc (B:tines e t d., 1957), the 1:wge class of substniices (see Boyer, 1959; Dixon and TVebl), 1964), which react avidly with free and bouiid SII groups i?i W L V O aiid ~n v i t w lias unfortunately not bccri tested cxtcnsively for carcinogenicity. Among the group arc the well-known SH reagents, iotloacetatc, chloroacetopliciioiie, :incl otlic’rs, tlie l a c l i r y m a t o r ~aiid vcsic:tnts, alloxan, ant1 tlic mnlcimides.
A. SI-I REAGEUTS Iodoxcetic acid and cliloroncctoplicnone have tumor-promoting activity (Gwynn ant1 S:il:ani:~n, 1953). 1oclo:tcetate is t: very well-known and !)on.crl‘ul inhibitor of S1I groups, both f r w nnd protein-bouiitl, :ifl’ccting hot11 iiioiio and clitliiol g i ~ u p Its . effect on SII crizyiiies may be frcqucntly riiotlifictl 1)y GS1-I and other SI-I conipouiitls (see TTebb, 19G3). Iodoncctic a c i d and iodoacetairiide coniplctcly inhibit the glutatliione ixxluctase of wlicat germ (Young and Colin, 1956) although iodoacetate is witliout eflcct on the enzyme of yeast extract (Raclter, 1955). Tlie iiietliioiiinc sulfosiclc-riictliioiiiiie reductase system in yeast is sensitive to iotlonccbtc, tv o closely approsimnted SH groups on tlie cnzynie being
278
J . S. HARINGTON
involved (see Black, 1963). Iodoacetamide has no effect on r a t liver GSH levels when given orally, perhaps because it reacts vigorously with stomach constituents (Johnson, 1965). Chloroacetophenone is a powerful and irreversible SH inhibitor and disturbs a SH SS cquilibrium in morphogenesis (Mulherkar and Sherbert, 1963). The teratogenic effects of this substance on the chick embryo can be completely rcverscd by subsequent treatment with CySH (Mulherkar et al., 1965), although the latter has no effect on chloroacctophenonc-induced abnormalities in the amphibian embryo (Beatty, 1951), perhaps because of difficulties in pcnetratioii of the embryo sur1965). face by the amino acid (Mulherkar et d.,
B. LACHRYMATORS AND VESICANTS Most lachrymators inhibit SII enzymes (see Peters, 1963; Johnstone, 1963) and generally rcact more readily with proteins containing SH groups than with simple thiols such a s CySH. The pyruvate oxidase system is particularly susceptible to the action of lachrymators and vesicants because of the reaction of the latter with lipoate (see Peters, 1963). Lewisite is a highly active vesicant, also exerting its toxicity on pyruvate oxidation. Dithiols, especially BAL, are effective antidotal substances, forming stable cyclic derivatives. Mustard gas combines with a large number of groups including SH, though this interaction does not play a principal part in the mode of action of the vesicant.
C. ALLOXAN This substance (see Laznrow, 1949; Falkmer, 1962) reacts with GSH and protein SH groups by oxidation or by forming new compounds (see Lazarow, 1949). I n certain circumstances its action can bc reversed by SH compounds. Alloxan-diabetic rats appear resistant to aminonzo dye-induced cancer (Salzberg and Griffin, 1952), and alloxan itself has an anticarcinogenic effect on implanted tumors induced by butter yellow (Gillman et al., 1958).
D. MALEIMIDES A N D RELATED COMPOUNDS N-Ethylmaleimide reacts rapidly, but not specifically (Smyth e t al., 1960) with SH groups. As far a s is known it is not carcinogenic nor an initiator of tumors (on the mouse skin), being very toxic in its action (Searle, 1964; Dickens and Jones, 1963). The uptake of oxygen by human erythrocytes is inhibited by N-ethylmaleimide ; the effect is rcversed by GSH (Sheets and Hamilton, 1958). Rfaleirnide reacts with rnicrosomal cytochrome reductsse through SH groups (Strittmattcr, 1959) and also inhibits the glutathione reductase of germinated peas
THE SULFHYDRTL GROUP A K D CARCIWOGESESIS
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(Rlnpsoii and Islierivood, 1963). The effect of tlic reagent on cell division in E . colt and the lily anther, where direct effects 011 GSI-I are involved, lias been discussed earlier (see Section IV). Xlaleiniidcs as a class arc effective plant-growth inhibitors aiid aritimitotic agents, probably because of their affinity for SH groups (Friediiiami et ul., 1949, 1952).
13. MALEICHI-DRAZIDE The action of this plant regulator in breaking chromosomcs sounded warning that it might be carcinogenic, but extensive tests in a number of systems demonstrated that this was not so (Barnes et ul., 1957). More recent evidence (Dicltens xiid Jones, 1965), however, implies that maleic liydrazitle (in the pure form and not as a salt) “definitely behaves as a carcinogen.” Onc inode of action of the substance lias been well elucichtccl by Hughes and Spragg ( 1958). Rlaleic hydrnzidc irreversibly affects ccrtitiii enzymes requiring S H groups for activity though it does not react with soluble S H groups in vitm or in tlic presencc of tissue extracts. An association between tlie inhibition of dirision hy liydrazidc and S H metabolism was found, in particular with GSH, which in the pea seedling participates in reactioiis specific t o cells preparing to divide. The amouiit of GSH in thc tissue increases as tlie coiicentr:~tioiiof maleic hydrazidc iiicrenses, although tlie normal furictioiiiiig of tlie GSH + GSSG retlox equilibrium does not seem to be affected. It was deduced that GSH iuiictions in a t least two difiereiit systems, one of wliicli is important t,o division. T h e main action of maleic hydrazide in inhibiting the growth of tlie pea seedling scciiis to be due to its reaction witli protein SH, so inhibiting tlie reduction of protein SS by GSH during mitosis as envisngetl by I2lazi:i’s proposed SH $ SS cycle in cell division, aiid 1e:ding to a n accumulation of GSH (Rlazia, 1961a). Hughes aiid Spragg (1958) suggest that during mitosis in the treated plant tissue there is either an increase in the activity of enzymes reducing glutathioiie relative to those oxidizing it, or t h a t other reactions Ixquiriiig GSH specifically arc inhibited. As anothcr alternative, increase in syiitliesis of GSH could be suggested. It was inferred that maleic liydrtzidc had a negligible effect on enzyme systems associated with tlic oxidi~tionor reduction of GSI-I such as glutathioiie reductasc. Clearly, of GSIi is not iiiliibitccl sirice this subst,:tnce :tccuniulated ; on the otlier h:iiid, stiiiiulatioii of GSH synthesis (oversyiitlicsis) could be respoiisiblc for tlie effect. Hughes aiid Spragg believe that some specific stage in the GSH-protein S H reaction of cell division is affected, so preventing tlie iiormal functioning of the reaction. One reductase :t
280
J . S. HARINUTON
system possibly susceptible to mnlcic hydrazidc is tlic protcin disulfide reductase found in yeast; this enzyme unit appcnrs to participate actively in tlie regulation of cell division in this organism (Nickerson, 1948; Nickerson and Fdconc, 1959; Nickerson and van Rij, 1949). The appnrcnt specificity of nialcic hydraeidc in its reaction on SH systems in the pea seedling, coupled with what should be regarded as its carcinogenicity (Dickeiis and Jones, 1965), emphasizes the need of further investigations of tlie mode of action of this substance on mammalian systems. A significant feature too, is the apparent failure of maleic hydrazide to affect glutathione reductase (Hughes and Spragg, 1958), which, according to recent views (Eldjarn e t nl., 1962; bee Black, 1963), may be tlie only SS rctluctabc present in nnimd tissues, t1iou:li not in plants (Hatch and Turner, 19GO), where w piotein disulfide rccluctase other than cystine and glutathione rcductasc is prexiit. Tlic cnzyme in plants appears to have a dithiol group, and unlikc the other reductase mentioned, is strongly inhiloitcd by low concciitrations of arsenite and Hg", Ag+, Cut+, Ctl++,and Zn++ ions (Hatch and Turner, 1960). Obviously, reaction with SH systems neccl not he the only way in which rnalcic hydrazidc worlis; Evans and Scott (1964) suggcst that tlie delay in cell development piotlured by this material is due t o severe depression in the rate of DNA synthesis. VIII. The SH Group in Carcinogenesis
The pnrticipntion a i ~ k)cli,ivior l of SH groups in c:iwinogcncsis have h e n discussed in some c1ct:iil in scvernl rcpoits (see Needham, 1942; Braclict, 1950; Stern and Willlieim, 1943; Cwlcutt, 1960, 1961 ; Calcutt and Coates, 1961 ; C:ilcutt and D o x y , 1962a,b; Calcutt ct nl., 1961). In general, it scenis true to say that no consistcnt pattern of changes in SH or SH-SS levels in established tumors has einerged. Quite frequently, high levels of SI-I compounds have been found in ninlignant tumors, but this finding is by no mcans uniform and tlie values may vary widely (Needham, 1942), probably because of secondary effects associated with tumors in various stages of development and growth. The value of serial studies of changes in SII levels in experimental carcinogciiesis over known periods of time is shown in several investigations. A. SKINCARCINOGENESIS Application of tlibcnz [a,h]nntlrrncene niitl 7,12-rlinicthyll~tm[ a ]antliraccne to tlic skins of inicc produces changes in SI-I levels soon after administration of tlic cnrcinogcns (DiPaolo and Nicdbala, 1957). During
T H E SULFI-IYDRYL GROUP A S D CARCINOGENEGIS
281
the first two hout*s protein and soluble SH levels rose, then fell, and hy the third hour 1i:td risen again to levels well above riornial for all the compounds tested. I n most cases the increased levels had not returned to noiiiinl n itliiii 5 days after treatment. Anthracene and acctonc were without effect on the SH levels. A subsequent histoc.hcniica1 examination employing the same carcinogens confirmed this pattern (DiPaolo and Chu, 1958). The carcinogens hnd either no ciffect on protcin SS levels or a t the most slightly iiicrcasetl them over tlie control valueh. An increase in SH levels after treatment of skin with carcinogens was also reported in histochemical studies by others (Setalii et nl., 1960, 1962). Both local and oral administration of 7,12-diinethylbcnz [ a ]aiithixene and 3-methylcholanthre~ie significantly incrcascd the aniount of S H present, as shown by intense and distinct staining. I n tlie above cases, the levels of SH were higher than those obtained after the local application of tumor promoters of the Tween type. I n licnign lierntinizing sliiii papillomas tlic distribution of S H a i d SS appeaiwl to hc nc:tily iiornial. Aftcr treatment with the carcinogens, the liistocliciiiically deiiionstr:tblc SS in the normal skin disappeared and staiiiiiig for SII gi'oups mas intensified, dimethylbenzanthracene producing tlic cflcct more strongly than methylcholanthrene. It is of interest that promoters appeared to have no effect on the SI-I-FS st:ttc, whciwis c:ircinogciis intensified the staining for SH and ahi05t complctcly tlirninished tli:it for SS. Qumtitative confirmation of 1,hcse results would be of considerable value, and may pinpoint a biochemical lesion in tlic glutnthionc rtductnsc system. Increases in SH levels were also reported in other studies. Whereas the application of acetone t o the n i o i i ~skin had no effect on soluble SH groups, the single wpplic:ttion of 7,12-dimethylbcnz [ a ]anthraccne or bciizo [ a ] pyrene produrcd n SII cwiitcmt (as determined chemically) greater than t h a t in untreated controls (DiPaolo, 1963). Soluble SH levels in the hamster pouch t i w t c t l n ith 7,12-dirnethyIbenz [ a ]anthracene slio.lr.ctl incrcnscb 36 hours nftcr trc:ttnicnt, but these had decreased a t ti0 hours (Rlorris et uZ., 1961). Pouches from female hamsters did not diow the early rise ant1 the later clccrcase was more profound. The soluble SH levels remained low until tumors had been produced when the lcvcls in the whole pouches slowly increased. The resulting tumor tissue contained about 40% of the concentration of soluble SH present in nontumor-bearing tissue. Changes in the levels of acid-soluble SH groups have I m n found in rat uteri treated with estradiol. I n hoth this and , it hiis been suggested (Morris et nl., 1961) t h a t SH (:is GS1-I) iiiny linve reacted with tlic horinone or have been involved in detoxific:ition reactions with the carcinogen.
282
J . S. HARINCTON
B. LIVERCARCINOGENESIS Three recent papers conveniently sumniarize the changes in S H levels occurring in the livers of animals administered hepatocarcinogenic substances, in particular aminoazo dyes. I n two of these (Neish and Rylett, 1963; Neish e t al., 1964), the possible importance of GSI-I in liepatocarcinogenesis has been emphasized; in the third (Dijkstra, 1964), the general lack of agreement among different workers about the effccts of hepatocarcinogens on SH levels has been discussed. Dijkstra’s study (1964)has also shown that the effect of a single dose of aininoazo dyes on the trichloroacctic acid-soluble SH content of the liver depends on the time that has elapsed after the administration of the dye; this may, he believes, help t o resolve some of the conflicting results in the literature. 1. Experimental Studies
Rats fed 3’-methyl-4-dimcthylaminoazobenzeneshowed a late incre:tse of ncid-solublc SII m:itcrinl+ (but not SS) and n considerable dccre:ise i n GSII in thc 1 : ~ :iftcr ~ s ti c~*itriiciit(Fiala a ~ i dF d a , 1959). The changes nppcnied to be part of a process occurring in two stages: ( u ) respiratory damage to thc cell by carcinogens, due to a gradual depletion of mitochondria, arid ( b ) a relatively sudden increase of glycolysis to supplement the insufficient rcspir‘‘t t‘ion. The second stage was accompanied by a sudden change in the distribution of nonprotein SH groups and a redistribution of cytopl,‘i Pmic RNA. All the changes seemed to occur a t about the same time ant1 were marked by massive proliferation of cells. The cell appeared to have entered a new adaptive equilibrium characterizing the transformation to malignancy. Another study (Ncish and Rylctt, 1963) showed that the level of liver GSH rapidly increased following the intraperitoneal injection of the strong hepatocarcinogen, 3’-methyl-4-dimethylaminoazobenzcne.A more gradual and less pronounced increase resulted from thc injection of a weaker carcinogen. The injection of a noncarcinogenic azo dye, 2-methyl-4-dimethylaminoazobenzene, depressed GSH levels in the liver to below normal (Neish and Rylett, 1963). I n a n extension of this study, Neish et al. (1964) found correlations bctwcen tlie relative c:ircinogenic activities of azo dyes and the ability of these compounds to increase the liver GSH content, and to form “boiind dyes.” A correlation was also reported between the carcinogenicity of an azo dyc and the liver cellularity 4 weeks from the start of tlie feeding of the dyc. Neish et nl. (1964) point out t h a t the compounds producing only an
T H E S VL F HT DRT L GROUP AND CARCINOGENESIS
283
elcvntion of liI.cr GSII need not necessarily be carcinogenic, but may be inc.oniplcte c:trcinogc.iis. In ntldit!on, the increase in GSH may be invoked for the dctosific:\tioii of carcinogen metabolites or may be due to derangements in cellular copper metabolism by carcinogens, possibly by chclatiou with the lattcr or thcir nict:ibolitcs. The feeding of a siiigle dose of the carcinogens 3’-rnethyl-4-dimethyl~i~iiiionzobciizrii~~ or 4-~11iii~~t~iy~:~iiiii~0:1eol~r1~ze1ie to rats causes a significant increase ill trichloroncctic Ltci(l-soluhlc SH groups in the liver after about 40 hours (Dijkstra, 1964). The iiicrease appears to be related t o the protein-binding potential of the aniinoazo dyes rather than t o their carcinogenic activity, since 3-iiiethyl-4-diinethylamiiioazobe1izeiie, which is not carcinogeiiic- n n r l wliich Lintls estriisirely, also incrcases livcr SH levels, though iiot to the same cxtciit as docs tlie curcinogenic 3’-mcthyl4-dii~ietliylainino:~eobenzcne.Thc iioncarciiiogen, aniinoazobcnzcne, which showed very little protcin hinding, caused 110 cliange in the soluble SH level. I n a parallel study (Dijltstra and Pepler, 1964), it was found that the levels of trichloroncctic acid-soluble SH in the livers of rats fed continuously with carcinogenic or noncarciiiogcnic aminoaeo compounds increased during the first 7 weeks of the experimcnt. This increase was not characteristic of the carciiiogciiic process although a further significant increase acconipanictl the appearance and development of tumors. No such change was found in rats fcd noncarcinogenic dyes. It is not yet possible to draw any conclusions regarding the fluctuations in SH lerels shown fairly consistently in the studies of skin and liver carcinogencsis described above. In all cases, a n increase in acidsoluble SH levels was reported as part of the changes occurring after the administration of the carcinogens, but whether this is part of a change associated specifically with carcinogenesis only cannot yet be ascertained. From a series of p a p e i ~(Calcutt, 1960, 1961 ; Calcutt and Coates, 1961 ; Calcutt and Doscy, 1962a,b; Calcutt et nl., 1961) dcscribing the cffccts of diffeimt cnrcinogcns :tiid noncarcinogens on animals, it was concluded that a rise in tissue SII levels is an essential prerequisite for tumor formation by a wide variety of carcinogens. Later studies led Calcutt and Doxcy (1962b) to believe that SH groups may not be directly involved in tumor initiation but rather in the cocarcinogenic or tumor development phase. Harrap and Speed (1964) were unable to discover any marked difference betwceii the mid-soluhle SH levels in the leucocytes of untreated leukemic patients and those of normal donors. However, a soluble SS material was found in the whole blood of patients with chronic gi-anulocytic leukemia who were undergoing treatment, but which had
284
1
J. S. HARINGTON
disappeared in remission. Patients could be classified into three groups according to the concentration and location of the SS compound(s) in the blood cells: ( a ) untreated patients with SS in the leucocytes; ( b ) LIyleran ( I ,4-dimethanesulfonyloxybutane) or X-ray-treated patients with the level of acid-soluble SH of the leucocytcs much reduced and SS much increased; (c) patients about to experience a n acute blast-cell transformation, and having SS in the erythrocytes. Patients suffering from acute granulocytic leukemia, however, did not fall into such clearly defined categories, and the activity of glutathione reductase in erythrocytes from these patients was not impaired or modified (Harrap, 1965). Since GSH appeared to be the main S-containing constituent prcscnt in the blood of patients with untreated chronic granulocytic lcukemia, it was concluded (Harrap and Speed, 1964) t h a t the lesion responsible for the abnormalities detected might have been in some way related to the glutathione reductase system of the cell. The results of the experimental studies of Dijkstra (1964) show t h a t the extent of the increase in trichloroacetic acid-soluble SH content during the first 7 to 10 weeks after the feeding of a singlc close of dye cannot be related to the induction of tumors because there is no apparent difference betwreii thc eff ects of the carcinogenic and noiicarcinogenic dyes used. However, a distinct increase in acid-soluble levels was found to accompany the development and appearance of tumors in animals fed continuously with carcinogenic dyes and this was not observed in animals fed noncarcinogenic ones (Dijkstra and Pcpler, 1964). This last observation may represent a real and specific alteration in the behavior of SH-compounds of a certain type during carcinogenesis and may point to some kind of adaptation that has occurred in the SH metabolism of those animals receiving continuous doses of carcinogcn. Such an adaptation might possibly lead to the maintcnaiicc of high levcls of intracellular, acid-soluble SH groups which could then initiate cell division. The nature of the SH material involved is, as Dijkstra (1964) indicates, of considerable importancc, particularly since very little is known of any specific SH compounds involved in the control of mammc‘11‘ian cell division. Preliminary studies suggvst that the SI-I m ~ t c r i a lassociated with the %on-specific” increase shown in Dij kstra’s study of hepatocarcinogencsis may be GSH. Of even greater interest would be to know the identity of the SH compound or cornpounds related to the action of liepatocarcinogens only (Dijkstra and Pepler, 1964). Neish and his collcagucs (1963, 1964) have describcd GSH as the SI-I compound involvcd in the carcinogcnic process in the r a t liver, although Fiala and Fiala (1959) in their experimental study of hepatocarcinogencsis re-
THE SULFHYDRYL GROUP AND CARCINOGEXESIS
285
ported increases in acid-soluble SH compounds but not in GSI-I, which showed a sustained decrease. Reconciliation of these views will be awaited with interest. I i t t l e seems to be known about the behavior of enzymes involvcd in SH or GSH mctnbolimi in carcinogenesis. I n one study (Rlalmgrcn nncl Sylvkn, 19601, glutathione retluctase activity in tumor cells was about twice that in normal cells although low activity was found in two tumor ccill populations. Higher glutathionc reductnse activity appeared in tlie plasma and livers of tiimor-i)e:ti~iiigr:tts tli:iii in the corresponding oi.gaiis of noi-ma1 anirn:ils (RIalmgrrn an(l S y l v h , 1960). Increased levels of the enzyme in the scmm of patients witli cancer have also been reported (see Douglas, 1963). GSH-syntliesizing activity in liver slices was detcctcd in 6570 of tumor-bearing aniinals and in 30% of riornials; hydrolytic activity for GSH w a s found in liver slices in all cases tc.sted hy Mardaslicv and Piklrat ( I 951). The rate of synthesis of GSH was much higher in tumor bearers and tlie rate of hydrolysis a little higher than in normal animals but these changes may he clue t o scroiidary requirements for SH during tumor giowth. The tumor tissue itself showed no in v2tro GSI-I-syiithcsiziiig activity and hormon:tl influences may therefore be active i n controlling the synthesis of the peptide in tlie intact animal. IX. SH Metabolic Systems Possibly Involved in Carcinogenesis
A necessary requirement in the hypothesis described in this review is that sustained increases of frcc (acid-soluble) S H would lead the cell into division, provided that all other conditions are satisfied. This adaptive increase of S H would act as an integral part of a mitotic initiating and coordinating mechanism for division. If GSH is considered as the principal free SH cornpound in cells and tissues (CySH being present in trivial quantities only), an increase in tlie level could be effected by increasing the activity of enzyme systems rcducing GSSG over those oxidizing GSH, by inhibiting systems specifically requiring GSH (sec Huglics and Spi-agg, 1958), or by increasing or activating the synthesis of GSI-I. A . THEGLUTATHIOSE REDUCTASE SYSTEM Glutathione reductase (see Long, 1961; Dixoii and ’CVehh, 1964) is responsible for the niaintcnancc of intracellular glutathione in the rcduced state and seems also to be capahlc of reducing many othcr small inoleciilar disulfides (Eldjarn, 1965) . The enzyme catalyzes irreversibly
286
J. S. HARINGTON
the reduction of oxidized glutathione (GSSG) to GSH in the presence of reduced pyridinc nuclcotides: NAD(P)H,
+ GSSG
--f
NAD(P)
+ 2 GSH
I t is widely distributcd in animal and plant tissues and in microorganisms, and is mainly confined to the particle-free supernatant fraction (the cell snp) although it may be found in the nucleus (Stern and Tiinoncn, 1954; 0 r d and Stockcn, 1963; Wane;, 1962; Eldjarn, 1965). No GSSG reductase sccms to be present in isolated mitochondria, perhaps because the mcrnl)rtine is impermeable to GSH (Eldjarn, 1965). A depcndcncc ol thc enzyme 011 SH groups for activity has been suggested, perhaps in the form of a dithiol prosthetic group (see Black, 1963; Mapson arid Isherwood, 1963). Tissue extracts arc capable of reducing a variety of disulfides other than GSSG, though GSH and glutathione reductase are part of most of the systems involved in the reduction of the disulfides (see Eldjarn e t al., 1962). Glutathione reductase appears to be the only well-characterizcd SS reduring system in mammalian tissues. An isocnzyme system also seems to hc implicatcd (Eldjarn, 1965). Both glucose-6-phosphate and 6-phosphoglucoriate, successive intermediates in the direct oxidation of glucose, have bccn used to form a coupled system by providing NAD (P)112 for the dcinonstration of glutathione rcductase (Venncsland and Conn, 1954). I n erythrocytes the main supply of N A D ( P ) H , comes from the dchydrogcnation of glucose-6-phosphate and 6-phosphogluconate, while in other tissues a n alternate source is found in isocitric dehydrogenase and malic dehydrogenase activity (Cohen and Hochstein, 1963). Disturbances in such coupled systems could, in the erythrocyte, according to recent suggestions (Scheuch et al., 1961; see Carson et al., 1956), lead to a decline in the metabolism of glucose, especially through hcxokinasc and other cnzymrs involved in the oxidative breakdown of this subst:~ncc. OF S Y S T E M S SPECIFICALLY REQUIRING GSH B. INHIBITIOS
The effect of malcic hydrazide on SH metabolism of the pea seedling is a good example of this type of process. Malcic hydrazide reacts with
protein (enzyme) SH, in this way preventing the normal reduction of mitotic protein SS so that GSH, which would normally reduce the SS, accumulates. Thc SH erizyme affected appears to be a protein SS reductslsc but apparently is not glutathione reductase (Hughes and Spragg, 1958). Intcrfcrcncc in coupled systems could also account for changes in GSH level.
THE SIJLFI-IYDRYL GROUP AND CARCINWENESIS
287
C. TIIESYSTHI:SIS OF GSH This occurs in two stages, each rcquiriiig a separate enzyme, though synthesis by other reactions cannot be excluded (see Long, 1961). Syntlietase I catalyzes the following reaction: 7-Glutaniic acid
+
+
cystcinc AT1’ -+ y-glutaiiiylcystcinc ADP
+
+ Pi
(inorganic phosphate),
and bynthctnse I1 the ncst: y-Glutaiiiylcy.teine
+ glycine + ATP -+ GSH + -4DP + Pi
Syntlictnsc I1 npl)cnrs to rcquiie c~sscntinlSH groups for activity; for syntlietase I, this is not yet l-~ I l O ~ \ ’ l l . The synthesizing enzymes arc widely distributed and appear t o be associated with the initoc1iontlri:i in heitns nncl pen-seedlings (Wehster, 1953). The ciicrgy for tlie syntlii.sis is derived from oxidntire activity lwxuse synthesis is mnrkcclly iiic~rcasc~cl by the addition of Krcbs’ cyclc inti~riiicdintcs and inhibited by respiratory inhibitors (cyanide mid azide) , by niiaci obiosis and by dinitrophcnol, an inhibitor of oxidntivc pliosphorylatioii i n plnnts as in otlicr orgmisms (sce Wcbster, 1953) . Coupling of respiration to GSH synthc. inay be mcdiated through ATP, which is itself capable of rel)lncing I(i*ebs’cyclc interniediates in causing GSII formation. Atlclecl A4TPalso cause> GSH syntliesis in tlic prrscncc of cynnitlc ( W c ~ l ) r, 1953). In ixthatioii &image to the leiis hot11 tlic reduction of GSSG and the syntlicsis of GSH are affected, 1e:ding to a rapid drop in GSIT. This coiiicidcs with a fall in tlic level of ATP after what seeins to be :in inipairnieiit of hotli the licsosc-iiionop1ioEphnte shunt arid glyrolysis (IJeriil:m, 1962, 1965). Tlic ATP-trapping nction of c$hioniiie (see F a r l ~ e r ,3963) could also be of interest in this regard (see Section VI,C). In two rccwit studies (Wernze e t al., 1965, and Zicha et nl , 19651, total hody iixidintioii was fouiid to increase tissue GSH which later returncti to normil. The fall in tissue ATP, nlbo fouiid after iimdiatioii (l lna ss :ind Timm, 1964) , W:LS ascribed by Wcrnzc et al. ( 1 965) to a stimulation of ATP-utilizing synthesizing processes, particularly those involving GSII. Yet anothrr c a w of concurreiit drop in hoth GSII and A T P ~ 1 seen after crythrocytcs were treated with ncctylplieiiylliydraziric (&lager et nl., 1964) ; here n Ixpi(l lobs of GS1-I was followed by a stccp fall in ATP content. Tliir pointctl to sonic claniage to the glycolytic systcrn which is directly rc.sponsiblc for -4TP replciiishment (see RIagcr c t al., 1964). Tlic III:LIII waction sec’nis to irivolvc, tlie iiiliihition of hclxokinase and is associated 111 soinc way with the oxidatioii of intracellular GSH.
s
288
J . S. HARINGTON
Glycolysis and the Synthesis of GSH
The finding that active glycolysis is required for optimal synthesis of GSH (Yannri et al., 1953; see McIlwain, 1959) may be important in any attempt to relate modifications of the control of GSH synthesis with carcinogenesis. Intermediates of glycolysis stimulate the synthesis of GSH by maintaining high concentrations of ATP which are optimnlly generated by the reactions of glycolysis. This process also assures a continued supply of energy-rich phosphate by countering the drain of AT€’ by adenosinetriphosphatase and by preventing the accumulation of A D P (Yanari et al., 1953). I n view of this association (between GSH synthesis and glycolysis) , it is tempting to think that a cell modified to increase its synthesis of GSH might also produce the most favorable conditions in which this would seem to occur, that is, at least in the pigeon liver, by glycolysis. GSH itself and SS reductase systems have other connections with glycolysis. GSH plays an important part in maintaining in the rcduced state the SH enzyme, triosephosphate dehydrogenase, the limiting enzyme in the glycolytic process, starting with hcxoscdiphosphate (O’Dell et al., 1961). Aerobic glycolytic activity in the pea seed extract is controlled by processes involving the iionenzymatic oxidation of protein SH groups and the reduction of disulfide groups of glyceraldehyde3-phosphate dehydrogenase by a protein disulfide reductase not cystine reductase or glutathione reductase (Hatch and Turner, 1960). On the other hand, however, association of mitosis with anaerobic hchavior is by no means a general principle for all cells: Swann (1957) and Stern (1960) have given lucid discussions of aerobic and anaerobic processes in mitosis. Studies of the oxygen consumption of the microspores of Trilliuna, especially in relation to the mitotic cycle, suggest that active division in this organism may be associated with anaerobic behavior (Stern and Kirk, 1948). The observed increase in soluble thiols of the dividing cells of this organism does not seem, however, to activate the glycolytic system (Stern, 1960).
D. POSSIBLE PARTICIPATION OF HORMONES IN GSIl METABOLISM T o what extent GSH metabolism is regulated by hormones is not known, although there is evidence that both the synthesis of GSI-I and the reduction of GSSG are affected in normal situations. The known mechanisms of adaptive induction of enzymes by substrate and hormones can both lead to an increased accumulation of enzyme protein by continuing synthesis (see Knox, 1963; Fiala and Kasinsky, 1963). According to Knox (1963), the hormone-type induction acts by causing the
T H E SULFHYDRTL GROUP AND CARCINOGENESIS
289
production of more of the limited essential species of RNA necessary for syiitliesis; in other words, additional eiizyinc synthesis is puahed through by the provision of additional RNA templates. One aspect probably rclatetl t o this is the iiiducetl synthesis of ciizynies by polycyclic arornatic hydrocarbons or drugs and the general effects of carcinogens on adaptive enzyme formation (Conney and Burns, 1963; Jucliau et al., 1965; see Boyland, 1964b). Several studies suggest t h a t pituitary factors are involved in thc etiology of experiinental carcinogenesis (Bielschowsky, 1958, 1961 ; Yai ei al., 1963; Miihlbock, 1963, 1964; Weisburger, 1964; Weisburger et al., 1964), although it is not yet possible to ascribe to any single hormone a central role in carcinogenesis. It seems conceivable t h a t growth hormoiie may he related to GSH synthesis, cell division, growth ~nhibition, :tiid carcinogencsis hecausc: (a) Administration of growth hoi*nioiie increases the level of GSH in tissues (Bartlctt et nl., 1956; see .Jocelyn, 1958), and is associated with incitascd synthesis of GSH in tinilor dc~c1o~)nient (Slincter and Law, 1956; see Jocelyn, 1959). ( b ) Growth hormone has an initiating effect on mitosis compared with inhihitoi~yeffects of corticotropin nntl coi~tisone(Hemingway, 1960, 1961). (c) Growth hormone is related to GSH synthesis in a way (Gregory and GOBS,1933; Painter, 1928; see Needhain, 1942) which may account for the growth-inhibitory effects of carcinogciii observctl hy flatldow (1937), Haddow and Robiiison (1937 1 , Hadtlow c t nl. (1937). ( d ) Growth hormone (and other liornioncs) :tppear to be esseiitial for aninials to brcoiiic susccptiblc to the action of certain C:LI cinogcris (Biel~cliowsliy,1958, 1961 ; Stern, 1960). ( c ) Growth hoi iiionc itsclf is carcinogenic aftcr p~~olongctl ntlministrstion (3loon e t al., 195Oa,h,c). X. Other SH Systems and Carcinogens
A. GESERAL The possible involvement of carcinogens with SH groups i n processes other than those conceining tlie metabolism of GSH slio~ddof course bc considerccl. SH-containing proteiiis participate in tlie transfcr of nctivatccl amino acids from soluble RNA to ribosomes (von tler Deckcri and Hultin, 1960; Hiilsiiiann :tiid Lipmann, 1960) atid this is inliibitetl by GSSG and iodoacetnte (Hiilsniann and I,ipninnn, 1960). GSH pnrticipttcs in the synthesis of proteins in E . coli and in yeast (Schmidt et nl., 1956) :tiid intact SH groups are necessary for tlie DNA-prinicd RNA
290
J . S. HARINGTON
synthesis in bacterial extracts (Chamberlin and Berg, 1962). I n some cases SH groups may be involved in chromosome breaks (Moutschen and Degrnevc, 1965). Thiol groups have bccn reported present in the D N A nucleotidyltransfcrasc of cert:tin tumor cells (Keir and Shepherd, 1965) and in calf-thymus histoiies (Pliillips, 1965). Nuclear protein contains a large :mount of SH groups which may he implicated in cell division (see Jellurn and Eldjarn, 1965, for references) ; these authors nmke the interesting point that the fluctuations of acid-soluble S H polypeptides seen during mitosis (see Section I V of thc prcscnt review) may be clue “to a release from, and a fixation to, certain nuclcoproteins.” Tlic position of GSH and other naturally occurring pcptidcs in biological processes has been dealt with rcccntly in a penetrating article by \Valcy (1966).
B. THERETINE-PRONINE COMPLEX Ilcporth hy Szcnt-Gyiirgyi ( 1965) ant1 his associates of the cocxistence of a growth-inhibitor (retine) and stimulator ( promine) in various animal tissues suggest the presence of a regulatory niechanisin for cell division. Rctiiie appears to be a incthylglyoxnl derivative, which brings it into the orbit of the glyoxalase enzyme system. The latter is responsible for changing mcthylglyoxal into lactic acid, with GSH involved as :I cofactor; for a long time a rather dark and undisturbed area in biochemistry. There are strong infcrcnces that the promine half of the coinplex has SH groups capable of reacting with rctine. This would explain the property of this subst:tnce to inhibit growth. Szcnt-Gyorgyi h:ts p o i n t d out that the work on retine and prominc may link cell division with participation of SH groups. It is not known whether there is any connection betwcen rctinc (or promine) and the synthesis of GSH, and further work on the postulatcd regulatory function of thc Szent-Gyiirgyi substances will be awaitcd with interest. I n the littest paper of the scrics available, Egyud and Szent-Gy8rgyi (1966) consider it possible that SH groups act as a common denominator for iiinny of the single processes involved in cell division. Furthcrmorc, it is felt t h a t some intimate relationship betwecn cancer and SH groups is iiidicatcd. I t has hccn found that a-ketoaldehydc ( a “model” derivativc of incthylglyoxal, used in the absence of pure rctine) inhibits cell t1iv:sioii in scvcral orgunisms in concentrations which do not affect respiration. Tlic inhihition is due to reaction of thc aldehyde with SH groups. Tlic attractive possihility exists that rornplexes of the retine-promine
THE SULFHYDRYL GROUP AND CARCINOGENESIS
291
type may serve a second function in which they permit the intraccllular storage of largc rcscrvcs of stimulating substance (or inhihitiiig) which can be called on without the necessity of de novo synthesis. The dorniant potato tuber has comp:ir:itirely littlc GSH, but :Iftcr it 1i:is heen treated with the vapors of ethylcne chlorohydrin, large amounts of tlie pepticlesufficient to be isolated-txcome available. I t is tliib liba-:itctl GSII which seems to be the causative agciit i n the hrcaking of tlic tlorniant state in the tuber. Sexton has given a concise account of this (1953, p. 388 e t seq.). Substances other than GSH are active in breaking dormancy and sulfur compourids are common among these. Sexton pointed out that growth-inhibitory unsaturated kctonic su1)stances (for cx,~nipIc,a,/I-iiiisaturated lactones) are known t o regulate seed germiii:ition nnd plant growth; SH compounds should inxctivatc thew inhibitors hy adtlitire reaction a t the double bond. From this it seems possible that cthylene chlorohydrin, in breaking dormancy, may act by dissociating a stimulator-iii1iil)itor complex of GSH and a lactone, perliaps of the hcscnohctonc type tliat r\lcclawnr et ul. (1943) found first in malt, and later in B witk variety of other plants. Inhibitory substances in amounts compnrable with that of tlic GSH released after dormancy has ljeeii hroken may thcrcxfore lw found in relatively high concentration in the potato tubcr a t this time. If such a complex is found, one may gums it possible that thcic may not be a single universal type of stimulator-iiilii1)itor coniplex--not that this has ever been claimed-hut that a 131oininc.-retiiic 1)alancc iiiay bc found in some tissues :mcl o r g : m + i i i ~:ind n thiol-lnctone complex i n komc othcrs. All this is speculative, however, ant1 the account can be cndctl with a l)r.icf mention that promine, the growth stimulator dehcribed by YzcntGyoigyi :inti his associates, may turn out to :L SH coni~~ouiid. 011cc:in deduce this from the results of the work doiie so far on retine and related methylglyoxal derivatives. I t has already hecn suggested that the promine half of the complex almost certainly contains SH groups which piobably react with the glyoxal-like retine ; i n this way the gi3owthinhibitory capacity of the latter could be nrcouiitcd for. Egyiitl ant1 Szent-Gyorgyi’s paper of 1966 clearly su1)ports this contention. I t riiny not be too surprising if promine transpires to be a re1:itiwly biiiiple thiol, possibly of the GSH or reduced lipoatc type. Ho~vever,the situution is none too simple. Neither GSH nor CySH give I?, \ ~ l u c scoinp:ir:tblc witli that of promine (itlcntificd hy its biological activity by Ilegycli e t al., 1963) when run in the same solvent systems and under the same conditions (Harington, unpublished). The R , value of GSSG is
292
J. S. HARIKGTON
close t o t h a t of promine but this oxidized derivative of GSH can be firmly excluded as being related to promine because of the lack of SH groups in the molecule. XI. Discussion
A. GENERAL DISCUSSION The participation of SII coinpounds and groups in the fundamental processes of growth and division has a long and interesting history. It is a tantalizing history too, in t h a t it has proved next to impossible to coordinate the many, and very often unrelated pieces of information on the subject which have become available over the years. Having started with the discovery of glutathione by Hopkins, one entercd the era of Hanimett and his associates, who, in the 1930s) conferred on this peptide the distinction of being a specific mitotic hormone. This claim was to be weakened some 25 years later, however, when a clearer perspective of SH function in cell division appeared from the work of hlazia, Stern, Swann, and several others which swung tlie pendulum in the other direction. Yet, while it must be accepted that the SH group is simply one factor of many in the “mitotic pool,” a number of careful studies were to show that SH compounds in certain conditions could stimulate cell division when ordinary nutritional effects could be excluded. Provided that a11 other requirements (nucleic acid synthesis, energy, respiration, etc.) were met, SII compounds added to the medium could tip the scales, :md division would follow. This made sense if seen against the working of SH-SS cyclcs in cell division first enunciated by Rapkine and then later elaborated by Mazia and his colleagues. Here, a cyclic oxidation-reduction process involving SS and SH appears to take place during clcavage of the egg, in this case, that of the sea urchin. The levels of free and bound SH alternate in a reciprocal fashion, with a high level of free SH coinciding with cleavage of the egg. The nature of the free SH compound (which, from our present vantage point in time, we could call “a switch”) in the cycle naturally commanded great interest. Believed a t first to be glutathione (as it is in the lily microspore), it later appeared that the nature of the free SH compound varies with cell type; in the sea urchin egg, ChZoreZZa, and yeast, it is a SH-containing protein or polypeptide. Changes in SH levels were later found in maminalian cells, but on a broader scale. I n 1942, Needham pointed out that SH concentration is related t o rate of cell proliferation, and this was later confirmed. Regenerating rat liver shows a peak of mitotic activity coinciding with
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one in the nonprotein SH level; the diurnal periodicity of mitosis in regenerating livers shows high and low lcvels of mitoses a t tiiiies wlicn the GSH levels are also high and low respectively. It is to tlie credit of Crabtree that the first serious study of S H groups in carcinogenesis was made from 1940 and onward over the next decade. From his work on anticarcinogenesis, he concluded that reaction between the carcinogen and suitably spaced SH groups was of the first importance in the early carcinogenic change. After this good start, however, the status of SH-gro~ipactivity in cancer seemed to diminish, no doubt clue to a large extent to the diffuse results obtained from work on established tumor tissue. Some careful serial studies of cliangcs of SH lcvels in experimental earcinogenesis, starting in the h t e 1950s, were to restore the balance. I n work on skin and liver cancer5 in animals, increases of free SH compounds were found to be related to the induction of the tumors in ways which could possibly he specific. Here again, as in the SH-SS cycle in division, the nature of the free SH compound, which could possibly be acting as the switch for division, is of much import:tnce; there is provisional evidence in the work on liver carcinogenesis that the compound coiiceriicd may he glutathionc. With this background in mind, a study of some of tlie reactions of a wide range of physical and cliemical carcinogens with SH groups was undertaken according to a hypothesis that a t least one form of the carcinogenic process might occur as a. result of cell division being inhibited by the primary interaction of the carcinogen with S H groups directly involved in the control of division. From this and other eviclencc, it was concluded tliat. the S H materials involved in carcinogenesis may be found within one of the systems in which glutathione is reduced, oxidized, or synthesized, in particular the last.
B. DISCUSSIOX OF
THE
REVIEW
After outlining the participation of S H groups in normal cell division and growth, and the working of SH-SS cycles in cell division in a few organisms, the peculiar property of growth inhibition by certain types of chemical carcinogens was next exainined. Some 30 years ago, Haddow showed that carcinogenic polycyclic hydrocarbons possess unusual and strong growth-inhibitory powers ; he went on to suggest t h a t cancer could result froiii the cell freeing itself from the inhibitory effects of carcinogens to produce a new cell race without control of cell division. How the carcinogens inhibit the growth is still unknown. Haddow thought that they might interfere with the normal functioning of the pituitai'y gland, perhaps suppressing the secretion of growth hormone.
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A part of the present review attempts to extend this proposition by coupling it with Needham’s suggestion that a connection exists not only between the action of growth hormone and glutathione Icwls but also between the latter and body size. After a review of the evidence, it is concluded in the present paper that the growth inhibitory action of the carcinogenic polycyclic aromatic hydrocarbons may be due to a general systemic suppression of the synthesis of glutathione and to an interference in the hormonal control of this process for the whole body, with gi owth hormone playing a key part. A substantial part of the present paper is concerned with the interaction of carcinogens with SH groups. I n view of the considerable reactivity of the latter, it is not surprising that multiple reaction with many carcinogens takes place. Some of the i n vivo and in vitro reactions between SH groups and the following carcinogens have been examined in some detail: polycyclic aromatic hydrocarbons hormones hepatocarcinogens alkylating agents ni trosamines 4-nitroquinoline-N-oxide
lactones, quinones and derivatives metals and metal derivatives arsenic polymer carcinogens radiation
Reaction of the SH group with the carcinogens listed above is extensive, but it is also extensive with certain SH-reactive substances which are either not carcinogenic or whose carcinogenic activity has yet to be determined. The well-known SH reagents, for example, iodoacetate, p-mercuribenzoate and N-ethylmaleimide, fall into this class. From the above, it is clear that mere nonspecific reaction of a carcinogen with SH groups is insufficient to account for the carcinogenicity of a large number of different substances. (Crabtree emphasized this in his work on anticarcinogenesis.) Therefore, if some step in SH metabolism is to be considered as a primary site in carcinogenesis, more specific SH systems will have to be looked at. As yet, it is not necessary to account for the behavior of avid SH reactants such as iodoacetatc and others because so far they have not been tested for carcinogenicity; such highly reactive members are probably inactivated by nonspecific SH reactions before reaching the site where carcinogenic action is to be initiated, or they may be “prematurely” detoxified. For a physical or chemical carcinogen to reach this site, the agent, the substance or its derivative should be physically and chemically stable and not over-reactive. Once having reached thc primary point, the material must act specifically on some system which is unaffected by noncarcinogens reaching the same site. As f a r as this locality is con-
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cenied, it seems clear that several particular enzyme systems should be capable of producing a sustained and overcompensatory increase of free SH in the cell or in that part of it that is directly involved in SH cell division processes. To validatc the hypothesis put forward in this paper, m e has to assume that such increases of SH must be capable of leading the cell into division provided that all other requirements for a cell about to divide have been adequately met. The manner in which high or increased levels of free (acid-soluble) SH may lead a cell into division is not yet known. The possibility is considered t h a t such SH could act in the normal state and in carcinogenesis as a switcli in some mitotic initiating or coordinating arrangements of the type described by S w a m ( 1957, 1958) in his reviews of cell division. But this would iiiiply that SH-SS cycles exist in niainmalian division, and that they are analogous with those t h a t have been found in the division processes of some lower organisms, notably the sea urchin egg and the lily microspore. However, this is not yet known although there is suggestive evidence that some kind of related cycle does exist in inariimalian cells. It is also not known whether glutathione is the main free (acidsoluble) SH compound involved in sucli a postulated mammalian cycle, or whether the key substance might be a type of SH-polynucleotide resembling t h a t which plays an essential part in the cell division of the alga, Chlorelln, arid of yeast cells. Evidence from other sources indicates that glutathione may play a cardinal part in physicochemical cycles in division. The peptide is neatly implicated in the sea urchin egg: Here it has a strong effect on the balance between the gelating or polyinerizing systeni on the one hand, and on that causing decrease in gelation or tlepolymerization on the other (Runnstrorn arid Kriszat, 1962) . Wilson and Heilbrunn (1952) concluded that if the oxidation of SH to SS groups was inhibited, mitotic gelation did not occur and division was prevented. The presence of free SH groups is essential for the induction of gels in proteins by urea (Huggins et al., 1951), and in Ehrlich ascites cells viscosity is increased following the addition of glutathione (DiPaolo, 1965). If a SH-SS cycle does occur in mammalian cell division, and assuming that glutathione is the principal free S H compound involved, there are three possible ways in which high levels of this material could be produced: ( a ) by increasing the activity of enzyme systems reducing glutathione (GSSG) over those oxidizing it (GSH)-the glutathione reductase system ; ( b ) by inhibiting systems specifically requiring glutsthioiic ; or ( c ) by increasing or activating the synthesis of glutathione. The review coricludes with a brief account of each of these systems
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and emphasizes in partJicular the possible importance of the synthesis of glutathione. Active glycolysis is required for the optimal working of this process. It is also pointed out that growth hormone may be implicated in the in vivo synthesis of the tripeptide, and not only with this synthesis but also with cell division, body size, growth inhibition, and carcinogenesis. Increases of acid-soluble S H compounds as seen in experimental carcinogenesis appear in some instances to be specific concomitants of this process. I n a t least two studies (Dijkstra, 1964; Dijkstra and Pepler, 1964) a distinct increase in levels of acid-soluble S H accompanied the development and appearance of tumors in animals fed continuously with carcinogenic dyes; this was not observed in animals fed noncarcinogenic dyes. These findings may represent a real and specific alteration in the behavior of SH compounds of a certain type during carcinogenesis and may point to some kind of adaptation which has taken place in the SH metabolism of those animals which received continuous doses of the carcinogen. Such a n adaptive change could possibly allow the maintenance of high levels of intracellular acid-soluble SH groups as a prerequisite for the initiation of cell division. This leads on to a discussion of a hypothesis of carcinogenesis in which SH groups are involved in a primary capacity.
C. A HYPOTHESIS
Definition of the Hypothesis At least one form of the carcinogenic process starts when cell division is inhibited by the carcinogen reacting with SH groups directly involved in the control of division. An ovcrcompensatory response by the cell to this inhibition produces a high level of free (or acid-soluble) SH which is subsequently maintained and which acts as a mitotic initiating and coordinating mechanism for division. The specific mechanism involved in this form of carcinogenesis is a loss of feedback control of glutathione synthetases, the enzymes originally inhibited by the carcinogen. This leads to a depressed state in the biosynthesizing chain which in turn causes an overproduction of GSH. Expressed in another form, the carcinogen stimulates the formation of glutathione synthetases by limiting the synthesis of GSH. As a result of evidence drawn from the present review, the hypothesis may be formulated in greater detail in the following way: (1) A wide variety of carcinogens, both physical and chemical, produce a t least one of their effects on the control of cell division.
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(2) Carcinogenesis begins with the interaction of carcinogen with the SI-I groups of an enzyme system directly associated with cell division and responsible for maintaining a high level of free (acid-soluble) SH which acts as a mitotic-initiating and coordinating mechanism for division (Swann, 1957, 1958). ( 3 ) Because of this interaction, the earlicst effect of single or repeated doses of carcinogen is usually a n inhibition of division and therefore of growth, although in some cases these are stimulated. (4) The response of the cell to this inhibition involves an adaptive adjustment of a feedback system governing the synthesis of thc inhibited SH enzyme so t h a t a n excess of enzyme is ultimately produced and maintained. The actual disturbance probably occurs in a system which exerts its control through alteration of enzyme amount rather than through altered enzyme activity. Examples of excessive synthesis of synthetase enzymes havc been well documented in studies of microorganisms (Moyed, 1960, 1961a,b; Moyed and Umbarger, 1962; Lester and Yanofsky, 1961 ; Shedlovsky and Magasanik, 1962), but not as yet to any great extent in multicellular organisms. One very relevant example in animals appears in the work of Siperstein (1965) and his colleagues on the disturbed biosynthesis of cholesterol in hepatomas. I n this first cxample of the deletion of a negative feedback system from a malignant tissue, it mas found t h a t the rate of cholcsterol synthesis in the tumors of cholcsterol-fed mice averaged 160 times that in the livers of the samc mice. Thus the tumors showed absolutely no response to dietary cholesterol, t h a t is, there was a consistent abscnce of the cholesterol feedback system. This was later shown to involve a single enzyme. (5) The feedback system concerncd in carcinogenesis may be localized in specific parts of the cytoplasm in which disturbances in SH metabolism would not be detected unless sought for specifically. It scciiis reasonable to believe that the cell contains several different SH or SS metabolic sites separated from each other and capable of fairly independent operation. (6) The postulated carcinogenic change takes place in a minority of cells which, a priori, have to be more sensitive than their fellows to feedback alterations produced by carcinogens. This postulate is nccessary if one is to account for the relatively small number of cells of the many exposed which become malignant after a tissue is trcated with carcinogen. (7) The transformation from inhibition to oversynthesis of enzyme, and ultiniately of product, must be a stable one transmitted from niothcr to daughter cells. It is proposed t h a t the change results
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from the cell adapting itself to an abnormal internal or external stimulus a t the cytoplasmic level although somatic mutation or a disturbance in the metabolism of nucleic acids could be involved. At first sight the hereditary transmission of an adaptive response in a cell seems to imply the inheritance of acquired characters but the situation in carcinogenesis as described here should be taken as being analogous to that seen in the differentiating cell. Here, as embryonic cells find themselves in different cell environments, then they begin to differ from their parcnt cells. The main difference between one kind of differentiating cell and another lies in which of its genes are active. This aspect has been discussed in another paper in some detail (Harington, 1967). From the work described there, it was contended that it would seem justifiable to believe that metabolic events in the cytoplasm of a cell can result in parts of a gcnc being switched on or off, and that this new pattern of potentiality could be expressed hereditarily without the occurrcncc of structural changes of the gene. Braun (1965) has provided experimental evidence to show how tumor growth in plants can be reversed, in a manner which implies that genes are switched on or off, and not in any way changed in structure. (8) The overproduction of a synthesizing enzyme, and hencc its product, both involved in the coordination of cell division, would be expected to result in the loss of control of this process, with conceivably, the development of cancer as an end result.
Alternatives to Oversynthesis of Glutathione as Possible Primary Biochemical Lesions in Carcinogenesis Oversynthesis of specific SH-producing enzymes thus becomes, according to the present hypothesis, an essential feature of the early carcinogenic change. Concomitant intcraction of carcinogen with the product of synthesis, glutathione itself, may also occur, in which case oversynthesis might again follow after suitable feedback adjustment. If oversynthesis is not involved, then SH production must be so modified that a constantly high level of free SH is produced and maintained in those specific parts of the cell associated with the control of cell division processes; these free SH groups must in some way, directly or indirectly, be affected by carcinogens. A second alternative to oversynthesis may lie in the possibility that SH-SS cycles in cell division are adjusted in such a way that the levels of free (or low molecular weight) SH materials are no longer diminished
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during tlic cyclical change; that is, t h a t the cycle is abolished and a constant pattern of higli free SH levels is maintained, this last becoming a pernianent feature of cell activity. Yet another possibility concerns the overactivation of SH-synthesizing enzyiiics alitady present or thc inactivation of systems which hydrolyze glutnthionc. Enzyme degradation, as opposed to enzyme synthesis, has tieen neglected in considerations of feedback adjustment (see Hsrington, 1967, for references) .
Possible Sites of the Priinnry SH Defect
It has already becn pointed out that the cell problably contains sevcral different SH or SS metabolic sites, separated from each other, and able to work fairly independently. Thus, a number of different SH-SS redox situations may exist in different loci (Eldjarn, 1965). As an example, only a sniall fraction of the total SH groups of certain cells appears to lw involved in ~ensitivityto radiation, and these may be localized in specific piirts of the cell (Scaifc, 1964). Modification of SH systems in such localities would not be reflected in changw in SH lcvcls in total tissuc or even in cells, and for this reason may cscapc notice. The detection of radiation effects on glutathione rcductase and SH in cell nuclei (Ortl and Stocken, 1963; Scaife, 1964) is a promising application of more refined approaches to S H nietabolisin in treated cells. Insofar as thc synthesis of glutathione is concerned, both the y-glutainylcystcinc syntlietaee (synthetasc I ) and glutathione synthetase (synthetase 11) of rabbit liver are present in the supernatant fraction (see Wttlcy, 1966). In beans and pea seedlings, mitochondria appear to be the sites of glutathione synthesis (Webster, 1953). Glutathione reductase does not seem to be present in these organelles because, perhaps, as Eldjarn (1965) has suggested, the membrane is impcrineable t o glutathione. Conclusion One last point coimrning a possible relationship between carcinogenesis and specific reaction with SH groups remains to be madc. Clearly, any inteiyrctwtion which suggests that a defect in the biosynthesis of certain crizyines in SH metabolism is onc of the primary events in carcinogcncsis need not, indeed cannot, exclude the participation of DNA and R N A . The priniary biochemical lesion could result from a failure in function or integrity of the nucleic acids, brought about in different ways. And any adaptive change in the rate or amount of enzyme bf.ing synthesized would have to be mediated through DNA or RNA,
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either by hormonal action or by feedback operations of the switch-on switch-off type discussed in the text. This raises the important possibility that even if the primary lesion were to be found in a specific SH-synthesizing system, thc carcinogen may not have acted directly on the synthetases themselves but rather on SH groups associated with the DNA and/or the RNA responsiblc for the actual synthesis and feedback control of the affected enzyme system. I n conclusion, recent evidence suggests that the biosynthesis of certain proteins is exprcsscd in an oscillatory form: If this is true, it should be possible to find out whether the patterns are altered by carcinogens or in carcinogenesis. The finding of a specific change in oscillation could help to define more clearly the dynamic attributes of the primary lesion in cancer. Considerations such as these may in turn lead to the developnient of a rational and more comfortable therapy for the patient. For instance, it may prove possible to re-set the disturbed equilibria in cancer cells by modifying tlierapcutically a specific feedback circuit. These themes have been developed more extensively in another paper (Harington, 1967) to which the reader is referred.
ACKNOWLEDGMENTS The author is indcbtcd to Dr. D. A, Gilbert and Dr. G. P. Warwick of the Chester Beatty Research Institute and Dr. A. G. Oettlk of the South African Institute for Medical Research, Johannesburg, for valuable criticism and interesting discussions. Sincere appreciation is expressed to Professor Sir Alexander Haddow, K.C.B., F.R.S. and Professor I?. Bergel, F.R.S. for their encouragement and interest, and to many colleagues who helped in various ways. In particular I wish to thank Mr. J. Dijkstra of the South African Council for Scientific and Industrial Research, Pretoria, for his constructive comments which proved invaluable during the planning of the final copy of the manuscript. The contribution of Miss Eone de Wet is gratefully acknowledged, as is the full support afforded to me by the Anna Fuller Fund during the course of this and other studies. I n expressing gratitude to the persons and Fund named abovc, I do not intend that they should share any rcsponsibility for any of tlic conclusions I have drawn in this paper.
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Szent-Gyorgyi, A. 1965. Science 149, 3437. Tamiya, H. 1963. J. Cellular Comp. Physiol. 62 (Suppl. I), 157-174. Tliimann, I<. V., and Bonner, W. D. 1949a. Science 109, 444. Tllimann, K. V., arid Bonncr, W. D. 1949b. Proc. Natl. Acad. Sci. U.S. 35, 272-276. Thompson, M. E., and Johnston, A. M. 1958. Nature 181, 647448. Tonge, B. L. 1962. Nature 194, 284-285. Tecn, C . C., and Collier, H, B. 1959. Nature 183, 1327-1328. Tsen, C. C., and Tappel, A. C. 1958. J. Biol. Chem. 233, 1230-1232. Upton, A. L. 1963. Exptl. Cell Res. Suppl. 9, 538-558. Valeri, C. R., and McCallum, L. E. 1965. Nature 205, 561-563. Vallee, B. L., Ulmer, D. D., and Wacker, W. E. C. 1960. A . M. A . Arch. I n d . Health 21, 132-151. van Heyningen, R., Pirie, A., and Boag, J. W. 1954. Biochem. J. 56, 372-379. Veldstra, H., and Havinga, E. 1944. Enzymologiu 11, 373-380. Vcnncdaiid, B., and Conn, E. E. 1954. In “Glutathione” (S.Colowiclr, A . Lazarow, E. Racker, D. R. Schxarz, E. Stadtman, H. Waelsch, eds.), pp. 105-126. Academic Press, New York. Vocgtlin, C., and Chalkley, H. W. 1930. Public Elealth R e p t . ( U S . ) 45, 3021-3063. von der Decken, 4.,and Hultin, T. 1960. Biochim. Biophys. Acta 40, 189-191. Waley, S. G. 1959. Biochem. Soc. Symp. (Cambridge, Engl.) 17, 79-92. Waley, S. G. 19%. Advan. Protein Che,m. 21, 1-112. Wang, T . 1962. Nature 195, 1099. Warwick, G. P. 1963. Cancer Res. 23, 1315-1333. \I’c4h, J. L. 1963. “Enzyme and Metabolic Inhibitors,” 1‘01. 1, 949 1’p. Academic Prrss, New York. Webster, G. C. 1953. Arch. Biochcm. Biophys. 47, 241-250. bYcisl)urger, J. H. 1964. I n “Cellular Control Moclianisms and Cancer,” Unio Intern. Contra Cancrum Conf. (P. Emmelot and 0. Miihlbock, eds.), pp. 300-306. Elsevier, Amsterdam. \I’(,isl)iirger, J. H., Pai, S. It., and Yamamoto, R . S. 1964. J . Null. Cancer Inst. 32, 881-895. Wellers, G., anti Aschkenasy, A. 1957. Compt. Rend. SOC.Biol. 151, 1861-1863. 126, 291-298. \Vcxmz.., H., Braun, H., and Koch, W. 1965. St~ahlcnthe~apie Wlieeler, G. P. 1962. Cancer Res. 22, 651488. \I‘lii~c?lcr.G. P. 1963. Cancer lies. 23, 1334-1349. White, J., and White, A. 1939. J. Biol. Chem. 131, 149-161. Whitehead, G. B. 1955. Indian J . Malarial. 12, 427432. Wliitchcad, G. B. 1959. J. S. African Vet. Med. Assoc. 380, 221-234. Whitehead, G. B. 1961. J . Insect. Physiol. 7, 177-185. Wilson, G. B., and Morrison, J. H. 1961. “Cytology,” 297 pp. Chapman Hall, London. Wilson, L. P. 1946. Growth 10, 361-373. Wilson, W. L., and Heilbrunn, L. V. 1952. B i d . Bull. 103, 139-144. Wolbach, S. B. 1937. A m . J. Pathol. 13, 662-663. U’olfson, N., and Fry, D. S. 1965. Bxptl. Cell RES.38, 66-74. Wood, J. L., and Fieser, L. F. 1940. J . A m . Chem. Soc. 62, 2674-2681. Wynder, E. L. 1952. New Engl. J. Metl. 246, 573-582. Yanari, S., Snolre, J. E., and Bloch, I<. 1953. J. Biol. Chem. 201, 561-571. Young, L. C. T., and Conn, E. E. 1956. Science 124, 628. Zicha, B., Diensthier, Z., Borovk, J., Benes, J., and Neuwirt, J. 1965. Strahlentherapie 126, 299-308.
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THE TREATMENT OF PLASMA CELL MYELOMA',' Daniel E. Bergsagel,3 K . M. Griffith,' A. Haut," and W. J. Stuckey, Jr.'
I. Introduction . . . . . . . . . . . . . . 11. Plasma Cell Neoplasms . . . . . . . . . . A. Etiology . . . . . . . . . . . . . B. Immunoglobulins . . . . . . . . . . . C. Course of the Disease . . . . . . . . . . 111. Antincoplastic Trcatnient of Plasma Cell Myeloma . . . A. Evaluation of the Response to Therapy . . . . . B. Effectiveness of Antincoplastic Agents in the Trcatnient of Plasma Cell Myeloma . . . . . . . . . . IV. Summary . . . . . . . . . . . . . . References . . . . . . . . . . . . .
. . . . . . . . . . .
.
.
.
311 312 312 315 321 329 329
. . 337 . . 353 . . 354
I . Introduction
The dcvelopnicnt of an animal model of plasma cell myeloma in BALR/c mice by Dr. Michael Potter and his associates a t the National Cancer Institute, and increased knowledge of the structure and metabolism of the iinrriunog1ol)ulins procluccd by plasma cells have improved our understancling of plasma cell tumors. Prior to 1959 chemotherapists The studies done by the Southwest Cancer Chcniotherapy Study Group (SWCCSG) reported in this paper were supported by the Cancer Chemotherapy National Service Ccntcr, National Cancer Institute, Public Health Service. 'The Adult Section of the SWCCSG, composed of the principal investigators (named first) and their associates at the following institutions, contributed clinical rnatcrial for myeloma studies: R. A. Hettig, P. Davis, and M. Lane, Baylor University College of Medicine, Houston, Texas; D. E. Bcrgsagcl, Universiby of Tcxas M. D. Anderson Hospital and Tumor Institute, Houston, Texas; W. C. Levin and W. E. Truax, IJnivcrsit,y of Texas Medical Branch, Galveston, Texas; E. P. Frenkel, University of Tcxas Soutlirvestcrn Medical School, Dallas, Texas; W. J. Stuckey, Jr., R. Stauh, and C. C. Sprague, Tulane University School of Medicine, New Orleans, Louisiana ; A. Haut, University of Arkansas Medical Center, Little Rock, Arkansas; W. F. Denny, Veterans Administration Hospital, Little Rock, brkansas; C. P. Alfrey, Jr., Veterans Administration Hospital, Houston, Texas; H. A. Buccliner and 0. T. Baker, Veterans Administration Hospital, New Orleans: Louisiana; and W. Whitcomh and C. Bloedon, Veterans Administration Hospital, Oklahoma City, Oklahoma. :' LTnivrrsit,y of Trxns M. D. Anderson Hospital and Tumor Instit,utr, Houston, : Princcss Margaret Hospital, Toronto, Ontario, Canada. omathematics, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. University of Arkansas School of Medicine, Little Rock, Arkansas. Tulanc University School of Medicine, Xew Orleans, Louisiana. 311
312
BERGSACEL, GRIFFITH, HAUT, AND STUCKEY, JR.
were discouraged by the failure of drugs such as urethane, stilbamidine, and the antimetabolites to alter the course of the disease; many malignant disease treatment centers did not encourage the referral of patients with plasma cell myeloma unless they had localized skeletal lesions which could be treated with X-irradiation. I n the past few years it has been recognized that treatment of plasma ccll myeloma with two different alkylating agents (cyclophosphamide or melphalan) results in objective improvement in 30-50% of patients, and prolongs survival significantly. The prognosis with this disease is now as good as, or better than, the prognosis of patients with chronic myelogenous leukemia. The objective of this review will be to interrelate the concepts which have developed froiii investigations of mouse plasma cell tumors, the structure of protciiis produced by plasma cell malignancies, and recent advances in the treatment of plasma cell myeloma. II. Plasma Cell Neoplasms
A. ETIOLOGY
A plasma cell neoplasm results when a cell, which normally differentiates and produces an immunoglobulin, undergoes a malignant transformation and the neoplastic daughter cells successfully establish a tumor clone. The agent(s) directly responsible for the malignant transformation are unknown, but certain factors such as the genetic constitution, antigenic stimulation, and exposure to X-irradiation have been found to influence the incidence of plasma cell tumors in animals and in man. The genetic constitution of mice is a most important factor for the induction of plasma cell neoplasms. Thus, 68% of BALB/c mice given three intraperitoncal injections of mineral oil of 0.5 ml. at 2-month intervals developed plasma cell neoplasms, with a peak incidence at 10 months of age (Potter and BOYCC,1962). Other strains of mice do not develop plasma cell tumors following the same schedule of mineral oil treatments (Potter and MacCardle, 1964). The possibility that genetic factors may play a role in the genesis of the human disease is suggested by the occurrence of plasma cell tumors in a t least nine pairs of siblings (Mandema and Wildervanck, 1954; Nadeau e t al., 1956; Herrell e t al., 1958; Castleman, 1959; Hirsh and Schwarz, 1959; Manson, 1961; Massari e t al., 1962; Thomas, 1964; Lconcini and Korngold, 1964), and the significantly higher incidence 01 myeloma in the Negro as compared with the Caucasian population of Brooklyn (MacRilahon and Clark, 1956). Factors which stimulate nonspecific proliferation of plasma cells
THE TREATMEKT OF P L A S M A CELL MYELOMA
313
induce plasma cell neoplasms in BALR/c mice, in mink, and inay also be of importance in man. The intraperitoneal injection of mineral oil into BALB/c mice causcs the formation of numerous mesenteric oil granulomas composed of oil globules containcd in single or multiplc cells, undifferentiated rnescnchyiiinl cells, fibroblasts, scattcrcd lymphoid cells, and plasma cells. Thc early neoplasms appear as focal islands of large, hyperchroniatic plasma cells within and on the peritoneal surface of the mesenteric granulomas (Potter and MacCardlc, 1964). The niineral oil appears to induce multiple primary plasma cell tumors, for the isolation and transplantation of the early, discrete lesions (“clones”) from a singlc animal has resulted in the establishment of several different protein-producing lines of plasma cell tumors (Pottcr, 1962). The variation in the proteins produced by tlie BALB/c plasma cell tumors is not related to host genetic factors, for these factors are uniform in this highly inbred strain. It seems likely that thc typc of protein produced by the tunior is determined by the stage of differentiation and the protein-producing apparatus of the plasma cell which undergoes a malignant transformation. Since a few plasma cell tumors do not produce any protein, some tumors protluce only light chains (Berire-Jones proteins), while others produce both hcavy and light chains, and the type of protein secreted by a ‘Lclo~iccl” tumor does not change during multiple transplant generations (Potter et al., 1964), it would appear t h a t the malignant tr:tnsformation may occur in cells which are undiffcrcntiated (cells producing no protcin) , partially diffcrentiatcd (cells producing only light chains), or completely differentiated (those producing both heavy and light chains), an(1 that tlie protcin-producing apparatus of each tumor “clonc” is an individual, stable, heritablc characteristic. Initially BALB/c mice injccted with mineral oil develop a diffuse, heterogeneous liyperglol~ulinemiw,but if the tumors arising in mineral oil granulomas are left undisturbed, they grow and becomc confluent, arid a homogcncous protein peak appears in the serum clertrophoresis pattern. Delayed tranhplantation usudly results in the identification of only one typc of protein-protlucing. neophsrn (Potter, 1962). Attempts to grow niixturcs of two plasnla cell tumors with differcnt growth rates usually results in the eniergcnce of only one line, but a systematic study has not been completed (Potter, personal communication). The cells of the more vigorous tumor appcar to replace or inhibit the growth of the other tumors. Thus, intrapcritoneal mineral oil in BALB/c mice stimulates the formation of mineral oil granulomas in which focal collections of plasma cells appear. During an early stage, multiple primary plasma cell neoplasms can be “cloned” from discrctc peritoneal nodules, but later a vigorous tumor clone overgrows, or inhibits the growth of thc
314
BERGSAGEL, CRIFFITH, HAUT, AND STUCKEY, J R .
others, and a plasma cell neoplasm producing only one type of protein emerges. A transition from a diffuse multiclonal proliferation of plasma cells, associated with an electrophoretically heterogeneous hypergammaglobulinemia, to a monoclonal proliferation, with a homogeneous myelomalike hyperglobulinemia and urinary Bence-Jones proteins, has also been observed in about 10% of mink affected with Aleutian disease (Porter et al., 1965). There is some evidence that genetic factors and agents which stiniulate the proliferation of irnmunoglohulin-producing cells may also be of importance in the pathogenesis of human plasma cell tumors. The occurrence of plasma cell tumors in siblings has been mentioned earlier. Two patients with malignant lymphomas have been observed t o show a slow transition from a heterogeneous hypcrglobulineniia to a “monoclonal gammopatliy” (Waldenstriim, 1964n) suggesting that a similar transition from a diffuse to a “monoclonal” plasma cell proliferation may also occur in this disease. This pattern of a diffuse reactivc response progressing to the emergence of a single clone of protein-producing cells is very similar to that observed in BALB/c mice injected with mineral oil, and in mink with Aleutian disease. The Occurrence of a “monoclonal gammopatliy” in 31 patients with an associated carcinoma, and the demonstration of a striking plasmacytosis in the stroma and surrounding tissues of 12 of these tumors, has prompted the speculation that the antigenic components of certain neoplasms may stimulate a “monoclonal” plasmacytic response under certain conditions (Ossernian and Takatsuki, 1963). Virus-like particles were found in the plasma cells of mouse myeloma 55563 by Howatson and McCulloch (1958) and were subscqueritly demonstrated in most spontaneous and induced mouse plasma cell tumors (Parsons et al., 1961a; Dalton et al., 1961). Similar, virus-like particles have been found in the plasma cells of 3 of 18 myeloma p:itients (Sorenson, 1965) ; all of the patients with virus-like particles produced proteins with /3 electrophoretic mobility ( ? A-myeloma globulins). Most of these virus-like particles are of the “A” type of Bernhard (1958), consisting of two concentric membranes surrounding an electronlucent center. Considerable evidence suggests that they arc neither infectious oncogenic nor passenger viruses, for they lack a riucleoid, which is the anatomical structure of a virus assumed to contain thr reproductive nucleic acid, and all attempts to induce plasma cell tumors with cell-free extracts of the mouse tumors have been unsuccessful (Parsons et al., 1961b). Aleutian disease of mink appears to be caused by a virus, for the
THE TREATMENT O F PLASMA CELL MYELOMA
315
disease is readily transmissible by cell-free extracts of affected mink tissue (Henson et al., 1962) and cell-free serum (Gorham et al., 1963), arid the infectivity is sedimentable by ultracentrifugation. However, only 10% of the mink affected with the disease develop a monoclonal type of protein abnormality (Porter et al., 1965), and the neoplastic nature of the monoclonal disease has not bcen established. The virus of Aleutian inink disease certainly stimulates a diffuse, intense plasmacytosis and heterogeneous hyperglohulinemia, but its role in initiating a monoclonal ( ? neoplastic) proliferation of plasma cells is unknown. The significantly increased frequency of deaths from myeloma among American radiologists during the years 1948-1961 (Lewis, 1963), and the increased prevalence of myeloma in the proximally located survivors of the atomic bomb a t Hiroshima (Anderson and Ishida, 1964) strongly suggest a causal relationship between exposure to X-irradiation and the incidence of myeloma.
B. IMMUNOGLOBULINS 1. Normal Immunoglobulins
A complete discussion of the structure and biological activity of the inimunoglobulins is beyond the scope of this paper ; fortunately this subject has been reviewed recently by Cohen and Porter (1964). Items which are of interest in a discussion of plasma cell tumors will be considered. The nomenclature recommended by an international committee attending a World Health Orgnnization meeting on Nomenclature of Human Immunoglobulins (1964) has bcen employed in this article (Table I ) . A newly described immunoglobulin (Rowe and Fahey, 1965b) NORM.4L
Immunoglobulin class
TABLE I IMMUNOGLOBULINS
Concentration (mg./ml.)
Antibody function ~
6.6Sy; y2; yss) y A (82A; y l A ) yM (82M; y l M ; 19Sy)
yC: ( y ; 7Sy;
12.4 2.2a 2 . 8 0.7" 1.23 5 0.35"
+
Median 0 . 03,b range < 0.003-0.4 a
Fahey and McKelvey (1965). Rowe and Fahey (1965b).
Most acquired antibodies Skin-sensitizing antibodies Saline isohemagglutinins, saline RH antibodies, cold hemagglritinins, many heterophile antibodies Unknown
316
BERCSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
has been assigned the symbol of yD. The Bence-Jones, or light chain proteins, previously referred to as Type I, 1, or B, are called type K, and Type 11, 2, or A, becomes Type L. The heavy polypeptide chains of yG, yA, yM, and yD are known by the Greek letters y , a, p , and 6, respectively; the light polypeptide chains are K (for type K) and h (for type L). Using these symbols for light and heavy polypeptide chains it is possible to write molccular formulas identifying thc types of polypeptide chains detectcd in pathological proteins. The pathological proteins are referred to as (a) G-, A-, or D-myeloma globulin; ( b ) Mmacroglobulin; ( c ) type K or type L light chain protein or Bence-Jones protein; and ( d ) heavy chain proteins with the appropriate class designation. The yD class of immunoglobulin was idcntified in normal serum following the discovery of a myeloma globulin (Rowc and Fahey, 1965a) composed of heavy chains with unique antigenic properties ( 6 ) and type L light chains. Normal serum contains yD in concentrations ranging from less than 0.003 mg./ml. to as much as 0.4 mg./ml.; since the values are not distributed in a Gaussian manner within this range (Rowe and Fahey, 1965b) the median value and range are given in Table I. The functions of yD are unknown. It is of interest to note that the relative frequency of the plasma cell tumors producing pathological proteins belonging to different immunoglobulin classes (see Table 11) is proportional to the relative concentration of each immunoglobulin class in normal serum. If one assumes that the number of plasma cells synthesizing each class of immunoglobulin is proportional to the serum concentration, this would suggest that all types of plasma cells are equally susceptible to malignant transformation. Gcnctically determined factors, called allotypes, have been discovered on y-chains (Gm factors) and on type K light chains (Inv factors) of immunoglobulins. No allotypic factors have been identified to date on a-, p-, or 6-heavy chains, or on type L light chains (see revicw of Fudenbcrg and Franklin, 1963). It has been postulated that the allotypic factors identify the products of genes which control the synthesis of the various heavy and light polypeptide chains of the immunoglobulins. Several Gm factors have been described, and there are three known Inv factors, Inv (1), Inv(a) , and Inv (b) . These allotypic factors have been useful in demonstrating the homogeneity of myeloma protenis, for no G-myeloma protein carries the product of more than one of the genes Gm", Gmb or Gmf, (MBrtensson, 1964). It is of some interest that the y-chain subgroups of myeloma proteins identified by antigenic differences (Grey and Kunkel, 1964; Terry and Fahey, 1964) show a relation-
TABLE I1 CLASSIFICATION OF PLASMA CELL NEOPLASMS BY TYPEOF PROTEIN PRODUCED Frequency Osserman and Takatsuki (1963) Pathological protein Classification
Bergsagel et al. Immunoglobulin types Molecular formulas (1965) myelomas
1. Myeloma, no paraprotein
None
2 . Myeloma with paraprotein
Light chain protein G-myeloma globulin A-m yeloma globulin D-m y eloma globulin
3. HY-chain disease
Hr-chain
4. Waldenstrom’s
M-macroglobulin
macroglo bulinemia
None
Myelomas
2/255 (0.8%)
3/262 (1.1%)
32/90 (36%) 142/262 (54.2%) 18/90 (20%) 9/90 (10%) 58/262 (22.2%) 9/90 . (10%) . ,_ None described to date Only one case described
HY(type C ) HY(type Z) (P27 K2)n
(p2, X2)n
All plasma cell neoplasms 3/306 (1 .O%)
259/306 (84.6%) d B
r
r
5td
r
4/5 1/5
3/306 (1 .O’%)
41/306 (13.4%)
0
*M
318
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
ship to certain Gm factors (Kunkcl et al., 1964; Terry et al., 1965). None of the eight Gm factors tested for were found on purified human Gmyeloma proteins of subclasses y2a and y2b. G-myeloma proteins of subclasses 7% and y2c were positive for a t least one G m factor; y2b myeloma protcins were either Gm (a+), Gm ( a x , or Gin ( b2f+); y2c myeloma proteins containcd combinations of factors Gm(b’), (b’), ( b 4 ) ,and (c) (Terry et al., 1965).
+ +,
2. Pathological Proteins The term “pathological protein” is used to refer to the protein produced by a plasma cell neoplasm; the term is not meant t o imply that these proteins arc abnormal, for conclusive cvidence of this is lacking. It would appear that the protein produced by a neoplastic plasma ccll is determined by the stage and type of differentiation of the ccll originally affected by a malignant transformation. Some of the evidence which supports this view was reviewed earlier. It would therefore seem that a classification of plasma cell neoplasms based on the type of protein produced by the tumor would bc a n excellent way to group patients. A classification based on this principle and the incidence of cnch type of tumor in two large series are shown in Table 11. The series reported by Osserman and Takatsuki (1963) included all specimens with 7-globulin abnormalities seen in their laboratory in a 10-year period, on which there was adequate data on the serum and urine and enough clinical information to allow classification. Of the 400 classifiable paticnts, the diagnosis was myeloma in 262 (65.5%), Hy-chain disease in 3 (0.80/0), and was of unknown etiology in 71 patients (17.8%). Thirty-one of the patients with a “monoclonal gammopathy” of unknown etiology had an associated neoplasm of a tissue not known to be associated with normal immunoglobulin production. All of the serum and urine specimens studied by Bergsagel et al. (1965) were obtained from patients with plasma cell myeloma studied by the Southwest Cancer Chemotherapy Study Group (SWCCSG). It is of interest to note the remarkable similarity in the incidence of the various protein abnormalities of the mycloma patients in the two series. The important clinical observation that a pathological protein can bc demonstrated in the serum or urine of 9 5 9 9 % of patients with plasma cell neoplams is well known. Light chain proteins only are produced by 22-2470 of the myeloma patients. More than 5070 of patients with iiiyeloma form a G-myeloma globulin, and 22% form an A-myeloma globulin. Only one D-myeloma globulin has been described (Rowe and Fahey, 1965a). In the series reported by Osserman and Takatsuki (1963) , there were only 3 patients with Hy-chain disease; the number
T H E TREATMENT OF PLASMA CELL MYELOMA
319
of patients with this disease was later increased to a total of 5 (Franklin et al., 1964; Osserman and Takatsuki, 1964) and subgroups termed type C and Z have been recognized (Takatsuki and Osserman, 1964). I n the Ossernian and Takatsuki (1963) series 35 of 101 patients with G-myeloma globulins, 11 of 41 patients with A-myeloma globulins, and 3 of 41 patients with M-inacroglobulins also excreted light chain proteins in the urine. I n each case, the antigenic type of urinary light chain was identical with the light chain of the serum myeloma globulin. Indirect evidence from iniinunofluorescence studies indicate that the urinary light chain protein and the larger serum myeloma globulin are produced by the same cell in patients who haye 110th a seiuni and a urinary myeloma protein ; the appearance of the Bence-Jones proteins in the urine secms to be the result of an excessive production of thc light chains by the neoplastic cells (A. Solomon et al., 1963). A great deal of evidence, derived from the studies of protein produced by human arid mouse plahma cell tumors, supports the view that most of these tumors arc monoclonal. The hypothcsis requires that the turnor shall produce a single, honiogerieous typc of protein. Many studies have shown that most pathological proteins migrate as discrete bands with synirnetrical boundaries on filter paper clcctrophoresis and in the ultracentrifuge, and irnniunochernical studies have demonstrated t h a t thcse proteins arc composed of a single antigenic type of heavy and/or light polypcptidc chain. A pathological protein containing more than one antigenic type of heavy or light chain has never been described. The allotypic specificity of the y-chains of G-myeloma proteins has also been demonstrated (Mirtensson, 1964). Studies of myeloma marrow, using the sensitive, indirect immunofluorescence technique ( A . Solomon et al., 1963), have shown that all, or almost all, of the marrow plasma cells contain protein of the same antigenic type as the mycloma protein found in the serum or urine of the patient. The work of Potter and hie associates, demonstrating that “cloned” mineral oil-induced plasma cell tumors of BALB/c mice produce a singlc typc of protein, and the ohservation that the clcctrophorctic and antigenic properties of this protein do not change during the course of numerous transplant gcncrations and appear to bc stable, heritable characteristics of the tumors, has been mcntioned earlier. Recent starch gel electrophoresis and Ultracentrifuge studies have dcmonstratetl two types of heterogeneity in some liiiman and mousc myeloma proteins. The commonest form of heterogeneity, due to the formation of polymers, frcquently occurs with A-myeloma globulins and M-macroglobulins. Polymer heterogeneity results in the formation of multiple peaks when the sera are examined in the ultracentrifuge
320
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
(Hermans et al., 1959; Fahey, 1963a; Kunkel, 19601, and broad, irregularly spaced bands on starch gel electrophoresis (Engle et al., 1961; Fahey, 19634. Starch gel electrophoresis has also demonstrated a second type of heterogeneity in many human and murine Bence-,Jones proteins and G-myeloma globulins, which is not due to polymer formation and has bcen termed “polymorphic heterogeneity” (Bernier and Putnam, 1964). This type of heterogeneity causes the formation of 2 to 5 discrete, narrow, regularly spaced bands in the starch gel electrophoresis pattern (Fahey, 1963a). Bernier and Putnam (1964) found that the starch gel electrophoretic heterogeneity of four Bence-Jones proteins could be explained by (a) contamination with other proteins (e.g., albumin, glycoproteins, 7Sy-globulins, and transfcrrin) ; ( b ) light chain monomers and dimers; and (c.) polymorphic variants. It was possible to isolate variant proteins chroniatographically from two samples. These variants had sedimentation coefficients and antigenic properties which were identical with the major component, but differed in starch gel mobility. The variant Bence-Jones proteins had slightly different thermosolubility curves, and differed by one or two peptide spots when examined by peptide mapping. The basis of polymorphic variants of Bence-Jones proteins has not been explained, but the great similarity of the variant t o the major component, with differences in only one or two peptides, suggests that the variant may arise from a mutation occurring in the original malignant plasma cell line. The data reviewed above are consistent with the hypothesis t h a t most myelomas arise from the malignant transformation of a single plasma cell which successfully establishcs a clone of neoplastic cells. The occurrence of polymer heterogeneity of the myeloma protein is not inconsistent with the view t h a t a clone of plasma cells should produce only one protein, and polymorphic heterogeneity could be explained by a mutation in one of the cells of the neoplastic clone, resulting in the establishment of a mutated clone producing a polypcptide chain with an altered amino acid sequence, and a change in the electrical charge of the myeloma globulin. A few myeloma patients have been found to produce two distinctly different proteins. Curtain and O’Dea (1959) described a myeloma patient with two abnormal serum components, one with a sedimentation constant of 7s and the other of 1 8 s . Kistner and Norberg (1964) reported a patient producing both a G- and an A-myeloma protein; Engle and Nschman (1964) found both type K and type L Bence-Jones proteins in the urine of a patient with plasma cell myeloma; anrl Imhof et al. (1964) noted the occurrence of two pathological proteins of different antigenic types in 6 of 350 patients with paraproteinemia. It seems
THE TREATMENT O F PLASMA CELL MYELOMA
32 1
likely t h a t the appearance of two proteins t h a t differ structurally and aritigenically in a patient with niyeloina is due to the proliferation of two neoplastic plasma cell clones. Support for this vicw has been provided by the ininiunofluorescent demonstration of a population of plasma cells responsible for the production of a 7 S pathological protein and another population of plasma cells producing an 18 S pathological protein in a patient previously reported to have two pxthological proteins (Curtain, 1964).
C. COGRSEOF
THE
DISEASE
The clinical, radiographic, pathological, and biochemical features observed in patients with plasma cell malignancies will not be covered completely in this section, for this mataerial has been admirably reviewed elsewhere (Adams et al., 1949; Osscrman, 1959; Waltlenstroni, 1961 ; Osserman and Takatsuki, 1963; Drivsholm, 1964; Snapper and Kahn, 1964). Instead, certain aspects of stages in the course of the disease, complications which require special treatment, and the principal causes of death will be considered. 1 . Clinical Stages
a. Diagnosis and the Preclinical Stage. The diagnosis of a plasma cell neoplasm requires the demonstration of an increascd proliferation of plasma cells, or of related cells which may have lymphoid features. Plasiiiacytosis is usually denionstrated in a bone marrow aspiration snicar, but increased numbers of plasma cells may also hc found in the splcen or lymph nodes, and occasionally thc primary tumor arises in a n extramcdullary site ( D o h and Dewar, 1956; Helnius, 1964). Shapiro and Watson ( 1953) dcmonstratcd an increased proportion of p1:tsnia cells in 9 of 10 splenic aspirations from patients with rnycloma. Unfortunately there are no pathogiiornonic cytological features which permit accurate differentiation of neoplastic from normal plasnia cells, or of the cells responsible for the production of the various pathological proteins (Brittin et al., 1963; Brecher et al., 1964). Although a plasma cell neoplasm is the commonest cause of a marked marrow plasniacytosis, the demonstration of 2070 plasma cells in the marrow is no longer considered diagnostic, for a reactive plasmacytosis of this intcnsity has been denionstrated in association with the following groups of diseases: ( 1 ) sensitivity to drugs or certain antigens; (2) collagen diseases; (3) infections, predominantly chronic and frequently of granulomatous type; (4) cirrhosis of the liver; and (5) disseminated malignant neoplasms (Fadem, 1952; Clark and Muirli~ad,1954; Aherne, 1958). A
322
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, J R .
patient who developed a plasmacytosis of 30% in the peripheral blood during an episode of serum sickness induced by equine tetanus antitoxin (Barnett et al., 1963) is a striking example of the reactive plasinacytosis that may occur in some patients. The occurrence of a homogeneous M-peak in a serum electrophoresis pattern probably represents the proliferation of a single plasma cell clone; however, it is not always possible to demonstrate that the lesion is neoplastic. Many investigators have reported groups of patients with a “n7onoclonal gammopathy” of unknown etiology (Ranstroln, 1946; Osserman, 1958; Hammack et al., 1959; Ogryzlo et al., 1959; Ossernian and Takatsuki, 1963; Waldenstrom, 1964a). I n some of these patients, the protein abnormality has re’mained stable for as long a s 9 years, and the failure of repeated attempts to demonstrate a neoplastic proliferation of plasma cells or a lymphoma suggests that in these patients the condition may be benign (Osserman and Takatsuki, 1963). However, the fact that many patients progress to frank myeloma makes it necessary to view this group with the suspicion that they are in a preclinical stage of one of the plasma cell malignancies. Because a malignant plasma cell disease cannot be established on the basis of a single pathognomonic feature, the diagnosis has been based on the association of several features. Most clinicians would agree with a diagnosis of a malignant plasma cell disease in one of thc following circumstances: (1) the association of ‘marrow plasmacytosis (greater than l o % ) , osteolytic lesions, and a homogeneous serum or urinary globulin; (2) the association of osteolytic lesions and marrow plasmacytosis, in the absence of other diseases such as metastatic carcinoma or bone tuberculosis, which could cause similar lesions; (3) the demonstration of tissue plasmacytosis, plus osteolytic lesions, or a pathological protcin; (4) the occurrence of a persistent tissue or niarrow plasmacytosis and a progressively increasing concentration of a homogeneous serum or urinary globulin; or (5) the formation of a plasma cell tumor, with invasion, distortion, or destruction of local tissue. Thus, most clinicians prefer to wait until there are clear indications of uncontrolled (i.e., neoplastic) growth evidenced by the formation of an invasive tumor, tissue destruction (osteolytic lesions), or a progressive increase in the concentration of the product of the tumor’s growth (pathological protein) in the serum and/or urine, before making a diagnosis of a malignant plasma cell disease. The studies of the “monoclonal gammopathies of unknown etiology” (Osserman and Takatsuki, 1963; Waldenstriim, 1964a) are of great importance, for a better understanding of the clinical significance of this disorder, and the clear identification of patients with neoplasia, may result in a definition of
T H E TREATMENT OF PLASMA CELL MYELOMA
323
iiialignant plasma cell diseases which will permit earlier diagnosis and treatment. b. “Solitary” Myeloma. The term “solitary” niyeloiiia or plasmacytoma has been used to describe the patients who prescnt an apparently single skeletal lesion. It seems likely that these patients 1-cpresent an earlier stage of plasma cell myeloma, for a group of patients with apparently “solitary” lesions treated with S-irradiation liad a better survival rate than patients with multiple lesions. Cohen et al. (1964) have published an analysis of 44 apparently solitary myelomas of the PERCENT LIVING
I
2
5
10 20
40 60
8 0 90 95
99
PERCENT DEAD
FIG.1. Survival from diagnosis of patients with “solitary” and “generalizcd” plasma cell malignancirs. L ~ ‘ S o l i t a r y , ”44 patients, mediim 52 months, from Cohen e t al. (1964) ; A--“Generalized,” 185 patients, median 8.5 months, Midwest Cooperative Chemotherapy Group (1964).I vertebral column (the marrow was normal in 13, and not examined in 31 patients), treated primarily with X-irradiation. The cumulative proportion of patients in this series surviving at yearly intervals has been plotted on logarithmic probability paper in Fig. 1. The median survival of’ 52 months from diagnosis is significantly better than the rnediaii survival of 81/2 months from diagnosis estimated for 185 patients with multiple myeloma (Midwest Cooperative Chemotherapy Group, 1964) shown on the same figure. The improved survival of patients treated when the disease appears t o be solitary indicates that these patients form a special group; the observation t h a t the disease progresses to the
324
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, J R .
disseminated form in almost all cases suggests that they are diagnosed in an earlier stage of the disease. c. Generalized Stage. The majority of patients with plasma cell myeloma present generalized disease. Of 255 inyelorna patients studied by the SWCCSG, 60% were males, and 40% of the 142 patients on whom race was reported were Negro. The median age was 59, with a range of 28 to 87 years; 83% wcre ovcr the age of 50 years. The frequency of various abnormal laboratory tests shown in Table I11 agrees with the TABLE I11 PLASMA CELLMYELOMA: STATVS AT FIRSTVISIT Abnormality Pathological protein, serum and/or urine Marrow plasmacytosis > 10% Hemoglobin < 11.0 g.70 Osteolytic skeletal lesions Bence-Jones proteinurea Blood urea nitrogen > 30 mg.% Serum calcium > 11. O mg.%
TO
SWCCSG
Number/number reported
Per cent abnormal
253r25.5
99 85 69 68 50 36 30
129/151 169/244 143/ 2 10 111/212 80/220 71/236
frequency noted by Adams et al. (1949). In addition to the abnormalitics listed in Table 111, it should be noted that hyperuricemia (serum uric acid greater than 6.0 mg.7.) occurs in about two thirds of patients, lymphadenopathy in about lo%, and splenomegaly in about 10%. Amyloidosis was dcrnonstrated in 29 of 150 myeloma patients reviewed pathologically by Magnus-Levy (1952), but was noted clinically in only 7 patients studied by the SWCCSG. Fever due to an infection is conimon in plasma cell myeloma. Adams et al. (1949) observed fever in 52% of their patients. Fahey et al. (1963) found a frequency of 0.17 to 0.19 infections per patient per month in a group of 27 myeloma patients followed a t the National Cancer Institute ; this frequency is significantly higher than the 0.03 infections per patient per month among the general patient population seen a t the Clinical Center in 1961. A marked increase in serum viscosity has been observed in many patients with M-macroglobulinemia (Waldenstrom, 1944; Fahey, 1963b) and in some patients with A- and G-myeloma globulins. The hyperviscosity syndrome will be discusscd later. Myeloma patients with certain manifestations have a very poor prognosis. Patients with a blood urea nitrogen of more than 40 mg."/., hypercalceniia resistant to adequate treatment with hydration and prednisone, those unable to maintain a hemoglobin of more than 9.0 g . F without repeated transfusions, and patients with a rapidly increasing
THE TREATMEST OF PLASMA CELL MYELOMA
325
number of plasiiia cells in the peripheral blood have a markcdly shortened survival.
2. Supportive T h e r a p y Therapy directed a t reducing the number of proliferating plasma cells is the iiiobt iiiiportant aspcct of the medical management of patients with plasma cell neoplasms; this therapy will be discusscd in the next scction. Supportive care desigiicd to prevent or relieve tlic niajor coinplic:itions of infections, pathological fractures, liypercalceniia, renal failure, and the hypcrviscosity syndroiiic will bc considercd in this bcctloll. a. Geneml. Patients should be encouraged to be as active as possible in order to reduce the osteoporosis and loss of muscle tone wliich accompany inanition. To accomplish this, the physician must relieve pain by the use of X-i~~r:iiliatioiito localized skvlctal lesions, splints or intramedullary pins to iiiiinobilize pathological fractures, crutches for support in walking, fitted corsets with steel back braces t o btahilizc a painful lumbar spiiie, :uid the use of aiinlgcsics. b. Infections. Infections occur frequently in patients with niyelonia. Piicunioriia is tlic coniinoiicbt type of iiifection, followed by urinary tract infections, cutaiieous infcctioiis, septicemia, sinusitis, and other less frequent forms (Zinnciiian and Hall, 1954; Glencliur e t al., 1959; F:ilicy e t al., 1963 ; RIidwest Cooperative Clieiiiothcrapy Group, 1964). Ittycatetl bacterial, especially pneuiiiococcal, infections are coninion. In tlic series reported by (;lcncliur et al. (1959), 28 patients (55%) ex1)ei.iciicecl at total of 76 cpisoclcs of pncumoiiix, or aii average of 2.7 bouts per patient; one pciticnt suffered 12 different episodes of pncuiiionia during the course of his discasc. Fahey et al. (1963) found that the frcquc 11 c y of 111fcc ti oiib 11I iii y e 1oiiia pat iciit b p i ~ ~ ling u c A - or G -my c 1oinii globulins or Bcnce-Jones proteins did not correlate with the type or aiiiount of patliologic:il protcin, or tlic s ( m m Icvels of noriii:il imniiirioglobdins, b u t was related to the ability to respond to several antigens. Infectious coiiiplications occurred less frequently in patients with RIniacroglobulinciiii:i, but no diffcrenccs in antibody responke or normal serum ininiunoglobulin levels could be tlciiionstrated bctwccn patients with ilI-iii:icrogloI~uliiiciiiia :ind thow with niyclonia. Tlic deficient antihody rcsponw is probably tlie niobt inlpo~tant of the factors responsible for the incrcxscd susceptibility to infections, hut other defects ni:iy alho be iiiiportant. Iiigrani (1960) found tlixt ICLICOcyte pliagocytic activity is inliibited in vitro by plnsiiia cont:iiniiig increased concentrations of myeloma globulins with 7 electrophoretic mobility, and RIarsh and Perry (1964) observed a poor grnnulocyte
326
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
response to endotoxin in myeloma patients. However, i t is not known whether these abnormalities of granulocyte function are important factors in determining the susceptibility to infections. All infections in myeloma patients require imnediate attention. Bacterial cultures should be obtained, and antibiotic therapy initiated. For patients with serious infections, antibiotic therapy is frequently recommended before the results of cultures and antibiotic sensitivities are available. The vigorous combination of methicillin, chloramphenical, and colistimethate sodium has been recommended (Silver, 1963) when infections with penicillin-resistant staphylococci and gram-negative organisms are frequent and there is a great danger of septicemia. y-Globulin administration should be considered for paticnts with recurrent infections. Pooled human 7-globulin (1576 solution) may be administered initially in a dose of 0.3 ml./lb. intramuscularly; a maximum of 5 i d . is given a t one site and the full dose may be given over several days. Maintenance injections of half the initial dose may be given evciy 3-4 weeks. Definite statements regarding the value of y-globulin therapy, or the dosage required cannot be made, becausc clinical studies in rnyeloina have not been completed. Fahey (196313) noted that the following factors should be taken into account in selecting the dose of y-globulin for myeloma patients: (1) the markedly shortened half-life of normal y-globulin in patients with G-myeloma globulins may increase their dosage requirements; (2) fever accclerates the rate of y-globulin catabolism; (3) some serum antibodies are among the y-macroglobulins, but these proteins may not be fully represented in commercial y-globulin preparations. The use of prophylactic antibiotic therapy should be considered in patients who experience repeated infections with sensitive organisms such as the pneumococcus. c. Hypercalcemia. An elevated serum calcium was noted during the initial evaluation of 30% of the patients seen by the SWCCSG, and frequencics of 43% (Bcntzel ct al., 1964) and 70% (Gutman et al., 1936) have been reported. Increased bone resorption appears to be tlic main factor responsible for the development of hypercalcemia (Lazor and Rosenberg, 1964), and this complication should be looked for in all patients with osteolytic lesions or osteoporosis, espccially in patients with skeletal lesions who are immobilized. The renal lesions produced by hypcrcalcemia and hypcrcalcurin, which begin as cellular lesions in the collecting ducts, and the later appearance of calcium precipitates, have been reviewed by Frci e t aZ. (1963). A prominent clinical feature of hypercalceinia is polyurea, and this may be followed by anorexia, nausea, vomiting, dehydration, and
327
THE TREATMENT O F PLASMA CELL MYELOMA
rapidly advancing azotemia. One third of the SWCCSG myeloma patients who presented hypercalcemia also had an elevation of blood urea nitrogen of more t h a n 40 mg.70. Rlild hypercalcemia may be controlled by adequate hydration, reducing calcium intake, and increased activity. In addition, corticosteroid therapy is frequently required, and successfully reverses the hypercal-
-rw I
, , , May. I965 -1mprovemeni
maintained
Doys ofter Melphalon started
FIG.2. The effect of melphalan therapy on a myeloma patient (56-year-old Cmcasian male) who presented with severe anemia, hypcrcalceniia, hypcrglobulinemia, palpable scalp plasmacytomas, and diffuse, painful, lytic skeletal lesions. Hydration alone failed to lower the serum calcium, but 2 weeks after starting melphalan, the serum calcium had fallen to normal (5.2 meq.iliter). T h e y-globulin level fell from lO.Og.% to a plateau of I.S-2.0g.% within 2 months and the M-peak disappeared later. Four transfusions were required during the first 25 days, but after this the hemoglobin level climbed slowly without transfusions, reaching a level of lZ.Og.r/, on day 440. The scalp plasmacytoinas flattened within 3 weeks, and pain disappeared, allowing the patient to become ambulatory and return t o full ztrtivity. The patient has takrn 1.5 mg./kg. in 4 days a t 2-month intervals, developing only moderate hcmatological toxicity after each course. Improvement is maintained.
ceinia in the majority of patients (Merigan and Hayes, 1961) by reducing boiic rctsorption (Bcntzel e t al., 1964). If the myeloma is sensitive t o the effects of a chcmotherapcutic agent, the hypercalcemia may be controlled by therapy directed a t reducing bone resorption by controlling the neoplastic osteolytic process. The correction of hypercalcemia, the fall in the myeloma serum and urinary protein, and rise in hemoglobin, which followed melphalan therapy alone, is shown in Fig. 2.
328
BEIIGSAGEL, GHIFFIT€I, NAUT, AND STUCKEY, JIt.
d. Pathological Fractures, The importance of relieving pain and promoting hcaliiig by tlie immobilization of fracturcs by splints or intramedullary pins and X-irradiation has bcen stressed earlier. The usefulness of sodium fluoride in strcngthcning tlie skeleton in patients wlth myeloma and thus relieving pain and preventing pathological fractures needs to be explored in clinical trials (Cohcn and Gardner, 1964). e. Renal Failure. The major factors involvcd in tlie pathogenesis of renal insufficiency in inyelonia patients have been reviewed by Frei et ul. (1963), and may be listed as follows: (1) the most important factor is tlic excretion of Bcnce-Jones protein, which may form tubular protein prccipitates resulting in obstruction and a foreign body reaction ; (2) Bcnce-Jones proteins m:ty also cause intracellular tubular daniage, for intracellular liyaline bodies (probably Bcnce-Jones protein aggregates) have bccn found early in the dcveloping nephropatliy ; (3) hypercalcernia occurs frequently and may lead to renal daniage (see abovc) ; (4) pyclonephritis occurs frequently in myeloma paticnts ; (5) amyloidosis inay occur in a sinall percentage of patients; and (6) rarely there may be plasma cell infiltration of the kidney. Each myeloma patient who develops renal insufficiency must bc carefully evaluated, for factors (3) arid (4) are revcrsiblc, and the maintenance of a good urinary output by adcquatc hydration may prevent or delay factor (1). Therc is no evidence that the renal insufficicncy resulting from tubular Bence-Jones precipitates or renal amyloidosis regresses following successful antineoplastic therapy. However, if the amount of Bcnce-Jones protein produced by the plasma cell tumor decreases after antineoplastic therapy, one would expect that the progression of the lcsions would a t least be slowed. f . Hyperviscosity Syndrome. A marked increase in serum viscosity develops in many paticnts with M-macroglobulinemia (Waldenstroni, 1944; Faliey, 1963b), and in some patients with A- and G-myeloma globulins. The increased serum viscosity observed in two myeloma patients was found to be caused by the formation of G-myeloma protein aggregates (Smith e t al., 1965). Scrum hyperviscosity causes a clinical syndronic with the following manifestations: (1) bleeding diathesis characterized by cpistaxis and bleeding from mucous membranes, a prolonged bleeding timc, abnormal clot retraction, and abnormal prothrombin consumption (even when the platelet count is normal) ; (2) retinopathy, with dilatation and segmentation of retinal veins, round hemorrhages, and papilledcma ; (3) neurological symptoms of vertigo and nystagnius, and electroencephalographic changes ; (4) cardiac failure; and (5) weakness, fatigability, and anorexia (Fahcy, 1963b; Smith c t al., 1965).
T H E TREATMEST O F PLASMA CELL MYELOMA
329
The :tbovc syndrome is causctl by macroglobulins, or protein aggregates wliicli hnvr :i 1ii;irkcd effect on beruin viscosity because of their size and shape. I3ccnusc~tlic offciitling protcins arc largely intravascular, :tnd are pi~ocluccila t a fixed rate hy nialignniit plasma cells, it is possible to lower thcir plusinn concentration cffwtivcly hy plasniuphercsis, and producc niarkcd clinical irnprovcmcnt (Solomon and Fahcy, 1963) . Tlic cffcctivencbs of plasniaplicrc~is is easily evaluatcd by following tlic cliangcs in scruiii viscosity and the clinical nianifestations. Hyperviscosity syniptoim are r u e when thc beruin viscosity falls to lcss than 3, rclative t o water (Fahey, 1963b). Plasmaphcrcsis is nccessary to rapidly relieve tlie disturbing hypcrviscosity syndrome, but it sliould be suppleinentcd by antincoplabtic therapy to shrink the tumor and reduce tlic amount of protein proiluced. 3. Cnzrses of Death
The actual inechanisni of death in patients with plasma cell myeloma is oftcn obscurc, as in all patients with neoplastic discascs. The Midwest Coopcrative Chemotherapy Group (1964) listed the principal factors contributing to death i n a series of 48 autop~iedpatients in the following scqucnce: (1) infcctions, primarily pncumonias, wcre listed in 56% of tlie cases; (2) noninfectious cardiopulmonary complications, such as pulmonary edcrna, heart failurc, and otlicrs were listed in 29% ; (3) uremia was lwlicvetl to 1)c the niajor factor in 17%; (4) myeloma in 1270 ; (5) hemorrhage iii lo?, ; and 16) miscellaneous other causcs accounted for thc rcmaindcr. I l l . Antineoplastic Treatment of Plasma Cell Myeloma
A.
EV.4LV.jTIOS O F T H E
RESPOSSETO THERAPY
C:lieiiiothrrapist6 have frecpcnt ly ( ~ x p r c ~ w tlic l opinion that a cliug wliicli is complctcly effertivc i i i (wring a maligiiant ilihc:ibe should bc easy to wcognize, for this diug n.oultl ca.~isc:ill Iii:iiiifCbt2itioii~of tlic tumor to disappear, and treatcd patienti would h a m a normal lifc expectancy. Unfortuniitely, this opinion has not bren tested in the tiw~tincwtof riiye1Oiiia, for a c l l ~ i gwhich has been used in a curativc way has not hecn reportcd The prohlciii~i n drug evaluation have bccii to ( I ) distiiiguish drugb that (~aiisctunior regression in a uwful proportion (c.g., in 20% or ~ n o r c )of patients, from those that arc incffective; and ( 2 ) caomparc the relntivc value of drugs that arc cffectivc i n inore than 2070 of patient?. Progress in these two arcas is rcquircd to guide the development of new drugs, and for thc estnblishnient of an animal
330
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
tumor screen that will detect active drugs for the treatment of plasma cell myeloma in man. 1. Direct and Indirect Manifestations of Myeloma
In the evaluation of the response to therapy, a clear distinction must be made between changes in the manifestations of the disease which are thought to be the result of tumor regression, and the changes which frequently accompany tumor regression, but may also occur as an indirect effect, such as a rise in hemoglobin and gain in weight following testosterone therapy. The Chronic Leukemia-Myeloma Task Force of the National Cancer Institute has adopted the following definitions to distinguish between objective effects on direct and indirect manifestations of myeloma: a. Objective Effects on Direct Manifestations of Plasma Cell M y e loma. Improvement which satisfies one or more of the following criteria is believed to represent tumor regression: (1) a t least a 50% regression in the product of the two largest diameters of palpable (or X-ray visualized) plasmacytomas ; (2) significant decrease in the serum myeloma protein, e.g., a fall to SO%, or less, of the prestudy value; (3) significant decrease in the urinary myeloma protein, e.g., if the prestudy value is 1.0 g./24 hr., or more, there should be a fall t o 50% or less; if the prestudy value is between 0.5-1.0 g./24 hr., the decrease should be to less than 0.1 g./24 hr. (this manifestation of the disease may not be a reliable indication of the response to therapy, if the prestudy value is less than 0.5 g./24 hr., or if there is renal insufficiency and increasing azotemia) ; and (4) definite radiographic evidence of skeletal healing. Considerable emphasis has been placed on the importance of the changes in the myeloma serum and urinary protein. There is good evidence that these proteins are produced by myeloma plasma cells (A. Solomon eE al., 1963), and studies of mouse plasma cell tumors have shown that the serum myeloma protein concentration associated with a particular tumor line is a direct reflection of the size of the tumor in the mouse being studied (Nathsns et al., 1958; Fnhey, 1962). The plasma cell tumors of mice differ in thc amount of protein present per gram of tumor, and it seems likely that varying amounts of protein are produced by plasma cell tumors of different patients. Thus, although the amount of myeloma protein present in the serum or excreted in the urine of different patients does not correlate with the amount of tumor tissue, changes which orcur in a single patient do reflect, in a general way, changes in the size of the tumor. It is theoretically possible for a drug to interfere with the protein-producing apparatus of the cell and
T H E TREATMENT O F PLASMA CELL MYELOMA
331
lower myeloma proteins without affecting the size of the tumor, but a drug which produces this effect on patients with myeloma has not becn dcscribed. It has also been observed t h a t patients who respond t o melphalan therapy with a decrease in the myeloma serum and/or urinary protein to 50% or less, of the prestudy value, survive significantly longer than those who do not (Bergsagel et al., 1965). b. Objective Effects on a n Indirect Manifestation of P l a s m Cell A l yeloma. Improvement in the following manifestations of myeloma usually occurs in patients who show tumor regression ; however, similar improvement may also occur as a n indirect effect when there is no shrinkage of the tumor. Improvements in these manifestations, which occur without improvement in a direct manifestation of the disease, are of unknown significance: (1) a significant rise in hemoglobin, e.g., an increase of 2.0 g.% or more; (2) a significant gain in weight, e.g., 10 pounds or more, in the abseiice of edema; (3) a fall in serum calcium from more than 12.0 mg.% to 10 nig.%, or less; (4) if dehydration is not a factor, a fall in blood urea nitrogen from more than 30 mg.% to normal ; ( 5 ) recovery of normal immunoglobulins ; (6) improvement in serum albumin to normal; (7) a reduction in the percentage of marrow plasma cells to less than 5% (although this parameter is occasionally valuable, it has proved to be unreliable because of the spotty nature of the plasmacytosis) ; (8) marked lymphadenopathy or splenomegaly in myeloma patients is usually caused by a plasma cell infiltration, and regression may be considered an objective effect on a direct nianifestation of the disease by some; however, lymphadenopathy may be due to other causes (e.g., infection), and splcnomegaly may result from hrmolysis. Only those agents which cause an objective effect on one or more of thc direct manifestations of myeloma should be regarded as bcing capable of causing tumor regression. Changes which occur in the indirect manifestations of myeloma may be helpful in rating the excellence of the response to therapy, but do not, by themselves, reflect changes in the size of the tumor. Similarly, subjective effects, such as the relief of pain, changcs in performance status, appetite, etc., are often useful in grading the response, but should not be relied on t o identify effective agents. 2. Comparison of the Effectiveness of Dijerent Methods of Treatment
a. Factors Influencing the Response Rate. It is relatively easy t o differentiate between compounds which produce objective improvement in more than 20% of myeloma patients, and those which are less active (Bergsagel et ul., 1962d; Bergsagel, 1962). The comparison of the rela-
332
BERGSA(;EL, GRIFFITH, HAUT, A N D STUCREY, JR.
tive effectiveness of drugs which have produced objective improvement in more than 20% of patients, arid of different dosage schedules of effective drugs, is much more difficult. Cornparisons may be made on the basis of the rcsponse rate and the survival of groups of patients. Some of the important factors which influence the determination of the response rate and survival of myeloma patients will be considered in this section. The frequency of objective improvcmcnt following niclphalan therapy, reported by different investigators, has varied from 15 to 85% (Table I V ) . The determination of the response rate in these studies was influenced by the following factors: the selcction of patients; the criteria used to ticfine “objective iriiprovcnient”; and the definition of a n “adequatc trial,” or the requirements for an lLevaluable”patient. The selection of patients for a clinical trial will obviously influence the rcsponse rate. When new agents are initially tested for evidence of activity in the treatment of myeloma patients, many study groups elect to test the drug on “good-risk” patients, because it is fclt that the prognosis of the “poor-risk” patients (see Section II,C,l,c) is so poor that they will not live long enough to be treated adequately. Other patients with n good pi,ognosis (c.g., “solitary” myelonin trcatcd with X-irradiation) may also be eliininated. When the SWCCSG began t o evaluate melphalan, patients who presented hypercalcernia and/or uremia were excluded because, a t that time, melphalan was of unproved value and it was felt that hypercalcernia required immediate treatment with hydration and prednisone (Bcigsagcl e t al., 1962d). Most of the patients treated initially with prcdnisoiic rcccivctl niclphalan a t a later date, aftcr the hypercalcemia had bccn controlled, but 10 of these patients expired before mclphalan could be started. The elimination of these patients favorably influenced the response rate observed by the SWCCSG. Similar selective factors have probably influenced the response rates observed in other studies. Waldrnstrom (196413) reported a scrics of 75 myclonia patients: 8 of these patients were not titated with mclphalan; 6 patients had advanccd disease, and 5 of thcse died within 2 weeks of admission to hospital. The diagnosis of myeloma was not cstablislictl prior to the dcath of one patient, and the eighth patient had mild disease, without pain, and is being followed without therapy. Myeloma patients who fail to improve following an adequatc trial with one type of alkylating :grnt are very likcly to fail to respond to treatment with another alkylating agent. Therefore, the inclusion of patients who have failed to improve following cyclophosphamide or chlorambucil therapy in a mclphalan trial would probably lower the melphalan response rate.
THE THEATMEST O F PLASMA CELL MTELOlIA
333
Selective factors may also influence tlic type of patient sccn and c:ttcd :it a hospital. An institution which aewpts a large nuiiibcr of patien ts referred from considcrahle distances probahly rcceives patients with less cxteiisi1.e tliscase tliaii n large coriiinunity hospital. Patients n itli advancctl dimisc, who woultl liavc difficulty traveling, would not be sent far from Iiome, but would be admitted to a local hospital. This typc of sclcctire factor inny 1i:ii e influenced the rcspoiise ratc ol)servcd at the I‘nivorsity of Tcms M. D. Ander~onHospital, wliicli iwcivcs only refrrred patients, tlie majority being referred from outside tlic c1ty [ W c ‘ 1)clon~). The criteria used to dcfinc what constitutes objcctivc improvement 1i:is :t ninrked effect 011 the response rate. The groups reporting the lowest rcsponsc rates shown in Table I V used tlic most conservative criteria for evaluating tlie rcbponsc. W:ddcnsti 6in (1964b) did not follow tlic quantitative cliangcs in protcinurcn following mclplialan therapy; since 22-2470 of rnyeloina patients produce only Bence-Jones proteins, tlic cv:iluation of tlic 1 esponsc to melphalnn of a largc proportion of patients in this series was incomplete. I n a series of 199 patients treated by the SWCCSG, the mclphnlan responses of 2276 (42 patients) were not cvaluable because the reports n’crc inndequatc ; the patients liacl received concurrent corticosteriod thci,apy; or tlie patients died, or were lost to follow-up, within 3 weeks of ~tnrtiiigthc drug. If thcsc noncvwlual)lc patients arc removed, the rcq)on>c rate improves from 34 to 4370. Similarly, one would expect the i cbpoiisc rate to ixnprovc. :is tlic, diirntion of trcatmcnt rcquiiwl for an adequate trial is prolonged. Ossernim (1965) evaluated the response of patients who ’vverc treated with nielplialan for 6 nionths or more, and reported n w r y Iiigh rcsponac rate of 8 5 F ) . I n the SWCCSG series, 53 of 199 patients (27%) died during thv first 6 inonths of therapy. JIost of tlitw tlcnths ocr~i~~rc~cl in p a t h i t s who failctl to iniprove, and on(’ m7ould t q c c t tlie rebponsc r:\tc to increase i f this group of “tioiirespoiiders” wcrc eliminated by requiring that, rvaluable patients complete 6 nionths of t rc ti t nicn t. Variations in tlic melpha1:m do.:tgc schedule may also influence the response rate. Rfany invc,stig:itors 1i:ivc used low-dose, continuous maintenance tlierapy, while other+ have used an intermittent schedule in whirli a largc dose (1.0-2.0 mg./kg.) is given over a 4-day period, and iqwatcd a t 6- to I 0 - ~ v e c kintervals (A.1LI.A. Council 011 Drugs, 1965), but as yet an atleqiiatc comparison of the effectiveness of the two >clictlulcs has not bcen reported. Sincc the factors nientioncd ahove were not standardized, it is not possible to coinpare the response rates observed by the different groups. ti
TABLE IV RESPONSERATESREPORTEDFOR PLASMA CELL MYELOMA TREATED WITH MELPHALAN Improvement in 1 or more objective manifestations Author and study group
Number
Number
yo
1. Ossermari (1965)
Total Evaluable
? 45
38
85
2. Iloogstraten (1964) Leukemia Group B
Total Evaluable
65 ?
?
54
Total Serum M-protein
67 37
? 18
49
3. Waldenstrijm (1964b)
Dosage schedule
Comment
10 mg./day X 7-10; no Evaluat.ed only patients treated for 6 months Rx for 3-8 weeks; maintenance, 2 mg./ or more day 0 . 1 5 mg./kg./day X 7; no Rx > 14 days; maintenance, 0.05 mg./kg./day
Evaluated results for patients on study 3 months or more. Objective improvement in 2 or more parameters
Similar to 1
No data on response of 8 patients producing BJPb only, and 22 others. Of 37 patients with serum M-protein 18 showed a decrease of > 40%
4. SWCCSG
lZIDAIIQTotal Evaluable SWCCSG Total Evaluable
61 56 190 157
34 34 68 68
56 61 34 43
High dose, intermittent
5. Costa (1963) Eastern Solid Tumor Group
Total
42
16
38
Range 10-100 mg./kg./ day), median 50
6. Brook et al. (1964) Western Cooperative Cancer Chemotherapy Group 7. Lee et al. (1965)
Total
36
13
36
Low dose, continuous 0.05-0.1 mg./kg./ day
Total “Adequately treated”
40 27
6 (1AP 6 (lA)c
15 22
Xot stat,ed
8. Bernard et 01. (1962)
Total
17
4
24
10 mg./day X 6
9. Videbaek (19621
Total
30
3
17
10. Speed ef 01. (1064)
Tot a1
20
8
40
Total Total evnluable 0
561 476
212 212
Similar to 1
-
Evaluated patients on study > 3 weeks
“Adequately treated” patients received drug for 3 months or more
Rise in hemoglobin in 8 Fall in myeloma protein in 4
38 45
The 1-niversity of Texas NI. D. Anderson Hospital and Tumor Institute, Houston, Texas.
* Bence-Jones proteins. c
1.4 status calls for improvement in all objective parameters.
W W
cn
336
BERGBAGEL, C;RlFFITI-I, IIAUT, AKD STUCKEY, J R .
b. Factors Influencing Survival. At the present time, a comparison of the survival of two coinparable groups of patients treated by different drugs or dosage schedules is the most reliable way of determining the relative effectivcncss of diffcrent, mcthotls of treatment. The two groups must be coniparablc. In thc clinical studies the random allocation of patients to the two groups is rclicd on t o avoid concentrating the goodor poor-risk patients in one group. The shortened survival of poor-risk patients has bccn discussed carlicr (5cc Section II,C,l,c) . Additional factors which may influencc tlic survival of groups of patients include the following: (1) patients who present “solitary” lcsions have a lo~igermedian survival than patients with gcneralizcd discase (sec Fig. 1) ; (2) in a melphalan-treated series, the mcdian survival time of patients producing only type L light chain protcins was shorter than the median survival of paticnts producing other types of niyelonia proteins (Bcrgsagel e t al., 1965); (3) the median survival for females is longer than for males (Feinleib and AllacMahon, 1960) ; (4) thc median survival of younger patients is longcr than that of patients over the age of 60 (Feinleib and Macillahon, 1960) ; and (5) the median survival of ,Jewish women has been reported to be morc than twicc as long as non-Jewish women (Feinlcib and MacMahon, 1960) . If the survival of a group of patients receiving a new form of treatment is compared with a prcviously treated group, i t is essential to eliminate all selective factors which would tend to bias favorably the group receiving the new form of therapy. Thus, if paticnts with hypercalcemia, uremia, leucopcnia, or thrombocytopcnia, and the early deaths are excluded from thc new therapy trial, the same type of patient must be eliminated in the estimation of the survival of the previously trcated group. Korst e t al. (1964) havc cornpared thc survival of a group of 162 myeloma patients who (lid not rcccivc cyclophosphamidc with thc total group of 207 patients trciited with cyclophosphamide, and 165 paticnts who rcccivecl an “ac1cqu:ttc trial” of thc drug. The survival figures arc shown in Tablc V. It will be notctl that 42 of thc cyclophospliamidc-treated group (2276) died, or wcrc lost, during the first 2 months, and the elimination of thcsc paticnts resulted in an improvement in the median survival from 24.5 to 32 months. However, thc survival of the group who rcccivccl cyclophosphamide for a t least 2 months cannot be compnrctl with the survival of the group who received 110 cyclophosphamidc, for the short survivors have not been eliminated from the latter group. For this reason the survival from onset of therapy of 165 myeloma patients treated with cyclophosphamidc for a t least 2 months was compared with the survival of 70 patients treated with urethane for the same minimum pcriod (Table V ) . The improved sur-
337
THE TREBTMENT O F PL.LSM.4 CELL hlYELOMA
viva1 of the cycloplios~~haiiiide-treatedgroup is statistically significant ( p < 0.01 1. The comparison of the survival of patients receiving a new form of thcrapy with a histoiical conti 01 group is sul)jcct to many pitfalls; some TABLE V ‘I’m EFFECTOF CTci,ormsrII I M I I ) E U N THE Sr RV I V A L 1’1, \SM 1 CELL
A . Survival from diagnosis No cydophosplianiide Cyclophosphamide, total series Cj’clophosphamidc-treated for more than GO days H. Survival from first therapy Urethane for more than 00 days Cyclopliosphamide for more than 80 days
OF
PATIENTS \\ITII
h1I ELOM la Niimbrr of patients
Median survival (months)
162 207 165
9.6 24.5 32 O
i0
13.5
165
24.5
From Korst el al. (1964).
uf thebe 1i:tr.c beeii discussed t)y Rlotlan 1965). Prospective, controlled, r:uitlomizcd clinical trials are vei y tiiiie-Coiibiiiiiiri~,but tlie great ndvantages are self-evident.
B. EFFECTIVEYESS OF ANTISEOPLASTIC ACEKTSI N THE TREATMENT OF PLASMA CELL h1YELoMA illany drugs have been given t o lxttients with plasma cell niyeloina since Alwall (1947, 1952) iiiadc the initial report of remarkable iinprovernent in a patient treated with urethane. RI:iny of these trials have been inadequate because too few patients were treitted, or the quantitative clinngcs in the niyelonia SCI uni :inti/or urin:iry I)rotein, palpable tumors, and bone recalcification have not been rcpoited. If the published reports arc \-icwed with leniency :tiid :icccpted as adequate trials, the agents tested in the trc:itment of p l a m a cell inyeloma may lie classified illto those wliicli are 131ob:il)ly iiic4fcetivc, those which may occasion:illy 111 otlucr ol)jcc.tive improvcniciit hut f a i l to prolong survival, :ind t h r :\lkyl:iting ageiith, nhicli protfucc~ ohlcic’tivc>i l l l J ~ l ~ ~ ~ ~i lnl ~30t hto i l ~ 50% of patients, ant1 also prolong survival. ~
i
i
i
(
8
I . Agents Which Are Probably Ineffective a. Diamidiires. (1) Stilbamidinc (Snapper, 1948); (2) A 1 JL B 938 (Ward, 1958; Skinner e t al , 1963).
338
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, J R .
b. Antimetabolites. ( I ) Folic acid antagonists (Wright et al., 1951) ; (2) purinc antagonists: 6-mcrcaptopurine (Ossernin1i and Iiines, 1954; Rundles and Dugdale, 1958), 6-thioguanine (Carbone et ul., 1964) ; (3) pyrimidine antagonists: G-azauriicil (Rundles and Dugdalr, 1958), 5fluorouracil (Runtllcs and Dugdale, 1958; Gold e t al., 1959), fluorodeoxyuridine and iododeoxyuridine (Papac et al., 1962) ; (4) hydroxyurea (Davis, 1964). c. Miscellaneous. 1-Aminocyclopentanecarboxylic acid (Bergsagel et al., 1962a; Krant et al., 1962; Mass, 1963) ; vinblastine (Costa et al., 1963) mitomycin C (Bergsagel et al., 198213) ; aeaserine (Holland et al., 1961). 2. Ayerits Which M a y Produce Objective Effects, but Fail to Prolong Survival
a. Urethane. The patient reported by Alwall (1947, 1952) was markedly improved by the first course of urethane. Prior to therapy this patient showed widespread lytic skeletal lesions, an elevated sedirncntation rate, marlccd plasma cell infiltration of the marrow, a severe aiicmia, and proteinurca. Following urethane therapy all of these abnormalities returned to normal except the skeletal lesions, which remained unaltered. The rcmission persisted for 2 years. On relapse, thc patient failed to respond to a second course of urethane. Other investigators have also observed objective improverncnt following urethane therapy (Rundles et al., 1950; Harrington and Moloney, 1950; Weder, 1950; Snapper et al., 1953; Osserman, 1958). Objective improvement occurs in fcwer than 2070 of pirticnts trcated with urethane. Luttgens and Bayrd (1951) observed some evidence (often slight) of objective improvement is only 20% of 47 patients who were able to continue urethane for more than 2 months; since 66 patients were started on the drug, this represents an ovcr-all frequency of 1476 improvement. Using intravenous urethane, Seibert et al. (1966) observed a sustained decrease in marrow plasma cells, myeloma protein, or tumor mass in 6 of 30 patients (23%). I n a study by thc Eastern Solid Tumor Group (Holland et al., 1966), myeloma patients were randomly allocated to urethane (47 patients) or a placebo (36 patients). Objective improvement was not observed in any of the patients receiving urethane or placebo, and the survival of the nonazotemic patients in the two groups was identical. From a review of the survival of 600 myeloma patients reported in the litcrature, Osgood (1960) was unable to show that urethane therapy had altered the median survival of patients. Thus, although urethane may occasionally produce objective improvement, the frequency of response is
THE TREATMENT OF PLASMA CELL MYELOMA
339
Iwobably less than 20%, and there is no evidence that the survival of myeloma patients has been improved by its use. b. Corticosteroids. Therapy with corticosteroids usually corrects hypeiwilcemia in myeloma patients by decreasing bone resorption (see Section II,C,2,c) and frcqurntly lowers tlie myeloma serum protein, clccreases proteinurea, and produces a substantial rise in hemoglobin. A decrease in marrow plasmacytosis and healing of skeletal lesions has not been reported. Adams and Skoog (1957) observed a marked decrease in the myeloma serum protein iii 18 of 26 patients (70%).Mass (1962) reported a decrease of a t least 1.5 g.% in serum globulin in 7 of 25 patients (28%) with an initial serum globulin of 4.5 g.% or more. However, despite the objective effects observed with this group of drugs, a study conducted by Western Cooperative Chemotherapy Group (Mass, 1962) failed to show a difference between the survival of 33 patients randomly assigned prednisoiie therapy, and 32 patients assigned to a placebo group. c. X-irradiation. This form of treatment is used mainly to treat "solitary" lesions, and to relieve the pain caused by localized destruct,ive bony lesions. Local tumor regression occurs, hut a reduction in the myeloma serum or urinary protein would only be expected if the bulk of tlie patient's tumor were in the irradiated field. X-ray therapy is very important in the managenlent of patients with myeloma, but there is no evidence that its use has improved survival (Osgood, 1960). d. Radioisotopes. A number of isotopes have been used, including the following: PJ2;SrS9 (Lawrence and Wasserman, 1950; Reinhard et al., 1946) ; I I 3 ' (Kriss et al., 1955; Kay, 1959) ; Ca" (Anderson et al., 1956) ; Ygo (Greenberg et al., 1962) ; and Lu'?~(Anderson et al., 1960). None of these isotopes has proved to be clinically useful. 3. Alkylating Agents
Melphalan and cyclophosphamide have had extensive clinical trials in the treatment of plasma cell myeloma. These drugs are reportcd to produce objective improvement in from 30-5070 of patients, and also to prolong survival; they will be discussed in the next section. It seems likely that most alkylating agents are capable of producing objective effects on manifestations of myeloma if they can be administcrcd in doscs which produce hcmatologicnl toxicity similar to that observed with effective dobcs of cyclophosphainide or melphalan. However, this hypothesis has not been tested adequately, and the relativc cffectiveness of many alkylating agents cannot be compared because of the limited nature of the clinical trials which have been conducted. The following alkylating agents have had limited clinical trials: nitrogen
340
BERGSAGEL, GRIFFITII, IIAUT, AND STUCKEY,
JR.
mustard (Jacobson et aZ., 1946) ; triethylcnc inclamine (Rundles and Barton, 1952; Wright et al., 1955) ; triethylene phosphorainide (Wright et al., 1955) ; quinacrine mustard (Jones et al., 1958) ; R-48 (Rider and Warwick, 1958) ; eponate (Hamniack and Fromineyer, 1963) ; AB-100 (Brown e t al., 1962) ; and n1et:tsnrcolysin (Austin et al., 1962; Lovina, ClC&-C\H, ClC&-CH,
/N e
THZ C
H
z
- CH-COH
8
FIG.3. Melphalan, ~ - 3 - { ptbis-(2-chloroethyl) aminolpheny1)-alanine.
et al., 1962). RIaiiy of thesc drugs havc produccd objective effects on direct manifestations of myeloma, but none of thcni appears to be more effective than melplialan or cyclopliospliamide, and they will not be considered further. 4. Melphalan and Cyclophosphamide
a. Structure. Bergel and Stock (1954) synthesized the DL-, L-, and D-forms of 3-{p- [ bis- (2-chlorocthyl) amino] plieny1)-alanine, and Larionov et al. (1955) indepcntlcntly p r ~ p a r e c lthe DL-form in Rloscow (Fig. 3 ) . Thcse compounds are known by variety of synonyms: t h e DL-fOl’111 is known as CB-3007, nicrphalan, or sarcolysin ; thc L-form as CB-3025, NSC-8806, L-phcnylalanine inustard, or inelphnlan (Alkeran) ; and the n-form :is CB-3026 or nicdphalan. In animal trials, thc L-form was a considerably more potent antituinor agcnt than was thc D-form. This finding emphasizes the iniportancc of tlic stereochemical configuration of thcsc agents. The clilorocthyl group 1ias also bccn attaclied in tlie nwta and ortlio position of DL-plienylalanine; both of these drugs were found to be less active than melphalan in the trcatnient of niyclorna (Austin et al., 1962; SWCCSG, unpublished observations). Cyclopliosphaniidc (cytoxan, entloxan, procytox, NSC-26271), shown in Fig. 4, is an alkylating agent of considcrnble interest, because it is innctivc in the form administered. Thc active form is relcased following hydrolysis within the body ( Urock, 1958) . b. Dosage Schedules and Toxicity. Two different schedules have becn reconimcnded for melphalan (A.M.A. Council on Drugs, 1965) . Rlany investigators have advocated starting patients on a dose of 6-10 mg./day for 2 to 3 weeks. The administration of thc drug is rcgulatcd on the basis of chngcs in the lcucocyte and platelet counts; the drug is stopped if the lcucocytcs fall to lcss than 2000/1nm.’, or platclets fall below 75,00O/m1n.~Maintennncc therapy of 0.01 to 0.03 nig./kg./day is started
THE TRE.IT.\.IEST O F PLASMA CELL MTELOhIA
34 1
when t,he leucocytcs and platclets begin to risc, and must be monitored by regular blood counts. The SWCCSG has eiiiployed intermittent total doses of 1.0-2.0 iiig./kg. given in 4 clays (0.25-0.50 iiig./kg./d:iy X 4 ) , repeated a t 6- to 8-n-eek intervals. It is felt that tlie use of large doses intermittently pi*ocluccs grc:itcr inhibition of tlic iiiycloiiia plasma. cells, without periiianently tl:ini:iging tioi~iii:tl 1iciiintol)oicsi~.After a dose wliich c:iuses riioclc~ratc 1icwi:itological tosicity, tlic iioriii:tl iii:irrow clciiieiits rccovcr iiioi~!rnpiclly than tlic tumor cells. In Fig. 5 , tlic cuiiiuliitive ~)roportionof patients developing Icuco1)enia (fall iii Iciicocytcs to lens tlian 3ooo/iiim.") and/or throinbocytopcnia ( f a l l ill 1)latelets to less t h i 100,000/iiiii~.3)at increasing total doses arc plot tctl on logaritliiiiic-prol,ability gr:ipli paper. Dos:tgc schedulc A, uscttl i i i tlic early S\VC'CSQ trial:: of this drug (I3crgs:tgel e t al., 1 9 6 2 ~ )coiisistetl of 0.2 iiig./kg./tl:iy rontiiiuetl until Icuc.opcnia or throni-
1)ocytopciii:i w : i b o l ~ t r v c ~Thc l . toxic cft'(& of iiie1ph:ilaii administered in tliia w:ty :ti.(> cuniiil:itivc ; t h f a l l in lcueocytes and platelets rcaclicd :i nadir al)out 14 &iy+ aftclr tlic drug w:ts discontinucd. With dosngc schedule A tlic Icucocytc~couiit fell to less th:m 1000/111m.3 in 30% of I)nticiits after iii(~l1)Ii:il:iii w:is tliscoiitiiiuc~l.This liematological toxicity is cxccssivc :m1 indicatcs that continuing thrral)y a t tliis dose rate until lcucocytcs f a l l I)clow 3000/niin. I , or platelets f a l l Ixlow 100,000/mm.3, rcsiiltb iii ovcrtlob.iigc. Tlic iiicclinn total dose adniinistercd to this group was 2.8 nig./kg. ; this figure pro1)ably overestimates the median dose required to producc iiiodcrntc>, acwptahle, liematological toxicity with bclictlule A. Because of the cxce4i.e Iicit1:ttoIogical toxicity observed with schedulc A, t l o ~ g escl~trlulcB w:is clc.viscrl. T h c tot:ll dose rcqumd to cause 1nod era t c 1icni:i t o log i c :i 1 t 0sic i t y 0 ..5-2 .O iiig./kg. ) w'a b ac 1iiiin ist e red i i i 4 days, and repeated wlicn kigiib of iii:irrow recovery were obberved 6 to 8 n-ccks latoi,. A few patietits ( 7 % ) tlc~velopctl Ieucopenia or tIiroin1)orytopciii:i with total doses of 0.75 irig./kg. or Its, but the iiiajority required 1 .O iiig./kg. or 11101'~.From these data with sclicdulc B, the median dose required to cause lcucopenia or thrombocytopenia is estiiiiatctl to be 0.93 nig./kg. Tlic hernatological toxicity observed was mod-
342
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
crate and acceptable; thc leucocytes fell below 1000/mm? in only 2% of patients. The leucocytc count falls to its lowest level in 14 to 21 days and rcturns to normal within 28 to 42 days after the initial dose. The cyclophosphamide dosage usually employed is 2.0 mg./kg./day orally, monitored by blood counts every 1 to 2 weeks, and adjusted to cause moderate leucopenia (Rivers et al., 1963; Korst et al., 1964). c. Effects on Plasma Cell Myeloma. Prospective studies comparing the effectiveness of cyclophosphamide and melphalan in the treatment 10.0
50 4.0 h
sF
-
30 29
x
. e
1.0
t?
total dose(0.5-20mg/kg) in 4days
0.5
I 2 5 10 20 4 0 60 80 95 99 999 Percent developing Leukopenio ond /or Thrombocylopenio
FIG.5. Melphalan hematological toxicity. The total doses of melphalan (mg./kg.) required to cause leucopenia ( <3000/mm?) and/or thrombocytopenia (<100,000/ mmP) was determined for myeloma paticnts treated with dosage schedule A (0.2 mg./kg./day to toxicity) and dosage schedule B (0.5-2.0 mg./kg./ in 4 days). The cumulative proportion of patients developing leucopenia, and/or thrombocytopenia at increasing total doses has been plotted on logarithmic-probability graph paper. The points fall close to a straight line, indicating that the range of doses required to cause hematological toxicity forms a log-normal distribution with both schedules. The median dose requircd to produce hpmatological toxicity (k.,in 50% of these myeloma patients) was 2.8 mg./kg. for schedule A, and 0.93 mg./kg. for schedule B (SWCCSG).
of myeloma are bcing done by the Veterans Administration Cancer Chemotherapy Study Group but this study has not been completed. Using cyclophosphamide, the Midwest Cooperative Chemotherapy Group (Korst et al., 1964) observed objective improvement in 80 of 207 patients (Table VI) . The cyclophosphamide response rate observed for all of the patients collected from literature reports is 33%; this does not differ significantly from the response rate of 36% for all of the melphalan-treated patients collected from the literature (Table IV) .
343
THE TREATMEKT OF PLASMA CELL MYELOMA
Total ntiniber of myeloma patients
Itcferencc Corenho and Alpert (1941) BergsaKrl and Levin (1960) Bethell el u1. (1960) Shnider el a!. (1960) .Matthias el a1. (1960) Foye et al. (1960) Coggins el ul. (1960) .J. Solonion el al. (1963) Rivers el al. (1903) Korst el ul. (1964)
Total
Improvement in one (or more) objective manifestation
5 3 12 3 14
0 0
5 1 7
0 0
0 0
1 6
“4 207 ( t o t d evdiiable 16.5)
SO [80/207 ( 3 8 . 7 % , ) ; S0/165 (48.5%>)]
280
Similarly, there is no evident difference between the effect of cyclophospliamidc and nielphalan on the survival of two large series of patients with plasnia cell mycloniu (Fig. 6 ) . The mcdian survival from diagnosis of patients treated without alkylating agents was 11.2 months for the SWCCSG (103 patients) and 9.6 months for the RIidwest Group of 162 patients. The iiiedian survival froin diagnosis of both treatment groups is significantly longer than the survival of patients who were not trcated with c.yclopliosl,haiiii(~e or niclphalan, and the survival ciirvc~sof tlic two treatcd groups were :tlmost identical. TABLE V I I
EFFECTO F
SURVIVAL IN P L A S M A SbVCCSG (1959-M~c11, 1965)
M E L I ’ I I A L A S ON
(:ELI.
MYELOMA
Median survival (months) from
Patient group
Number dead/ total number
Onset of symptoms
Diagnosis
84/103 104/199 1061203
19 7 3‘2 7 30 3
11 2 23 6 20 9
31/68 73/128
46 0 25 1
38 s 21 4
~
S o nielphalan All melphalan-t reated Melplialan consecutive series (no melphalan-42) Melphalan responders hlelphalan nonresponders and iioirevaluable
344
BERGSAGEL, GHIFI‘ITII, I-ISVT, AND STUCKEY, J R .
The effect of mclphalan on thc smvival of patients with plasma cell myeloma is considered in inorc detail in Tablcs V I I and VIII. The survival figures for the total SWCCSG scries arc shown in Table V I I ; the survival figures a t the University of Texas RI. D. Aiitlersoii Hospital and Tumor Institute (MDAI-I) are shown in Table VIII. The figures given for the median survival wcre cstimatcd from a maximum likclihood fitting to a log-noi~naldistribution ; thcsc values agree quite well with median survivals cstimatcct by tlic life-table method. P L H C E N ~LIVINC~
I 2
5
40 6 0 80 90 9 5 PERCENT DEAD
10 20
99
FIG.6. Survival from diagnosis of patients with plasma cell myeloma treated with melphalnn ( 0 ) or cyclophosphamide (A).[Mephalan, 196 patients, median 23.6 months, SWCCSG ; c.~clol,liosl)lianii~r, 207 piticats, i n t d n n 24.5 nionths, h‘oisl, et (11. (19641.1
Most of the patients in tlic iho-melplialan’’ group were seen after 1959; thcse patients werc treated with stcroids, S-irradiation, urcthanc, and various new agcnts. This group was not sclcctctl randomly, and does not constitutc a good “control” sclrics. Ilowcver, tlicse patients were trcxtcd during the s:me interval as tlie carly melphalan series, and tlie median survival does not diffcr significantly from the rncdinn survival of 17 months clctcrrnined by Osgood (1960) from an analysis of GOO cases rcportctl in tlic literature. The duration of survival for every patient wlio received a single dose of melphalan was usccl to estimate the mcclian survival of the melphalantreated group listcd in thc second line of thc tables. The survival of
345
THE T R E A T M E N T O F PLAShl.1 ( X L L M Y E L O M A
3Iedian survival (months) from
Patient gruiip
No melpliltlan A11 melphalan-treated RIelphalnn consecutive series (no melphalan, 9) Melphalan responders Melphalan nonresponders and nonevaluable
No. dead/ total no.
Onset of symptoms
1)iagnosis
3/39 26/61 25/59
17.5 55.2 49.2
10.0 45.1 39.1
13/34 13/27
58.2 40.1
47.3 28.0
a From studies carried out a t the University of Teuas %I. D Anderson Hospital and Tumor Institnte, Houston, Texas.
tliis group is significantly better tliaii that of the p:ttients who received no rnclphalnii (from onset of symptoms, p < 0.01 ; from diagnosis, p < 0.01). It is recognized that thc total mclplinlan-treated group was favorably biased by the initial admission of good-risk patients (initially patients with hypercalcemia or uremia were not treated with melphalan) and a n increased proportion of longer siirvi~orswlio had been followed for some timc prior to the initintioil of thc melp1i:ilaii study. For this reason, the survival of a consccutivc >cries, trcnted primarily with melphalan, is shown. The patienth w r e arranged in clironological sequence by tlic date they were first m m . A date, following which the majority of patients were trcntctl with niclphalan, was selected for each institution. The duration of survival of a11 patients admitted following this date was used to estinintc thc mec1i:in survival of tlic consecutive series. Included in this consecutive beries arc 42 patients wlio received no melphalan. The survival of the mclphalan consecutive series was significantly Ixttcr than tlw “no-incl~~lial:~ii” gioup ( p < 0.01), but did not diffcr significnntly from tlw total mclplinlan group. I n the absence of other effective niethotls of treating plasma cell inyelornn, rrpcntcd c o ~ r s c sof melp1i:ilan were givcn a t 6- to 8-week intervals to both the responders and tlic nonresponders. Repeated courses of melplialan were given consistently t o nonresponder groups a t the 11. D. Anderson Hobpitnl, and less consistently by other members of tl1c gl’oup. At the 31. D. Anderson Hospital the survival of the nonresponder group was significantly better than the “no-inelphalan” group, with a p w l u e of less than 0.05 when survival was estimated from the onset
346
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, JR.
of symptoms. The improved survival of the melphalan nonresponders was significant at the 0.01 level when the survival of these two groups from time of diagnosis was compared. For the total SWCCSG series, the survival of the melphalan nonresponders did not differ significantly from the survival of the “no-melphalan” group. The survival data for patients treated a t M. D. Anderson are shown separately in Table VIII, because the melphalan-treated group a t this institution lived significantly longer than the group of patients treated a t other institutions of the SWCCSG. Of particular interest is the fact that the median survival of the melphalan nonresponders was significantly longer than that of the group who received no melphalan (from onset of symptoms, p < 0.05; from diagnosis, p < 0.01). The reasons for the improved survival a t this institution are not clear. Thc patients a t this hospital were similar to those seen a t other institutions with respect to age, sex, the duration of symptoms prior to starting melphalan, the incidence of hypercalcemia, and the melphalan dose rate (Table IX). The patients treated a t M. D. Anderson differed from those treated ::t other SWCCSG institutions in thc following respects: the proportion of Negro patients was smaller; fewer patients had a blood urea nitrogen (BUN) of greater than 30 mg.%; the mean hemoglobin level was slightly higher; and the frequency of early deaths was much smaller. A preliminary analysis of the SWCCSG results showed that Caucasians with myeloma were objectively improved significantly more frequently than Negro patients. Thus, with a smaller proportion of Negro patients, the group treated a t M. D. Anderson was more likely to respond, and to live longer. The higher hemoglobin level, the reduced frequency of uremia, and the smaller proportion of early deaths suggest that these pttients, as a group, had less advanced disease than the patients seen a t other institutions; this is probably the most important factor contributing t o the improved survival of patients treated a t this institution. A planned, prospective study is required to determine whether maintenance melphalan therapy prolongs the survival of the nonresponder group. If the improved survival of the nonresponders is confirmed, it will suggest that the criteria for distinguishing responders from nonresponders employed by the SWCCSG (Bergsagel et al., 1962d) are too rigid. 5 . Af ye lo rna Pro te iris and M e l p h a lnn B esponsiven ess
Normal plasma cells probably differentiate through a t least three stages. An uncommitted, undifferentiated stem cell probably proliferates so as to maintain the size of the stem cell pool constant; since all
THE TREATMENT OF PLASMA CELL MYELOMA
347
immunoglobulins contain light chains, it seems likely t h a t one of the initial stages in differentiation requires a decision as to whether the cell will produce antigenic type K or type L light chains. The next stage involves the selection of the type of heavy chain to be produced. The majority of plasma cell myelomas produce both a heavy and a light chain, 22-24% producc only type K or type L light chains, and 1% do not produce any protein (Table 11).The pattern of tumor protein synthesis does not change sigiiificaiitly during the course of the disease in man, or following numerous transplant generations of mouse myeloma. Thcsc observations suggest that the malignant transformation which initiates the tumor may occur in plasma cells a t different stages of differentiation ; the malignantly transformed cell has an unlimited capacity to proliferate, but the pattern of protein synthesis by all of the cellular descendents remains fixed. An alternative hypothesis t o explain these observations would be that a malignant transformation converts an undifferentiated stem cell into a malignant cell, and a t the same time initiates changes which determine the pattern of differentiation and protein synthesis of all daughter cells. The important feature of either hypothesis is that the pattern of differentiation and protein synthesis is fixed for all neoplastic daughter cells. The classification of patients with plasma cell neoplasms on the basis of the type of protein produced probably groups tumors that have differentiated to the same stage. This classification may also group tumors with similar clinical manifestations, growth rates, therapeutic responsiveness, and prognosis; if this is the case, the classification will have considerable clinical significance. The clinical patterns observed with several plasma cell neoplasms are shown in Table X. The patients producing types K and I, light chain proteins, and G- and A-myeloma protein were studied by the SWCCSG (Bergsagel et al., 1965). It will be noted t h a t patients with M-macroglobulinemia, and I-Iy-chain disease frequently develop lymphadenopathy and splenomegaly, but these clinical features were not observed in patients producing only type K or type L light chain proteins, and were uncommon in patients producing G- and A-myeloma proteins. On the other hand, osteolytic lesions were common in patients producing the various myeloma proteins, but are uncommon in patients with M-macroglobulinemin and Hy-chain disease. There also appear to be differences in the prognosis of patients producing myeloma-type proteins and those producing M-macroglobulin. Estimates of the survival of patients producing light chain proteins, or G- arid A-myeloma proteins prior to thc use of melphalan therapy are not available. However, Osgood (1960) has estimated the median
COXIPARISOS
OF
TABLE IX TREATED WITH &IELPHALAN AT THE UXIVERSITY OF TEXAS >I. D. HOSPITAL (hlDAH) ASD OTHERSRCCSG ISSTITUTIOSS (1956-D~c., 1965)
h1YELOMA
PATIEXTS
Number of patients" Age in years: mean median Sex: yo female Race: yo Negro (Segro/other) Duration of symptoms (months) prior to melphalan: mean median Hypercalcemia: no. > 11 0 mg. %/total (%) Uremia: no. BUNc > 30 mg. %/total (%) Hemoglobin (g.%) mhen melphalan started: mean median Melphalan dose rates (mg./kg./month) : mean median Early deaths: deaths in 3 weeks/total no. (%) followed
MDAH
Other institutions
65 60.1 60 45 20 (13/52) 16.4 11.0 6/30 (20%) 8/37 (22%) 10.6 10.6 0.73 0.56 1/65 (2%)
179 58.4 60 37 47 (82/91) 16.0 8.3 18/114 (16%) 47/127 (37%) 9.6 9.3 0.90 0.48 20/173 (1170)
AkSDERSON
n m
Significance
8m E
t
=
0 . 8 9 (N. S . ) b
1 . 0 8 (N. S.) t = 4.38 ( p < 0.01) t = 0 . 1 2 (N. S.)
t
=
t = 0.52
(N.S.)
t
< 0.1) = 1 . 9 7 ( p < 0.05)
t
=
t = 1.92 ( p
1.25 (N.
t = 3.43 ( p
S.)
< 0.01)
The data in this table were analyzed a t a later date than the material in Tables IV, VII, and VIII, and for this reason the number of patients differs. b i Y . S.: not significant. BUN: blood urea nitrogen.
P
c
E 7
2;
7 5:
! w +
;P
C
e ;P 2,
t,
z
35 *ia
TABLE X CLINICAL
.3
z
PATTERNh: PL.4hM4 CELL ;?;EOPLASRlS
m 1
s
Pathological protein Clinical features J,ymphadenopathy Spleiiomegaly Osteolytic lesions Survival from onset of symptoms
Type I< Type L light chain protein light chain protein S o n e (0/12)0 Xone (0/12)" Common 1T months (median).
Sone (O/9)a Sone (0/9)" Common 17 months (median)c
G- and hmyeloma protein Pncwmmon (l/i'O)a I~ncommorr(4/70). Common 17 months (median)
3
+-
2 &I-macroglobulin
Hy-chain protein
Common Common Uncommon 38-40 monthsd
Common ( 5 / 5 ) b Common ( 5 / 5 ) b None (O/5)b 4-36 monthsb
+
0 "! CJ
P
%
*2 3
Patients studied by the SIVCCSG (Bergsagel et nl., 1965). The figires in parentheses refer to the numbers of patients producing the indicated pathological protein, who manifested lymphadenopathy or splenomegaly, and the total numbers of patients in this group. b Patients with Hy-chain disease reported by Franklin et al. (1964) and Osserman and Takatsuki (1964). The survival figures indicate the range observed for 5 patients. c Median survival est,imated for 600 patients with plasma cell myeloma treated prior t,o the use of alkylat,ing agents (Oagood, 1960). Average survival (Kappeler et al., 1958). a
m
F
zI:
mr
2P
350
BERGSAGEL, GRIFFITH, I-IAUT, AND STUCICEY, JR.
survival for 600 myeloma patients producing all protein types collected from the literature prior to the use of alkylating agents; the median survival of the total group was 17 months. The survival of patients with M-macroglobulineniia and Hy-chain disease is not known with as great accuracy as for plasma cell myclomn. However it is the clinical imprcssion of many investigators that M-macroglobulinemia is a more benign disease than myeloma, and many patients are known to survive for long periods of time. The average survival of patients with M-niacroglohulinemia has bccii reported to be 38-40 months (Kappeler et al., 1958). The average survival noted for M-macroglohulinemia cannot be cornparcd directly with the median survival reported for myeloma patients, but this figure does suggest that the prognosis of M-macroglohulinemia is better than for myeloma. The proteins produced by 91 patients with plasma cell myeloma treated with rnclphalan by nicmbers of the SWCCSG, were availahlc for antigenic typing. This material provided the unique opportunity for determining whether the tumors which produced different types of myeloma protein differed in their response to nielphalan (Bergsagel et al., 1965). The data obtained from this study are summarized in Table XI. The data available on the response of patients with Mmacroglobulinemia and Hy-chain disease to mclphalan therapy are inadequate for this comparison. Patients were classified as being objectively improved (responders) by melphalan therapy if one, or more, of the following criteria were satisfied: (1) a decrease in the myeloma serum protein to 50% or lcss, of the prestudy value; (2) a decrease in the amount of urinary protein excreted per 24 hours to 50% or less of the prestudy value; (3) an increase of 2.0 g.% or more in hemoglobin; (4) shrinkage of palpable TABLE kI EFFECT OF MELPHALAN ON PLASMA CELL TUMORS 'PRODUCING OF MYELOMA PROTEINSQ
VARIOUS
TYPES
Melphalan response
Responder
Myeloma protein type
TypeK light chain proteins
G-and Amyeloma proteins
11/11
30/65
0/9
68 43-109 4/12
55 45-67 25/70
23 18-31 6/9
No. objectively improved/no. evaluable Survival from onset of symptoms Median (months) 95% confidence interval (months) No. deadjtotal no.
Intermediate Nonresponder
Patients studied by the 8WCCSG (Bergsagel et al., 1965).
TypeL light chain proteins
T H E TREATMENT OF PLASMA CELL MYELOMA
35 1
plasmacytomas of 50% or more; and ( 5 ) a decrease in serum calcium from more than 6.0 to 5.0 meq./liter or less. These criteria were selected arbitrarily with the helief that the changes arc clinically significant. The effect of melphalan therapy was not evaluated if the changes in the myeloma proteins in serum or urine were not determined, or if the patient died or wits otherwise lost in less than 3 weeks. The patients can be divided into threc groups on the basis of the response to melphalan therapy (Table XI). All of the evaluable patients producing only type K light chain proteins responded ; these patients are listed as the responder group. I n the group of patients producing G- or A-myeloma proteins, 30 of G5 were objectively improved; these patients are listed as an intermediate group. None of the patients producing only type L light chain proteins were objectively improved; these patients are listed as nonresponders. The estimates of survival are significantly different ( t test) for the comparison of the survival of the nonresponders with the intermediate group (from onset of symptoms, p < 0.02; from diagnosis, p < 0.01 ; froin start of mclphalan, p < 0.05). With the sample size available for comparing the survival of the responders and nonresponders, the differences are not significant, although for the survival from the onset of symptoms the difference is close to significance a t p = 0.05. The differences between the survival of the responder and intermediate group are not significantly different. Since less than 50% of the patients are dead in the responder and intermediate groups, these estimates of survival are unstable, and, in view of the small sample size of the responder and nonrcsponder groups, the estimates should not be interpretated a s being well established. Other investigators (Osserman, 1965; Lee et al., 1965) have reported that objective improvement occurred in 5 of 10 patients producing only type L light chain proteins, and 3 of 6 patients producing only type K light chain proteins failed to improve following melphalan therapy. These observations clearly indicate t h a t the correlation of melphalan response to the type of protien produced by the tumor is not absolute, but the data are inadequate to disprove the thesis t h a t paticnts producing only type K light chain proteins respond more frequently than those producing only typc L light chain proteins. Since the difference.: in the frequency of response to melphalan observed in these three series may be due to many factors, it is difficult to compare them. Two important differences are the dosage schedules employed and the selection of evaluable patients. Ossermm (1965) only evaluated the response of patients treated for 6 months or more, and Lee et nl. (1965) report on “adequately treated” patients (27 of 40) observed on therapy for a minimum of 3 months. The SWCCSG
352
BERGSAGEL, GRIFFITII, HAUT, AND STUCKEY, JR.
evaluated the response of all patients followed for a t least 3 weeks; it was found that 3 to 9 months of treatment were required for optimum improvement, but in most cases it was possible to rate the response within 3 weeks after the first dose of the drug. I n the SWCCSG series, 14 of the total group of 91 (15%) [2 of 12 patients (17%) producing only type K light chain proteins, and 3 of 9 patients (33%) producing only type L light chain proteins] died within the first 6 months. The elimination of patients who die early would probably result in the loss of more nonrespontlers than responders from the series of Osserman and of Lee e t al.; this malies their series quite different from the SWCCSG series. If i t is assumed that the different melphalan dosage schedules employed did not influence the results, and also assumed t h a t there were no early deaths in the Osserman or Lee e t al. series, there is still convincing evidence t h a t patients producing only typc K light chain proteins respond more frequently (14 of 17) than those producing only type L light chain proteins (5 of 19) when the results of the three series are combined. Further studies of the response of patients producing different types of myeloma protein to melphalan therapy are required. Thew observations suggest t h a t the classification of plasma cell malignancies on the basis of the type of protein produced is useful for grouping patients with similar clinical manifestations, prognosis, and melphalan rrsponsiveness. Why should plasma cell tumors producing only type K light chain proteins respond inore frequently to melphalan therapy than those producing only type L light chain proteins? It is possible that an extracellular factor, such as the myeloma protein, may react with the alkylating agent and inactivate it. If the type L light chain proteins could be shown to react more readily than type K light chain proteins, it would be possible to explain the different response rates. This possihility was checked in two ways. First, the rate a t which melphalan reacts in vitro with type K and type L light chain proteins was checked (Fig. 7) and no differences in the reaction rate could be demonstrated. Second, if type L light chain proteins inactivate melphalan more readily than type K light chain proteins, one would expect to find less severe hematological toxicity in the patients producing only type L light chain proteins. The heniatological toxicity observcd in patients producing either type K or type L light chain proteins was checked carefully, and no difference in the severity of the hcmatological toxicity could be demonstrated (Bergsagel, 1966; Bergsagel et al., 1965). Thus, it seems unlikely that the different response rates can be explained on the basis of an extracellular factor.
THE TRWl'MEST OF PLASMA CELL MYELOMA
353
It s e e m more likely that inelphalan responsiveness is determined by an intracellular factor related to the stage of differentiation of the malignant plasma cell or to the growth characteristics of the tumor. Dr. Bruce and his absociates a t the Ontario Cancer Institute have shown that dividing cells are much inore sensitive to iiiaiiy types of chemotherapeutic agents than are cells in the resting ( Go ) phase of the ccll cycle (Bruce and Alccltcr, 1965; Bruce et nl., 1966). It is possible that tumors I)roducitig only typc I< light cliain proteins 1i:ir.e a larger
r
b
50t
"
I
I
I
1 (Patient) 4 5 0 mg. (W.R.H
450mg.(C.N.1 300mg.(M.O. ) 3 0 0 m g . (J.C.W.)
0
1
2
3
4
5
Time (hrs.)
FIG.7 . Reaction of melplialan with types I< and L Bcnce-Jones Iwoteins. Type
B and 1, Bence-Jones proteins were precipitated from urine samples by 50% saturation with ammonium sulfate. Tlic protein precipitates were dissolved in distillrtl wat,rr, dialyzctl against distillrtl \vatcar f o r 4 days, :ind tlrrn lyopliilizcd. Weighed samples were incubated at 37°C. with 20 mg. mrlphalan in 0.15M NnCl ;it, pI5 7.0. A t t l i c tinics int1ir:rttd om IIir almc L, tlic llrotrin was precipitatcd with ethanol. Free melphalan, including the adsorbed drug released from the prot,rin in the conrse of c~thanol privipi(:ttion, \\:is inrnsurrd sprctrol,lroinctrirally. The reaction of melpli:ilan with Bencc-Jones protcins wzs studied by Dr. J. H. Iinford and Miss Jennie Hmi:itle, Department of Medicine, University of Manit.oba, Winnipeg. Canada, using mctliotls dcvelopcd in his laboratory (Israels anti Idinford, 1963).
proportion of tlivicling t*c~Ilsth:w 1)labiiin ccll tumors produciiig only type I, light c11:tiits, and this inci.casccl proportion of divicling cells may make the tumor more sensitive to the effects of inelphalan. Unfortunately, there is no direct evidence that the proportion of dividing cells in plasma cell tuniors producing only type K light chain proteins is greater than in tuinorb producing only type I, light rh:iin proteins. IV. Summary
A plasnin cell tumor develops when a ccll, destined to produce an is affected hy a inalignant traiisfoi-mation, antI the ininiunoglo~~uiin, ncoplastic daughter cells successfully establish a tumor clone. The genetic constitution, antigenic stiniulation, and exposure to );-irradiation
354
BERGSAGEL, GRIFFITH, HAUT, AND STUCKEY, J R .
appear to be of sonie importance in the etiology of plasma cell neoplasms of animals and man; there is no direct evidence that the malignant transformation of plasma cells is caused by a virus. The tumor protein synthesis pattern reflects the stage of differentiation of the majority of the tumor cells; the classification of plasma cell tumors on the basis of the typc of protein produced is useful for grouping patients with similar clinical manifestations, prognosis, and melphalan responsiveness. The diagnosis of a plasma cell neoplasm requires the demonstration of a neoplastic (i.e., uncontrolled) proliferation of plasma cells evidenced by the formation of a tumor, destructive tissue invasion (e.g., osteolytic lesions), or a progressive increase in the pathological protein. The prognosis of patients with apparently solitary lesions is better than that of patients with generalized disease, and the development of heniatological or rcnal failure indicates a shortened life expectancy. Infections lead the list of factors contributing to the death of patients with plasma cell myeloma. Supportive care directed a t preventing or relicving the major complications of infections, hypercalcemia, pathological fractures, renal failure, and the hyperviscosity syndrome is of great importance in the management of patients. Alkylating agents (melphalan and cyclophosphamide) are the only antineoplastic drugs capable of producing objective improvement in direct manifestations of plasma cell tumors, and also prolonging survival significantly. Melphalan and cyclophosphamide appear to be equally effective in producing objective improvement and prolonging survival.
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T H E TREATMENT O F PLASMA CELL MYELOMA
355
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T H E TREATMENT OF PLASMA CELL MYELOMA
357
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358
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AUTHOR INDEX Numbers in italic indicate the pages on which the complete references are listed.
A .ih:icIi, A,, 338, 357 .Ibelev, G. I., 75, 76 .4bcll, C. W., 71, Y G Acs, A. C., 35, 56, 7 8 .1c.~, G., 87, 116 .4tlnms, W. S., 321, 324, 339, 343, 354, 356 Adnmson, R. H., 29, 30, 31, 7G, 78, 177, 185, 242 Sdclberg, E. rZ., 193, 198, 238 24yriipiiA,O., 281, 306 ilfkliam, J., 173, 178, 179, 180, 188, 192, 194, 240 Afzelius, 13. A., 174, 213, 214, 241, -144 bgpar, J., 85, 113, 114 Aherne, W. A., 321, 564 ;\isenberg, A. C., 119, 120, 129, 130, 144, 155
Alam, O., 227, 238 Alberts, A. W., 3, 61 Alclerson, T., 193, 200, 236 Aldridge, W. N., 216, 246 Allen, M. J., 50, 76 Allcnde, C. C., 85, 11s Allende, J. E., 84, 85, 88, 107, 113, 115 Alexandcr, J. A., 130, 161 Alexander, M. J., 343, 350 Alexandt~,P., 266, 273, 274, 300, 3'01 Alrxander, V., 255, 263, SO1 Algire, G. H., 145, 169 Al-Kassnh, S., 269, 300 Alhrd, C., 124, 150, 151, 155 .Allen, J. C., 318, 357 dllfrey, 5'. G., 111, 113 Alling, E. L., 321, 324, 354 illpcrs, J. B., 126, 127, 141, 142, 143, 157,
Anderson, J., 339, 354 Anderson, L. E., 236, 240 rlndrrson, R. E., 315, 354 Angclrtti, P. U., 133, 135, 146, 150, 15s Arcos. J. C., 204, 227, 228, 236 ,4rcos, J. S., 6, 7, 8, 9, 22, 23, 56, 76 Arcos, M., 204, 227, 2%' ilrffm:in, ,E., 190, 238 Argus, M. F., 175, 176, 177, 184, 185, 187, 191, 223, 227, 228, 229, 5'8s Arias, I. M., 19, 76 Arison, R . N., 128, 129, 131, 134, 135, 142. 160
ilrlinphaus, R., 84, 88, 90, 92, 96, 109, 113, 114, 116
Amdt, F., 235, 238 Arnstein, H. R.. V., 84, 113 Arrhcnius, E., 92, 114, 174, 209, 210, 211, 213, 214, 248, 2& .2rvin, J. M., 218, 242 Aschlienasy, A., 264, 309 Ashlry, L. M., 176, 185, ,941 Ashmore, J., 129, 130, 140, 160, 161 rhc>r, A . W., 255, 301 Aricrl)ac.li, V. H., 123, 146, 147, 149, 150, 156, 15S, 159 Aiigcnstein, I,., 277, 301 .4ristin, A. T., 166, 236 Austin, C., 340, 341, 354, 555 Avanzi, S., 266, SO1 .\xclrad, ii. A,, 270, 301 .2selrod, D., 68, 76 Asrlrod, F., 5, 19, 20, 78 .Iselrod, J., 166, 2 f G
B B:icq, 2. M., 274, 301 Bndcr, J. I?., 74, 76 Batlgrr, G. M., 263, 901 Blir, [J., 129, 131, 132, 135, 136, 15s Bitilic, M. J., 210, 211, 215, 216, 236, 239 Baker, D. T., 338, 355 Ball, H. A,, 140, 156 Hallicux, R. E., 320, 357 B:znnasch, P., 179, 183, 184, 239
1GO
Alpert, L. K., 343, .?55 Althoff, J., 177, 187, 243 Alrinp, -4,S., 286, 903 .4lwall, N., 337, 338, 354 Arnes, B. M., 3, 76 Andrrs, M., 234, 241 hndcrson, A . W., 276, 305 Anderson, G. E., 264, 301 361
362
AUTHOR INDEX
Barnes, I. C., 264, 301 Barnes, J . M., 164, 171, 172, 174, 175, 176, 185, 187, 191, 223, 239, 243, 277, 279, SO1 Barnctt, E. V., 322, 354 Barnctt, W. E., 85, 114 Barondes, S. H., 91, 115 Barron, E. S. G., 134, 160, 274, 303, 340, 357 Bartlctt, P. D., 257, 289, 301 Bartlctt, R. G., 263, 301, 307 Barton, B., 340, 358 Bartsch, K., 126, 133, 146, 158 Bateman, J. R., 335, 355 Bates, R. R., 43, 73, 74, 7s Battle, J. D., Jr., 343, 355 Baucr, J. M., 146, 149, 151, 161 Bnutzc, E., 229, 241 Bautze-Frccse, E., 229, 241 Bayrd, E. D., 312, 338, 356,357 B:de, G. H., 71, 76 Bcatty, R. A., 278, SO1 Bcck, L. V., 255, 263, 276, 301, SO5 Bcck, W. S., 125, 127, 134, 141, 158 Bcckcr, Y., 88,115 Berrs, L., 257, 289, 301 Behr.man, E. J., 147, 159 Bcinwt, H., 253, 265, 305, YOG Ben, T., 58, 60, 61, 62, 75 Bender, M. A., 314, 358 Benlich, A., 229, 239 Benedctti, E. L., 174, 183, 214, 240 Benes, J., 275, 287, 309 Bennett, T. P., 84, 85, 114, 115 Ben-Porat, T., 28, 75 Bentzel, C. J., 326, 327, 328, 354, 356 Bcnzer, S., 85, 116 Berenhlum, I., 66, 67, 76 Berg, P., 85, 86, 87, 114, 115, 116, 290, 302 Bcrgel, I?., 340, 354 Bergmann, F. H., 87, 114, 116 Bergquist, P. L., 85, 86, 114, 234, 239 Bcrgsagel, D. E., 317, 318, 331, 332, 336, 337, 338, 340, 341, 343, 346, 347, 349, 350, 352, 354, 355, 35s Bergstrand, H., 255, 276, 307 Berlin, C. M., 5, 35, 36, 37, 56, 76, SO Bernard, J., 335, 355 Bernhard, W., 314, 366 Bcrnicr, G. M., 320, 355
Bcsch, P. K., 264,303 Bcthell, F. H., 343, 355 Bcuthncr, H., 228, 236, 240 Biancifiori, C., 229, 239 Bielschowsky, F., 257, 289, 301 Bicrbaum, 0. S., 339, 355 Bicrman, M. R., 339, 357 Bicsele, J . J., 249, 301 Bigelow, B., 319, 349, 356 Birns, M., 150, 155 Bishop, J. O., 88, 114 Black, S., 263, 278, 280, 286, 301 Blackburn, N. R., 6, 17, 22, 53, 54, 55, 7s Blake, R., 35, 80 Blanchard, B., 41, 80 Blickenstaff, D. E., 50, 77 Blobcl, G., 109, 116 Bloch, K., 288, 309 Block, J . B., 326, 328, 356 Bloemcndal, H., 100, 114 Blom, J., 338, 358 Blum, G., 173, 178, 179, 180, 181, 188, 192, 194, 239 Boag, J. W., 275, 307, 309 Borresen, H. C., 280, 286,303 Bojarslri, T. B., 5 , 76 Bolding, F. E., 322, 356 Bolton, E. T., 68, 76 Bonner, W. D., 270, 309 Bono, V. H., Jr., 338,355 Bont, W. S., 100, 114 Borek, E., 86, 114,234, 23.9, 241, 245 Borenfrcund, E., 229, 239 Bornstein, S., 212, 213, 243 Borovk, J., 275, 287, 309 Borsos-Nachtnebel, E., 37, 80, 265, 307 BOS,c. J., 216,240 Bosch, L., 100, 114, 134, 155 Botkin, C. C., 177, 185, 242 Bott, K. F., 276, 301 Bottomley, R. H., 34, 76, 146, 147, 149, 158, 160 Bovcri, T. H., 232, 239 Bowdon, B. J., 164, 246 Boxer, G. E., 126, 127, 128, 129, 130, 131, 133, 134, 135, 136, 142, 146, 15S, 1GO Boycc, C. R., 312, 358 Boycr, P. D., 277, 301 Boyland, E., 50, 51, 76, 178, 179, 239, 259,
AUTHOR INDEX
262, 267, 268, 260, 277, 279, 289, 300, 301
Brachet, J., 251, 252, 255, 259, 263, 280, 301
Brand, R., 64, ?G Brandt, E. L., 265, SO4 Bras, G., 174, 2.j5 Braun, A , C., 298, 301 Braun, A. D., 265, 901 Br:inn, H., 275, 287, 30c’l Braun, H. A., 261, 305 Bray, G. A,, 50, 51, 77 Brecher, G., 251, 301, 321, 355 Brcmcr, J., 266, 280, 286, 301, 303 Brenncr, S., 02, 116 Brcsnick, E., 64, ?G, 150, 158 Brevcr, C. B., 88, 07, 114 Brindlcy, C. O., 338, 343, 356, 355 Brittin, G . M., 321, 355 Brock, N., 340, 355 I3rotiic. B. B., 27, 78, 166, 228, 239, 246 Brody, S., 85, 99, 114 Brook, J., 335, 555 Brookes, P., 67, 71, 76, 233,250 Brouwers, J. A. J., 166, 208, 220, 239 Brown, C. L., 340, 355 Brown, D. M., 229, 2,79 Brown, J. M., 181, 244 Brown, R. R., 12, 13, 38, 39, 40, 76, 79, 80 Browning, M., 340, 357 Bruce, W. R., 353,355 Brim, D., 147, 159 Bucciarelli, E., 229, 239 Budnick, I,. E., 146, 157, 160 Bucher, T., 126, 133, 146,158, 160 Buchnar, F., 182, 183, 239, 243 Bulba, S., 234, 246 Bullough, W. S., 277, 279, 301 Burdette, W. J., 232, 239 Burk, D., 145, 15S, 265, $01 Burley, R. W., 272, 301 Burnet,, F. M., 232, 239 Burns, J. J., 18, 19, 29, 30, 48, 49, 50, 51, 56, 76, 77, 289, SO? Uuii-Hoi, X. P., 6, 7, 8, 9, 22, 23, 41, 56, 76, 79
C Calcutt, G., 255, 259, 260, 261, 262, 267, 276, 280, 283, 301, 309
363
Calendar, R., 85, 114 Cnlva, IC., 150, 158 Cameron, G., 273, 303 C:impbell. P. N., 84, 113, 114 Canning, L. C., 92, 96, 98, 100, 101, 102, 104, 106, 108, 109, 110, l l 4 Cantarow, A,, 277, 302 Cant,ero, A , , 124, 140, 142, 150, 15S, 159 Carbon(,, 1’. P., 326, 327, 338, 354, 355, 356 C x e y , R. W., 338, S57 Carlcton, J . H., 177, 184, 244 Carson, P. E., 286, 30-7 Caruso, S. J., 262, 307 Case, A . A,, 236, 239 Cason, J., 260, 302 Caspersson, O., 276, 302 Castillo, G. B., 320, 356 Castleman, M., 312, 955 Cecere, M. A,, 88, 116 Ceh, L., 236, $40 Chalkley, H. W., 250, 273, 302, 30.9 Cliambcrlin, M., 290, 502 Chan, S., 147, 149, 158, 159 Chang, S. H., 254, 302 Chapman, C. G., 343, 356 Chatagner, F., 147, 159 Chen, G. C. C., 193, 198, 238 Chcng, T. Y., 85, 97, 116 Cho, Y. S., 30, 33, 76, 79, 147, 15s Christie, G. S., 182, 210, 211, 215, 216, 23s, 239 Christman, D. R., 263, 303 Cliristoffcrsen, T., 5, 10, 15, 54, 55, 63, 78 Cliu, C. H., 281, 302 ciari{, C. rr., 166, 24s Clark, D. W., 312, 357 Clark, H., 321, 355 Clark, J . M., Jr., 97, if4 Clark, W. H., 147, 160 Claudatus, J., 147, 158 Clayson, D. B., 229, 239 Clayson, D. P., 66, 76 Clayton, A. B., 65, 78 Clayt,on. C. C., 265, 305, 30s C k m , J . M., 171, 172, 948 Cleveland, P. D., 216, 239 Clifford, G . O., 336, 337, 342, 343, 344, 3<55, 357 Coates, J., 255, 260, 280, 283, 309
364
AUTHOR INDEX
Coffcy, G. L., 236,240 Coggins, P. R., 343, 355 Cohen, D. M., 323, 355 Cohen, G., 286, 302 Cohen, P., 328,555 Cohcn, P. P., 71, 80, 146, 149, 150, 158, 160 Cohen, S., 315, 555 Collier, H. B., 271, 309 Collins, A,, 264, 305 Collom, A,, 338, 358 Colsky, J., 338, 343, 356, 358 Conn, E. E., 277, 286, 309 Conn, J . W., 264, 302 Connclly, C. M., 85, 97, l l 4 Conney, A. H., 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 25, 29, 30, 48, 49, 50, 51, 52, 53, 54, 55, 56, 77, 78, 289, 302 Connors, T. A,, 252, 267, 302 Conway, T. W., 84, 89, 105, 114, 116 Cook, J . W., 258, 508 Cooke, J., 269, 302 Cooper, D. Y., 26, 27, 77, 79 Cooper, T., 338, 355 Corenho, U., 343, 355 Cori, C. F., 118, 158 Cori, G. T., 118, 158 Costa, G., 335, 338, 355 Cowdry, ,E. V., 86,114 Cox, R. P., 29, 80 Cmbtrce, H. G., 260, 261, 502 Craddock, V. M., 86, 114, 221, 222, 223, 224, 226, 228, 229, 230, 231, 239, 246, 268, 302 Craig, L. C., 85, 114 Cram, R. L., 289, 305 Cramer, J . W., 5, 7, 8, 17, 18, 19, 20, 39, 52, 77 Cramer, W., 258, SO2 Crane, R. K., 2, 77 Crisler, C., 177, 184, 244 Cristofalo, V. J., 131, 168 Cronkite, E. P., 251, 501 Cruirkshank, C. N. D., 277, 279, 301 Culvenor, C. C. J., 266, 302 Curtain, C. C., 320, 321, 35-5
D Dahlin, D. C., 323, 355 Daiher, D., 170, $39, Z44
Daisley, K. W., 275, 302 Dallam, R. D., 271, 305 Dalton, A. J., 314, 355 Dan, K., 252, SG'7 Daniel, M. R., 271, SO4 Danishefsky, I., 72, 79 Dann, A. T., 266, 502 Danon, F., 335, 355 Dao, T. L., 5 , 6, 17, 22, 43, 46, 47, 50, 53, 77, 81 Dnrtlcn, E. B., Jr., 314, 555 Darlington, C. D., 232, t?39 nnmcll, J. E., 88,116 Dashman, T., 36, 77 Daucheva, K. I., 142, 159 Daudel, K., 41, 79 Dimphinee, J . A,, 322, 368 Davidson, J. N., 28, Y7 Davirs, H. M., 265, 282, 284, 306 Davies, M. C., 88, 97, lf4 Davies, W., 269, 300 Davis, P., 338, 355 Dnvison, C., 29, 48, 49, 50, 56, 77, SO Dayton, P. G., 50, 76 Deakin, H., 275, 302 Dcderick, M. M., 338, 343, 556 Degraeve, N., 290, 306 De la Haba, G., 53, 81 de Larnirande, G., 124,150,151, 158 Delbruck, A,, 126, 133, 146, 158 de Man, J . C. H., 174, 184,239 Dcssel, B. H., 343, 355 Devlin, T. M., 134, 158 de Vries, G . C., 212, 213, 243 Dcwar, J. P., 321, 355 Dcxter, S. O., 265, 272, 606 Dick, A. T., 266, 302 Dickens, F., 120, 131, 134, 158, 230, 239, 269, 270, 278, 279, 280, 302 Dieckmann, M., 87, 114, 116 Dick, G. F., 340, 357 Diencr, E., 210, 217,244 Dienstbier, Z., 275, 287, 309 Dijlrstra, J., 255, 265, 282, 283, 284, 296, 302 Dillion, E. S., 338, 358 Dillion, M. I,., 338, 358 Dingle, J . T., 271, 304 Dingman, C. W., 68, 71, 80 DiPaolo, J. A., 280, 281, 295, 302, 303 Dische, Z., 275, $03
365
AUTHOR INDEX
Dison, F. J., 314, 315, 358 Dixon, G . H., 3, Y9 Dixon, M., 277, 285, 305 Doctor, B., 85, 97, 114 D o h , S., 321, 555 Donnclly, A. J., 131, 1GO Donnclly, W. J., 343, 555 Dontmivill, W.,177, 179, 187, 188, 193, 290 Iholan, 1'. I)., 264, 304 Dose, I<.,277, 303 Iloty, P., 88, 116 Donblc, J. A , , 267, SO2 l)ouglas, W. R., 285, 303 Doxry, D., 255, 260, 280, 283, $0' Dreyer, W.J., 313,358 Drivsholm, A , , 321, 356 Druckery, H., 164, 165, 167, 173, 176, 177, 178, 179, 180, 181, 184, 186, 188, 190, 191, 192, 103, 194, 206, 227, 225, 229, 230, 235, 236, 237, 230, 240, 949 D u l h , D. R., 28, 77, 70 DuboiP, K. P., 19, 50, 7.9 du Buy, H. G., 145, 15s Dudley, H. C., 339, 356 Diingcr, M., 170, 237, 244 Dugdalc, M., 338, 35s Dulbecco, R., 28, Y7 Duncan, B., 276, 301 Dunluim, L. J., 177, 186, 187, 238, 242 Dunn, T. F., 97, 114 Dutton, A,, 166, 167, 169, 202, 203, 226, 240, 241 Dutton, G. J., 20, 77 Dycr, H. M., 147, 150, 156, 157, 158
E Eagle, H., 72, 77 Ebaugh, F. G., Jr., 338, $58 Edwards, S. W., 147, 158 Egyud, L. G., 290, 291, 303 Ehrlich, J., 236, 240 Eignrr, E. A , , 97, 115 Eirich, F. R., 72, 70 Eisenstark, A,, 198, 2$0 Eiscnstnrk, R., 19S, 240 Eisman, S. H., 343, 355 Eistcrt, B., 235, 238 l
Ellilter, P. R., 276, SO5 Elliott,, K. A. C., 273, 304 Elson, L. A,, 256, 260, 263, 266, 267, 301, 302, 303 Elwood, J. C., 131, 168 Emcry, E. W., 339, 354 Emmclot, P., 134, 158 Em.mrlot, l'., 166, 167, 174, 183, 194, 208, 211, 213, 214, 216, 220, 224, 239, 240, ?.p,243 I h d r r , F., 236, 240 Endo, H., 268, 269, 303 Englc, R. L., Jr., 320, 356 English, F. L., 170, 240 Enomoto, M., 8, 18, 20, 39, Y9 Ensfield, B. S., 150, 158 Epstein, P. R., 263, SO3 Erickson, B. A., 288, ,306 Erirsson, J. 1,. E., 53, 55, 56, 71) Eriksson, T., 197, 199, 242 Ernster, I,., 26, 27, 53, 55, 56, 75, Y9 Errora, M.,277, 303 Eschenbrenner, A. B., 145, 159 Essner, E., 150, 158 Estabrook, R. W., 26, 27, 7Y, 79 Eston, J. K., 236, 240 Evans, C., 50, 51, Y6, 7Y Evans, H. J., 280, 30(? Evans, H. M., 257, 289, 30G E v ~ r c t t G. , A,, 85, 114 Eyzagnirre, J. P., 97, 114
F Fadem, R. S., 321, 356 Fagan, V. M., 109, 116 Fnhry, J. L., 315, 316, 318, 319, 320, 321, 324, 325, 326, 328, 329, 330, 355, 356, 355, 359 Fnlimy, M. J., 195, 196, 200, 241 Fahmy, 0. G., 195, 196, 200, 241 Falvonc, G., 251, 274, 280, 306 Falk, H. L., 17, 41, 42, 47, 7Y, Y9, 80, 260, 261, 303 Falkmer, S., 278, 303 Fangninn, W. L.,99, 114 Farhcr, E., 52, 67, 71, 79, 81, 86, 115, 147, 160, 221, 234, 241, 245, 266, 287, YO3 Fare, G., 265,303 Farmer, F. T., 339,354 Feigelson, P., 5, 35, 36, 7Y, 78
366
AUTHOR INDEX
Feinleib, M., 336, 356 Fcldman, H., 87, 114 Feldman, W., 236, 240 Ferger, M. F., 91, 115 Fcssenden, J. M., 88, 114 Fetterinan, I’. I,., 71, 75, 80 Fiala, A. E., 255, 265, 282, 284, 303 Fiala, S., 255, 265, 282, 284, 288, 303 Fieser, 1,. F., 165, 241, 259, 260, 261, 301, 303, 309
Fieser, M., 165, 241 Fine, J. M., 312, 557 Fishrr, M. W., 236, 240 Fiume, L., 206, 213, 242 Flanagan, C. L., 286, 302 Flaxoman, B., 50, 77 Flecher, A. A., 322, 358 Fleissner, E., 234, 241 Flrsch, P., 277, 303 Florini, J. R., 88, 97, 114 Flynn, L. M., 288, 306 Fodor, P. J., 149, 151, 159 Fohn, C. H., 147,160 Fong, C. T. O., 263, 303 Forbes, W. F., 277, 303 Ford, E., 19, 43,47,78 Fodds, I,., 143, 155 Fout,s, J. R., 14, 19, 29, 30, 31, 56, 76, 77, 75, 289, SO5 Fowler, W. M., 336, 337, 342, 343, 344, 357 Foye, L. V., Jr., 343,556 Fraenkel-Conrat, H., 91, lf6 Frank, W., 340, 357 Franklin, X. C., 316, 319, 349, 356 Franklin, R. M., 55, 80 Fransen, B., 234, 241 Fransino, A,, 343, 558 Frearson, P. M., 28, 77 Freese, E., 229, 233, 241 Frei, E., 111, 326, 328, 338, 343, 355, 356, 558 Freidrich-Freksa, H., 183, 241 Fricke, H. H., 50, 7:) Friedell, H. L., 274, 307 Friedman, M., 72, 75 Friedmann, E., 279, 303 Friend, C., 29, 50 Fritz-Niggli, H., 217, 241 Frommeyer, W. B., Jr., 322, 340, 556 Fruton, J. S., 278, 305
Fry, D. S., 252, 309 Fndenberg, H., 316, 356 Fujimoto, J. M., 50, 77 Fukunishi, R., 21, 41, 43, 46, 47, 78 Fukuoka, F., 268, 506 Fukuyarna, T., 262, 307 Furman, M., 19, 76 Furst, C. I., 259, 303 Fnrtli, J., 143, 169
G Gadekar, K., 177, 185,242 Gallagher, C. H., 215, 241 Galton, D. A. G., 335,569 Gardner, F. H., 328, 355 Garry, B. J., 3, 76 Gartner, L., 19, 76 Gasior, E., 90,107, 114 Gastel, R., 48, 49, 50, 56, 77 Gehan, E. A,, 338, 356 Geissler, E., 193, 196, 197, 241 Gelboin, H. V., 6, 17, 20, 22, 43, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 72, 73, 74, 77, 75, 79 Gerhardt, J. C., 3, 78 Gichner, T., 194, 198, 241, 2& Gierer, A., 229, 241 Gilbert, W., 86, 115 Gillette, J. A., 27, 78 Gillette, J. R., 5, 6, 7, 8, 9, 10, 13, 14, 15, 18, 19, 26, 77, 78, 228, 239 Gillman, T., 278, 303 Gilman, A. G., 21, 54, 56, 77, 78 Ginori, S., 147, 155 Glenchur, H., 325, 356 Glock, G. ,E., 134, 135, 15S, 159 Gijrlich, M., 217, 2’41 Gijssner, W., 183, 241 Gold, G. I,., 338, 343, 355, 356, 355 Cold, M., 86,114, 234,241 Coldberg, I. H., 68, 50 Goldblatt, H., 273, 303 Goldstein, J., 84, 85, 114,115 Goldstein, L., 147, 159 Goldzieher, J. W., 263, 264, 305 Goldzieher, M. A., 263, 264, 503 Gonzales, E. L., 274, 303 Goodall, C. M., 265, 303 Goodman, H. M., 88, 116 Griffin, A. C., 92, 94, 96, 97, 98, 100, 101, 102, 104, 106, 108, 109, 110, 114, 115
AUTHOR INDEX
Goranson, E. S., 142, 158 Gordon, S. A,, 274, 303 Gorham, J. R., 315, 366 Gorrod, J. W., 178, 179, 239 Goss, H., 257, 289, 304 Gottfried, B., 264, 303 Goulden, F., 256, 303 Gracc, J. T., 234, ,043 Graffi, A,, 177, 180, 186, 190, 241, 242 Grand, L., 41, 7 s Gray, I,. H., 255, 273, 274, 275, 303 Grrcn, H., 74, 81 Grcenbaum, A. L., 216, 945, 256, 306 Greenbaum, L. M., 289, 308 Greenberg, D. M., 266, 301 Greenberg, J., 198, 943, 339, 35G Grcenc, H. S. N., 143, 169 Greengard, O., 5, 35, 56, 77, 78, 122, 123, 152, 159 Grccnstcin, J. P., 66, 78, 118, 119, 120, 121, 122, 125, 135, 144, 145, 146, 147, 149, 150, 151, 169 Greenwood, D. T., 218, 241 Gregory, P. W., 257, 289, 304 Grcver, B., 219, ,949 G r r y , H . M., 316, 318, 356, 357 Griffin, A. C., 137, 150, 265, 278, 304, 307 Griffit,li, K. M., 317, 318, 331, 336, 341, 347, 349, 350, 352, 355 Grimmett, P., 257, 289, 301 Grover, P. C., 50, 7G Grubb, R., 318, 367 Grundmann, E., 182, 239, 241, 242 Guglielminctti, R., 199, 200, 247 Gullino, P. M., 147, 150, 156, 157, 158 GustdTson, R. G., 174, 213, 214, 241, %$'/t4 Gutman, A. B., 326, 35G Gutman, E. B., 326, 356 Gwynn, R. H., 277, 304
H Habel, I<., 68, 7G Hatldctl, J. R., 29, 80 Hadjiolov, A. A , , 142, 150 Haddow, A., 230, 232, 241, 256, 263, 277, 279, 289, 301, 3'04 Hagrrman, D. D., 124, 150 Haggith, J. W., 339, 5.54 H:iines, J. A., 221, 241 Hniss, H., 166, 206, 244 Hrtkim, S. A. E., 273, 304
367
Halbreicli, A,, 212, 213, 243' Hall, R . H., 234, 2$3 Hall, T. C., 338, 340, 343, 356, 367, 358 m i , w. H., 325,357, 369 H:ilvcr, J. E., 176, 185, 941 Htdvorson, H . O., 70, 78, 91, 114 Hamilton, H. ,E.,278, 308 Hammack, W. J., 322, 340, 358 TInnimctt, F. S., 249, SO,$ H:mpcrl, H., 175, 176, 245 Hmsrn, M. A,, 236, 246 H:irtlwty, B., 92, 114 Harington, J. S., 262, 272, 298, 299, 300, 304 Harlcy, J. D., 262, 304 ITarrqi, K. H., 283, 284, 304 Hnrrington, W. J., 338, 358 Harrison, B., 172, 2.14 H:trt, L. G., 30, 31, 78 Hartj(,, J., 182, 183, 2~44 Hitrtniann, W. W., 168, 2.11 Hartwoll, J. L,, 37, 78, 228, 241, 24.5 H:trtn.cll, L. H., 28, 77 Hasimoto, M., 146, 160 Hut(-11,M. D., 280, 288, 304 Hathorn, M., 278, 303 Halt, H . H., 168, 241 H:iving:L, E., 269, 309 H a w , , C., 236, 240 Hayrs, D. M., 338, 358 Hnyrs, R,. E., 111, 327, 367 H > x ~ I1,. ~ PJ.,~269, , 304 Hcatl, M. -4., 273, 308 Hcnth, D. F., 166, 167, 168, 169, 170, 172, 173, 178, 202, 203, 204, 205, 206, 207, 214, 226, 228, 241 Hrath, J. C., 271, 304 Hrftrr, R. W., 51, 81 Hcgycli, A,, 291, 304 Ilc~itirlbcrgrr,C., 70, 71, 76, i s , 80, 156, 160, 231, 232, $41, 244, 261, 304 Hrilhrunn, L. V., 295, 309 Hcintz, It,. 84, 116' Hcisr, E., 217, 741 Hckhuis, G. L., 146, 150, 1 6 s Hrlgrbostad, A,, 236, 2.f0 Hrlldrom, I., 29, SO Helmus, C., 321, 356 Heniberg, T., 265, 270, 304 Hcmingway, J . T., 264, 289, $04
368
AUTHOR INDEX
Hendrrson, E., 264, 308 Henderson, J. F., 5, 10, 14, 78, 156, 159 Hendry, J . A,, 230, 246 Hengy, H., 170, 244 Henke, H., 197, 199,241 Henriques, 0. B., 263, 264, 304 Henriques, S. B., 263, 264, 304 Henry, M. C., 141,161 Henwhler, D., 237, 241, 246 Henson, J . B., 315, 356 Heredia, C. F., 91, 114 Hermnns, J. F., 320, 356 Herrrll, W. E., 312, 356 Herrmann, A,, 236, 242 Herrold, K. M., 177, 186, 187, 238, 24.8 Hersulto, A,, 287, 305 HPSS,W. C., 264, SO4 Hmvett, C. L., 263, 302 Hintt, H. H., 5 , 76 Hickey M. D., 289, 308 Hickinhottom, W. J., 165, 242 Higa, H. H., 168, 243 Hill, I<., 183, 2.13 Hill, 1., M., 340, 359 Hill, M., 339, 354 Hill, W. T., 41, 78, 80 Hillegns, A. B., 236, 240 Hillman, R.W., 264, 301 Hines, M. S., 338, 358 Hirnmoto, Y., 253, 304 Hirono, I., 267, 304 Hirsh, W., 312, 356 Hishizawa, T., 63, 78 Hitchings, G. R., 150, 15s Hoagland, M. B., 105,114 Hobik, H. B., 182, 242 Hoch-Ligeti, C., 175, 176, 177, 184, 185, 187, 191, 218, 223, 228, 229, 238, 242 Hochstein, I)., 26, 27, 78, 286, 302 Horhster, G., 263, 307 Hiihne, G., 197, 199, 241 Hoffmann, F., 177, 180, 186, 190, 241, 248 Hoffmann-Ostenhof, O., 270, 304 Hogg, J. F., 142, 159 Holland, B. H., 92, 96, 98, 104, 106, 108, 109, 110, 114 Holland, J. F., 338, 343, 356, 358 Holland, J. J., 65, 78 Holley, R. W., 85, 113, 114, 11G Holmer, J. H., 176, 185, 187, 191, 223, 246
Homer, R. F., 230, 246 Hoogstraten, B., 334, 357 Hopkins, F. G., 273, 304 Homing, E. S., 273, 300 Hoslry, H., 338, 356 Howard-Flanders, P., 276, 304 Howatson, A. F., 314, 357 Hoyer, B. H., 68, 75, 7 9 Hrndec, J., 259, 304 Hreschyshyn, M., 343, 358 Hsu, T. C., 229,245 Hulsmann, W. C., 289, $04 Huggins, C., 19, 21, 41, 43, 46, 47, 78, 295, 304 Hughes, C., 253, 254, 255, 279, 280, 285, 286, 304 Hultin, T., 5 , 10, 14, 15, 18, 19, 57, 81, 92, 114, 209, 210, 211, 213, 214, 220, 221, 242, 243, 289, 309 Hurter, P., 171, 245 Hurwitz, J., 86, 114, 234, 841
I Ichii, S., 147, 159 Ickcs, C. E., 286, 302 Ida, N., 66, 79 Ilri, H., 146, 159 Imnmoto, F., 85, 97,114 Imhof, J. W., 320, 357 Ingram, M., 325, 357 Ingram, V. M., 84, 85, 87, f l 4 Inscoe, J . K., 5, 6, 7, 8, 9, 10, 14, 15, 18, 19, 20, 77, 7 8 Irlin, I. S.,75, 76 Irwin, L., 18, 78 Isherwood, F. A., 272, 279, 286, 305 Ishida, I<., 315, 354 Israds, I,. G., 353, 567 Iszsrd, D. M., 338, 357 Ivankovic, S., 164, 167, 175, 176, 177, 178, 179, 180, 181, 186, 188, 190, 192, 193, 194, 206, 229, 230, 840, ,942 Izak, G., 287, 305
J Jacob, F., 3, 70,79, 232, 248 Jacob, T. M., 91,92, 115 Jacobs, E., 338, 358 Jacohsen, K. H., 171, 172, 24s Jacobson, L. O., 340, 357
AUTHOR INDEX
,James, A. P., 277, SO$ .James, T. W., 250, 304, S o t i ,Jarvis, J. A. E., 170, 202, 207, 241 .Jnsmin, G., 176, 185, 186, 191, 230, 242, 245 ,Jcllinck, P. H., 18, 78 ,Jelliim, E., 290, 304 Jeney, A,, Jr., 267, YO2 ,Jcnsm, E. V., 19, 43, 47, 7S, 295, SO.$ Jenscn, I<.A,, 201, 242 .Jcrvell, D. F., 5, 10, 15, 54, 55, 63, 78 .Jocelyn, P. C., 257, 263, 289, SO/,, 305 Johnson, C. I,., 176, 185, 241 Jolinson, M. I<., 278, 305 Johnston, A. M., 272, 509 Johnston, M. W., 264, 302 Johnston, T. P., 164, 245 Johnstone, R. M., 272, 278, 305 Jollks-Bergcret, B., 147, 165 Jontlorf, W’. I<., 50, 51, 58, GO, 61, 62, 76, 7s Joncs, D. S.,92, 115 Jonc,s, H. E. H., 230, 259, 260, 270, 278, 279, 280, 302 Joncr, 0. W., 91, 115 Jones, It,, 338, 366 Joncs, I<., Jr., 340, 343, 357, 35s Jones, R. S., 37, 79 Jonsson, U., 340, 357 Joshi, S. S., 278, 30G Juchau, M. It., 289, 305
K Kadenbach, B., 133, IOU Kagi, J., 85, 116 ICulin, A. I., 321, Y6S Kahrig, C., 286, 507 Kaji, A,, 111, 114, 115 Iiaji, H., 111, 114, 116 Kalscr, S. C., 255, 263, 301, 305 Kanc, J. F., 227, 23s Kano-Sueokn, T., 99, 1 f G Iiaplan, A. S., 28, 78 Iiaplan, H. S., 232, 242 Kappeler, V. It., 349, 350, 3 2 Karlson, A. G., 236, ,0$0 Kasbekar,D. K., 263, 305 Kasinsky, E., 288, 303 &to, B., 50, 57, 58, 60, 61, 62, 78 K:iufinan, S., 4, 26, 79 Kawitniot,o, S., 66, 79
369
IGIY, H. E. M., 339, 557 Kay?, .I. M., 29, 81 I<eii., 11. M., 290, 305 I
370
AUTHOR INDEX
I
ill, H. F., 269, 270, 305 Iircbs, A,, 349, 350, 557 Krebs, H. A,, 137, 159 Krieg, D. R., 233, 242 Kriek, E., 166, 167, 211, 240, 242 Krim, M., 229, 239 Kriss, J. P., 339, 357 ICriszat, G., 295, 307 Kritrhrvsky, I)., 75, 79 Iiriiger, H., 219, 242 Kunkcl, H. A., 197, 198, 199, 241, 242, 240 Kuff, 11:. L.,112, 115, 313, 555 Kuhn, R., 265, 305 Kunltel, H. G., 316, 318, 320, 356, 367 Kurland, C. G., 90, 115 Kurl;md, L. T., 164, 181, 184, 185, 190, 2.$2 Kyle, L. H., 264, 304
L Larassagne, A,, 41, 79 Lackey, M. D., 145, 158 La Du, B. N., 27, 78, 228, 239 Laird, A. I<., 124, 159 I,ajtha, L. G,, 252, 307 Lamar, C., Jr., 34, 60 IJ:unborg, M. It., 85, 92, 116 I,;imont,, N. McE., 278, 503 Lan, T. H., 145 159 Lancaster, M. C., 270, SO7 Langdon, R. G., 263, 305, 306 Lange, R., 262, 305 Langley, B. W., 167, 171, 235, 242 I,nqueur, G. L., 164, 181, 184, 185, 190, 215, 242, 246 Lardy, H. A,, 123, 150 Larionov, L. I?., 340, 357 Larsen, A. E., 314, 315, 358 Imson, H. O., 168, 243 Imagnn, I,., 338, 343, 356, 358 Imnitzki, A,, 131, 160 Laster, W. R., 164, 245 Lavate, W. V., 263, 305 Law, L. W., 257, 289, 306 Lawlry, P. D., 67, 71, 76, 233, 239 Lawrence, J. H., 339, 357
I,nwrrnrr, J . S., 321, 324, 56.i I,aztirow, A , , 263, 278, .$Oh L;izor, M. Z., 326, 357 I,cder, B. W., 315, 95(i I,c~blonti,C. P., 270, Yfll I,ol)oursw, J., 147, 16:) Lcdrr, P., 91, 116 Lee, B. J., 335, 351, 357 Lee, J. S.,276, 305 Lee, K. Y., 169, 223, 224, 225, 228, 242, 243 T,rhninger, .4.I,., 256, 263, 505 Lriter, J., 151, 161 Lron, H. A,, 92, l l 4 I,eoncini, D. I,., 312, 357 Leong, J . L., 6, 7, 17, 20, 21, 22, 42, 45, 51 Lcopold, A. C., 270, 305 Le Page, G. A,, 119, 125, 169 Ide Page, R. N., 182, 216, 230 Lerman, S., 275, 287,305 Lessner, H., 340, 357 Lrster, G., 297, 305 Lcuthardt, F., 145, 149, 151, 159 Leutee, C. J., 227, 235 Levin, J., 276, 304 Levin, W. C., 340, 343,555 Levintow, L., 146, 159 Levy, R. N., 334,366 Lewis, C. E., 172, 242 Lewis, ,E. B., 315, 357 Li, C. H., 257, 289, 506 Licbrrman, I., 3, 79 Lijinsky, W., 169, 223, 228, 242 Lin, E. C. C., 123, 156, 159 I&, Y., 131, 168 Lindsley, D. L., 314, 558 Linford, J. H., 353, 367 Lingens, F., 229, 242 Linkenheimer, W. H., 263, 301 Lipmann, F., 84, 87, 88, 89, 99, 100, 107, 113, 114, 115, lie, 289, 304 Littauer, U. Z., 86, 115 Littbrand, B., 276, 307 Littlefield, J. W., 92, 97, 115 Litwack, G., 75, 79 Lobl, L. T., 218, 242 Lochmann, E. R., 229, 242 Locb, L. A., 57, 58, 60, 61, 62, 63, 64, 65, 70, 78 70 Lijw, H., 209, 210, 211, 213, 214, 242 Loftfield, R. B., 97, 115
AUTHOR INDEX
I,og:~n,M , 310, J59 I , o n l l ~ ~ ~ lH~ l, l215, , 2 ;$ I,onrlgail, 1' , 50, 77 I,ongeiirc hi.1, H. W., 50, ?!I r , o l ~ l l ~ l l ou., , 104, 199, 200, 242 I,otIiL~ir,1'. D , 8, 18, 20, 39, 79 L ~ U I >J ,, 33G, 337, 342, 343, 344, 355, 357 Louis, I,. H., 264, 302 LOVC,W. C., 140,161 J,ovina, T. O., 340, S57 Lowrnstcin, J., 319, 349, 35G Lowrnstein, J. M., 150, 158 I>uchbangll, C., 340, 357 I,iintlgren, D. G., 276, 301 I,usky, L. M., 261, 305 I,iittgcns, W. F., 338, 357 I,uttich, W., 237, 244 Lylhgor, B., 167, 171, 235, 24.2
M Maass, H., 275, 287, 305 McAlistc~,J. M., 339, 354 McBride, J., 142, 159 McCafferty, D. E., 37, 79 McCaleb, G. S., 164, 245 McCallum, L. E., 262, 309 MacCardle, R. C., 312, 313, 358 McCarthy, B. J., 65, 68, 75, 78, 79 McClintock, B., 70, 79 McCoy, T. A,, 147, 158 McConnell, L. I<. P., 271, 305 McCiie, M. M., 71, 75, 80 McCulloch, E. A,, 314, 357 MacDonald, H. L., 140, 142, 159 MacDonald, J. C., 38, 39, 40, 79 McDonnell, J., 264, 301 McIlwain, H., 288, 305 McIntire, K. R., 112, 115, 313, 358 McKelvey, E. M., 315, 35G Maclachlan, M., 322, 358 M(.Lauglilin, J. A., 291, 304 McLean, A. E. M., 174, 243 McLran, E., 174, 243 McLean, P., 135,159 MacMahon, B., 312, 336, 356, 567 McMnllen, A. I., 264, 301, 305 Madison, J. T., 85, 114 Madsen, N. B., 271, 305 Madsen, R., 236, 240 Magnlini, S. I., 312, 357 Magastnik, B., 297, SOY
371
hl.igcr, P X I 67, 71, 79, 86, l l & ,115, 164, 165, 169, 171, 172, 174, 175, 176, 178, 180, 181, 184, 185, 187, 188, 191, 202, 207, 208, 209, 210, 211, 213, 214, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 234, 259, 241, 242, 243, 2$5, 216, 268, 277, 279, 301, 305 Magcr, J., 212, 213, 243, 287, 305 Magnns-Levy, A,, 324, 357 Majiina, H., 128, 129, 131, 134, 135, 136, 142, 160 Makarova, S. I., 200, 24G Malick, B., 92, 104, 106, 108, 109, 110, 114 Mitlmgrcn, H., 285, 305 Malmgren, R. A,, 319, 321, 330, 555, 369 Mantlel, M., 193, 198, ,038 Mandclbaum, F. R., 263, 264, 304 Mandcll, J., 198, 243 Mandema, E., 312,S57 Manson, D. I., 312, 357 Manson, L. A,, 146, 151, 161 Mapson, L. W., 272, 279, 286, 505 Mardasliev, S. R., 285, 305 Margolis, F., 36, 77 Marks, P. A,, 3, 79 Marquardt, H., 194, 195, 196, 197, 198, 199, 200, 201, 243, %$G Mnrquisee, M., 85, 114 Marraccini, A., 263, 301 Marrian, D. H., 279,303 Marroquin, F., 234, 241 Marsh, J. C., 325, 567 Marston, H. R., 272, 305 MHrtcnsson, L., 316, 318, 319, 357 Martin, D. B., 3, 81 Martin, G. R., 50, 51, 76, 79 Martin, R. G., 3, 76, 79, 91, 115 Masayanis, T., 146, 159 Mass, R. E., 338, 339, 357 Massari, R., 312, 357 Masasso, J., 195, 196, 200, '41 Mathison, J. H., 227, 2,98 Matsumoto, H., 167, 168, 171, 242, 243 Matthaci, J. H., 65, 79, 91, 115 Matthews, R. E. F., 86, 114, 234, ,039 Matthias, J. Q., 343, 357 Mattocks, A. R., 168, 169, 241 Maxwell, E. S., 111, 115 Mayer, S.H., 274, 505 Mazcl, P., 5, 10, 14, 78
372
AUTHOR INDEX
Mazia, D., 249, 251, 252, 253, 254, 279, 306 Medawar, P. B., 291, 306 Meechan, R. A., 37, 79 Meeker, B. E., 353, 355 Meier, D., 217, 241 Meier, F. ,E., 277, 306 Meier, Chase, F. E., 277, 306 Meister, A., 147, 159 Melhuish, A. H., 256, 306 Meltzer, M., 319, 349, 356 Mennel, H. D., 173, 176, 177, 178, 179, 186, 193, 206, 240 Merigan, T. C., Jr., 327, 357 Merits, I., 55, 79 Merker, H. J., 56, 80 Merrill, S. H., 85, 113, 114 Merwin, R. M., 314,355 Metais, R., 312, 357 Michaelis, A., 194, 198, 241 Michaelson, I. A., 19, 77 Michel, E., 199, 200, 242 Michel, M. R., 28, 81 Mickelsen, O., 164, 181, 184, 185, 190, 242 Migliore, P. J., 317, 318, 331, 336, 347, 349, 350, 352, 355 Mihara, S., 253, 254, 304 Miller, A. J., 47, 79 Miller, E. C., 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 29, 38, 39, 40, 52, 53, 71, 76, 77, 79, 80, 86, 116, 124, 160, 220, 227, 231, 243, 265, 306 Miller, J. A,, 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 29, 38, 39, 40, 52, 53, 71, 76, 77, 79, 80, 86, 115, 124, 160, 168, 220, 227, 231, 243, 265, 306 Miller, S. P., 338, 355, 356 Mioduszewska, O., 264, 306 Mirsky, A. E., 111,113 Misiewicz, J. J., 343, 357 Mitchley, B. C. V., 178, 179, 239 Mittelman, A., 234, 243 Mize, C. E., 263, 306 Mizrahi, I. J., 167, 194, 208, 211, 212, 213, 214, 224, 240, 248 Modan, B., 337,357 Modig, H., 255, 276, 307 Miilbert, E., 183, 243 Mohr, U., 177, 179, 187, 234, 239, 243, 246 Moldave, K., 88, 90, 107, 114 Moldcnhavcr, M. G., 71, 78, 261, 304
Molomut, N., 264, 303 Moloney, W. C., 338, 356 Moment, G. B., 251,SVG Monder, C., 151, 160 Monier, R., 97, 115 Monod, J., 3, 70, 79, 232, 242 Monro, R., 88, 107, 113 Montgomcry, J. A., 164, 245 Moon, H. D., 257, 289, 306 Moore, B. W., 133, 135, 146, 150, 158 Moore, C. V., 339, 358 Moore, S., 339, 358 Morland, J., 5, 10, 15, 54, 55, 63, 78 Morris, A . J., 92, 115 Morris, A. L., 281, 306 Morris, H. P., 30, 31, 33, 34, 76, 78, 79, 80, 109, 11G, 126, 128, 129, 130, 131, 135, 136, 140, 142, 146, 147, 149, 150. 156, 157, 158, 159, 160, 161 Morris, I., 252, 253, 306 Morrison, J. H., 252, 309 Mortenson, L. E., 253, 306 Morton, M. J., 99, 115 Moscovitz, H. L., 338, 358 Moutschen, J., 290, SVG Moycd, H. S., 297, 30G Muhlbo .k, O., 289, 300 Mullcr, A. J., 194, 198, 200, 243, 244 Muller, E., 166, 206, 244 Mueller, G. C., 55, 80 Muller, H. A., 187,244 Mullcr, I., 277, 304 Muller, M., 173, 177, 178, 179, 180, 192, 227, 229, 239, 240 Muench, K., 86, 115 Muirhead, E. E., 321, 355 Mukhcrjee, T., 174, 213, 214, 244 Mul, N., 320, 357 Mullierkar, L., 278, 306 Muller, H. A,, 179, 183, 184, 239 Mundry, K. W., 229, 241 Munro, A,, 55, 79 Murphy, S. D., 19, 50, 79
N Nachman, R. L., 320, 356 Nachmanson, D., 2, 79 Nadeau, I,. A., 312, 357 Nadel, E. M., 177, 184, 244 Nadkarni, M. V., 29, 80 Nagahama, T., 167, 168, 243, 244
373
AUTHOR INDEX
Nagamatsn, A,, 278, 308 Najjar, V. A , , 143, 159 Nak:tliara, W., 268, 306 Knltanioto, T., 84, 89, 125 hrarnsimhdu, S., 27, 77 K:ls:itir, M., 252, 253, 254, 306 h-aslicd, N., 230, 240 Kathans, D., 84, 88, 99, 100, 115, 330, 358 -h;eetlhnm, J., 251, 257, 263, 280, 289, 292, 300
Keitlliardt, F. C., 99, 114 Neish, W. J. P., 255, 265, 282, 284, 506 Ncincth, A,, 36, 79 Nery, It.,267, 301 Neuhert, D., 256, 263, 305 Neufcld, E. F.,252, 806 Nenrath, G., 170, 237, 2.44 Keiiratli, H., 3, 79 Keuwirt, J., 275, 287, 309 h’cvinny, H. B., 343, 358 Newbcrne, P. M., 288, 306 Kcw.pl1, R. R., 339, 357 Sewton, H. P., 8, 18, 20, 39, 79 Nicliol, C . A,, 146, 157, 1C0 Nickel, E,, 210, 217, 2.41, 24.4 Nicltrrson, W. J., 251, 271, 274, 280, SO6 Nicdbala, T. F., 280, 503 Nignm, V. N., 140, 142, 159 h’irenberg, M. W., 65, 79, 91, 116, 142, 158 Nishida, I<., 167, 244 Nishimura, S., 91, 92, 115 Nishizuka, Y., 89, 115 Niskanen, ,E. E., 281, 508 Noll, H., 87, 109, 115, 116 Norberg, It., 320, 357 Norris, A. T., 86, 115 Novclli, G. D., 85, 111, 114, 115 Koviboff, A. B., 94, 115, 150, 168 Sovikova, M. A,, 340, 357 Nyholm, M., 281, 30s
0 Oi-.hoa, M., Jr., 93, 97, 111, 115
Oi:hoa, S., 88, 91, 97, 98, 100, 115, 116 OI~licl,E., 286, YO7 O’Conncll, D . J., 262, SO7 O’Dca, J. F., 320, 355 O‘Dcll, 13. L., 288, 306 Oelilert, W., 182, 183, 239, 244 Ofengaud, E. J., 87, 114, 116
O’Gara, R. W., 177, 185, 242, 244 Ogryzlo, M. A,, 322, 558 Oiltcniiis, .4.,172, 244 O’Learp, J. F., 172, 244 Olson, K. B., 338, 355 Omura, T., 27, 79 Ontlrc~j,M., 195, 196, 200, 240 O’Xed, Pvl. A , , 94, 96, 114, 115 Ono, T., 30, 33, 79, SO, 135, 160 Oota, K., 184, 24G Oppenheiniei-, B. S., 72, 79 Oppenheirner, E. T., 72, 79 Ord, M. G., 275, 276, 286, 299, 302, 306 Ord, M. J., 199, ,944 Orrenius, S.,53, 55, 56, 79 Osborn, S. B., 339, 354 Osgood, E. E., 338, 339, 344, 347, 349, 568 Osscrnian, E. I?., 314, 317, 318, 319, 321, 322, 333, 334, 338, 349, 351, 35S, 359 Otnni, T. T., 150, 159 Otsrika, H., 63, 7S, 253, 254, 304 Ott,, M. G., 71, SO O w n s , A. H., Jr., 338, 343, 355, 356, 358 Oyama, V. I., 72, 77
P Padilln, G. M., 250, 506 Pai, S.R., 289, 306, 309 Painter, T. S., 257, 289, 307 Papnc, R., 338, 358 Paradis, A. D., 151, 161 Pardoe, 4. B., 3, 78 Parish, J. H., 219, 244 Parsons, D. F., 314, 368 Passcy, R. D., 277, 279, 301 Pastcrnak, L., 194, 196, 197, 199, 200, 244 Patnki, J., 47, 78 Patno, E., 342, 343, 358 Patt, H. M., 274, SO6 Pcers, J. H., 251, 301 Peggie, K. S., 267, 301 Penman, S.,88, 115 Pensmick, J. R., 85, 114 Pcpler, W. J., 265, 283, 284, 296,302 Peraino, C., 34, 35, 54, 55, SO, 109, 116, 146, 147, 149, 160 Perova, S. D., 75, 7C Perry, J., 234, 246 Perry, S., 325, 357 Pert, J. H., 320, 366 Petcmmn, M. L., 92, 115
374
AUTHOR INDEX
R ~ ~ rr. I I ,w., 3, so Petrrs, R. A., 261, 272, 273, 278, 907 lt;inst,rGm, S., 322, YGS Pfeflwkorn, I,. C., 105, 114 Rao, K. V., 278, 306 PhellJS, H. I,., 71, SO R;ipkine, L., 252, 272, 307 Pliillip~,1).M. P., 290, dfj7 Kapoport, J . A,, 193, 198, 200, 201, :?dd Phillips, R., 168, 541 Itapoport, S. M., 286, 307 Piekurski, I,. J., 28, 79 Rapp, H. J., 177, 184, 244 Piez, K., 72, '77 Rasmussen, H., 263, 307 Pihl, A,, 251, 273, 274, 307 Ravdin, R. C., 343, 355 Pikkat, A. K., 285, 305 Rawls, W. B., 263, 264, 303 Piric, A., 252, 275, 307, 309 Razin, A., 287, 305 Pirniann, B., 170, 237, 244 Recknagcl, R. O., 215, 244 Pisciotta, A. V., 343, 355 Pitot, H. C., 30, 33, 34, 35, 51, 55, 70, 76, Rceb, B., 41, SO 79, 80, 109, 112, 116, 135, 146, 147, Reed, H. S., 270,307 149, 150, 156, 157, 158, 160, 232, 2-44 Rees, K. R., 215, 216, 241,244 Reese, C. B., 221, 241 Pizzo, A,, 41, 78, 80 Reese, W. H., 177, 185, 242 Plaa, G. L., 50, 77, 289, 305 Reeve, P.M., 265,282, 284,306 Plaine, H. L., 252, 307 Rege, D. V., 263, 305 Plowright, W., 237, 2'44 Regelson, W., 338, 356 Poen, H., 320, 357 Register, V. D., 263, 301, 307 Popper, H., 176, 185, 187, 191, 223, L4G Porter, D. D., 314, 315, 358 Rcich, E., 55, 68, 80 Reid, E., 144, 160 Porter, R. R., 315, 355 Posner, H. S., 5 , 6, 7, 8, 9, 10, 14, 15, 18, Reid, J. D., 219, 244 Reif, A. E., 13, SO 19, 77 Postnikova, Z. A,, 75, 76 Reinhard, E. H., 339, 358 Reiskin, A. B., 281, SO6 Potee, K. G., 343, 358 Potter, M., 112, 115, 312, 313, 314, 330, Remmer, H., 26, 50, 56, SO 356, 358 Rendi, R., 88, 97, 98, 100, 116 Potter, V. R., 13, 30, 33, 34, 76, 79, SO, R ~ v ~ sI,z.,, 255, 276, 302, SOY 109, l l G , 120, 135, 147, 160, 231, 244, Rcyers, I. H. M., 216, 240 265, SOT Rhoads, C. P., 265, 272, 305 Pratt, G. T., 314, 368 Rich, A., 87, 88, 116 Preiss, J., 87, 116 Richardson, H. I,., 37, SO,265, 307 Preussman, R., 164, 165, 166, 167, 168, Richmond, H. G., 272, 307 170, 173, 175, 176, 177, 178, 179, 180, Ridd, J. H., 166, 244 181, 186, 190, 192, 193, 194, 206, Rider, W. H., 340, 358 229, 230, 235, 237, 239, 240, 242, 24.1, Rieck, V. D., 276, 301 Riegel, B., 41, 78, 80 245 Price, C. A., 270, 306 Ricger, R., 194, 198, 241 Price, C. C., 340, 357 Rieselbach, R., 326, 328, 356 Price, J. M., 124, 160 Riggs, N. V., 167, 168, 171, 235, 245, 244, Prigot, A., 338, 340, 359 245 Pullman, B., 232, 233, 244 Riley, J. F., 219, 244 P u h a m , F. W., 320, 355 Riley, P., 277, 301 Riopelle, J. L., 176, 185, 186, 191, 230, R 24% 245 Racker, E., 126, 127, 133, 134, 141, 142, Riva, G., 349, 350, 367 143, 157, 160, 161, 277, 307 Rive, D. J., 167,245, 268,308 Rai, K. R., 339, 356 Rivers, S. L., 342, 343, 358 Rajewsky, B., 277, 303 Robbins, A,, 343, 366
AUTHOR Ih'DEX
R(iti(~rts,I). C., 230, 246
tiobcllt,.i,rc.. 151, Roberts, ,lC. H., 146, 149, 151. lC1 t < o h i ~ t , sJ. , .I.. 71. SO, 267, 307
Itobr~rlson,J . M., 85,114 Robc,rt,ron, I,. S., 237, 2j5 Robinson, A. M., 256, 263, 289, 3 / 1 , 304 Robinson, D. S., 215, 245 Robinson, G. M., 291, 301; Robinson, R.. 291, 306 Ro(*k~~il):ii~ti, ,J., 264, 30s Roc, 17. .J. C., 178, 179, :?N, 267, 270, 272, :XI.$,307 Roffia, I,,, 206, 213, 2.jl r{ogcrs, I,. A,, 14, 19, 56, 77 Rogers, S.,29, SO Rogers, W. I., 99,115 Rondoni, P., 227, 245, 259, 307 Rosc, F. L., 204, 230, 246, 2 $ 0 Rosm, I?., 146, 157, 100 Roscnbcsrg, I,. E., 326, 327, 364, 357 liosenllial, O., 26, 27, 77, 79, 131, 160 Row, S. W., 331, 332, 338, 346, 355 Ross, W., 237, 241, 245 Ross, W. C. J., 252, 266, 267, 302, 907 Roth, J. S., 35, SO, 150, 1G0 Rotlinian, S., 277, 303 Roulston, W. J., 272, 307 Rous, P., 232, 245 Rowc, D. S., 315, 316, 318, J5S Itridali, G., 41, 7.9 Riissnian, W., 133, 160 Ruff, J. D., 312, 356 Runtlel, W., 166, 206, 2.$.$ Rundles, R. W., 338, 340, 558 Runnstrijni, J., 295, 307 Ruschni;inn, G. K., 28, Sf Hylctt, h.,255, 265, 282, 284, 306
s S:ik:ii, H., 252, 307 S:iksliaug, J., 236, 245 Snlnman, M . H., 267, 277, 279, 301, 304, so7 Salas, M., 88, 216 Salerno, P. R., 274, 307 Salzbrrg, D. A , , 278, 307 Sanders, C., 140, 15s Sanford, K. I<., 273, 307 Yantilli, I?. I<., 229, 23!)
375
SW, h ~ I)., . 262, .ro S;tto, (;,, 27, 7!/
Sitto, H., 27, ?!I t l o , T., 262, SO7 ltlll~l~ it:., , 2.i.'
tvigc, LV, R., 277, 505 i\vit,sky, A,, 339, 356 Sits, H. J., 201, 246 sits,I<., 201, 246
Sraifc, J. F., 273, 274, 276, 290, 307 Si*li:tt)ol,F. M., 164, 245 Sc~liarlav,C., 338, 356 Solicltl, G., 177, 178, 184, 245 SCllCll, I',, 220, 239 S(~lic~partz, B., 277, 302 S c l i c r r ( ~I<., , 88, 115 Sclicucli, D., 286, YO7 Scliildl),zc.ti, A,, 167, 176, 192, 24@ Srliiinnusek, H., 126, 133, 146, 25S, IGO Scliiinlw, R. T., 5, 35, 36, 37, 56, 7fi, SO, 150, 152, 157, 1GU S~~lin~iilil, D., 164, 165, 167, 172, 173, 177, 178, 179, 180, 184, 185, 187, 191, 192, 227, 229, ?:W, ?.{O, 245, 246 Schniitlt,, C. H., 173, 177, 178, 181, 186, 193, 206, 240 Scli.niidt, I<., 129, 131, 132, 135, 136, 15s. 1GO Scliniidt, F. W., 129, 131, 132, 135, 136, 158, 160 S ~ ~ l i n i i dG., l , 289, SOS Si,hncitler, H., 228, 236, 210 S(,linc,itlcr, W. C., 124, 100 Srlioental, It., 165, 166, 167, 172, 173, 180, 181, 187, 188, 189, 190, 192, 227, 231, ,?$ 245, I, 262, 264, 268, 273, 305, dOS
Si.hocz;sl(,r, M. A , , 263, 5/17 Scliranini, C; ., 228, $45 Srliuelor, P. W.,50, 77 S(~linntncr,C. A , , 272, 307 Sclinstcr, H., 228, 245 Schwticr, R., 194, 195, 196, 197, 198, 199, 200, 201, 243, 245, 24G Scli\vnrtz, I. I,., 263, 303, 907 S[aIiwnrz:, G., 312, 356 Schwect, R. S., 84, 88, 90, 92, 96, 109: 113, 114, 116 Scoggins, R., 324, 325, 351; Srornik, 0. -4,, 105, 114 Scott, C. M., 256, 289, 3/.;
376
AUTHOR INDEX
Scott, D., 280, 303 Scott, D. B. McN., 281, 506 Scott, J. D., 256, 289, 304 Scott, R. B., 343, 357 Screenivason, A,, 263, 305 Seakins, A,, 215, 245 Searle, C. E., 268, 269, 278, 308 Segal, H., 36, 80 Seibert, D. J., 338, 558 Selawry, 0. S., 338, 343, 356, 358 Seligniann, M., 335, 355 Sen, A. K., 340, 357 Seneca, H., 264, 308 Seraidarian, K., 289, 308 Serfontein, W. J., 171, 245 Sctala, K., 281,308 Setter, V., 265, 304 Scxton, W. A., 263, 269,270, 291, 308 Shack, J., 145, 160 Shactcr, B., 257, 289, 30s Shaeffcr, J., 84, 88, 90, 92, 113, 114, 116 Shannon, R. N., 171, 172, 242 Shapiro, H. D., 321, 358 Sharma, C., 131, 180 Sharma, R. M., 131, 160 Shatkin, A. J., 55, 80 Shedlovsky, A. E., 297,308 Sheets, R. F., 278, 308 Sheid, B., 150, 160 Shelata, S., 257, 289, 301 Shelton, E., 124, 160 Shepherd, D. M., 219, 244 Shepherd, J. B., 290, 306 Sherbert, G. V., 278, 500 Shimkin, M. B., 267, 269, 270, 505, 308 Shkodinskaja, E. N., 340, 357 Shnider, B. I., 338,343, 356,558 Shonk, C. E., 126, 127, 128, 129, 130, 131, 133, 134, 135, 136, 142, 146, 158, 160 Shotlander, V. L., 215, 216, 244 Shrift, A,, 271, 308 Shubik, P., 41, 78, 80, 176, 184, 223, 228, 245, 246 Shull, K. H., 52, 81 Shvembcrger, I. N., 177, 246 Sieburg, H., 182, 241 Silber, R., 29, 80 Siler, R. A., 51, 81 Silver, L., 263, 303 Silver, R. T., 326, 358 Simer, F., 120, 158
Simon-Reuss, I., 279, 303 Simonsen, D. G., 151, 1G0 Simpson, M. E., 257, 289, 300 Singcr, E. J., 176, 185, 187, 191, 223, 24G Singhal, R. L., 126, 129, 138, 139, 157, 1G1 Sinha, K. P., 216, 244 Siperstein, D., 109, 110 Siperstrin, M. D., 297, 308 Sjogren, H. O., 29, 80 Skinner, W., 337, 358 Skipper, H. E., 164, 245 Skoog, W. A., 338, 339, 354, 358 Slater, T. F., 216, 245 Smit, J. A,, 276, 308 Smith, .E., 328, 358 Smith, M., 88, 116 S.mitli, M. A,, 35, 56, 78 Smilh, P. K., 29, 80 Smith, T., 340, 357 Smucklrr, E. A,, 216, 259 Smyth, D. G., 278, 308 Snapper, I., 321, 337, 338, 358 Snoke, J. E., 288, 509 So, B. T., 173, 181, 190, f?40 Sobrr, E. K., 150, 158 Siignen, E., 236, 245 SGrbo, B., 276, 308 Sokoloff, L., 57, 58, 78 Solomon, A., 319, 329, 330, 359 Solomon, D. K., 146, 157, 160 Solomon, J., 343, 359 Sols, A., 2, 77 Somcrs, C. E., 229, ,945 Sorcnson, G. D., 314, 359 Sorof, S., 71, 75, 80 Sorokin, C., 308 Spahr, P. F., 86, 115 Spain, J. D., 265, 305, 308 Spatz, M., 181, 246 Spccd, D. E., 283, 284, 304, 335, 369 Spcncer, K., 224, 242 Spencer, R. P., 124, 160 Spielgelman, S., 62, 63, 81 Sporn, M. B., 68, 71, SO Spragg, S. P., 253, 254, 255, 279, 280, 285, 286, 304 Sprague, C. C., 331, 332, 340, 341, 346, 354, 355
Spurr, C. L., 340, 357 Spyrides, G. J., 84, 89, 115, 116
377
AUTHOR INDEX
Srinivasan, P. R., 234, 246 Srivastava, S. K., 126, 129, 138, 139, 157, 161
Stacey, K. A., 266, 300 Staehlin, T., 87, 116 Stahl, K. W., 173, 177, 178, 186, 193, 206, 240 Stangcr, D. W., 41, 7S, SO Stanley, W. M., Jr., 88, 116 Stanton, M. F., 177, 185, 24G Starikova, V. B., 259, 30s Stefanini, M., 312, 357 Stein, W., 229, 246 Steinberg, A. G., 318, 350 Steiner, P. E., 41, SO Steinfeld, J. L., 335, 343, 355, 359 Steinhoff, D., 176, 177, 178, 180, 184, 190, 191, 192, 228, 236, 239, 240 Stelrol, J. A , , 234, 246 Stent, G. S., 99, 116 Stephenson, M. L., 97, 115 Stern, H., 249, 250, 251, 252, 253, 254, 255, 286, 288, 289, 30G, 308 Stern, I<., 280, 30s Stevenson, I. H., 20, 77, 218, 241 Stewart, G. A., 234, 241 Stewart, H. L., 37, 78, 145, 150 Stier, 8.R., 37, SO, 265, 307 Stirpc, F., 216, 646 Stjernvall, L., 281,908 Stock, J. A., 340, 35$ Stocken, L. A,, 275, 276, 286, 299, 302, 306, 30s Stollar, V., 29, SZ Stone, G., 322, 354 Stoner, H. B., 214, 246 Stout, A. P., 72, 79 Stowell, R. E., 258, 302 Strand, P. J., 6, 17, 20, 21, 22, 80, 81 Strauss, B. S., 231, 246 Stretton, 4.0. W., 92, 116 Striebirh, M. J., 124, 160 Strittniatter, P., 278, 30s Strong, F. M., 167, 171, 643 Stulberg, M. P., 85, 115 Stumpe, W. M., 264, 301 Sueoka, N., 85, 97, 99,114, 116 Siiss, R., 226, 229, 246 Sundararajm, T. A., 88, 116 Suntseff, V., 133, 135, 146, 150, 168 Sutherland, E. W., 3, 80, 81
Sutter, J. L., 252, 305 Suzulii, T., 262, 307 Svirn, H. J., 323, 355 Swan, A,, 335, 359 Swann, M. M., 249, 250, 251, 252, 288, 295, 297, 308 Sn.ann, P. F., 224, 230, 246' Smccney, E. W., 5, 35, 36, SO Swccnry, M. J., 129, 130, lG0 Swiaher, S. N., 322, 354 Sydow, G., 217, 218, 246 Sylvkn, B., 285, 305 Szcnt-Gyorgyi, A,, 290, 291, 303, 304, 309 Szwed, C. F., 324, 325, $66
T Takanami, M., 91,92, 103, 106, 11G Talcttsuki, K., 314, 317, 318, 319, 322, 349, 35S, 359 Tnliayams, S., 184, 246 Taller, D., 50, 7G Tamnoki, T., 55, 80 Tamiya, H., 253, 254, 304, 309 Tanaka, K. K., 151, lG0 Tanaka, T., 151, 160 Tanaka, Y., 43, 47, 77, 81, 321, 955 ~ ~D. F., ~295, 304~ i Tappel, A. C., 271, 309 Tatum, E. L., 55, 80 Taylor, G., 66, 79 Taylor, R., 338, 356 Teas, H. J., 201, 246 Temin, H. M., 112, 116 Terayama, H., 63, 78 Terracini, B., 176, 185, 224, 246 TCLrry, W. D., 316, 318, 359 Tharh, R. E., 88, 116 Thannhauser, S. J., 289, 307 Thcriot, L., 276, 304 Thimann, K. V., 270, 909 Thomas, C., 172, 173, 175, 176, 177, 179, 180, 181, 184, 185, 186, 187, 191, 240, 245, 246 Thomas, S. F., 339, 357 Thomas, T. F., 312, 359 Thompson, M. E., 272,309 Thompson, S., 17, 41, 42, Y7, 260, 303 Timm, M., 275, 287, 305 Timonrn, S., 286, 308 Ting-Kai, L., 85, 116
321,
~
178, 190,
261,
~
,
378
AUTHOR INDEX
Todaro, G. J., 74, 81 Todd, Lord A., 221, 241 Toledo, J. D., 189, 246 Tornatis, L., 176, 184,246 Tompkins, G. M., 4, 81 Tonge, B. L., 262,309 Toth, B., 176, 184, 223, 246 Tousler, O., 51, 81 Trninin, N., 66, 76 Trains, E. C., 5 , 6, 7, 8, 9, 10, 14, 15, 18, 19, 77 Trams, A,, 197, 198, 199, 246 Trams, E. G., 29, 80 Troosheikina, V. I., 340, 357 Truax, W., 337, 358 Tsen, C. C., 271,309 Tsugitn, A., 91, 116 Tung, T. C., 146, 149,160 Turner, C. J., 255, 301 Turner, J. F., 280, 288, 304 Trirnrr, I,. B., 338, 355 Tyson, T. L., 326,356
Villa-Trevino, S., 52, 81, 212, 213, 846 Villavicencio, M , 134, 160 Vinograd, J., 69, 81 Viollier, G., 146, 160 Voegtlin, C., 250, 273, 302, ,909 Vogt, M., 28, 77 von der Decken, A . , 5, 10, 14, 15, 18, 19, 57, 81, 289, 30% von Kreybig, T., 180, 190, 246 von Lacr, U., 194, 198, 200, 246, 246
W
Wackcr, W. E. C., 272,300 Waddington, C. H., 70, 81 Wagenknecht, C., 286, 307 Wagle, D. S., 141, 161 Wagle, S. R., 130, 141, 160, 161 Wagner, B. M., 315, 356 Watiba, A,, 88, 116 Waisman, H . A,, 146, 147, 149, 150, 151, 158, 160 Waldenstrom, J., 314, 321, 324, 328, 332, 333, 334, 350 U Waley, S. G., 255, 275, 290, 299, 300 Walpole, A. L., 230, 246 IJdcnfricnd, S., 166, 246 Walter, W., 235, 235 IJlmer, D. D., 272, 309 Wang, D. Y., 216, 245 Umbnrger, H. E., 297, 306 Wang, T., 286, 309 Upton, A. L., 258, 277, 309 Waravdekar, U. S., 151, 161 Utz, J. P., 324, 325, 356 Warburg, O., 118, 120, 161 V Ward, H. W. C., 337,359 Warner, J. R., 87,88,116 Vnerman, C., 320, 356 Warren, F. L., 256, 303 Vaerman, J.-P., 320, 356 Wartman, W. B., 41,78,80 Vagelos, P. R., 3, 81 Warwick, G. P., 71, SO, 266, 267, 307, 309 Vnleri, C. R., 262, 309 Warwick, 0. H., 340, 358 Vnlcriotc, I?. A., 353, 356 Wasserman, L. R., 328, 339, 357, ,955 Vallcc, I3. L., 85, 115, 272, 309 Vandcknr, M., 164, 207, 208, 220, 224, Watrous, R. M., 172, 246 243 Watson, J. D., 84, 87, 91, 112, 116 mi1 Heyninger, R., 252, 275, 307, 309 Watson, R. J., 321, 358 van R,i,i, N. J. W., 251, 271, 274, 280, 306 Wattenbcrg, L. W., 6, 7, 17, 20, 21, 22, 42, Van Sickle, R., 198, 240 45, 80, 81 van Vals, G. H., 134, 155 W d h , E. C., 277, 285, 305 Vnrcla, R. M., 46, 53, 77 Wcbb, J. I,., 277, 309 Vasiliev, J. M., 259, 3’08 Webb, M., 271, 304 Vasinn, 0. S.,340, 357 Wcbb, T. E., 109, 116 Vaughan, J. H., 322, 354 wI+)cyr, G . , 33, 51, 94, 116, 123, 124, 126, Veldstra, H., 269, 309 129, 130, 135, 138, 139, 140, 141, 142, Velez, M. E., 264, 303 144, 157, 159, 160, 161 Vennesland, B., 286, 309 Webster, G. C., 287, 299,309 Videbaeck, A., 335, 359
379
AUTHOR INDEX
Weder, C., 338, 559 Wcil, R., 28, 69, 81 Wcil-Malherbe, H., 131, 168 Weiler, E. C. W., 283, 246 Weiner, M. J., 335, 351, 357 Weinhouse, S.,120, 123, 131, 144, 158, 160, 161 Weinstein, I. B., 93,97, 111, I I5 Weintranh, S., 338, 369 Weisblum, B., 8.5, 110 Weisburger, E. K., 227, 246 Weisburger, J. H., 227, O.$G, 280, 300, 308 Weiscl, L. L., 264, 301 Weisfuse, L., 339, 356 Well, J., 343, $55 Wellers, G., 264, 309 Wells, R. D., 92, 115 Werne, J., 145, 159 Wernzr, H., 275, 287, 309 Westergaard, M., 201, 242 Wettstein, F. O., 87, 1lG Wheeler, G. P., 130, 161, 164, 246, 266, 267, 268, 308 Wheelwright, H. J., 171, 172, 242 White, A,, 256, 309 White, J., 256, SO0 Whitehead, G. B., 272, 500 Whiting, M. G., 164, 181, 184, 185, 190, 242, 244, 246 Whittington, R. M., 342, 343, 55s Wichern, H., 237, 244 Wick, A. N., 140, 15s Wiebeckc, B., 188, 238 Wiel, J., 336, 337, 342, 343, 344, 357 Wilclervanck, L. S., 312, 357 Willren, D. R., 254, 302 Willett, F. M., 343, 35G Willheim, R., 280, 308 Williams, J. N., 151, 160, 215, 2dG Williams, K., 267, YO1 M'illianis, R. T., 277, 279, 301 Williams, W. J., 146, 151, 161 U'illiams-Behinan, H. G., 134, 161 Wills, J. H., 172, 244 Wilson, C. B., 252, SO9 Wilson, H. E., 336, 337, 342, 343, 344,357 Wilson, I. B., 2, 79 Vv'ilson, L. P., 252,909 m'ilson, R. G., 72, 78 Wilson, W. I,., 295, 309 Winocour, E., 29, 81
Winzler, R. J., 265, 301 Wittmnnn, H. G., 91, 116 Wittmann-Liebold, B., 91, 110 Wogan, G. N., 71, 72, 78, YO Wolbach, S. B., 258, 909 Wolfson, X., 252, 309 Wolfson, S.,19, 76 U'ong, F., 338, 35s Wood, D. A., 338, 358 Wood, J . I,., 259, 260, 261, 300 Woodard, H. Q . , 145, 1Gl Wootlhouse, D. I,., 268, 269, 308 Woods, K. R., 320, 350 Woods, M. W., 145, 158 Woodward, G., 261, $05 U'ortliam, J. S., 72, 78 Wosilait, W. D., 3, 81 Wrba, H., 177, 187, 243 Wright, B. P., 338, 359 Wright, J. C., 338, 340, 969 Wright, 1,. T., 338, 350 Wrigley, F., 172, 246 Wu,C., 146, 149, 151, 161 Wu, R., 126, 127, 133, 134, 141, 142, 143, 157, 160, 161 Wulf-Lorentzm, G., 170, S4.g IYyndcr, E. L., 270, 909
Y l7:tmamolo, R. S., 289, 306, 300 Yaiiiane, T., 85, 97, 114, 116 Y n n , Y., 92, l l G Yanari, S., 288, 309 Yankofsky, S. A,, 62, 63, 81 k'anofeky, c., 85, 91, 99, 114, l l G , 297, 306 Tarmolinslry, M. B., 53, 81 Yielding, I<. I,., 4, S t Togo, H., 6, 17, 22, 46, 77 Yolin, n. S.,234, 243 Tokoyama, T., 146, 159 Yonmans, A. S.,236, 240 Yoiinians, G. P., 236, 240 Yoling, .E. M., 71, 75, 80 Young, T,. C . T., 277, SOD
2 Zavhau, H. G., 87, 114, 116 Zagcl, M., 177, 187, 290 Zak, 17. G., 176, 185, 187, 191, 223, 246 Zamecnik, P. C., 84, 85, 97, 115, 116
380 Zamir, A,, 85, 114 Zetterberg, G., 199, 200, 242, 246 Zicha, B., 275, 287, 509 Zil, H., 275, 303
AUTHOR INDEX
Zimmerman, I?. K., 194, 195, 196, 197, 198, 199, 200, 201, 245, 245, 24G Zinncman, H. H., 325, 556, 569 Zubrod, C. G., 343, 558
SUBJECT INDEX RNA ribosomal system from, 101A 109 Acenaphthene, effect on enzymes, 7, 9 Ascorbic acid, metabolism, hydrocarbon Acctanilide, effect on enzymes, 7 cffcct on, 50-51 2-Acetylaminofluorene, as enzyme snbragine-Iretoarid aiiiinotrniirfcrase, in st.rate, 7-8, 17-18, 19-21 neoplastic tissue, 149 as MC inhibitor, 40 Actinomycin D, in carcinogcncsis stud- Aspnrtate aminotransferase, in nc,oplnst ic tissuc, 150, 152 ies, 73-74 in nroAdrcnal necrosis, hydrocarbon-inducc,d .Isll:wtate ca~I~amoyltransferaxc~, Illastic tissue, 150, 152 protection against, 43-48 2-.4znfl1ioranthcne, effect o n enzymes, 9 ..llaninc-n-kctagliitarate aminotrnnsfcr4-Azafluorcnc, effect on cnzyinm, ’3 ase, in neoplastic tissue, 146, 148 Alkaline phosphatasc, in neoplastic tis- Azo reduction, cwrinogm effect on, 5 , 10, 15-16 sup, 145 Alkylating agents, in myeloma therapy, dzoetlianc, as carcinogim, 181 -lzulcnc, cffect on enzymes, 9 339-340 SH groups and, 266-268 B illlosan, interaction with SH groups, 278 Bcnntlryl, as enzyme substrate, 9 A.niino acid metabolism enzymes, in 1,2-Bcnznnthrurenc, effect on enzymes, neoplastic tissue, 150 7, 9, 16, 22 ~ - B m i n oacid oxidase, in neoplastic tisBenzrnc, effect on enzymes, 8, 9 sue, 145 Benzo[clcinnoliiie, effect on enzymes, 9 p-Amino hippnrate synthetase, see GlyBcnzofluorence, cffect on enzymes, 9 cine acyltransferase Aniinonryl transfer rihonucleic acids, 3,4-Benzophenanthrene, cffcct on csnzpmes, 9, 23 transfer enzymes and, 99-101 l,Z-Bmzopyrcne, effect on enzymes, ‘3 w, see Dehydropepticlase 3,4-Benzpyrenc, effect on enzymcs, 6, 7, Aminoazo dye reductasc, carcinogen cf8, 9-10, 12, 14, 16, 21, 22 fect on, 15-16 inhibition of, 41 Aminopcptidase, in neoplastic tissue, 151 .4nalgesics, effect on enzymes, 23-26 C Anesthetic gases, effect on enzymes, 24 Cancer. (See also Neoplasms) 2’,l-Anthra-l,2-anthracene,effect on cnprophylaxis, enzyme induction in, 41eymcs, 6, 9 43 Snthracene, effect on enzymes, 6, 9, 22 Carboxylcsterase, in neoplastic tissne, Anticarcinogenesis, 260-262 151 Anticonvulsants, effect on cnzyrncs, 23Carcinogenesis, anti-, 260-262 26 gene action and, 66-76 Ant,iliistamines, cffect on enzymes, 23sulfhydryl group in, 247-309 26 Carcinogens, effect on ascorbic acid I\ntiinflarnmatory agents, cffect on ennictabolism, 50-51 zymes, 23-26 enzyme induction, gene action and, 1Llrginase, in neoplastic tissne, 150, 153 81 A rsrnic, in t c m c t ion with snlf Iiydryl growth effects of, 255-250 ~ ~ O U ~ I272-273 S , nitroso compounds as, 163-246 roartion with SH groups, 259-277 Ascites tumor CC~IIP, aminoacyl transfer 381
382
SUBJECT INDEX
Cell division, sulfhydryl role in, 249-255 Chlorpromazine, as enzyme substrate, 10 Chlorzoxazone, effect on enzymes, 7 Chrysenc, effect on enzymes, 9 Cigarette smoke, SH groups and, 262 Coronene, effect on enzymes, 9 Cortisone, effect on enzyme induction, 33-36, 37 C-oxidation, carcinogen effect on, 5, 10 Cupferron, as mutagen, 195 Cycads, carcinogenicity, 164 Cycasin as carcinogen, 181 chemistry of, 167-168, 171 as public hazard, 235-236 as mutagen, 201 Cyclophosphamide, in myeloma therapy, 337, 339-346, 353 Cysteine, effect on cell division, 251 Cysteine desulphhydrase, in neoplastic tissue, 146
D Dehydropeptidase 11, in neoplastic tissue, 145 N-Demethylases, carcinogen effects on, 9, 10, 12-14, 30 N-Demethylation, carcinogen effects on, 5, 8-9 0-Demethylation, carcinogen effect on, 5, 10, 14-15 S-Demethylation, carcinogen effect on, 5, 10, 14-15 Diazomethane, as carcinogen, 181, 187 as mutagen, 201 Dibenzanthracenes, effect on enzymes, 6, 8, 16, 23 Dibenzopyrenes, effect on enzymes, 9 Diethylnitrosamine, biochemistry of, 217 as carcinogen, 174, 175, 176-177, 182, 183-185, 186, 187, 188, 191 as mutagen, 196197 reactions with cell constituents, 225 /3-Diketonase, see Fumarylacetoacetase 4-Dimethylaminoazobcnzene, as enzyme substrate, 10, 15 3,9-Dimethylanthanthrene, effect on enzymes, 6, 9 Dimethylbenzantliracencs, in adrenal necrosis, 4 3 4 8 effect on enzymes, 6, 7, 8, 9, 10-11, 14, 16, 17, 19, 21, 22, 36-37
Dimethylbenzophenanthrcnrs, effect on enzymes, 9 6’,7’-Dimethylnaphtho (2’,3’:3-4) pprrnc, effect on enzymes, 9 Dimethylnitrosamine, acute toxicity, 171-172, 173 biochemistry of, 213-217, 218-219 as carcinogen, 164, 175, 176, 184-185, 191 metabolism of, 202-206, 207-209 as mutagen, 196 in protein synthesis, 209-213 reactions with cell constituents, 220224, 226-227 N,N‘-Dinitroso-N,N‘-dimeth ylcthylcncdiamine, as carcinogen, 180 N,N’-Dinitrosopiperazine, as carcinogen, 179 as mutagen, 198 Dipeptide hydrolascs, in neoplastic tissue, 151 Diphenylnitrosamine, as mutagen, 195 DNA in carcinogcnrsis, 68-69, 76 “DOPA oxidase,” ,see Tyrosinase Drugs, action of, hydrocarbon effect on, 48-50 Durene, effect on enzymcs, 9
E Elaiomycin, toxirity of, 236 Enzyme induction, in canwr prophylaxis, 41-43 factors affecting, 2-5 gene action, carcinogcns and, 1-81 oncogenie virus effcrt on, 28-20 polycyclic hydrocarbon cffrct on, 5-23 inhibit,ors of, 52-56 mechanism, 51-66 in rat hcpatoma, 29-36 dietary factors, 32-36 phenobarbital effects on, 29-32 Enzymrs, “allostcric inhibition,” 3 carcinogcn effects on, 1-81 control mechanisms, 2 “isoteric inhibition,” 2-3 inicrosomal drug-metabolizing, nature of, 26-28 of neoplastic tissue, 117-161 p a t t x n of, 143-154 Estrogen, effect on enzymes, 7
SUBJECT INDE X
383
Glutiithione, effect on crll division, 251255 metabolism of, hormones in, 288-289 ovrmynthesis, in carcinogenesis, 298299 synthesis of, 287-288 F tems, inhil)ition of, 286 FDP phosphatusc, in ncoplastic t i w i r , Glutathione reductase system, in car137, 138 cinogcncsis, 285-286 7-Fluoro-2-acctylainmofluorene, as MC Glycr~ralticliydc~~I~osp~iatP tlrhydrogenasr, inhibitor, 40 in neoplastic tissue, 126, 127, 129Fluoranthrenc, effect on enzymcq, 8, 0 134, 136, 142 Fluorene acrnophtlienr, (affect on rnG1ycerolpliosphate dchydrogcnasr, in zymes, 8 neoplastic tissue, 130, 131, 133, 134, Fluorene phrnanthrene, effect on cn146 zymes, 9 Glycine aoyltransfrrasc, in nrolilastic tisFrirnd leuhernia virus, effect on e m \ in(% sue, 146 induction, 29 Glycogen formation, rnzgnics of, in nroFructose-1,6-dipliosphatnsr, in neoplastic plastic tissue, 137-143, 149 tissue, 139, 140 Glgcogrn sgnthrtase, in neoplastic tisFructose dil)liosphate altlolas~,in nrosue, 140, 141-142 plastic tissw, 126, 127, 130, 131 Glycolytic enzymes, in neoplastic tissue, F~imnrylacctoacrtasc, in neoplitstlc tls125-134, 150 sue, 147 “GOT,” see Aspartate aminotransferasr Greenstein’s generalization on ncoplastic tissue, 120-122, 132, 155 Gene action, carcinogencsis and, 66-7G enzyme i n d u d o n , carcinogens and, 1H 81 Gluconrogenesis, enzymes, in neoplastic Hepatic enzymes, carcinogen rffects on, 12-23 tissue, 137-143, 145, 146 Glucose-6-phosphatase, in neoplastic tis- Hcpatocarcinogenic substances, SH groups and, 264-266 sue, 137-140 Glucose-6-phosphate dehydrogenasc, car- Hcpatoma, enzyme induction in, 29-36 Hexobarbital, effect on rnzymrs, 7 cinogen effection, 11 in neoplastic t,issue, 134-137, 150 Hexokinase, in neoplastic tissue, 126, 127, Glucose-6-phosphate isomerase, in nco128, 130, 131 plastic tissue, 126, 127 Histidase, in neoplastic tissue, 146 Glucuronide formation, carcinogen effect Hormones, effect on enzymes, 23-26 on, 10 S H metabolism and, 263-264 Glutamate dehydrogenase, in neoplastic Hydralases, in neoplastic tissue, 151 tissue, 150 Hydroxylation, carcinogen effect on, 5, Glutaminase, in neoplast,ic tissue, 151 6 4 , 1618 Glutamine-kctoacid aminotransferase, in p-Hydroxyphenylpyruvate hydroxylmr, neoplast,ic t,issue, 149 in neoplastic tissue, 146 Glritamine synthetase, in neoplastic tis5-Hydroxytryptophan decarboxylase, in sue, 146 neoplastic tissue, 147 Gliitnminyl-sRNA synthetase, in neoHypnot,ics, effects on rnzymes, 23-26 plastic t.issue, 149 Hypoglycemic agent,s, effect on enzymes, o-Glut,amylt,ransferase, in neoplastic tis23-26 sue, 151 Ethionine, carcinogen action, 234 in rnzyme-induction studies, 52-53 Ethyl diazoarc>tate, as carcinogen, 181 as mutagen, 201
384
SUBJECT INDEX
I Immunoglobulins, normal, 315-318 pathological, 318-321 Insecticides, rffcct on enzymes, 23-26 Isocitric dchydrogcnasr, carcinogcn effrct on. 11
1 Lachrymators, interaction a i t h SH groups, 278 Lactate dehydrogcnasr, in neopla’tic tissue, 126, 127, 134 Lactic di~hjdrogcnasc,carcinogen effect on, 11 Lactones, intrraction with SH groups, 269-270 Liver microsomal rnz) iiirs, drug and insccticidc effects on, 23-26
M Malcic hydrazidc, interaction with SH groups, 279-280 Malrimidcs, interaction with SH groups, 278-279 Malic dehydrogcnasr, cni cinogeii effcct on, 11 Malignant cells, protein synthcws in, 83116 Melphalan, in rnycloma therapy, 327, 333-335, 339-354 Mcnadionc ieductase, carcinogen effrct on, 10-11 Mcpcridinc, as cnzjme substrate, 9, 14 Metals, intrraction with SH groups, 270272 4-Mrthoxyacctanilide, as cnzyme substrate, 10, 14-15 N-Methylaniline, as enzyme subbtiatc, 9, 14 Mcthj lazoxyinrthanol, as carcinogrn, 18I Methylazoxymrtliario1 glycosides, rhcmistry of, 167-168, 171 Ptlcth\ Ibcnaanthraccncs, effect on enzymes, 6, 8, 9, 16 2-Mi~tliyll~enzo[clphc~nanttirene, effwt on enzymrs, 16 3’-Mvth\ I-4-dinirthylaminoazobenzcne, as carcinogen, 37-39
3-Methyl cholanthrcnc, effect on enzymes, 6, 7-8, 10, 13-15, 17, 19-23, 29-30, 37-41 effect, on nuclcar RNA, 63 20-Mcthylcliolanthrcne, effect on enzymes, 8 Mctliyldihmzopyrcnes, effect on cnzgmrs, 7, 9 3-Mrthylnionomcthylamino azobcnzcnc, as cnzymc sithstrat,e, 8-9, 13, 14, 21 6’-MethylnaphtJho (2’,3’:3,4) pyrene picrnc, rfffxct on enzymes, 9 N-Mctlryliiitro~nniides, as niutagcnn, 194 hJ-Mcthyl-N-ni troso-N‘-nitrogiianidinr, as cnrcinogcn, 180, 192 as mutagen, 193, 194, 197 Microsomal electron transport cnzymrs, carcinogrn effect on, 18-19 Monoamine oxitiasr, in neoplastic tissuc, 149 Monoinethyl, 4-aminoantipyrine, as enzyme substrate, 9, 13, 14 Muscle relaxants, cffcct, on rnzymcs, 2326 Myeloma, plasma cell, seP Plasma cell myeloma
N NAD cytochronic bn rcductase, carcinogen effrrt on, 10 NADH cytochrome c reductase, cmcinogrn effect on, 10 NADH diapliorase, carcinogen effect on, 10 NADPH cytoclirome b;, carcinogcn effrct on, 10 NADPH cli torhrome c reductase, 2628 carcinogen effrct on, 10 NADPII diaphorase, carcinogrn effect on, 10 Naplitliac~rnc, cffcwt on rnzymrs, 6, 8 Naphthnlrnr, effcct on enzymes, 6, 7, 9 9-( I-Naphthyl) -1,2-henzanthracrne, effert on cnzymes, 9 10-(2-Xaplit hj 1) -1,2-benzan thracrnr, rffcct on rnzymrs, 9 Nrop!:isms, enzymrs of, 117-161 disprnsahlr and pcrsistrnt, 144-152 gluconeogencsis, 137-143, 145, 146 glycolytic, 125-134, 150
SUBJECT INDEX
nicasurcnient, 123-124 pattern, 143-154 pentose pathway, 134-137 Grccnstein’s generalization on, 120122, 132, 155 Warburg’s generalization on, 118-120, 132, 155 Xicotinamirk adenine dinucleotidc, in neoplastic tissue, 133-134 4-Sitroquinoline N-oxide, interaction with SH groups, 268-269 Nitrosamines, interaction with SH groups, 268 .Nitroso carcinogens, 163-246 acritc toxicity, 171-174 pathology, 174 alky1:ition of nucleic acids Iiy, 233-234 nnalytical methods, 168-171 biochrmical effects, 209-219 on protein synthesis, 209-213 crllular targets of, 231-233 c~licmistryof, 165-171 tlccxoinposition products, 228-231 isotopically labeled, 169 in foods, 236 mcchnnisms of action, 227-234, 233234 nictabolism of, 202-200 in vitro, 207-209 in vivo, 202-207 as iiiiilagcns, 193-201, 233 ncoplnstic chnngrs by, 175-191 in alimenl ary cmal, 188-190 in bladder, 186 in Ititlntby, 185-186 in liver, 175, 183-185 in lungs, 187-188 in nervous system, 190 in nose, 186 in skin, 190 oxiclntive decomposition, 166-167 photosensitivity, 165-166 1)rcp:tration of, 168 public I i c d t l i aspects, 234-238 rcnctions, with cell cwnstiturnts. 220-227 with sulfhydryl compouncls, 167 as t,cr:it,ogcns, 190-101 in t,ob:icm, 237-238 S-Sitrosoallylmcthylilmine, as carcinogen, 178
385
N-Nitrosoanabasine, as carcinogen, 179 N-Nitrosobenzylmcthylaniinc, as carcinogen, 178 as mutagen, 197 AT-Nitroso-n-butylethylamine, as carcinogen, 178 N-Nitrosobutyl-4-hydroxybut?.lainine, as rnrcinogen, 178 N-Nitroso-n-butylmethylamine, as carcinogen, 178 rcaction with cell constit.uents, 225 N-Nitrosodi-,1-amyIaminc, its carcinogen, 178 N-D;itro~odi-n-butylninine, as carcinogen, 177, 192 metabolism, 206-207 N-~itrosocli-~~-pro~~ylamine, as carcinogrn, 177 ~V-Nitrosoetli~l-2-li~d~o~yctliylatiii~i1~, :is cnrcinogrn, 178 1V-Nitrosoc.tIiylisopropylamiiic, as carcinogcn, 178 S-Sitrosoctliylurca, :is inritagcn, 193 S-Nitroro-~v-ctliylLii(,tliniii~,:is mrcinogcn, 181, 190 as mutagen, 195, 200 A~.-Sitrosoethylviiiyl~iiiiii~~, :is carcinogen, 178 N-Sitrosoiiictliylplrciiylamine, as carcinogen, 178 XJ miitagcn, 197 N-Nit,roso-N-mctliylurc~a, as carcinogen, 180, 189-190, 191 :is niutagcn, 193, 194, 200 rcwtions with cell constitrirnts, 22.5 l\T-~itroso-N-niethylurothane,acute toxicity, 172-173 :is carcinogen, 180, 189, 268 :is mutagcn, 194, 198-199 ~~J-NitrosomrthyIrinyl:~miiic, as carcinogen, 178 :is mutngen, 197 S-Sitrosomorpliolinc, biochemistry of, 219 its carcinogen, 179, 183, 187 ns mutagen, 197 iv-D;itrosopipeiidine, as cnrcinogrn, 179, 188 ns mutagcn, 197 S-Sitrososarcosine, as carcinogen, 179
386
SUBJECT INDEX
N-Nitrososarcosinc cstliyl estrr, as carcinogen, 179 p-Nitrotolurnc, as enzyme substrate, 10
0 Oncogmic viruscs, effect on enzyme induction, 28-29 Ornithine earbamoyltransferase, in neoplastic tissue, 149
P P-450 pigment, in liver microsomcs, 2728 Prnt,aeene, effect on enzymes, 9 Pent,osc pathway enzymes, in neoplastic tissue, 134-137 Pepsin, in neoplastic tissue, 145 Perylcne, effect on enzymes, 7, 9 Phenanthrenc, effect on enzymes, 6, 8, 22 Phenobarbit.al, cffcct on enzyme induction, 29-32, 56-57 Phenothiazincs, rffcct on benzpyrene liydroxylase, 4445 Plienylalanine, in protein synthesis studies, 60-63 Plienylalanine 4-hydroxylase, in neoplastic tissue, 146 Phenylalanine-pyruvate aminotransferase, in neoplastic tissue, 149 lO-Phcnyl-1,2-bcnzanthraccne, effect on enzymes, 9 Plienylbutazonc, effect on enzymes, 7 PhenyImcthyInitrosaniine, as mutagen, 195 Pliosphoenolpyruvate carbonylkinase, in neoplastic tissue, 139 Phosphofructokinase, in neoplastic tissue, 125, 126, 127, 128, 129, 130 Pltosphog.lucomutasct, in ncoylastic tissue, 140, 141, 142, 143, 150 Phosphogluconate dehydrogenasr, in ncoplaslic tissue, 134 Pliospltohexose isomerase, in neoplastic tissue, 125 Pliosphoglyccraldchyde dehydrogenasc, in neoplastic tissue, 125 3-Phosphoglycerate kinasc, in neoplastic lissue, 126, 127 3-Phosphoglyccrate phosphomutase, in neoplastic tissue, 126, 127
~’lios~)liopyruvaterarboxylasr, in nroplastic tissue, 137, 138 Phosphopyruvate hydratasc, in ncoplastic tissue, 126, 127 Phosphorylasr, in neoplastic tissuc, 140, 141, 142 Plasma cell myeloma, manifestations of, 330331 treatment of, 311-359 with antineoplastic agents, 337-353 Plasma cell neoplasms, c1inic:rl patterns in, 349 course of disease, 321-329 etiology of, 312-315 immunoglobulins of, 315-321 therapy, 325-353 Polycyclic hydrocarbons, effects, on carcinogcnesis, 3 7 4 3 Polycyclic hydrocarbons, effects, on carcinogcnesis, 37-43 on drug action, 48-50 on growth, 256-258 on protein synthesis, 56-63 enzyme induction and, 5-23 interaction with SH groups, 259-262 pharmacological and physiological significance, 36-51 Polymer cancers, SH groups and, 273 Polyoma virus, effect on enzyme induction, 28-29 Polyuridylic acid, in protein-synthesis studies, 59-63 “Proline oxidase,” in neoplastic tissue, 149 Protein synthesis, activating mechanisms, 8547 genetic code in, 91-92 in tumor cells, 110-111 in malignant cells, 83-116 nitrosaminc effect on, 209-213 polycyclic hydrocarbon effect on, 5663 rclcasc mechanisms, 92 ribosomal-polysomal complex in, 87-88 in tumor cells, 109 transfer reaction in, 88-91 LRNA in, 88-91 Puromycin, in enzyme-induction studies, 53-55 in carcinogenesis studirs, 73-74 Pyranthrcnc, cffcct on enzymes, 7, 9
387
SUBJECT INDEX
in norm:il w I I division and growtli, 249-250
T
Q Quinolinc, cffc.ct on en zy ~~ic’ s, 7 Quinonvs, iIilcraction with SH groups, 289-270
R Rutliation, SII coiii~Jorintlcffcbcts in, 274276 Iimnin, in neoplastic tissue, 145 I i e t m e , effect on enzymes, 9 RNA, in isolated nuclei, 64 mcwrnger, t e m p l a k stal)ility, in tumors, 34-35 niiclmr, nietabolism t l i c t l ~ y l r l i o l ~ ~ ~ i t(’ffcct l l ~ e ~011, ~ e 63-66 I
S SfLdativc’s, cffrcls on c.nzymrs, 23-26 SH I,cagetits, 277-278 Sulfonaiiiides, effect on enzymes, 23-26 Snlfoside formation, carcinogcm cffrLct on, 10 Siilfliydryl groiip, in c:ircinogenesis, 247300 1iyl)otllcsis of, 296-298 in liver, 282-285 in skin, 280-281 coll-division stimulation by, 250-252 intcrxction with carcinogens, 250-277 intrmction with vnrioiis siihstnnc*r~s, 277-280 metabolic systems of, and carcinogrnrsis, 285-289
Twt,ostc~ronr,c.fCc7c.t on onzyiiit’s, 7 Tlirc~oninedcliydrat:tsc, i n nroplastic: tissue, 147 Tobncco, nitrosamincs in, 237-238 r , , 1 1 h- H cytoclirorne c reductasc, natnrc of, 27-28 Triinc~t,h~lnitrosour~a, acute toxicity, 173 ;is carcinogrn, 180, 190 in neoplastic T r i c i s c ~ ~ ~ l i o s ~isomerasc, ~l~atc tissue, 126, 127 r . , I riplic~nylenc,cffcct on enzymes, 9 t I t S A , in protein synthesis, 88-91 Trylitoplian pyrrolase, in neoplastic tissue, 146, 156-157 ‘I’iimors, see Neoplasms Tyrosinnsc, in neoplastic t.issue, 145 Tylosine-cu-l
U IJDP g1uc:uronyl trausfrrzsc, carcinoycn cffect on, 19 UDPG glncuronyl transfcrasc, carcinogen effcct on, 19 Ultraviolet radiation, effect on SH groups, 277 IJrrtlr;ine, in myelo.ma theraliy, 338-339
V Vrsic:;uitr, interaction w i t h SH groups, 278 Violanthwne, cffcct on enzymes, 7, 9 Viruses, oncogenic, see Oncogcnic viruses
W Warburg’s generalization on neolilastic t,issrie, 118-120, 132, 155
Z Zos;izol:iminc, cffcct on enzymes, 6-7 inctaliolisrn, Iiydrocarbon effect on, 48-50
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