Advances in Cancer Research, Volume 8

ADVANCES IN CANCER RESEARCH VOLUME 8

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ADVANCES IN CANCER RESEARCH Edited by

ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital, London, England

SIDNEY WE INH'OUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania

Volume 8

@ ACADEMIC PRESS INC.

1964

NEW YORK AND LONDON

COPYRIQHT 0 1964 BY

ACADEMIC PRESS INC. All Rights Reserved N o part oj this book m a y be reproduced i n any form b y photostat, microfilm, or a n y other means, without written permission from the publishers. ACADEMIC PRESS

INC.

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Library of Congress Catalog Card Number 52-13360 United K i n g d o m Edition Published by ACADEMIC PRESS INC.(LONDON) LTD. BERKELEY SQUARE HOUSE, LONDON w.1

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 8 Numbers in parentheses refer to the page on which the author’s contributions begins.

HARRISBUSCH(42), Department of Pharmacology, Baylor University College of Medicine, Houston, Texas DIETRICH HOFFMANN (250), Division of Environmental Carcznogenesis, Sloan-Kettering Institute for Cancer Research, N e w York, f l e w York A. F. HOWATSON ( l ) ,Department of Medical Biophysics, University of Toronto, and Division of Biological Research, Ontario Cancer Institute, Toronto, Canada

M. J. KOPAC(122) , All-University Department of Biology, Graduate School of Arts and Science, N e w York University, N e w York, N e w York

H. F. KRAYBILL (1911 )* National Cancer Institute, Bethesda, Maryland GLADYS M. MATEYKO (122) All-University, Department of Biology, Graduate School of Arts and Science, N e w York University, N e w York, N e w York M. B. SHIMBIN(191),t National Cancer Institute, Bethesda, Maryland

WILLIAM.J. STEELE(42), Department of Pharmacology, Baylor University College of Medicine, Houston, Texas

ERNESTI,. WYNDER(250), Division of Environm(enta1 Carcinogenesis, Sloan-Kettering Institute for Cancer Research, New York, N e w York

* Present

address: Bureau of Environmental Health, U S . Public Health Service, Department of Health, Education and Welfare, Washington, D.C. t Present address: Temple University School of Medicine, Fels Research Institute, Philadelphia, Pennsylvania V

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CONTENTS CONTRIBUTORS TO VOLUME 8

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V

The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General

A . F. HOWATSON I. Introduction . . . . . . . . . . . . . 11. Modern Concepts of Virus . . . . . . . . . I11. Tumor Viruses: Structure and Mode of Development IV. Classification Scheme . . . . . . . . . . . V . General Discussion . . . . . . . . . . . References . . . . . . . . . . . . . . .

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1 3 14 28 32 36

Nuclear Proteins of Neoplastic Cells

HARRISBUSCHA N D WILLIAMJ . STEELE

I . Introduction . . . . . . . . . . I1. Isolation of Nuclei . . . . . . . . 111. Isolation of Nuclear Components . . . . IV . Enzymes of the Nucleus . . . . . . . V. The Acidic Nuclear Proteins . . . . . VI . Nuclear Globulins . . . . . . . . . VII . The Nuclear Ribonucleoproteins . . . . VIII . Acidic Proteins of the Deoxyribonucleoprotein IX . The Histones . . . . . . . . . . X . Discussion . . . . . . . . . . . . References . . . . . . . . . . . .

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Complex .

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112

Nucleolar Chromosomes: Structures. Interactions. and Perspectives

M . J . KOPAC AND GLADYS M . MATEYKO I . Introduction . . . . . . . . . . . . . . . . . . . I1. Nucleolar Bodies . . . . . . . . . . . . . . . . . . I11. Nucleolar Chromosomes . . . . . . . . . . . . . . . . IV . Experimental Studies on Nucleoli . . . . . . . . . . . . . V . Lampbrush Chromosomes . . . . . . . . . . . . . . . VI . Polytene Chromosomes . . . . . . . . . . . . . . . . VII . Experimental Modification of Puffing Patterns in Salivary Gland Chromosomes . . . . . . . . . . . . . VIII . Perspectives Involving Nucleolar and Non-Nucleolar Chromosomes . . References . . . . . . . . . . . . . . . . . . . . vii

122 123 132 143 150

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CONTENTS

Vlll

Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites

H . F. KRAYBILL AND M . B . SHIMKIN I . Introduction . . . . . . . . . . . . . I1. Processed Rations and Trout Hepatoma . . . . I11. Role of Fungal Metabolites in Diet and Cancer . . IV . General Discussion . . . . . . . . . . . . References . . . . . . . . . . . . . . .

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191 194 208 240 246

Experimental Tobacco Carcinogenesis

ERNESTL . WYNDER A N D DIETRICH HOFFMANN I . Historical Aspects . . . . . . . . . . . . . . . . . . 250 I1. Objective of Laboratory Studies . . . . . . . . . . . . . 251 I11. Some Characteristics of Tobacco and Tobacco Smoke . . . . . . . 252 IV . Biological Tests for Tumorigenic Activity . . . . . . . . . . 269 V. Certain Constituents of Tobacco Products . . . . . . . . . . 308 VI . Reduction of Tumorigenic Activity . . . . . . . . . . . . 372 VII . Interpretation of Experimental Findings . . . . . . . . . . . 387 397 VIII . Postscript . . . . . . . . . . . . . . . . . . . . 435 References . . . . . . . . . . . . . . . . . . . .

AUTHORINDEX .

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495 475

THE STRUCTURE OF TUMOR VIRUSES AND ITS BEARING ON THEIR RELATION TO VIRUSES IN GENERAL A

. F. Howatson

Department of Medical Biophysics. University of Toronto. and Division of Biological Research. Ontario Cancer Institute. Toronto. Canada

I . Introduction . . . . . . . . . . I1. Modern Concepts of Virus . . . . . . . A. Structural Elements: the Virion . . . . B. The Virion: Symmetry Properties . . . . C . Virions with Icosahedral Symmetry . . . . D . Virions with Helical Symmetry . . . . . E. Complex Virions . . . . . . . . 111. Tumor Viruses: Structure and Mode of Development . . . . . . A . General Considerations B . Polyoma Virus . . . . . . . . C. Rabbit Papilloma (Sliope) Virus . . . . D . Human Papilloma (Wart) Virus . . . . E . Simian Virus 40 (Vacuolating Virus) . . . F . Luckk Kidney Tumor Virus . . . . . . G . Adenovirus Type 12 . . . . . . . H . Mammary Tumor Virus . . . . . . I . Mouse Leukemia Viruses . . . . . . J . Avian Sarcoma-Leukosis Viruses . . . . K . Poxviruses . . . . . . . . . . IV. Classification Scheme . . . . . . . . A . Basis for Classification . . . . . . . B. Place of Tumor Viruses . . . . . . V. General Discussion . . . . . . . . Acknowledgments . . . . . . . . References . . . . . . . . . . I

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36

Introduction

'

It has long been known that some viruses that produce the acute infective type of disease commonly associated with such agents are also capable. in certain circumstances. of stimulating abnormal. though usually temporary. cell proliferation (Rivers. 1928) . What may be regarded as the converse of this. the production of necrotic lesions by a tumorinducing virus. has also been recognized for many years . It was shown by Duran-Reynals (1940) t h a t Rous sarcoma virus when injected intravenously into very young chicks does not produce tumors but results in a fatal hemorrhagic type of disease . It is only comparatively recently. however. that the importance of the phenomenon of two radically differ1

2

A. F. HOWATSON

ent types of cell response to a single viral species has become widely appreciated and subjected to intensive investigation. Interest was aroused to a large extent by the discovery of polyoma virus (Gross, 1953; Stewart, 1955; Stewart e t al., 1957, 1958) which exhibits very clearly the two kinds of virus-cell interaction both in infected animals (Ham e t al., 1960; McCulloch e t al., 1960) and in cells growing in vitro (Dawe and Law, 1959; Vogt and Dulbecco, 1960; Sachs and Medina, 1961). I n the latter system, complicating factors such as the immunological response of the host are eliminated, and the phenomena can be investigated by the quantitative methods that have been developed for the study of viruses in general (Stoker and Abel, 1962). Recently, interest in this subject has been further stimulated by the discovery that two viruses, simian vacuolating virus SV40 and human adenovirus type 12, that were previously regarded as typical viruses of the classical type causing cytolytic changes in cells, can induce tumors in hamsters (Eddy e t al., 1961, 1962; Trentin e t al., 1962). The fact that several viruses have been found to act in some circumstances as ordinary cytopathogenic agents and in other circumstances as tumor-inducing agents raises the question whether tumor viruses are a class of viruses basically different from other viruses or whether the ability under appropriate conditions to induce neoplastic changes in cells is a common property among viruses. The answer to this question will require more information about the biological potentialities of viruses and a better understanding of the mechanisms whereby they induce neoplastic changes. However, even with the information at present available, some light may be thrown on the question by considering the basic properties of known tumor viruses in relation to those of viruses in general. A comparison of this sort is greatly facilitated by separating viruses into families with properties in common. This brings up the subject of the classification of viruses. Viruses can be classified in many ways. I n the past it has been customary to rely to a large extent on the effects of viruses on their hosts, such as disease symptoms, host range, and cytological changes in infected cells. These criteria, however, are unsatisfactory for classification purposes since they are subject to much variation. It has already been noted, for example, that the same virus can induce different types of disease and have different effects on cells. I n recent years the application of new biophysical and biochemical techniques to the study of viruses has greatly increased knowledge of the physicochemical properties of the particles themselves and has led to increasing use of these properties as a basis of classification (Horne and Wildy, 1961; Andrewes e t at., 1961; Howatson, 1962b; Lwoff e t al., 196213). Presumably the ultimate ob-

STRUCTURE OF TUMOR VIRUSES

3

jective in characterizing any virus would be to determine the particular sequence of the bases in the viral nucleic acid, as this is the form in which all the information that determines the hereditary properties of the virus is believed to exist. This objective is not yet in sight, although recent progress in determining such properties of viral nucleic acid as molecular weight and base composition suggests that data of this sort may soon be used in characterizing viruses (Wildy, 1962). However, a t present more is known about the constitution of viruses a t a grosser level, the level of the organization of the macromolecules that form the framework of the particle. The study of the symmetry properties of the structural units of viruses has been greatly advanced in recent years by the development of new techniques for examining viruses in the electron microscope (Valentine and Horne, 1962). To a large extent recent attempts at virus classification have stemmed from this new knowledge of viral architecture. It is not the purpose of this article to discuss in detail the numerous problems in viral classification that are currently under consideration. However, the main classes into which viruses can be divided on the basis of structural symmetry and other physicochemical properties now seen1 to be fairly well defined and one might expect a study of the distribution of viruses among the classes to be of value in suggesting similarities and relationships that might not otherwise be perceived. I n particular i t will be of interest to examine the place that known tumor viruses occupy among the various families of viruses. The plan of this article is, first, to review briefly the basic properties of viruses in general, stressing particularly the structural features that have been revealed recently by electron microscopy. For fuller discussions of the present state of knowledge of virus structure the reader is referred to articles by Horne and Wildy (1961), Wildy and Watson (1962), and Caspar and Klug (1962). Then, the structure of known tumor viruses, insofar as i t has been elucidated a t the time of writing, will be discussed in more detail. Attention will also be paid to the manner of their assembly in infected cells. Finally, there is a discussion of the conclusions regarding the nature of tumor viruses that can be drawn from such a survey. II. Modern Concepts of Virus

A. STRUCTURAL ELEMENTS: THE VIRION Viruses consist essentially of an organized combination of nucleic acid and protein. The nucleic acid of any viral species is of a single type, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) ; the protein may consist of molecules of one or more different kinds. Larger viruses

4

A. F. HOWATSON

may contain additional materials (e.g., carbohydrate, lipid). The special type of combination of protein and nucleic acid that is characteristic of viruses has been discussed by Lwoff et al. (1959, 1962a,b), and the terminology that they introduced, with some modifications, is now generally accepted (see Caspar et al., 1962). The smaller viruses consist of a core containing the nucleic acid surrounded by a shell of protein, the capsid, the combination being termed nucleocapsid. This is essentially a loosc combination in which one element (the nucleic acid) is enclosed in a cage formed by the other (the protein) in a manner similar to that of the clathrate type of compounds (Powell, 1948). A combination of this type, depending as i t does on the goodness of fit of the elements, may confer a high degree of specificity on the arrangement. The capsid is composed of an ordered array or surface crystal of capsomeres which are the units observed in electron micrographs. These in turn consist of clusters of smaller elements, structural units, which are probably protein macromolecules. These remarks apply in the main to the smaller viruses which are of relatively simple construction. Many of the larger viruses are more complex, having additional structures such as envelopes and specialized appendages. The primitive mode of reproduction of virus particles involves loss of the structural integrity of the particles during the intracellular phase. In considering the structure of viruses we shall be concerned in the main with particles in the morphologically complete, potentially infectious, but biologically inert form in which they normally exist outside the cell. The virus particle in this form has been called the virion. The terms virus and virus particle will, however, sometimes be used where there is no ambiguity.

B. THEVIRION: SYMMETRY PROPERTIES The first indication that viruses possess symmetrically arranged substructures came from X-ray diffraction studies of viruses that could be obtained in crystalline or paracrystalline form. The pioneer studies of Bernal and Fankuchen (1941) on tobacco mosaic virus were followed by those of Watson (1954), Franklin (1955), Caspar (1956), and others on this and other plant viruses. This work has been reviewed a t length by Klug and Caspar (1960). Evidence for the existence of viral subunits was also obtained from chemical analysis of viral components (Harris and Knight, 1955). The significance of these results was discussed by Crick and Watson (1956, 1957). They pointed out that the amount of nucleic acid contained in the smaller viruses is insufficient to code the information necessary for the synthesis of more than a limited number of different types of protein molecule of average size. They concluded that the pro-

STRUCTURE OF TUMOR VIRUSES

5

tein coats of the smaller viruses probably consist of many identical subunits arranged in regular fashion, and discussed the types of symmetry that would be expected. Their predictions have since received ample experimental support from further X-ray diffraction studies and especially from electron microscopy. The contributions from the latter source are quite recent. Until five or six years ago the electron microscope had given little information about viral symmetry, its main application being in the investigation of the shape, size, and intracellular location of the particles (Williams, 1953, 1957; Bang, 1959). The advances in the past few years have been due in large measure to the introduction of new methods of enhancing contrast, especially the negative staining method. This was first used in electron microscopy by Hall (1955) and by Huxley (1957), but its exploitation in the study of viral structure was due largely to Home and his colleagues (Brenner and Horne, 1959). The method has revealed many features of viral architecture not detectable in any other way, The advantages and limitations of the technique are discussed by Valentine and Horne (1962). One of the first viruses to be studied by negative staining was adenovirus (Horne e t al., 1959a). The choice was a fortunate one for this virus shows more strikingly than any other that the surface is composed of subunits disposed in a very regular manner. It is now clear that the subunits or capsomeres are not identical with the structure units envisaged by Crick and Watson but are most likely clusters of such units. However, the basic symmetry is of a type discussed by these authors. This is a form of cubic symmetry, icosahedral symmetry, and i t is characteristic of a whole series of viruses; these will now be considered.

C. VIRIONSWITH ICOSAHEDRAL SYMMETRY The different ways in which a viral capsid, considered as an approximately spherical (polyhedral) shell composed of identical structure units, can be constructed has been considered in detail by Caspar and Klug (1962). The problem can be treated in a simpler, though Iess rigorous, way by taking as the construction units the capsomeres revealed by electron microscopy. From electron micrographs it appears that these are in the form of hollow hexagonal or pentagonal prisms which project from the surface of the virion. They probably consist of groups of five or six of the structure units considered by Caspar and Klug. Capsomeres appear to be stable physical entities in that they preserve their structural integrity when the capsid is disrupted (Wildy e t al., 1960a; Breedis e t al., 1962). If the height of the capsomere prisms is ignored, the problem reduces to that of finding the different ways in which hexagons and pentagons can be arranged in a symmetrical manner to cover R closed

6

A.

F. HOWATSON

surface. This was considered first as a problem in pure mathematics by Goldberg (1937), who derived a general solution. Horne and Wildy (1961) independently arrived a t some special cases of the general solution in their discussion of viral symmetry. It is evident that plane and cylindrical surfaces can be formed by an array of close-packed hexagons, but to form a closed surface, elements other than hexagons, e.g., pentagons, are required. A simple packing arrangement consists of twelve pentagons alone which results in the regular geometrical figure, the dodecahedron. This figure is closely related to the icosahedron and possesses the same type of symmetry (see discussion following). The icosahedral symmetry is retained and many other close-packing arrangements are possible if specific numbers of

FIG.1. Micrographs of three icosahedral DNA viruses (left to right, human papilloma, varicella, and adenovirus type 12) with (below) corresponding models constructed of hexagons and pentagons. The icosahedral shape becomes more pronounced 88 the number of capsomeres increases ( X 400,000) (Howatson, 1962b).

STRUCTURE OF TUMOR VIRUSES

7

hexagons are added to the twelve pentagons. It was shown by Goldberg (1937) that the general solution which gives the possible values of n, the total number of elements (hexagons and pentagons) on the surface is given by the formula n = 10(a2 ab b 2 ) 2, where a and b are integers. Of the arrangements represented by this formula, only a few have so far been shown to occur in viruses. Of these all but one are obtained by setting b = 0, which gives the series n = 10a2 2, where a = 1, 2, 3, 4, etc. Corresponding values of n are 12, 42, 92, 162, 252, etc. In one plant virus (turnip yellows mosaic) the arrangement of the capsomeres corresponds to the one obtained by setting a = 1 and b = 1, which gives n = lO(3) 2 = 32. Viruses may exist with structural elements arranged in accordance with other values of a and b, but there is reason to believe that these arrangements are likely to be less stable (Horne and Wildy, 1961). The types of structures under consideration can be illustrated by models. Figure 1 shows, on the lower half, models constructed from cardboard pentagons and hexagons joined along the edges by elastic bands. These correspond to members of the series above with b = 0 and a = 2, 4, and 5, giving total elements of 42 (12 pentagons and 30 hexagons), 162 (12 pentagons and 150 hexagons), and 252 (12 pentagons and 240 hexagons), respectively. It will be noted that as the number of surface elements increases the shape of the structure approximates more closely that of

+ + + +

+

15

FIQ.2. Diagram of an icosahedron showing axes of 5-fold, Sfold, and %fold symmetry.

an icosahedron. Figure 2 shows a diagram of an icosahedron, one of the five regular solids; i t has 20 triangular faces, 12 vertices where 5 triangular faces meet, and 30 edges. The icosahedron has axes of rotational

a

A.

F. HOWATSON

symmetry of three types, 5-fold, 3-fold, and 2-fold, passing through opposite vertices, the centers of opposite faces and the mid-points of opposite edges, respectively. Thus, during one complete rotation of the figure about an axis of 5-fold symmetry there are five positions which give identical configurations, for the 3-fold axis there are three such positions, and for the 2-fold, two. All the structures shown in Fig. 1, and indeed all those represented by the general formula given above, possess icosahedral or 532 symmetry. It is this property rather than the icosahedral shape (which is evident only in structures consisting of large numbers of elements) that is common to all the shell structures that have been considered. So far we have discussed models, and we have now to consider how these are related to the structure of actual viruses. The simplest way to do this is to make a direct comparison of a micrograph of a virus particle with a model in a suitable orientation. Examples of comparisons of this type are shown in Fig. 1, in which micrographs of each of three types of virus particles can be compared with the model below. I n the particle on the right (adenovirus) there is a clear one-to-one correspondence between the morphological units on the surface and the elements of the model. The viral subunits, however, are in the form of hollow prisms and are not in contact with one another. As in the model, each structural element is surrounded by six others except those a t vertices which are surrounded by five. Two of the latter are clearly seen and the triangular facets of the icosahedral shell are well defined. There is no difficulty in establishing that the arrangement corresponds to that of the member of the series n = 1 0 2 2 for which a = 5 (note that the number of units along the edge of the triangular face is a 1). The total number of elements, n, on the surface, from the above formula, is 252. The arrangement of the capsomeres on two triangular faces for this case ( a = 5 ) is shown in Fig. 3 (left) in which, however, no attempt has been made to represent the third dimension of the capsomeres. Another comparison between virus particles and model is illustrated

+

+

FIQ.3. Arrangement of capsomeres on two faces of icosahedron for series

n

= 1 0 2 + 2; a = 5

(left), a = 4 (right).

STRUCTURE OF TUMOR VIRUSES

9

by the middle pair in Fig. 1. In this case the virus is varicella, a member of the herpes group. The fine structure of herpesvirus was first described by Wildy et al. (1960a). I n this instance the correspondence between particle and model is not as close as is the case with adenovirus, but triangular faces can be observed, a t the vertices of which are subunits surrounded by five others, indicating that they are on axes of 5-fold symmetry. By counting the number of capsomeres along the edge of the triangle one obtains the value of a (here a 1 = 5, and a = 4) from which the total number of capsomeres, 1 0 2 2, is deduced to be 162. It will be noted that the capsomeres of this virus are distinctly hollow and are larger than those of adenovirus. The capsomere arrangement is shown in Fig. 3 (right). Figure 3 is diagrammatic, no attempt being made to represent the relative dimensions of particles and capsomeres. By using a similar procedure of direct comparison with models, other viruses have been found in which the capsid structure corresponds to the arrangement in which a = 3 and the total number of capsomeres is 92 (Vasquez; and Tournier, 1962; Jordan and Mayor, 1962; Bils and Hall, 1962). The next arrangement in the series, with a = 2 , gives a total of 42 capsomeres; i t has been assigned to several viruses-polyoma, human wart, SV40, and K virus. However, controversy has arisen concerning the validity of this conclusion (Mattern, 1962; Mayor and Melnick, 1962; Caspar and Klug, 1962). The determination of capsomere number and arrangement by direct comparison of micrographs with models is more difficult in the smaller viruses with fewer capsomeres. It is often not possible to detect with certainty capsomeres on two adjacent axes of 5fold symmetry, the location of which is of great value in determining the exact arrangement. The reason for this is simply that in the smaller viruses if one capsomere on a 5-fold axis (i.e., surrounded by five others) is clearly visible, adjacent similarly situated capsomeres are seen so obliquely that it is almost impossible to determine whether they are surrounded by five or six others. Further factors that may contribute to the difficulty in interpretation of the micrographs are flattening and distortion of the particles during drying and superposition of the images of the upper and lower surfaces. Recent experiments carried out by the author in collaboration with L. V. Crawford (Howatson and Crawford, 1963) leave little doubt that the capsomere number in the instance of polyoma and the papilloma viruses is indeed 42. In the method used, the capsomeres from individual virus particles were dispersed in such a manner that the number could be directly counted. Figure 4 is a field from a preparation of polyoma virus showing several groups in which the capsomeres are sufficiently distinct

+ +

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A. F. HOWATSON

Fro. 4. Disintegrated polyoma virus capsids. I n several of the groups the capsomeres are sufficiently well dispersed to be countable ( x 250,000) (Howatson and Crawford, 1963).

to be easily counted. The distribution of capsomere numbers per disintegrated particle shows a distinct peak a t 42 (Fig. 5 ) and rules out the possibility suggested by Mattern (1962) that the number of capsomeres

NUMBER OF CnpsoMEREs

FIG.5. Number of groups of capsomeres from polyoma virus particles having capsomere complements in the range 30-55. There is a distinct peak a t 42 (Howatson and Crawford, 1963).

on this virus is 92. Similar results were obtained with human and rabbit papilloma viruses. It should be pointed out that although most virus particles of a particular species conform to a definite structural pattern,

STRUCTURE OF TUMOR VIRUSES

11

aberrant forms are by no means unusual. This is especially true of the polyoma and papilloma viruses, in preparations of which abnormal particles, including elongated forms of variable length, are frequently found (Howatson and Almeida, 1960b; Williams e t al., 1960; Howatson, 1962a). There are two other viruses with fewer than 42 capsomeres for which the number is well established, but neither of these is an animal virus. They are the plant virus, turnip yellows mosaic, which, as already mentioned, fits the series with a = 1, b = 1, and n = 32 (Huxley and Zubay, 1960; Nixon and Gibbs, 1960) and the small bacteriophage +X174 which has 12 subunits ( a = I, b = 0) (Hall et al., 1959; Tromans and Horne, 1961). Many other small animal viruses have been examined by negative staining and it is apparent that the surfaces are formed of regularly arranged capsomeres but it has not so far been possible to determine the exact number and arrangement. For example, although it is known from X-ray diffraction evidence that the capsid of poliovirus has icosahedral symmetry, the exact distribution of the capsomeres on the surface still eludes the electron microscopist.

D. VIRIONSWITH HELICAL SYMMETRY A second type of symmetry, helical symmetry, is characteristic of cylindrical or filamentous viruses. Of these the most extensively studied is tobacco mosaic virus (TMV), about which there has been accumulated a great deal of structural and other physicochemical data. Information on the structure of this virus has been derived almost entirely from X-ray diffraction studies which are discussed fully in a recent review (Klug and Caspar, 1960). Some of the main features have been confirmed by electron microscopy (Huxley, 1957; Hall, 1958), which shows the virus particles as rod-shaped units 3000 A. long, of mean diameter 150 A. and having an axial hollow of diameter 40 A. By means of X-ray diffraction it has been deduced that the protein component or capsid is composed of an array of identical subunits arranged in the form of a tight helix. The pitch of the helix is 23 A. and there are 49 subunits in every three turns. There is evidence that the RNA is also in the form of a helix that runs between the protein subunits a t a distance of 40 A. from the axis. The protein subunits are not generally resolved in electron micrographs of negatively stained preparations but have been seen in preparations in which the virus particles were partially disintegrated (Horne and Wildy, 1961). I n rods obtained by reconstituting viral protein a periodicity has been observed corresponding closely to the pitch deduced from X-ray diffraction (Nixon

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A. F. HOWATSON

and Woods, 1960). Hart (1961) has reported a similar periodicity in shadow-cast specimens of intact particles. The particles of TMV are rather rigid structures ; other elongated plant viruses such as sugar beet yellows appear to be constructed in much the same way but are more flexible (Horne e t at., 1959b). I n viruses having helical symmetry the protein structure units do not appear to be grouped into capsomeres, the structural, morphological, and chemical units being identical. For this reason it has been recommended that the use of the word capsomere to describe the structural units of helical viruses be discontinued (Caspar et at., 1962). All the viruses with helical symmetry mentioned so far are plant viruses. The first evidence for the presence of similar structures in animal viruses was obtained by Horne and Waterson (1960) by the negative staining method. These studies revealed that some viruses of the myxovirus group have a component resembling closely the helical plant viruses, especially the flexible ones. The helices, however, are not naked as in the plant viruses but are enclosed in a membrane or enveIope with characteristic fine projections on the outer surface. The helical component is best seen in partially disintegrated viruses; in the intact particle the helix is presumably coiled up inside the envelope, but the configuration is diffi-

FIQ.6. Part of the filamentous internal component of a measles virus particle showing helical symmetry of capsid ( X 300,000). Micrograph courtesy of J. D. Almeida.

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cult to determine owing to superposition effects and distortion during drying. The length of the helices in these viruses is not known since they are usually fragmented during the preparation of the specimen, but portions longer than a micron have been observed in preparations of measles virus (Fig. 6 ) . Not all viruses show the helical component as well as measles. I n the case of influenza virus, for example, treatment with ether is required to release the internal component which is then observed in the form of fragments of diameter 90 A. (Hoyle e t al., 1961). It is known that the nucleic acid of this virus is associated with the internal component and it is possible that the RNA in this and similar viruses is disposed within the helix in much the same way as i t is in TMV.

E. COMPLEX VIRIONS The term complex virion is used to describe viruses that are structurally more complex than those already considered. Little is known about the symmetry properties of these viruses. Examples of viruses that belong to this category are vesicular stomatitis, an animal virus of unusual architecture, and in a wider field the many species of bacterial viruses that possess specialized tail-like appendages. In the present context, however, the most important members are the poxviruses, which form a group having many characteristics in common. The best studied of the poxviruses is vaccinia. The complex architecture and the disposition of the various chemical components within the virus have been the subject of extensive investigations (see, for example, Peters, 1960). The application of the negative staining method has revealed some previously unobserved structures a t or near the surface of the particles (Herzberg et al., 1961; Nagington and Horne, 1962). The most striking feature is a network of tubules of diameter about 90 A. covering the particle (Fig. 7 ) . There is some indication that the tubules may be helices formed of discrete subunits, and it has been suggested that they enclose the nucleic acid of the virus which is of the DNA type. It. is unlikely, however, from other studies, that the DNA is located near the periphery of the particle. An alternative interpretation is that the tubules consist of protein and correspond to the capsomeres of other viruses. I n vaccinia and most of the other poxviruses that have been studied the tubules appear to be in discrete lengths that project from the surface or run parallel to it for varying distances. However, it has been shown (Nagington and Horne, 1962; Nagington e t al., 1962) that in two members of the pox group, orf and bovine papular stomatitis viruses, thc tubules form a continuous structure that crisscrosses the surface of the particles.

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FIG.7. Negatively stained vaccinia virus particles showing irregularly arranged filaments on surface ( x 200,000). Ill. Tumor Viruses: Structure and Mode of Development

A. GENERAL CONSIDERATIONS The structure of some tumor viruses has already been referred to in connection with the general discussion of viral symmetry. We shall now discuss in more detail the structure of a number of viruses that have been described as oncogenic (cf. Gross, 1961). The manner of assembly of the particles in infected cells will also receive attention. The electron microscopy of tumor viruses has been the subject of several recent reviews (Bernhard, 1960; Dmochowski, 1960; Dalton and Haguenau, 1962). Our objective is to summarize previous work and to bring it up to date by the inclusion of recent findings, with especial emphasis on symmetry or other structural properties that may help in elucidating the relation of tumor viruses to other viruses. B. POLYOMA VIRUS The basic morphology of polyoma virus was first reported by Kahler et al. (1959), who examined shadowed preparations of purified virus obtained from supernatants of infected cell cultures. The particles were approximately spherical and of average diameter 44 mp; no details of fine structure were reported. Shortly thereafter, in several laboratories, specific particles that were identified as polyoma virus were observed in

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thin sections of cells from infected mice (Dourmashkin and Negroni, 1959; Howatson et al., 1960; Edwards et al., 1960), hamsters (Howatson and Almeida, 1960a,c) ; and also in mouse cells infected in vitro (Bernhard et al., 1959; Banfield et al., 1959; Dmochowski e t al., 1959). The particles were round and uniformly dense for the most part, although some appeared hollow. They were first reported to have a diameter of 27-28 mp but later observations (Howatson and Almeida, 1960b) indicated that the particles had a lightly staining peripheral layer and an over-all diameter of about 38 mp. Occasionally, long rod-shaped forms were observed in association with the spherical particles. Rather rarely the spherical particles were observed invested in a membrane, the total diameter then being 50-60 mp. The fine structure of the polyoma virion was investigated by Wildy et al. (1960b) by the negative staining method. As previously mentioned it was concluded that the capsid possessed 42 capsomeres in an arrangement having icosahedral symmetry. The average diameter was 45 mp. The structure of the rod-like forms has also been examined by negative staining (Howatson and Almeida, 1960b). It was found that the surface was covered with an array of capsomeres similar to those of the spherical capsids. The rods were of approximately the same diameter as the spherical particles but there was some variation, part of which was probably due to different degrees of flattening during drying. I n preparations of polyoma virus treated with specific antiserum, mixed aggregation of particles and rods has been observed (Almeida e t al., 1963), indicating that they have common surface antigens. The rods vary greatly in length; some are ellipsoidal, the length being only slightly greater than the diameter; others are a micron or more in length. The ends are usually hemispherical but sometimes the end is ragged as if incomplete or broken. Horne and Wildy (1961) have given a possible explanation of how the rods are constructed in terms of the models discussed in Section I1,C. A cylindrical surface of any length can be formed from hexagonal elements, pentagonal ones being required only to close the ends. Thus i t is conjectured that in the absence of pentagonal elements (capsomeres) there might be a tendency for the particle to grow indefinitely in length. Wildy et al. (1960b) noted that in their preparations there were some particles in which the centers were dense and only the peripheral capsomeres were visible. These they interpreted as “empty capsids”particles in which the core was missing and the central portion had been penetrated by phosphotungstic acid (PTA) . By subjecting partially purified preparations of polyoma virus to equilibrium density gradient centrifugation in rubidium chloride Crawford e t al. (1962) obtained two

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opalescent bands; one a t a density of 1.32-1.33 which consisted almost entirely of particles of normal appearance and which retained most of the infectivity; the other, which had a density of 1.29-1.30, possessed little infectivity and contained particles that appeared “empty.” These findings are consistent with the interpretation that the empty particles are devoid of the nucleic acid containing core. It was noted that in areas where the PTA was thin, capsomeres were visible over the whole central area of the empty particles so that care had to be exercised to avoid interpreting them as “full.” The empty particles probably correspond to the ring forms with translucent centers that are sometimes seen in sections. Nearly all the elongated forms are found in the empty fraction, indicating that they contain no DNA and are therefore not infectious. We have already mentioned the existence of particles of larger diameter which appear to consist of 45 mp particles invested in an envelope. Dourmashkin (1962) has investigated the nature of these and concludes that they are formed when the 45 mp particles are ingested by cells. The entry of the virus is effected by a process akin to pinocytosis. The cell membrane invaginates and closes round the particle forming a small vesicle in which the particle is transported toward the interior of the cell. Sometimes more than one particle is enclosed within the same envelope. Treatment of the virus with specific antibody inhibited the uptake of the virus and no membrane-bound particles were observed under these conditions. It would seem that the envelope should not be regarded as part of the polyoma virion, being essentially cellular material. The growth cycle of polyoma virus has been studied by several authors by examination of thin sections of cells both from infected animals and from cultures infected in vitro. Determination of the exact sequence of events during the infective process has been hampered by a marked asynchrony in response of the cells that has been demonstrated both by morphological and by biological methods, Only a small proportion of the cells exposed to virus show evidence of viral multiplication at any particular time. However, the broad outline of the infective cycle seems clear and is essentially the same whether studied in mouse cells in culture (Dourmashkin and Negroni, 1960) or in hamster cells in vivo (Howatson and Almeida, 1960a). As previously mentioned, polyoma virus enters the cell by pinocytosis and is transported toward the interior of the cell enclosed in a small vesicle. The fate of the particles following this stage is obscure. The next visible change that has been reported is the appearance some 40 hours after infection of typical virus particles in the nuclei of some of the cells exposed to virus. These are found in association with dense masses of chromatin which is often closely applied to the nuclear membrane. Enormous numbers of particles ( 105-10G) may be found in a single nucleus without any trace of virus in the

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cytoplasm. Eventually, however, with breakdown of the nucleus or nuclear membrane, particles are released into the cytoplasm. Large aggregates of virus particles are also found within cytoplasmic vesicles or in association with membranous networks. These are probably the result of phagocytosis by the cell of the remains of other infected cells, Extracellular aggregates are also commonly observed. Cells in which there are large concentrations of virus in the nucleus probably cannot survive, and indeed there is much evidence of cellular necrosis. It is not known whether some degree of viral multiplication is compatible with cell survival or whether i t occurs in cells that become transformed into tumor cells. However, the general impression is that the nuclei of cells are either heavily loaded with virus or else are unaffected.

(SHOPE)VIRUS C. RABBITPAPILLOMA This virus is present in variable but often considerable quantities in papillomatous growths that occur in wild cottontail rabbits (Shope, 1933, 1935). It can be rather easily separated by differential centrifugation from tissue components, a fact that enabled the virus to be purified and its basic physical and chemical properties to be determined a t a relatively early date (see Sharp, 1953; Hollman, 1962). The virus was shown to contain DNA and protein, with only small amounts of other substances, possibly impurities. In early shadow-cast preparations the particles appeared as spheres of 70 mp mean diameter (Kahler and Lloyd, 1952) ; later, lower values were obtained. Williams (1953) noted that the surfaces of such particles appeared to be covered with little knobs but the number and arrangement of these was not determined. Detection of the particles in infected papillomatous tissues proved to be difficult and was not achieved until 1959 (Moore e t al., 1959b; Stone e t al., 1959). These investigators observed uniformly dense, round particles of diameter approximately 33 mp in the nuclei of infected cells. Purified virus preparations were examined after embedding and sectioning by Haguenau et al. (1960), who observed similar dense particles (diameter 26-29 mp) and also particles that had light centers, suggesting that they were empty. Recent studies by negative staining have given additional information about the fine structure of this virus (Fig. 8). Williams e t al. (1960) fractionated a preparation of the virus by rate zonal centrifugation in sucrose and glycerol density gradients. They observed two main components; one consisting predominantly of empty particles with low infectivity and phosphorus content, the other consisting mainly of intact particles with high infectivity and phosphorus content. Breedis et aZ. (1962), using a different method of separation, equilibrium sedimentation in cesium chloride density gradients, obtained a similar type of separa-

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FIG.8. Rabbit papilloma virus from a preparation banded in a rubidium chloride equilibrium density gradient by L. V. Crawford. Most of the particles appear “full” ( X 200,OOo).

tion into predominantly empty and predominantly full components. Both groups noted that the particles were covered with regular arrays of capsomeres but no definite conclusion was reached regarding their number and arrangement. However, according to Melnick (1962), published micrographs of the virus indicate that the arrangement is similar to that of polyoma virus-42 capsomeres arranged in accordance with icosahedral symmetry. This conclusion is supported by recent data on the distribution of the numbers of capsomeres in collapsed capsids, already referred to (Howatson and Crawford, 1963). Elongated structures, covered with small projections similar to the capsomeres of the spherical particles, have been observed in preparations of the virus (Williams et al., 1960). These are similar to the elongated forms associated with polyoma virus. Breedis et al. (1962) found that after exposure to cesium chloride, the viral capsids, especially the empty ones, became very fragile, breaking down readily into the constituent capsomeres. The appearance of these indicates that in three dimensions they have the form of hollow prisms closed a t one end. The development of the papilloma virus in the skin of the rabbit has been described by Stone e t al. (1959). This site is a favorable one for

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studying viral development since the stages can be correlated with easily recognizable stages in the differentiation of the cells as they move through the various layers of the epidermis to the surface. The virus particles are first detectable as distinct entities in the nuclei of cells in the lower stratum spinosum. At the earliest stages there is a close association of the particles with the nucleolus. The virus later spreads from the nucleolar region to fill the whole nucleus and may spill into the cytoplasm. As the cells approach the surface they become keratinized, cytoplasmic detail is lost, and eventually only heavily keratinized shells containing pools of virus are seen.

D. HUMANPAPILLOMA (WART)VIRUS Particles believed to be the human papilloma virus were first observed by Strauss e t al. (1949) in aqueous extracts of wart tissue prepared for electron microscopy by the shadowcasting method. A few years later Bunting (1953) observed tightly packed aggregates of particles in thin sections of wart tissue. More recently the structure of this virus has been described by Williams et al. (1961) in thin sections of infected cells and in purified preparations of virus after metal shadowing and negative staining. By the last method, which revealed most structural detail, the virus particles were shown to be approximate spheres of average diameter 55 mp. The capsid consisted of a well-defined array of capsomeres showing evidence of icosahedral symmetry (Fig. 9). By comparison with

FIG.9. On the left, a single human papilloma virus particle (negatively stained with contrast photographically reversed); on the right a model having 42 projections arranged in accordance with icosahedral symmetry. The correspondence between particle and model is close (Williams et al, 1961).

models the number and arrangement of the capsomeres was deduced to be the same as that characteristic of polyoma virus. As in the instance of polyoma virus, this conclusion has been questioned by Caspar and

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Klug (1962) and by Mattern (1962). However, in experiments similar to those previously described in connection with polyoma and rabbit papilloma viruses, the capsomere complements of collapsed capsids were counted directly (Howatson and Crawford, 1963). The distribution of capsomere numbers confirmed the interpretation of the structure given by Williams et al. (1961). Particles of abnormal size and shape, especially elongated rodlike forms similar to those seen in rabbit papilloma preparations, are observed quite frequently in the human virus preparations, espccially those from the less dense band separated by equilibrium density gradient centrifugation. I n fact, in size, fine structure, and types of abnormal forms, the human papilloma virus resembles very closely the rabbit virus. This similarity extends also to the manner of development of the viruses in the cells of the epidermis, which is next discussed. A study by thin-section electron microscopy of the development of human papilloma virus in the epidermal layers of wart tissue has been reported recently by Almeida e t al. (1962b). Virus particles were not observed in cells of the basal layer where proliferation occurred, but were first encountered in cells of the lower stratum spinosum. As in the case of rabbit papilloma, the virus was closely associated with the nucleoli in the early stages of its formation. I n succeeding layers the particles were more numerous and occupied most of the nucleus; finally, as the cells became increasingly keratinized and lost their normal structures, they were seen as large aggregates surrounded by keratinous material. VIRUS) E. SIMIANVIRUS40 (VACUOLATING This virus (SV40) is present in apparently latent form in many rhesus monkeys (Sweet and Hilleman, 1960). When cultures of kidney cells from Cercopithecus aethiops monkeys are inoculated with the virus it causes a characteristic type of cytopathogenic effect. Eddy and her colleagues (Eddy et at., 1961, 1962) discovered that if the virus is injected into newborn hamsters, i t induces, after a lapse of several months, sarcomata a t the site of injection. The virus particles are very similar in size and structure to polyoma virus. Melnick (1962) and Mayor and Melnick (1962) concluded from examination of negatively stained preparations of the virus that, as in polyoma and the papilloma viruses, the capsid consists of 42 capsomeres arranged in icosahedral symmetry. Bernhard et al. (1962) reached a similar conclusion. The latter authors also demonstrated a filamentous form, the surface of which was covered with structures similar to capsomeres. Electron microscope studies of the development of the virus in cells infected in vitro have recently been

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reported from the same laboratory (Tournier e t al., 1961) and also by Gaylord and Hsiung (1961). Virus particles of uniform size (33 mp) and density were found in large numbers in nuclei of infected cells. The mode of development was very reminiscent of that of polyoma virus in mouse cells. VIRUS F. LUCK^ KIDNEYTUMOR Some years ago Fawcett ( 1956) examined by thin-section electron microscopy the fine structure of cells in adenocarcinomas found in the kidneys of leopard frogs (Rana pipiens). In about one-third of the tumors there were distinctive particles that were thought to represent the virus associated with these tumors. The particles were described as hollow spheres of diameter 90-100 mp having a thick capsule and a dense inner body of diameter 35-40 mp, eccentrically placed within a central cavity. The initial site of development appeared to be the nucleus but particles were found in the cytoplasm and also extracellularly. The extracellular particles were enclosed in an envelope derived from the plasma membrane. I n structure and mode of development the resemblance to the herpesviruses was very striking. No electron microscope studies of this virus have since been reported, so i t is not known whether it possesses a capsid with structure similar to that of the herpesviruses.

G. ADENOVIRUS TYPE12 This particular type of human adenovirus was reported by Trentin et al. (1962) to be capable of inducing malignant tumors when injected into newborn hamsters. Other types of adenovirus were ineffective.l Thin sections of tissue culture cells infected with the type 12 adenovirus showed intranuclear arrays of particles of the size and morphology associated with other members of the adenovirus group. The structure of the type 12 particles made apparent by negative staining (Fig. 1, right) is identical to that described for several other types of adenovirus that have not been shown to have oncogenic capabilities. The symmetry of these particles is discussed in Section 2,C. H. MAMMARY TUMOR VIRUS Positive identification of this virus has proved extremely difficult. This has been due to the lack of a simple and accurate titration procedure, to the presence of more than one type of particle in tumor cells and active extracts, and to difficulties in purifying the particles. Another 'Recently, however, type 18 has also been shown to be capable of inducing tumors in hamsters (Huebner et al., 1962).

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problem that is still unresolved is the presence of particles of the type associated with infectivity in tumors induced in mice that by biological test are free of virus (Bernhard et al., 1956a; Dmochowski, 1957). However, there is a considerable body of evidence to the effect that the type B particles of Bernhard (1958) represent the mature virus, although, as Moore e t al. (1959a) have shown, some infectivity may be carried by a smaller component which is probably the nucleoid of these particles. The structure of the type B particles is intimately related to the way they are released from the cell, and has been studied mainly by thin sectioning methods. These and other particles (type A) commonly observed in and around mammary tumor cells have been described by numerous authors (for recent reviews see Moore, 1962; Bernhard and Granboulan, 1962) and will be discussed here only briefly. The type A particles are found intracellularly, often in the form of large cytoplasmic inclusions in the Golgi region of the cell. The particles appear in sections as “doughnut” bodies of diameter about 70 mp with translucent centers and a double-layered envelope. The nature of the type A particles is uncertain; they are found in many different types of mouse tumors but no biological activity has ever been associated with them. The type B particles are found in variable but sometimes large numbers in spaces between cells and also within cytoplasmic vesicles. A very probable sequence of stages in the development and maturation of the particles has been deduced from micrographs. The particles form by a budding process a t a cytoplasmic membrane, usually the plasma membrane. When the particle is first released from the cell it has an appearance resembling that of a type A particle surrounded by an extra (double-layered) membrane; this being originally the portion of the cell surface where the budding occurred. De Harven (1962) refers to these particles as type A3 to distinguish them from the intracellular particles which are called type A2. Bernhard (1958) has suggested that the A3 particles can be formed by A2 particles moving up to the cell surface and being extruded, enclosed in a portion of cell membrane. However, this is not the usual mode of formation, the majority of the A3 particles being assembled entirely from submicroscopic components a t the cell surface. After severance from the cell there is a redistribution of material within the particles which results in the formation of a dense nucleoid. This is enclosed in a fine membrane and usually takes up’ an eccentric position within the particle. I n sections the nucleoid has a diameter of 30 to 40 mp and the particles an over-all diameter varying from 100 to 120 mp. The structure of the virus has been studied more recently by negative

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staining. Lyons and Moore (1962), using density gradient centrifugation as the final step in purification, obtained preparations containing many particles similar to those shown in Fig. 10 (lower right). The particles

FIG.10. Mammary tumor in C3H mouse. Spread cell preparation, negatively stained, showing two types of particles, type A (center and upper right) and type B (lower right and left) ( X 144,000) (Parsons, 1963~).

were predominantly spherical of average diameter 130-160 mp and had on the surface distinctive projections about 10 mp long similar to those observed on influenza virus (Horne e t al., 1960). The particles were, however, larger than influenza virus particles (average diameter 13& 160 mp compared with 80-100 mp for influenza) and showed no evidence of possessing an internal helix such as is found in influenza and other myxoviruses. The outline of a central body, presumably the nucleoid seen in sectioned particles, was visible in many particles. These particles correspond to the type B particles, seen in sections; indeed on close inspection of high quality micrographs, the array of projections a t the periphery of sectioned particles can be clearly seen. Parsons (19634 examined the virus without purification in thinly spread areas of mammary tumor cells treated with phosphotungstate,

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and observed two types of particles corresponding to the types A2 and B seen in sectioned material (Fig. 10). The A2 particles had a circular outline of diameter 70 mp and appeared to be spherical shells with a mottled surface which, however, was obscured by penetration of the phosphotungstate. The B particles were similar to those described by Lyons and Moore (1962).

I. MOUSELEUKEMIA VIRUSES Under this heading are grouped a number of viruses connected with different types of leukemia in mice. The most fully characterized morphologically are those associated with the names of Gross (1951), Friend (1957), and Moloney (1960). In structure and mode of development the mouse leukemia viruses have much in common. Like the mammary tumor virus they appear to develop by a budding process from cytoplasmic membranes. The virus particles differ morphologically from mammary tumor virus particles, falling into two classes that have been designated type C and type A1 according to whether the central area (nucleoid) is or is not electron dense (De Harven, 1962). The A1 particles are intermediate in size (90 m,u) between the A2 (70 mp) and A3 (100 mp) particles already mentioned in connection with mammary tumors. They are extruded from the cell membrane in much the same way as the A3 particles but differ from them in showing only two or sometimes three concentric dense rings instead of four. It is probable that they are precursors of the type C particles; these are smaller on the average than B particles of mammary tumors and have larger, less dense, and more centrally placed nucleoids. The relationship between the various mouse leukemia viruses has not been clearly defined. The three viruses discussed above are antigenically distinct (De Harven, 1962) ; they produce different disease patterns and have different ranges of susceptible hosts. These differences are complemented by minor but probably significant differences in size and structure of the viruses. The Moloney virus in particular has several interesting features. Measurements by Dalton (1962) suggest that the mature virus is significantly larger (by 10 mp) than the Gross and Friend viruses which are of about the same size (90 mp). Elongated forms are found in association with the usual roughly spherical Moloney virus particles. Particles of dumbbell shape having two nucleoids within a single envelope are also encountered; other particles have tail-like appendages. This virus has recently been obtained in a highly purified state from the plasma of infected animals (Moloney, 1962). Extracts consisting almost entirely of type C particles show a high degree of infectivity, leaving little doubt concerning their identity with the Moloney virus. A peculiarity of the leukemia virus particles is that they appar-

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ently multiply not only in the malignant cells (lymphoblasts) but also, and to a greater extent, within niegakaryocytes which do not appear to be malignant cells (Dalton, 1962). The appearance of negatively stained Gross leukemia virus has been studied by Parsons (1963a) both in purified preparations and in thinly spread areas of cells from leukemic animals. The particles revealed little regular structure; they consisted of round flattened sacs of diameter 110160 mp, in some of which a central body of diameter about 75 mp, presumably the nucleoid, could be discerned. Occasionally a few fine projections were observed on the outside surface of particles but there was no array of well-defined spikes such as are observed on the mammary tumor virus. This difference may account for the different appearance in sections of the outer membrane of the mammary tumor and leukemia viruses. Dalton e t al. (19.62) examined purified preparations of Moloney virus after negative staining and observed particles of remarkable appearance. They consisted of two main parts, a “head” that was often hexagonal in outline, and a tail-like appendage, with a resulting appear-

FIG.11. Negatively stained particle from preparation of Moloney leukemia virus, showing hexagonal head and tail-like appendage ( x 150,000). Micrograph courtesy of A. J. Dalton (Dalton et al., 1962). ance superficially resembling that of the T-even bacteriophages (Fig. 11). The appendages seem to be more elaborate structures than the fragments of cell membrane that, are sometimes left attached to other viruses that

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form by a budding process (Dourmashkin and Simons, 1961; Howatson and Whitmore, 1962). Although osmotic effects occurring during preparation and forces exerted during drying probably alter the appearance of the particles, the fact that particles with tails are observed in thin sections of megakaryocytes indicates that the tail-like structures are not entirely artifactual. What functional role, if any, that they play is completely unknown.

J. AVIANSARCOMA-LEUKOSIS VIRUSES The virus particles associated with a variety of neoplastic diseases of the chicken-the avian sarcoma-leukosis complex-will now be discussed. Of these diseases the best known and most intensively studied are the Rous sarcoma and the leukemias, myeloblastosis and erythroblastosis (Haguenau and Beard, 1962). There is increasing evidence that these viral diseases are closely related and that they may represent different host or host cell responses to a single infective agent rather than the effects of a multiplicity of different agents. However this may be, the virus particles associated with the different diseases cannot bc morphologically distinguished from one another. They closely resemble the mouse leukemia viruses in structure, and are elaborated by a budding process similar to that described in connection with these viruses. An exception appears to occur in the case of myeloblastic cells transferred to tissue culture from the blood of diseased chickens. I n these cells the virus particles are reported to form inside viroplasts, membrane-bound cytoplasmic bodies (Bonar e t al., 1959, 1960). However, the particles in these cells are not morphologically different from those produced by the budding process in other types of cell infected with the same strain of virus (Heine e t al., 1962). I n those cases where preparations showing a high degree of purity have been obtained, there is good correlation between particle numbers and infectivity. The particles associated with infectivity resemble the type C mouse leukemia particles. I n sections they appear as round bodies of diameter 70-80 mp limited by a doublelayered membrane and containing a dense nucleoid 30-40 mp in diameter. Aggregates of intracellular particles of smaller diameter are occasionally seen in Rous sarcoma cells (Bernhard e t al., 1956b; Haguenau e t al., 1961). The relation of these to the mature virus particles is not clear. Dourmashkin and Simons (1961) examined the structure of Rous sarcoma virus by negative staining and compared it with the appearance in sections. As in the case of the Gross mouse leukemia viruses the particles were rather irregular in shape and size, suggesting a collapsed sac

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or envelope. There were sometimes indications of a central body, presumably corresponding to the nucleoid seen in sections. The outer surface of the envelope was smooth or in some cases fringed with a fine layer of filamentous material. In a few particles thought to be viral, a hexagonal arrangement of hollow ring-shaped units with an approximate centerto-center spacing of 75 A. was observed, but it was not clear what relation this had to viral structure.

K. POXVIRUSES The poxviruses constitute a well-defined group, the members of which have many characteristics in common. They are among the largest of the animal viruses (maximum dimensions in the range 250-300 mp) and have a distinctive bricklike shape when in the dried state. They contain DNA and have a complex developmental cycle, visible steps of which are confined to the cytoplasm. The structure of the poxviruses as it is revealed by negative staining has already been described (Section I1,E). Many of the poxviruses are capable of inducing abnormal cell proliferation but the extent and duration of this varies from one member to another. Some, such as vaccinia, produce only slight and transitory cell proliferation in wiwo; others, such as molluscum contagiosum, are capable of inducing benign tumors. I n one instance, Shope fibroma, malignant tumors can be induced if the virus is inoculated into young animals 01 adult animals treated with cortisone (Duran-Reynals, 1945). The properties of Shope fibroma virus have recently been reviewed by Febvre (1962). The cycle of development and the structure of molluscum contagiosum virus has been studied in thin sections of infected tissue by Dourmashkin and Bernhard (1959). I n cells infected with these viruses the mode of development is essentially the same as that of the other poxviruses that have been investigated, and will he described here only very briefly. The earliest manifestations of viral synthesis occur in the cytoplasm where areas consisting of dense matrix material (viroplasm) appear. Portions of this material become encircled by membranes; the bodies so formed are immature virus particles. Many of these contain a dense region or nucleoid. As maturation proceeds the particles become slightly elongated, the nucleoid takes up a more central position, changes to a dumbbell shape and is flanked by two smaller areas of high density. This is the now classical appearance in sections of the mature poxvirus particle as described by Peters (1956) and others. A peculiar feature of the mature molluscum contagiosum particle, not shared by all poxviruses, is a small dense granule that is present in many of the particles (Banfield, 1959).

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IV. Classification Scheme

A. BASIS FOR CLASSIFICATION

I n accordance with the emphasis placed in this article on the structural characteristics of virus particles, the first broad division of viruses is made on the basis of the symmetry properties described in Section 11. The use of these properties for general viral classification was suggested by Horne and Wildy (1961). I n this article only animal viruses are considered. There are three main categories; viruses having capsids with icosahedral symmetry, viruses having capsids with helical symmetry, and viruses in which the type of symmetry is complex or has not been determined. These categories are subdivided according to the type of nucleic acid the virus contains. A further subdivision is made according to whether or not the capsid is enclosed in an envelope. Finally, the site of assembly of the virion within the infected cell is taken into account, since this property correlates well with the others and is a useful guide in classification. The classification scheme, which is essentially the same as one previously described (Howatson, 1962b), is shown in Table I. I n the last column some typical examples of viruses belonging to the various categories are given, with capsomere numbers where known. Among the viruses arranged in this way there are many that have been grouped together in the past on other grounds, such as similarities in disease pattern or host cell response. On the other hand, there are examples of viruses, such as measles, which were previously difficult to classify and which by reason of recently discovered structural patterns can now be assigned to a definite category. I n the first main category, icosahedral RNA viruses, there occurs the large group of enteroviruses, all of which are small unencapsulated viruses of similar size (25-30 mp) and morphology. A typical member of the group is poliovirus, the capsid of which is known from X-ray diffraction studies to have icosahedral symmetry. However, the capsomere number and arrangement of this and other enteroviruses have not been definitely determined. Another member of the category is RE0 virus (Sabin, 1959), formerly classed as an enterovirus but now regarded as distinct since it is considerably larger (60-70 mp) and has a well-defined capsid structure (92 capsomeres). The viruses in this category are assembled in the cytoplasm of infected cells and are never enclosed in an envelope. This feature is probably related to their mode of release from the cell which occurs by a rapid expulsion or burst rather than the relatively slow release characteristic of membrane-bound viruses.

STRCCTURE OF TUMOR VIRUSES

29

The second category, icosahedral DNA, has been subdivided into viruses that have naked capsids and viruses in which the capsid is invested in an envelope. The importance of this distinction is not entirely clear. Preparations of the herpesviruses which are in the enveloped class usually contain a high proportion of unenveloped capsids. Wildy and Watson (1962) have produced evidence to the effect that naked herpes simplex capsids are capable of infecting susceptible cells. The envelope may therefore be a nonessential component of the virion, its function being to afford additional protection to the viral nucleic acid and perhaps to increase the efficiency of adsorption of the virus to cells. On the other hand, Epstein (1962a) stresses the importance of the envelope and considers that naked and membrane-bound viruses differ fundamentally. The icosahedral DNA viruses with naked capsids are assembled and appear to complete their development within the nuclei of infected cells, although mature particles may pass from the nucleus to the cytoplasm before being released from the cell. The encapsulated viruses are also assembled in the nucleus and pass into the cytoplasm. At this stage the particles consist of naked capsids, hut particles that emerge from the cell before lysis occurs do so by a budding process that invests the capsid in an envelope derived from the cell membrane (Epstein, 1962b). I n the icosahedral DNA category, considerable success has been achieved in determining capsid structure. Included in the category are the L'papova'' viruses, which have a number of properties in common, including the possession of 42 capsomeres (Melnick, 1962). Another homogeneous group consists of the various human adenoviruses and related animal viruses such as infectious canine hepatitis (ICH) and the simian virus SV39. These viruses are closely similar in structure having capsids of well-defined icosahedral shape and symmetry, and a total of 252 capsomeres (Horne et al., 1959a; Davies e t al., 1961; Archetti et al., 1961). The subsection of encapsulated viruses consists of viruses having 162 capsomeres. It is of interest that varicella (chicken pox virus) has recently been shown to have this configuration (Almeida et al., 1962a), thus confirming a t the level of capsid structure its relationship to the herpes rather than the pox\.''iruses. The second main category consists of viruses having capsids with helical symmetry. Of these only plant viruses have naked helical capsids. In all known animals viruses the capsid consists of a helical component coiled up inside an enveloping membrane. There appear to be two main types of helix, one of diameter 90 A., the other 170-180 A. (Waterson, 1962). I n rabies virus evidence for a double helix has been found (Pinteric et al., 1963). As far as is known all viruses in this category contain

w

0

TABLE I VIRALCLASSIFICATION SCHEME' Site of assembly of virion

Naked (N) or encapsulated

Examples of virusesb

Nucleic acid

Icosahedral

RNA

Cytoplasm

N

DNA

Nucleus

N

DNA

Nucleus and cell membrane

E

RNA

Cell membrane

E

Influenza, mumps, measles

DNA

-

-

None known

RNA DNA

Cell membrane Cytoplasm

E N

Vesicular stomatitis Vaccinia, ectromelia, fowl pox

Helicale

Complex

(El

____

________

Symmetry of capsid

Non-oncogenic Poliovirus, coxsackie, ECHO, (92) R E 0 (42) K (252) Adenovirus, SV39, ICH, GAL (162) Herpes simplex, varicella, pseudorabies

Oncogenic

(42) Polyoma, papilloma, SV40 (252) adenovirus (types 12 and 18) Luck6 kidney tumor?

Mammary tumor, mouse leukemia' avian leukemia, Rous

Myxoma, fibroma, molluscum contagiosum, monkey pox

a Some of the commoner animal viruses classified according to physicochemical properties. Viruses known to have oncogenic capabilities are listed separately in the last column. b Numbers in parentheses indicate number of capsomeres. c In the helical RNA category the site of assembly and type of nucleic acid have not been definitely established in all cases.

? 9

x

0

2

8Z

STRUCTURE OF TUMOR VIRUSES

31

RNA. The site and mode of development of these viruses has not been determined in every instance, but in those in which i t is known (e.g., influenza) the assembly of the virus takes place a t a cell membrane, usually the plasma membrane, and the particle is extruded by a budding process. In the third main category are placed viruses of complex structure. It may well be that as more is learned about their properties some of these will be transferred to one of the categories already discussed. However, a t present it seems better to form a separate category for the structurally complex virions. An example of an RNA-containing virus of this type is vesicular stomatitis. The virion is encapsulated and is formed a t a cytoplasmic membrane. The DNA-containing subsection is represented by the poxviruses which are assembled and mature in the cytoplasm.

B. PLACE OF TUMOR VIRUSES We now consider where the tumor viruses discussed in Section I11 fit into the classification scheme outlined in the previous section. 1. Icosahedral R N A No animal viruses in this category have been shown to have oncogenic capabilities. 2. Icosahedral D N A

I n this category there are several tumor viruses-the papilloma viruses, polyoma, and the recently discovered SV40-all of which have capsids consisting of 42 capsomeres. It also includes human adenovirus type 12 which has 252 capsomeres. All of these viruses are unencapsulated. The Luck6 kidney tumor virus has been tentatively assigned to the subsection of encapsulated viruses because of its resemblance to the herpesviruses.

3. Helical R N A No tumor virus has been shown to possess a capsid having helical symmetry and consequently none can be assigned definitely to this category. However, the mammary tumor virus and also the mouse and chicken leukemia viruses have some features in common with the helical RNA viruses. So far as is known they all contain RNA, and like other members of the category they form a t the cell surface by a budding process. In the case of mammary tumor virus there is a striking resemblance between the intact, negatively stained particles and influenza virus particles (Fig. 12). Because of these similarities and despite the

32

A. F. HOWATSON

FIG.12. Influenza virus, negatively stained. Note similarity to type B mammary tumor virus particles (Fig. 10) ( x 200,000). Micrograph courtesy D. H. Watson.

lack of evidence of helical symmetry, these tumor viruses have been tentatively placed in this category. 4. Helical D N A

No viruses, oncogenic or other, have been assigned to this category. 5. Complex R N A

There are no tumor viruses in this Category. 6. Complex D N A

This category includes several poxviruses associated with tumors that are generally benign-myxoma, fibroma, molluscum contagiosum, and a monkey poxvirus (Niven e t al., 1961). V. General Discussion

It is clear from the distribution of tumor viruses in the categories shown in Table I that there is considerable variety in their structure, nucleic acid type, and mode of development. However, they are not distributed at random, being concentrated in some categories and absent in others. Among the icosahedral viruses there is a striking difference with respect to tumor induction between the RNA- and DNA-containing

STRUCTURE OF TUMOR VIRUSES

33

types. I n the latter category the 42 capsomere (papova) group accounts for most of the tumor-inducing viruses which include the classical papilloma viruses (rabbit, human, and very probably similar viruses affecting other species), polyonia, and SV40. However, other viruses in this group, for example, K virus (Kilhain, 1952), which is structurally very similar, have so far failed to show any tumor-inducing activity (Parsons, 196313). The discovery of the oncogenic properties of adenovirus type 12 added yet another to the list of viruses in the icosahedral DNA category that have tumor-inducing activity and showed that the property is not confined to viruses with the 42 capsomere configuration. Other types of adenovirus with apparently identical structure, however, have not shown any tumor-inducing activity, so that structure alone provides no clue regarding the apparently special properties of the type 12 variant. It is possible that the resemblance of the Luck6 tumor virus to the herpesviruses may extend to its having a capsid of similar construction. If this proves to be so i t will add a third type of capsid configuration to those possessed by viruses with tumor-inducing properties. It is not known whether capsid structures of the types discussed are confined to DNA viruses. At present no icosahedral RNA viruses are known to have similar structures. However, since the number of different capsid configurations is limited, i t is possible that viruses otherwise quite different in their properties might have the same capsomere arrangement. It is thus not wise to place too much stress on capsid configuration as a distinguishing feature of tumor viruses. It is much more likely that the different behaviors of the RNA and DNA icosahedral viruses with regard to tumor induction are related to their different modes of replication in host cells. This matter will be discussed in a later paragraph. A second high concentration of tumor viruses occurs in the helical RNA category (with the reservation that the evidence supporting their allocation to this category is incomplete). Detailed information on the structure of these viruses is scanty and it is not possible to relate their tumor-inducing capacity to any special structural features. The tail-like appendages described by Dalton in a member of this group are unique in animal viruses but their significance is a t present completely unknown. Finally, the complex DNA category contains members of the pox family that have tumor-inducing potentialities. These represent a third group of tumor viruses with properties that are very different from those of the other two. As mentioned in a previous section, there are minor differences in the structure and development cycle of different poxviruses but in no instance has any specific characteristic been definitely linked to capacity for tumor induction. It is apparent that segregation of viruses in accordance with the

34

A. F. HOWATSON

physicochemical properties of the particles does not result in any clearcut separation of oncogenic from non-oncogenic viruses. It indicates, rather, that capacity for tumor induction exists in viruses of widely varying characteristics. However, this capacity is invested much more commonly in some types of virion than in others. It seems likely in view of the recent additions to the number of viruses that have been shown to have tumor-inducing capabilities that many more such viruses remain to be discovered. One value of a classification scheme such as that discussed here is in suggesting areas in which the search for such viruses should be concentrated. It must be conceded that improved knowledge of virus structure has added little to the understanding of the nature of tumor viruses. Perhaps this is not surprising in view of the relatively gross level of the available information. Furthermore, the viral property of greatest interest, the mechanism whereby a virus transforms a cell into a tumor cell, is not as obviously related to structure as is the manner in which the virus replicates itself. However, the two types of activity are probably closely interrelated, so that some discussion of the different modes of viral replication is relevant. All viruses must be able to propagate themselves in order to perpetuate the species; in fact, their biological activity is almost entirely directed toward this end. Since viral replication occurs only inside a susceptible host cell and requires the cooperation of the metabolic machinery of the cell, viral multiplication necessarily involves an alteration or disturbance of the host cell metabolism. As we have noted earlier, there exist a number of different patterns of viral synthesis, and these will be associated with a corresponding variety of disturbances to cell metabolism. These disturbances are usually severe enough to be incompatible with cell survival, and lysis of the cell sooner or later ensues. This is the classical conception of the effect of viral invasion of a cell and it holds true for many viruses. For example, i t would appear that the mode of replication of the icosahedral RNA viruses is such that the resulting disruption of cell metabolism invariably leads to cell death, However, other types of virus with different patterns of synthesis may multiply with less drastic effects on cell metabolism, allowing the cells to survive for longer periods or perhaps indefinitely. I n the latter case, virus and cell may attain an equilibrium, cells multiplying and producing virus a t a slow rate. There is evidence that this is so for Rous sarcoma virus in cells propagated in vitro (Temin and Rubin, 1959), and it may apply also to some of the other viruses that form by a similar budding process. It has been surmised that the presence of the virus stimulates the cells to Bbnormal mitotic activity or releases them from normal growth re-

STRUCTURE OF TCTMOR VIRUSES

35

straints, thus providing an explanation for the tumor-inducing action of such viruses. According to this hypothesis the continuing presence of virus within the cells is necessary to maintain the growth stimulus. I n still other circumstances the stimulus to metabolic activity initiated by the invading virus affects the normal synthetic processes of the cell and there is an initial phase of hyperplasia. This is usually followed rapidly by necrosis as viral multiplication gets under way, but the outcome depends very markedly on the particular virus-cell system in question. When certain poxviruses (fibroma, molluscum) are applied to the appropriate host cells, viral synthesis is delayed or inhibited, but the cells are altered by the virus in such a way that they proliferate abnormally. A similar situation may arise in the case of other viruses which replicate in a different way, for example, some members of the icosahedral DNA category. It has recently been shown that infection of cultured cells with adenovirus results in increased synthesis of protein and DNA without much damage to the normal synthetic mechanism of the cells (Green, 1962). It is conceivable that some cells or types of cell when invaded by such a virus do not succumb in the usual way. They may be able to resist viral multiplication and its sequelae but suffer damage to the synthetic mechanism of such a nature that growth regulation is lost. The defect, if passed on to the cell’s descendants, would give them a growth advantage. According to this view, abnormal proliferation occurs because the virus damages the cell’s growth-controlling mechanism without killing the cell. Another possibility that has been much discussed is that the virus plays a more positive role, integrating its own genetic material with that of the cell and so altering its hereditary characteristics. Whatever the role of viruses of the icosahedral DNA type in effecting the transformation from normal to tumor cell, i t seems clear that the continuing presence of infective virus in the transformed cells is not necessary to maintain the abnormal growth pattern, This has been shown most clearly in the case of polyoma virus where persistent efforts have failed to reveal the presence of infective virus in some of the cells transformed in vitro by the virus (Dulbecco and Vogt, 1960) and in some tumors induced by i t in v i m (McCulloch e t al., 1960). It follows from the above discussion that the likelihood of a particular virus being able to cause cell transformation and induction of tumors is closely related to the manner in which the particles normally replicate in host cells. This in turn is related to the structure of the particles. No distinctive pattern of structure or mode of replication is uniquely associated with capacity for tumor induction, which appears to be a property of viruses that is much more widespread than was suspected only a short time ago.

36

A. F. HOWATSON

ACKNOWLEDGMENTS The article was written while the author was on leave of absence from the Division of Biological Research, Ontario Cancer Institute, and the Department of Medical Biophysics, University of Toronto, and a guest of the Department of Virology, University of Glasgow. Grateful acknowledgment is made to Professor M. G. P. Stoker and his colleagues for hospitality and many helpful discussions.

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Mayor, H. D., and Melnick, J. L. 1962. Science 137, 613-615. Melnick, J. L. 1962. Science 135, 1128-1130. Moloney, J. B. 1960. J . Natl. Cancer Znst. 24, 933-951. Moloney, J. B. 1962. Federation Proc. 21, 19-31. Moore, D. H. 1962. Zn “Tumors Induced by Viruses: Ultrastructural Studies” (A. J. Dalton and F. Haguenau, eds.), pp. 113-150. Academic Press, New York. Moore, D. H., Lasgargues, E. Y., Murray, M. R., Haagamen, C. D., and Pollard, E. C. 1959a. J . Biopkys. Biochem. Cytol. 5, 85-92. Moore, D. H., Stone, R. S., Shope, R. E., and Gelber, D. 195913. Proc. SOC.Exptl. Biol. Med. 101, 575-578. Nagington, J., and Horne, R. W. 1962. Virology 16, 248-260. Nagington, J., Plowright, W., and Horne, R. W. 1962. Virology 17, 361-364. Niven, J. S. F., Armstrong, J. A., Andrewes, C. H., Pereira, H. G., and Valentine, R. C. 1961. J . Pathol. Bacteriol. 81, 1. Nixon, H. L., and Gibbs, A. J. 1960. J. Mol. Biol. 2, 197-200. Nixon, H. L., and Woods, R. D. 1960. Virology 10, 157-159. Parsons, D. F. 1963a. J . Natl. Cancer Inst. 30, 569-583. Parsons, D. F. 1963b. Virology 20, 385-387. Parsons, D. F. 1963c. J. Cell Biol. 16, 620-626. Peters, D. 1956. Nature 178, 1453-1455. Peters, D. 1960. Intern. Kongr. Elektronenmikroskopie 4, Berlin, 1968, Verhandl. 2, 552-572. Pinteric, L., Almeida, J. D., and Fenje, P. 1963. Virology 2Q, 208-211. Powell, H. M. 1948. J. Chem. SOC.1948, 61. Rivers, T. M. 1928. Am. J . Pathol. 4, 91-124. Sabin, A. B. 1959. Science 130, 1387-1389, Sachs, L., and Medina, D. 1961. Nature 189, 457459. Sharp, D. G. 1953. Advan. Virus Res. 1, 277313. Shope, R. E. 1933. J. Exptl. Med. 58, 607-624. Shope, R. E. 1935. Proc. SOC.Exptl. Biol. Med. 32, 830-832. Stewart, S. E. 1955. J . Natl. Cancer Znst. 15, 1391-1415. Stewart, S. E., Eddy, B. E., Gochenour, A. M., Borgese, N. G., and Grubbs, G. E. 1957. Virology 3, 380-400. Stewart, S. E., Eddy, B. E., and Borgese, N. 1958. J. Natl. Cancer Zmt. 20, 12231243. Stoker, M., and Abel, P. 1962. Cold Spring Harbor Symp. Quant. Biol. 27, 375-386. Stone, R. S., Shope, R. E., and Moore, D. H. 1959. J . Ezptl. Med. 110, 543-546. Straws, M. J., Shaw, E. W., Bunting, H., and Melnick, J. L. 1949. Proc. SOC.Exptl. Biol. Med. 72, 46-50. Sweet, B. H., and Hilleman, M. T. 1960. Proc. SOC.Exptl. Biol. Med. 105, 420-427. Temin, H. M., and Rubin, H., 1959. Virology 8, 209-222. Tournier, P., Granboulan, N., and Bernhard, W. 1961. Compt. Rend. 105, 420-427. Trentin, J. J., Yale, Y., and Taylor, G. 1962. Science 137, 835. Tromans, W. J., and Horne, R. W. 1961. Virology 15, 1-7. Valentine, R. C., and Horne, R. S. 1962. I n “The Interpretation of Ultrastructure” (R. J. C. Harris, ed.), Vol. I, pp. 263-278. Academic Press, New York. Vasquez, C., and Tournier, P. 1962. Virology 17, 503-510. Vogt,, M., and Dulbecco, R. 1960. Proc. Natl. Acad. Sci. U. S. 46, 365-370. Waterson, A. P. 1962. Nature 193, 1163-1164. Watson, J. D. 1954. Biochim. Biopkys. Acta 13, 10-19. Wildy, P. 1962. Symp. SOC.Gen. Microbiol. 12, 145-163.

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Wildy, P., and Watson, D. H. 1062. Cold Spring Harbor Symp. Quant. Biol. 27, 25-47. Wildy, P.,Russell, W. C., and Horne, R. W. 1960a. Virology 12, 204-222. Wildy, P.,Stoker, M. G. P., Macpherson, I. A,, and Hoine, R. W. 1960b. Virology 11, 444-457. Williams, R. C. 1953. Cold Spring Harbor Symp. Quant. Biol. 18, 185-195. Williams, R. C. 1957. Ciba Found. Symp. Nature Viruses 1957, 19-38. Williams, R. C., Kass, S. J., and Knight, C. A. 1960. Virology 12, 48-58. Williams, M.G., Howatson, A. F., and Almeida, J. D. 1961. Nature 189, 895-897.

NUCLEAR PROTEINS OF NEOPLASTIC CELLS* Harris Busch and William J . Steele Department of Pharmocology. Boylor University College of Medicine. Houston. Texas

I . Introduction . . . . . . . . . . . . . . . I1. Isolation of Nuclei . . . . . . . . . . . . . A . Isolation of Nuclei of Liver Cells . . . . . . . . . B. Isolation of Nuclei of Tumors . . . . . . . . . . C . Isolation of Nuclei of Other Tissues . . . . . . . . D . Nonaqueous Techniques . . . . . . . . . . . I11. Isolation of Nuclear Components . . . . . . . . . . A . The Nucleoli . . . . . . . . . . . . . . B. The Nuclear Ribonucleoprotein Network . . . . . . . C . The Chromosomes . . . . . . . . . . . . D . Nuclear Ribosomes . . . . . . . . . . . . E . Deoxyribonucleoproteins . . . . . . . . . . . F. The Nucleolochromosomal Apparatus . . . . . . . . IV . Enzymes of the Nucleus . . . . . . . . . . . . A . DNA and RNA Nucleotidyl Transferase . . . . . . B. D P N Synthetase . . . . . . . . . . . . . C . Glycolytic Enzymes . . . . . . . . . . . . D . Citric Acid Cycle . . . . . . . . . . . . . E . Enzymes of Protein Synthesis . . . . . . . . . . F. Other Enzymes . . . . . . . . . . . . . V . The Acidic Nuclear Proteins . . . . . . . . . . . A . Quantity in Nuclei . . . . . . . . . . . . B. Nuclear Lipoproteins . . . . . . . . . . . . VI . Nuclear Globulins . . . . . . . . . . . . . A . Amino Acid Composition . . . . . . . . . . . VII . The Nuclear Ribonucleoproteins . . . . . . . . . . A. Ribosomal Proteins . . . . . . . . . . . . B. NHz-Terminal Amino Acids of the Saline-Soluble Proteins . . . C . Electrophoretic Studies . . . . . . . . . . . D. Cytonucleoproteins . . . . . . . . . . . . VIII . Acidic Proteins of the Deoxyribonucleoprotein Complex . . . . A . Effects of Antitumor Agents on Acidic Nuclear Proteins . . . B. Labeling of Acidic Nuclear Proteins . . . . . . . . C . Fractionation of Acidic Nuclear Proteins . . . . . . . D . Amino Acid Analysis . . . . . . . . . . . . E . NHz-Terminal Amino Acids . . . . . . . . . .

42 44 45 45 47 47 48 48 50 50

51 52 55 57 57 58 50 59 60 60 61 61 62 63 64 65 66 66 68 69 70 70 72 73 74 75

* The original studies by the authors reported in this manuscript were supported in part by grants from the Jane Coffin Childs Fund, the National Science Foundation, the U S. Public Health Service, the American Cancer Society, and the Anna Fuller Fund .

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IX. The Histones

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A. Nomenclature and Definitions . . . . . . . B. Physical Relationships of DNA and Histones . . . C. Functions of the Histones . . . . . . . . D. Origins of the Histones . . . . . . . . E. Chemistry of the Histones . . . . . . . F. Extraction of the Histones . . . . . . . G. Separation of the Histones . . . . . . . H. Purification of Histones of Tumors. . . . . . I. Structural Analysis of the N-Proline Histone (Fraction 2b) J. Metabolism of Histones . . . . . . . . K. RP2-L . . . . . . . . . . . . X. Discussion . . . . . . . . . . . . References . . . . . . . . . . . .

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I. introduction

Now that extensive evidence has been accumulated that the nucleic acids are both the repositories and transmitting agents for genetic information, the crucial question posed by biochemists interested in specialization of mammalian cells as well as oncologists is, “What are the types and the nature of the control mechanisms that define the segments of the genome that are permitted to function in individual cells?” Although experimental evidence is not yet satisfactory, it has been suggested that RNA (ribonucleic acid) may control gene expression (Jacob and Monod, 1961) or that histones may be involved (Stedman and Stedman, 1943). Recent studies by Yanagisawa (1962) have provided evidence that along with the accumulation of repressors in bacterial systems, there is also accumulation of RNA that may be the repressor substance for some facets of gene expression. At present, it is not clear how RNA might function to repress the activity of DNA (deoxyribonucleic acid) in the sense that quantitatively there is relatively little RNA associated with nucleoproteins, i.e., RNA comprises less than 207% of the amount of DNA in the nucleoprotein complex. One possibility that has been suggested is that newly formed RNA remains on the surface of DNA until it is released by appropriate substrates or proteins and then the DNA surface is derepressed. Although the evidence is not yet complete, it is difficult to state with any degree of certainty that suppression of the genome is either accounted for in part or entirely by combination of RNA with DNA. On a quantitative basis, however, the former would seem more likely if such mechanisms operate. The very large amount of protein in the deoxyribonucleoprotein complex, approximating 60% of the total mass, makes protein a more likely candidate for control of gene function, a t least on a quantitative basis. Although the nuclear protein composition of cells has not been studied in

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such controllable biological systems as stimulation of uterine function by estrogens and progesterone derivatives, or in the testis stimulated by testosterone, some studies have been made on the effects of hepatectomy on metabolism of nucleoproteins of liver cells. The high rate of biosynthesis of both histones and acidic nuclear proteins in these growing tissues as well as neoplastic cells has been associated in part with the rapid biosynthesis of DNA. However, even in resting cells, there is an appreciable, if not rapid, biosynthesis of all nuclear proteins. Such findings, along with the quantitative studies, have aroused much interest in the nuclear proteins recently, both in connection with their possible role in genetic control as well as their function in specialized and growing tissues. It has been apparent for some years that the proteins and enzymes of the nucleus were of transcendent importance both to the synthetic reactions involved in neoplastic cells and to the aberrations of growth that characterize these cells (Busch, 1962). However, it has been difficult to isolate and purify the proteins of the nucleus and it is only in recent years that enzymatic activities of some of these proteins have been elucidated. In the last few years, major advances have been made in the field of nuclear proteins both from the standpoint of methodology and in evaluation of the similarities and differences of the nuclear proteins of tumors and other tissues. Some of the major recent advances include the following: 1. Improved methods for isolation of nuclei and nucleoli (Busch e t al., 1959c; Chauveau e t al., 1956; Siebert, 1963; Muramatsu e t al., 1963b). 2. The development of methods for isolation and fractionation of the histones of the Walker tumor and other tissues (Johns, 1962; Johns and Butler, 1962; Johns e t al., 1960, 1961; Hnilica and Busch, 1963). 3. Amino acid analysis and amino terminal-amino acid analysis of the acidic proteins of the nucleus (Steele and Busch, 1963a). 4. Evidence for the presence in neoplastic tissues of a nuclear protein complex, coded as RP2-L, that differs in composition or chromatographic characteristics from proteins of other cells (Busch, 1962). 5. Evidence that histones may function in resting cells by suppression of the biosynthesis of RNA (Huang and Bonner, 1962; Allfrey e t al., 1963). 6 . Evidence that histones may suppress the synthesis of DNA. 7. The finding that DNA nucleotidyl transferases or polymerases are indeed nuclear, and not cytoplasmic, proteins (Keir e t al., 1962; Smellie, 1963). 8. Evidence that the alkylating agents and other antitumor drugs suppress the biosynthesis of the acidic nuclear proteins (Busch et al., 1959a, 1961; Nyhan, 1960; Strozier and Nyhan, 1962) and indeed, bind the acidic proteins to complexes of DNA and RNA (Steele, 1962).

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9. The presence of some cytoplasmic proteins, such as hemoglobin, in the nuclei of some species (Tooze and Davies, 1963). II. Isolation of Nuclei

I n studies on the nuclear proteins, it is obviously of particular importance that the nuclei be isolated in such a manner that ( a ) the nuclear proteins remain in the nucleus, and ( b ) the proteins of the cytoplasm do not enter the nucleus or contaminate the preparation. The manifold difficulties inherent in almost all procedures for isolation of nuclei have been emphasized in recent reviews by Dounce (1963) and Roodyn (1963), who concluded that the technology for isolating nuclei from either nontumor or tumor cells has not yet been perfected. Nuclei of tumor cells have not yet been isolated with morphology essentially identical to that of nuclei in tumor cells of tissue cultures, as seen by phase microscopy, although nuclei with virtually the same morphology as that seen in intact cells have been isolated from liver cells. Among the problems that exist in isolation of nuclei of tumors is the remarkably tight adherence of ribonucleoprotein strands a t the nucleocytoplasmic junction. Extensive losses of nuclear enzymes from cells treated with aqueous media for disruption of the cell have been noted by a number of authors. Unfortunately, however, i t is not possible a t present to be precisely certain which enzymes are normally present in the nucleus and which are not. The criteria generally used to evaluate the purity of nuclei are those set out earlier (Busch and Davis, 1958): ( a ) The nuclei should be anatomically identical with those of the whole cell; ( b ) the contents of the nuclei as they exist in the cell should all be present in the isolated product; (c) the isolated nuclei should not contain cytoplasmic constituents. The most critical problem in evaluation of these criteria for any method of preparation of nuclei is the definition of the normal state of nuclei in the whole cell. It is of particular importance to note that the nuclei in the whole cell are in a medium that has not yet been duplicated in isolation studies. It must be recognized that disruption of the cytoplasm undoubtedly influences the nuclei (Dounce, 1963 ; Roodyn, 1963). Moreover, the effects of nonaqueous systems (Behrens, 1932) and of cold temperature conditions remain to be ascertained (Siebert, 1963). Direct staining is of uncertain value in the definition of the structure of the nucleus, since staining immediately alters the affinity of nuclear components for ions that define the physical shape of nuclear structures such as the nuclear ribonucleoprotein (RNP) network. I n studies in this laboratory (Muramatsu e t al., 1963b) it was noted that Azure C actually has a higher affinity for the nucleoli than Ca++and indeed, could not be displaced from R N P receptors by Ca++.Electron microscopic studies on nuclear isolation procedures suffer from the fact that denaturation of

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nuclear components occurs and it is not yet certain which of the procedures employed for fixing and embedding the samples is least likely to produce artifacts in a given tissue sample. Apparent distortions of nuclear configuration may result (Smetana, and Busch, 1963) from either thin sectioning or may reflect the true state of cut nuclei. I n msny instances, however, electron microscopy provides the only true measure of contamination. A. ISOLATION OF NUCLEIOF LIVERCELLS One of the interesting facets of isolation of nuclei from cells hss been the general finding that isolation of nuclei from liver cells was very simple by comparison with isolation of nuclei from other tissues (Dounce et al., 1950, 1955; Busch et al., 1959c; Chauveau et al., 1956; Dounce, 1950, 1954, 1955, 1963; Hogeboom e t a,?., 1953; Roodyn, 1963; Wilbur and Anderson, 1951). Using any one of a variety of techniques for homogenization of liver masses (Hogeboom et al., 1953; Dounce, 1963) it is readily possible to largely remove the cytoplasmic components that adhere t o the nuclear membrane of liver cells. By differential centrifugation of the nuclei (Schneider and Hogeboom, 1956) and then by centrifugation of suspensions of the nuclei in force fields of 40,000 g for 30-60 minutes, the nuclei were sedimented from 2.0-2.2 M sucrose while other contaminants rose toward the top and side of the suspension medium (Chauveau et al., 1956). Nuclei from liver have been described as “clean,” meaning that there is little, if any, adherent cytoplasm. However, the microscopic examinations are generally made with the aid of phase microscopy and it is clear to all workers in the field that many cytoplasmic contaminants may not be visible by phase microscopy. One can visualize mitochondria, some of the cell components such as the nuclear membrane and the “membrane” of the cell surface as well as large “ribonucleoprotein strands” without any difficulty. However, it is quite difficult to visualize the smaller elements of the endoplasmic reticulum and, hence, one cannot be assured by means of such studies that the nuclei are truly devoid of cytoplasmic contamination. Indeed, electron microscopy, which is capable of providing more evidence that the nuclei have been satisfactorily isolated, still has limitations in the sense that the nuclei may be surrounded by amorphous masses of enzymes or proteins that have not been visualized by the techniques usually employed. With negative staining, some proteins may be found.

B. ISOLATION OF NUCLEIOF TUMORS The methods for isolation of liver nuclei simply do not provide uncontaminated nuclei from tumors. With the aid of phase microscopy, marked contamination of the nuclei with cytoplasmic materials is ap-

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HARRIS BUSCH AND WILLIAM J. S!l'EELE

parent (Takahashi et al., 1963). These contaminants may be masses of ribonucleoproteins visible by staining with Azure C or Toluidine Blue, or mitochondria adherent to cytoplasmic tags attached to the nuclei. I n some instances, the contamination is so gross that it is difficult to distinguish between the nuclear preparations and preparations of whole tumor cells with or without a cytoplasmic membrane. I n a number of cases, osmotic shock has been utilized as a means for improving the quality of the nuclear preparation (LettrB, 1951 ; Sauer et al., 1960; Samarina, 1961; Hudack and Brummond, 1961, 1963; Hudack et al., 1961). Preliminary soaking of the cells in either distilled water, one-quarter isotonic saline, or similar hypotonic solutions results in swelling of the cell membrane or its rupture and swelling of the cytoplasm. Under these conditions, the cells are said to be more susceptible to homogenization. One of the obvious disadvantages of this procedure is the fact that, as the cells are infiltrated with water, many nuclear components can also be extracted. Another problem is that lysozomes may rupture and hydrolytic enzymes may produce partial autolysis of the preparation. As Dounce has emphasized (1963), the technique of homogenization largely determines the nature of the product obtained. I n this sense, the study of the conditions under which the nucleus is freed from the surrounding cytoplasm has been particularly necessary. Of special iniportance in cytoplasmic adherence to the nucleus is the presence of divalent ions in the homogenizing medium. I n studies in this laboratory with the Walker tumor, it has been shown that in the presence of 0.003 to 0.005 M Ca++or Mg++in the medium, it is not possible to shear the cytoplasmic tags, largely ribonucleoproteins, from the nuclear membrane (Busch et al., 1963b,c). This is very different from results obtained with liver cells and has been ascribed (Smetana et al., 1963) to the large amount of ribonucleoproteins a t the nucleocytoplasmic junction in tumor cells. Apparently the amount of these ribonucleoproteins is much smaller in liver cells than in tumor cells. Accordingly, in studies in this laboratory with tumor cells, 0.25M sucrose without added calcium has been used as the homogenizing medium for isolation of tumor nuclei. The homogenizer employed in this laboratory for final shearing of the cytoplasm from nuclei of tumor cells is an all-glass unit, or a Kel-F pestle in glass test tube, with a pestle clearance of 0.003 inches. A larger pestle clearance results in the persistence of many tumor cells in the suspension, and a smaller pestle clearance results in damage to the nuclei. Many attempts have been made in this and in other laboratories to obtain nuclear preparations with a variety of homogenizing systems, including Poort's panker (Poort, 1957), the French press, and other procedures.

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but the results have not been satisfactory. The pestle clearance is not the only factor involved in the release of the nuclei from the cytoplasm, since there is apparently a factor of hydrodynamic shear that is dependent on the speed of the homogenizer and the viscosity of the medium. I n addition, there is an abrasive factor that is best illustrated by the case of the all-glass homogenizers, in which there is both glass-to-glass contact of the pestle and the tube as well as abrasion by tiny glass particles released by the rubbing of the pestle against the tube. I n the case of the Kel-F pestle, disruption of cells and freeing up of nuclei apparently is related to the shearing force of the hard pestle rubbing against the glass surface; this system works best with a motor providing a higher torque as well as a higher speed than the usual homogenizer motor. The process of release of the nuclei from the cytoplasm of tumor cells clearly involves a complex series of events.

C. ISOLATION OF NUCLEIOF OTHERTISSUES I n experiments on other tissues, the procedure for isolation of nuclei has been studied most extensively for the thymus (Allfrey, 1963a,b; Roof and Aub, 1960) and the results from different laboratories seem to vary. The thymocytes have cytoplasmic rims of varying sizes, some of which are extremely small. The question of cell viability as well as the effect of differences in technique have not been fully evaluated and precise data remain to be accumulated for recovery and quality of nuclei of the thymus and other tissues. I n the studies in this laboratory on the spleens, kidneys, lungs, and intestines of rats, satisfactory nuclei were not ohtained by any of the techniques employed. A number of workers (Lett$, 1951; Fisher and Harris, 1962; Hymer, 1963) have reported that detergents accelerate the disruption of ascites tumor cells and also enhance the purification of their nuclei. However, the biochemical characteristics of such nuclei remain to be elucidated. The nuclei of Hymer (1963) are reported to have biosynthetic activity, but their appearance suggests that some of the nuclear mass was removed in the extraction procedure. A procedure designed to block degradative reactions in nuclei is the phenol procedure designed by Georgiev et al. (1960a,b).

D. NONAQUEOUS TECHNIQUES The development of a technique for isolation of nuclei in organic solvents (Behrens, 1932) had the potential virtue of permitting the nuclear components soluble in aqueous media to remain in the nucleus and, moreover, to remain in specific loci comparable to those of the living cell (Allfrey et al., 1952). However, i t was apparent that the lipid barriers in the cell would be markedly altered by such a procedure, that lipopro-

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HARRIS BUSCH AND WILLIAM J. STEELE

teins might be removed, and that morphological alterations would occur in some structures. Siebert (1963) has suggested that the nonaqueous technique is of particular value in the isolation of nuclei for studies on enzyme localization. Using improved solvent systems and rapid freezing procedures for preparations of tissues, nuclear preparations have been obt,ained in high degrees of purity with active enzyme systems. Unfortunately, studies have not been carried out with this system on nuclei prepared from tumors. Ill. Isolation of Nuclear Components

It is outside the scope of this review to delineate in detail the many new findings regarding all of the components of the nucleus (Busch e t aZ., 1963d). Since the possibility exists that the individual substructures of the nucleus of tumor cells contain proteins important both to their structural and functional characteristics, it is essential to consider the kinds of structures from which the nuclear proteins may originate, as well as the present state of methodology for their isolation. A. THENUCLEOLI The largest single nuclear structures visible in nuclei of cells in interphase are the nucleoli, which have been studied in great detail by cytochemical techniques as well as by electron microscopy (Busch e t al., 1963c; Bernhard and Granboulan, 1963). Recently, two methods have been developed for isolation of nucleoli from cells of the Walker tumor and rat liver. I n one of these procedures, nuclei or nuclear preparations are sonicated (Monty e t al., 1956; Maggio e t al., 1963) in a medium of 0.25M sucrose or 0.88M sucrose containing 0.0033M CaCl, for intervals ranging from 25 to 40 seconds (Muramatsu e t al., 1963b). Under these conditions almost all of the nuclear membranes are broken and the nucleoli are released into the medium. They are then selectively sedimented in force fields of 100 to 25009 for purification. Another procedure employed (Busch e t d., 1963c) is compression of nuclei in a French press under pressures of 6000-8000 pounds/square inch and rapid decompression. I n this procedure, the selective “hardening” of the nucleoli with Ca++is of special importance and CaCl, was present in the suspending medium in a concentration of 0.005 M . The nuclei are broken and the nucleoli with some nucleolus-associated chromatin are released into the medium. Differential centrifugation through a sucrose gradient provides a nucleolar preparation in which the nucleoli are obtained in high yield from the Walker tumor and in a size and shape that are somewhat more similar to those seen by phase microscopy or by staining techniques in

NUCLEAR PROTEINS O F NEOPLASTIC! CELLS

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tumor cells than the nucleoli obtained by the procedure employing sonication. In any of the procedures employed in this laboratory, considerable destruction of the nucleoli occurred, but the percentage of nucleoli recovered has been as high as 60% on some occasions; the usual range is 20-30%. The nucleoli recovered from the isolation procedures in which sonication was used to disrupt the nuclei are rounded or oval, despite the fact that intracellularly, as seen by phase microscopy, they have odd, multicornered shapes (Kopac and Mateyko, 1958). The nucleoli isolated by high pressure compression and rapid decompression of nuclei have a morphological appearance very much the same as the nucleoli in situ; the yield is also higher than that obtained by sonication. This procedure provides a preparation that is chemically very similar to that obtained by the use of sonic oscillation (Desjardins et al., 1963). Although the compression procedure would appear to have a number of advantages over the procedure employing sonication, the method needs to be developed further. One of the complexities of isolation of nucleoli is the difficulty of purification of these dense bodies by differential centrifugation. Some of the nuclei have a density equivalent to that of the nucleoli, although the majority are considerably less dense. Accordingly, either virtually all of the nuclei must be broken before the nucleoli are subjected to centrifugation, or differential sedimentation employing low and then high force fields must be employed (Muramatsu e t al., 1963b). Under the latter conditions, the losses of nucleoli of large size are very substantial. The procedure of choice embodies the isolation of nucleoli by a single step centrifugation after disruption of the nuclei that is almost, if not quite, complete initially. The procedure utilizing compression and rapid decompression has resulted in products with a ratio of 4000 nucleoli to one nucleus (Desjardins et al., 1963). Many histochemical studies have been made on the nucleoli using special techniques for histones and other proteins. Alfert (1956-1958; Alfert and Geschwind, 1953) could not find evidence for the presence of histones in nucleoli and some electron microscopic evidence obtained in this laboratory (Smetana and Busch, 1963) have suggested that the fibers of DNA present in nucleoli may be essentially not covered by histones or other proteins. On the other hand, there is much protein in the nucleoli as evidenced by the fact that 85% of the dried, defatted nucleolar preparations consisted of protein. Although studies are preliminary a t the moment, these proteins appear to be part of the group of acidic nuclear proteins that will be described in more detail in a later section. Histo-

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HARRIS BUSCH AND WILLIAM J. STEELE

chemical studies have suggested that the nucleolar proteins are rich in groups (de Albertini, 1959; Hyde, 1961).

-SH

B. THENUCLEAR RIBONUCLEOPROTEIN NETWORK The nucleoli of tumor and liver cells appear to be part of a larger structural entity of the nucleus described as the nuclear ribonucleoprotein network (Smetana e t al., 1963). This network consists of an interlacing of many tubules, possibly containing some fibers and particles that are apparently in various stages of synthesis and transfer from the nucleoli to the cytoplasm through what are apparently defined nuclear channels. As much data now suggest, the nucleoli are probably the active sites of synthesis of the basic elements of the polysomal ribonucleoproteins (Busch e t al., 1963b). Because the channels of the nuclear RNP network are small and fragile and apparently run a tortuous course, electron microscopic studies have suggested that they were merely accumulations of dense bodies in the nucleus (Bernhard and Granboulan, 1963; Swift, 1963). Although the channels do not seem to have a well-defined membrane, any more than does the nucleolus, the particles appear to be mutually adherent in some sort of a loosely organized framework. Methods have not yet been developed for the isolation of the nuclear ribonucleoprotein network as a n organized entity. Rather, i t would appear that elements of the network are extracted either as part of the “nuclear ribosomes” or with elements of the nucleoli. C. THECHROMOSOMES

It is of paramount importance to the study of the over-all metabolism and functions of the nucleus of the cancer cell that methods be developed not only for the isolation of chromosomes, but also for their separation and biochemical analysis. Although this goal has been a n obvious one (Mirsky and Ris, 1947-1948a,b), only recently have Somers et al. (1963) succeeded in isolating a preparation that contained morphologically identifiable elements. Using either acidic or hypotonic media, chromosomes of Chinese hamster fibroblasts were released from nuclear preparations obtained from cells in metaphase that were incubated with colchicine in tissue culture systems. It was found that media containing various sucrose M MgC1, and CaC1, were satisfactory for concentrations and 5 x preservation of the morphology of the chromosomes. The chromosomes were sedimented in force fields of 1500g. Although purification of the chromosomes from the products obtained in the course of the procedures employed is a n obvious necessity, the basic methodology for isolation of chromosomes would seem to be a t hand and available for further analytical procedures.

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51

The presence of such impurities as unbroken nuclei and segments of broken nuclei in such preparations limits their usefulness for the studies of the proteins of the chromosomes. It seems likely that improved isolation procedures will permit more satisfactory biochemical studies on the chromosomes in the future. Previous reports have appeared that have indicated the authors’ belief that they had isolated chromosomes (Mirsky and Ris, 1947-1948a,b, 1949, 1950-1951). These reports suggested that the chromosomes had a composition similar to that of the chromatin isolated by usual procedures indicated above and also that there was some residual material that accounted for about 8-100/0 of the total material. As one reviews the experimental study and the marked lack of either form or structure of the material isolated, one is not surprised a t the quick and essentially unanswered questioning of these reports by Lamb (1949), who suggested that the material isolated by the above workers was really chromatin broken up in the Waring Blendor used to “homogenize” the nuclei.

D. NUCLEAR RIBOSOMES The nuclear ribosomes have been obtained by extraction of nuclei with isotonic saline solutions or by extraction with dilute Tris buffers (Frenster e t al., 1960; Wang, 1960, 1963a,b; Allfrey, 1963a,b). Froin all of the studies made thus far, these ribosomes are identical with the cytoplasmic ribosomes studied by other authors in either plants, bacteria, or the cytoplasm of mammalian cells in their requirements for Mg”, ATP, etc., for incorporation of labeled amino acids into proteins. The electron micrographs of the nuclear ribosomes have established that those isolated from calf thymus nuclei are structurally similar to cytoplasmic ribosomes of other tissues. Most of the studies of the nuclear ribosomes have been made with nuclei isolated from calf thymus. Other studies are in progress on isolation of similar particles from the nuclei, with buffered saline solutions or Tris buffers (Logan and Davidson, 1957; Allfrey, 1963a,b; Wang, 1963a,b). I n part, the difficulty experienced by some workers in extraction of the nuclear ribosomes from tissues other than calf thymus may be due to the presence of the ribosomes or their precursors in the nuclear ribonucleoprotein network. The channels of this network are apparently filled with tubular structures that may be dispersed to form the RNP particles. If these structures were easily broken, the ribosomes formed could be reisolated as part of the nuclear ribosome complex. A difference in friability of the nuclear RNP network could explain the ready isolation of nuclear ribosomes froin calf thymus and the difficulties in similar isolations from other tissues.

52

HARRIS B U S C H AND WILLIAM J . STEELE

There are alternative possibilities to the question of the presence of R N P particles of the type described by Allfrey’s group and by Wang in tissues other than thymus. These include the possibility that the nuclei of the thymus are similar to those of the tumor cells in the very strong adherence of the cytoplasmic R N P particles to the nuclear membranes. If such was the case, the so-called nuclear ribosomes may merely be cytoplasmic ribosomes that have not been freed by the homogenization procedures. Secondly, the possibility exists that the presence of the R N P particles in the nuclei of the calf thymus represents a case of nuclear specialization that is not duplicated in many other tissues. The chemical identity or difference of the proteins or the nucleic acids of the nuclear and cytoplasmic ribosomes of calf thymus to one another or to those of other tissues, has not yet been investigated or reported. The RNA content of the ribonucleoprotein extracted with Tris buffer from the nuclear preparations of the Walker tumor was 26% (Steele and Busch, 1963a) , a value very similar to that reported by Pogo et al. (1962). It has been found that Tris extracts not only components of the nuclear ribonucleoprotein network, but also extracts many of the components of the nucleoli. The proteins extracted in these preparations appear to be largely of the group classified as the acidic nuclear proteins (see Section V ) .

E. DEOXYRIBONUCLEOPROTEINS As defined by over-all protein content, the nuclear component with the greatest amount of protein is the deoxyribonucleoprotein complex (Table I ) . All of the values presented in Table I1 have been obtained with nuclei treated with saline solutions for extraction of proteins (Table I ) . Since, in the procedure employed, very soluble proteins of the nuclei are extracted in the homogenization procedure, the values will probably be revised downward in the future. The fraction extracted with 2 M NaCl contains virtually all of the deoxyribonucleoprotein (DNP) of the nucleus (Table 11). However, it requires a t least two extractions to carry this procedure to completion and the possibility exists that some of the other nuclear components are also extracted with 2 M NaCl. This procedure for extraction of deoxyribonucleoproteins has been employed with reasonable success since it was introduced by Mirsky and Pollister (1942, 1946; see also Mirsky, 1947, 195CL-1951, 1956; Samarina, 1961). Studies of Crampton et al. ( 1954a,b, 1957) have shown that the solubility of the DNP is such that employment of salt solutions of this concentrat,ion is sufficient to ensure total extraction of the DNP. Less certainty attaches to the use of 1 M NaCl since this is a borderline concentration and if the preparation contains a significant amount of a solution with

63

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

TABLE I CHEMICAL PROCEDURE FOR ISOLATION OF NUCLEAR PROTEIN FRACTIONS Isolated nuclei

I

0.14 M NaCl, 2 times I L

L

7

Preci itate 0.1 M Tris pH 7.6

“Nuclear sap” proteins I

I

J

J

Precipitate I 2.0 M NaCl

“Nuclear sap” proteins

1

Deoxyribonucleoproteins-1 1

7

Preci itate 2.0 M NaCl

0.5 N H2SOa;extract twice with 0.2 N HZSOa 8 L 1 .1 J. Deoxyribonucleoproteins-2 Precipitate Histones Precipitate I 0.05 N NaOH, 3 times 6% PAS-phenol; phenol extract, 3 times I .l 1 c cI “Residual proteins” Acidic ribonucleoproteins aqueous phase phenol phases 5% TCA, 9O”C., methanol 2 times ___ (2volumes) 1

I

I

1 DNA

.1 Phenol-insoluble proteins

I

1 Acid-insoluble

RNA products

proteins

TABLE I1

FRACTIONATION OF ISOLATED NUCLEIOF LIVERAND WALKER256 CARCINOS.ARCOMA AND NUCLEICACID CONTENT OF INDIVIDUAL FRACTIONS~ Liver Fraction 0 . 1 4 M NaCl 0 . 1 0 M Tris 2.0 M NaCl (1st extract) 2 . 0 M NaCl (2nd extract) 0 . 0 5 N NaOH Residual

Walker tumor

Recovery

DNA

RNA

Recovery

DNA

RNA

17.0 5.3 54.0

1.46 6.80 31.1

3.40 10.9 5.16

12.0 14.0 54.0

0.14 1.47 27.2

5.02 26.1 4.80

10.0

12.2

5.12

3.0

8.4

5.80

8.91 0.57

12.0 2.0

0.29

12.6 0.33

5.6 2.2

0.30 0.0

0.0

The values for recovery are percentages of the lipid-free dry weight of the isolated nucleus. The values for nucleic acids are percentages of the dry weight of the individual fractions. The data represent averages of duplicate determinations on preparations of three to five separate experiments. The total recovery of dry weight in the nuclear fractions averaged 94% for liver and 97’% for Walker tumor.

54

HARRIS BUSCH AND WILLIAM J. STFXLE

a low salt concentration or water, much of the D N P may be left in the unextracted material (Klyszejko and Khouvine, 1960; Holbrook et al., 1962; Evans e t al., 1962). It is a matter of considerable uncertainty whether the D N P extracted with 2 M NaCl is identical to the chromatin, either in the spireme form of the resting cell or in the condensed chromosomes of the metaphase stage of mitosis. Although one can be certain that essential elements of the chromatin are extracted from the spireme of the resting or nondividing cell, experiments on the extraction of the chromosomes have not been reported. It has been suggested that extraction with 2 M NaCl may “denature” the deoxyribonucleoprotein or extract too much with the DNA. An alternative procedure is to extract the nuclei with water and precipitate the DNP with 0.15 M NaCll. It now appears that the aqueous extraction procedure produces a product that more closely resembles some of the D N P in its physiological state than the product extracted with 2 M NaCl (Kawade, 1957; Zubay and Doty, 1959). Although, theoretically, isolation of nuclei should precede the extraction of the nuclear proteins or deoxyribonucleoproteins, in many instances the nuclei have been “isolated” by a “once-over-lightly” procedure such as treatment with the ball-mill in a dilute citric acid solution (Davison and Butler, 1954, 1956) and preparations have been washed, with or without blending, a number of times with saline solution (0.15 M NaCl). Under these conditions, a preparation referred to as deoxyribonucleoprotein has been obtained, but this preparation is really simply the residue of the cell or LLnuclearpreparation” that contains the saline-insoluble components. Electron microscopic studies carried out in this laboratory The chemical relationship between deoxyribonucleoproteins in the nucleus and materials obtained by extraction techniques has not been clarified. There are differences between deoxyribonucleoproteins extracted with water and “recombined nucleoproteins” obtained by extraction with high salt concentration and precipitation on dilution with water. Relatively few experiments have been done on fractionation of the crude aqueous extract of nucleoprotein. However, some workers have observed that water extraction provides two fractions of nucleoprotein (Dounce, 1955; Fredericq, 1962; Zubay and Doty, 1959)-a soluble component and a gel component that are separable by high speed centrifuging. The identity of the proteins associated with these fractions remains t o be determined. Histones are a main protein component of the soluble nucleoprotein (Crampton, 1957), but the protein components of the gel nucleoprotein have not been extensively studied. I n different tissues, different amounts of the nucleoprotein may be present. I t is possible that the nonhistone protein component of the DNP complex (the acidic deoxyribonucleoprotein) may be an important part of the gel form. It is also possible that tissues engaged in the rapid synthesis of protein may possess a larger proportion of the DNA in combination with acidic proteins (perhaps as depressed DNA) than tissues, such as the thymus and sperm that have large amounts of histone or protamine in combination with DNA.

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

55

(Smetana and Busch, 1963) and in others (Zbarsky et al., 1962) have demonstrated that much of the “ground substance” of the nucleus remains unaffected by this treatment. Moreover, the nucleolus is not very much affected by this extraction procedure. Nonetheless, it should be noted that this procedure has provided the histones with the highest degrees of purity yet obtained (see Section IX,H). Preliminary isolation of nuclei has also been followed by direct acid extraction for obtaining the “acid-soluble nuclear proteins” that probably include the histones, although other components also accompany the histones (Phillips, 1961; Busch e t al., 1963a,d), On chromatographic analysis of the histones, the fractions obtained from the deoxyribonucleoproteins differed from the fractions obtained from those of the “nuclear sap” on chromatography and in quantity (Busch et al., 1963a). The “acidsoluble proteins” of the nuclear sap adhere more tightly to the CMC (carboxymethylcellulose) columns and also are present in much larger amount in the nuclei of most tissues. In essence, these proteins probably represent the acid-soluble components of the aqueous phase of the nuclear sap, the nuclear ribonucleoproteins and the acid-soluble components of the nucleoli and the nuclear ribonucleoprotein network. Another procedure employed occasionally for the isolation of the deoxyribonucleoproteins (Hammarsten, 1924) is extraction of nuclei or other preparations with distilled water. The high degree of solubility of deoxyribonucleoproteins in distilled water is exemplified by the fact (Crampton e t al., 1954a,b, 1957) that about 95% of the total cellular deoxyribonucleoprotein is extractable with water. The nature of the remainder of the D N P has not been investigated although it would be of interest to ascertain whether the turnovers, base composition, and protein characteristics were the same for the insoluble as the soluble group.

F. THENUCLEOLOCHROMOSOMAL APPARATUS Some reports in the literature have stated that the nucleolochromosoma1 apparatus is the residue remaining after initial extraction of the nuclei with 0.15M NaCl followed by extraction with 1 or 2 M NaCl (Zbarsky and Georgiev, 1959). When saline solutions are employed without the concurrent use of Tris buff ers, the “nuclear ribonucleoprotein network” is best demonstrated. The extraction with 2 M NaCl removes almost all of the basic nuclear proteins. Electron microscopic analysis shows that elements of the fibrils of the nuclear ribonucleoprotein network as well as the substructures of the nucleoli are not removed by extraction with saline solutions and hence the “nucleolochromosomal apparatus” would appear to have much in common with the nuclear ribonucleoprotein network. Whether elements of the chromosomes other than

56

HARRIS BUSCH AND WILLIAM J. STEELE

DNA and histones remain after extraction has been made with 2 M NaCl is not yet clear and i t is essential that further studies be made on this point. Following the extraction of nuclei with 2 M NaCl, a residue remains that is generally extracted with dilute alkali to obtain a fraction referred to as the acidic proteins (Table I). The acidic proteins of the residue and the acidic proteins of the chromatin have remarkably similar amino acid analyses and amino terminal analyses (Steele and Busch, 1963a). The proteins of the nucleolus as well as the ribosomes also have a similar over-all amino acid analysis and amino terminal analysis (see Section

VIII,D,E). The so-called “residual fraction,” about which there is much lore and relatively little data, has been claimed to be the nuclear membrane and other things. However, recent studies of Steele and Busch (1963b), have established that the so-called residual fraction is probably a mixture of denatured and insoluble acidic proteins and collagen that arises from TABLE I11

A SUMMARY

O F PROCEDURES FOR ISOLATION O F

NUCLEAR COMPONENTS

A. Following initial isolation of nuclei 1. Isolation of nucleoli: Either by sonication of nuclei prepared in 0.0033-0.005 M CaC12 and 0.25 M sucrose or by compression and rapid decompression of nuclear preparations in the French pressure cell; purification by differential centrifugation 2. Isolation of chromosomes: By gentle procedures employing cells in metaphase; aspiration and ejection from syringes followed by centrifugation in sucrose solutions 0.0005 M with respect to Mg++ and Ca++

3. Isolation of nuclear ribonucleoproteins: Extraction from nuclei of calf thymus by 0.15 M NaCl or 0.01 M Tris buffers containing 0.001 M MgClz followed by differential centrifugation 4. Isolation of deoxyribonucleoproteins: Either by extraction of nuclei with 2 M NaCl

or with water followed by precipitation of the deoxyribonucleoproteins from solutions 0.104.20 M with respect to NaCl

5. Nucleolochromosomal appaTatus: A residual fraction obtained after successive extraction of nuclei with 0.15 M NaCl and 2 M NaC1; bears many similarities to the nuclear ribonucleoprotein network, but probably contains other components

B. Without initiaI isolation of nuclei 1. Nucleoli: Obtained from tumors without extensive nuclear preparation inasmuch as i t is not possible to remove cytoplasmic components in the presence of calcium ions a t concentrations required to maintain the integrity of the nucIeoli. Techniques are the same as in A,1, above 2. Deoxyribonucleoproteins: Obtained as a residue following prolonged treatment of nuclear preparations with isotonic saline solutions

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

57

concentration of tissue contaminants in the course of the extraction procedures. Whether contractile elements of the spindle are present is not known (Zbarsky and Perevoschchikova, 1951a,b). From the procedures already presented, it is apparent that a number of methods exist for fractionation of the nuclear proteins, both by procedures employing morphological criteria and by procedures employing direct extraction methods, as shown in Table 111. IV. Enzymes of the Nucleus

Although many studies have been made on the enzymes of the nucleus (Stern et al., 1952; Schneider and Hogeboom, 1956; Siebert, 1963; Siebert and Smellie, 1957; Siebert et al., 1953), most produced more questions than answers. Among the important questions were (1) Were the enzymes really in the nucleus or were they simply contaminants that resulted from adherence of cytoplasmic components to the nuclear membranes? (2) Were some enzymes important to the functions of the nucleus wen though they were present as a relatively small proportion of the total cellular concentration? ( 3 ) Were enzymes of the nucleus that were apparently isozymes of cytoplasmic enzymes the same proteins or were there differences in either protein structure or enzymatic properties? One of the important findings that provided a kind of marker for nuclear enzymatic activities was that of Hogeboom and Schneider (1952) , who found that DPN-synthetase was localized in the nucleus of liver cells. One important consideration is that some nuclear proteins are extractable in the aqueous media employed for homogenization of cells. Although relatively little is known of these substances, the recent studies of Keir e t al. (1962) and Smellie (1963) have established that the important enzyme DNA polymerase or DNA-nucleotidyl transferase is so readily extractable from the nucleus that it was initially thought to be a cytoplasmic enzyme. It seems likely that many other proteins and enzymcs normally present in the nucleus are extracted by the sucrose, citric acid, or glycerin solutions commonly used for preparation of the nuclei. A. DNA

AND

RNA NUCLEOTIDYL TRANSFERASE

It may now be presumed that many of the enzymes of the nucleus would be extracted either in the dilute extraction media used for the initial preparation of the nuclei or by 0.14M NaC1. Although the enzymes of the nucleus are still far from adequately defined, it is apparent that messenger RNA (m-RNA) formation is dependent upon the activity of the RNA polymerases or nucleotidyl transferases, and further that the nuclei of a number of tissues, such as regenerating liver, are rich in DNA nucleotidyl transferases (Bcssman et al., 1958). The evidence provided

58

HARRIS BUSCH AND WILLIAM J. STEELE

by Keir et al., (1962) and Smellie (1963) shows that the evidence initially presented for the cytoplasmic localization of the DNA nucleotidyl transferase was based upon procedures that employed aqueous media for preparation of the nuclei. When the Behrens technique, as modified by Siebert (1963), was utilized, the activity of the enzyme was found to be largely localized to the nucleus. Although the localization of the DNA polymerase or nucleotidyl transferase has been something of a mystery (Prescott et al., 1962), it seems that there is no doubt that the RNA polymerase or nucleotidyl transferase is functionally localized to the nucleus in mammalian cells, if for no other reason than that the enzyme requires either DNA or highmolecular-weight RNA for its templates. Since these templates are limited to the nucleus and there is no evidence that RNA synthesis proceeds in any other part of the cell (Prescott, 1961; Busch et al., 196313) any of the enzymes for RNA polymerization found in the cytoplasm would necessarily be either precursor enzyme or nonfunctional protein. One of the critical questions emerging from the studies of Smellie (1963), working in Davidson’s group, is that of the site of synthesis of TTP (thymidine triphosphate) and the factors that set off this synthesis. In their view, i t is most likely that this substrate is the rate-limiting factor for biosynthesis of DNA, although this view is by no means generally accepted (Busch et al., 1963d). Apparently two pathways are involved in the biosynthesis of TTP, one involving transformylation of deoxyuridylic acid to form T M P (the thymidylic pathway) and the other, a kinase reaction involving the direct phosphorylation of thymidine. Another possibility for the format,ion of T M P is the deamination of deoxycytidylic acid, but the precise quantitative relationships of these pathways for formation of TMP are not yet clear. In any event, either a one- or two-step transphosphorylation or pyrophosphorylation of T M P occurs and this reaction is apparently the one that is rate-limiting for the availability of TTP (Smellie, 1963). Parenthetically, i t should be noted that these workers have found a nuclease t o be present in preparations of liver nucleotidyl transferases that apparently blocks the synthesis of DNA, or indeed may destroy the product before the preparation has time to accumulate much of the product.

B. D P N SYNTHETASE Perhaps the first enzyme for which there was universaI agreement for localization in the nucleus is one that catalyzes the formation of diphosphopyridine nucleotide (DPN) from the two precursors, ATP and pyridine mononucleotide (Hogeboom and Schneider, 1952, 1955’1. It now appears that one of the main factors for the persistence of this enzyme

NUCLEAR PROTEINS O F NEOPLASTIC CELLS

59

in the nucleus was that the enzyme is quite insoluble in any of the conventional aqueous media used for isolation of the nuclei (Schneider, 1963). This enzyme is also very stable and resists destruction a t a time when other more sensitive enzymes are being denatured. I n a sense, it can be used as an enzymatic standard for the preparation of the nuclear enzymes or proteins.

C . GLYCOLYTIC ENZYMES Siebert (1963) has carried out a number of studies on nuclear glycolytic enzymes empolying modifications of the Behrens nonaqueous technique (1932) for isolation of nuclei. He has found a variety of enzymes in the nuclei of liver cells that have not been found by other workers using conventional aqueous media. The over-all rates of glycolysis have been studied (Siebert, 1963) and have been found to be similar in the nucleus and in the whole homogenate of the cells. Although such data are not construed as representing a high degree of nuclear concentration of the enzymes of glycolysis, they certainly are strongly suggestive that the nucleus can carry out glycolytic reactions for production of energy. Evidence has also been presented that glycolytic enzymes and substrates are present in calf thymus nuclear preparations (Allfrey, 1963b). I n an attempt to answer the critical question whether the nuclear enzymes are the same as, or are different from, those of the cytoplasm, Siebert and Hannover (1963) have studied a variety of characteristics of the nuclear and the cytoplasmic lactic dehydrogenases of ra t liver cells. By paper electrophoresis, thermal denaturation, reduction of a-ketobutyrate, D P N reduction, or inhibition of the reaction by sulfite, significant differences were not found between the nuclear and cytoplasmic enzymes. On the other hand, the rates of reduction of D P N analogs, temperaturedependent changes of kinetic properties and the pH-dependent binding of pyruvate as studied with substrate inhibitions were different for the nuclear and cytoplasmic enzymes. This point is one of considerable importance since i t suggests that there is not a free exchange or identity of protein structures of the lactic dehydrogenases of the nuclei and the cytoplasm of liver cells. One possibility is that the nuclear or cytoplasmic enzymes are linked to specific cellular components that are different in different intracellular locations. D. CITRICACID CYCLE The presence in nuclei obtained by the Behrens technique (Siebert, 1963) of such enzymes as malic dehydrogenase, isocitric dehydrogenase, and glutamic dehydrogenase suggests that either these enzymes are formed in nuclei, that mitochondria1 precursors are formed in nuclei, or

60

HARRIS BUSCH AND WILLIAM J . STEELE

that the procedure somehow permits the leaching of such enzymes from mitochondria and absorption by nuclei. Using this technique, Siebert (1963) has not been able to find a-ketoglutarate oxidase or succinic dehydrogenase in the nuclear preparations. Evidence for the presence in nuclei of enzymes for hydrogen transport (Stern and Timonen, 1954) and of malic and isocitric dehydrogenases as well as a-ketoglutaric acid has been presented (Allfrey, 1963b) in studies on calf thymus nuclei.

E. ENZYMES OF PROTEIN SYNTHESIS For synthesis of proteins, ATP is an essential component of the amino acid-activating system as well as for other energy-requiring reactions. In studies on nuclear preparations from calf thymus, Allfrey (196313) has shown that rapid synthesis of ATP occurs and apparently by reactions that differ in a number of respects from the synthetic reactions that occur in mitochondria. Evidence has been presented (Webster, 1960; Rendi, 1960; Allfrey e t al., 1955a,b, 1957, 1963) that amino acid-activating enzymes are present in the nuclear preparations of calf thymus as well as enzymes for the transfer of amino acids from the amino acyl-AMP complexes to s- or t-RNA (soluble or transfer RNA) (Hopkins, 1959). I n addition to the evidence that has suggested that the amino acids are incorporated into proteins on the nuclear ribosomes, recent studies have shown that the labeled protein shifts in its ribosomal localization from the core to the coat in the course of the biosynthetic reactions. F. OTHERENZYMES

A variety of hydrolytic enzymes such as p-glucuronidase, acid phosphatase, glucose-6-phosphatase, triphosphatases, adenosine deaminases, esterases, DNase's, 5'-nucleotidases, phosphatases, and some special enzymes such as tyrosinase have been reported to be present in nuclear preparations by Siebert (1963). The precise significance of most of these enzymes for nuclear function is not clear a t the present time. Since the nucleus is a specialized organelle that is presumably in close contact with the remainder of the cell, there is no reason to assume a priori that most enzymes would not be found within the nucleus. The chief question is whether such enzymes are important ta the functional role of the nucleus. Although progress is being made in this field, improved methodology has a t least clarified the point that enzymes for synthesis of some of the nucleotides and for synthesis of nucleic acids are definitely present in the nucleus and are related to the role of the nucleus in synthesis of the macromolecules essential for transfer of genetic information from mother to daughter cell as well as transfer of genetic information to the remainder of the cell.

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

61

From the standpoint of the composition of the enzymes of the nuclei, it is regrettable that, until the present time, none of the enzymes has been isolated in sufficient amounts or purity to determine either the amino acid composition, NH,-terminal amino acids, or other structural information. Such data would help to clarify the relationships between the proteins isolated by the standard fractionation techniques from the nuclear sap, the group soluble in 0.14M NaCl, and the remainder of the nuclear acidic proteins to be discussed below. V. The Acidic Nuclear Proteins

The acidic nuclear proteins are found as residues after the extraction of the acid-soluble proteins from any of the fractions isolated by salt solutions. The relationship of the morphology of the structures from which these proteins are isolated to the chemical nature and functions of these proteins is essentially a mystery a t the present time. The problems of dealing with these rather insoluble proteins by methods currently available have been so great that few workers have had the courage to attack the problem of their isolation or composition. A. QUANTITYIN

NUCLEI

The results of early studies on the acidic proteins were couched in rather metaphysical terms. Stedman and Stedman (1943; Stedman, 1944) suggested that their (‘chromosomin” might be the genetic material of the cell and indeed that the DNA was rather inert; of course, the concept that DNA was a storage material was widely held a t that time. “Chromosoinin” was isolated from the nuclear residues after previous extractions with acid and was initially described as a fraction insoluble in acid, but slowly dissolved in dilute NaOH. I n addition to the presence of both acidic and basic amino acids, “chromosomin” was reported to contain tryptophan. Later studies revealed that relatively less L‘chromosominll was present in the nucleus than had been thought to be present initially (Stedman and Stedman, 1947) and only about 30% of the nuclear dry weight was (Lchromosomin.” 1. Tr.Pr.

Mirsky and Pollister (1942, 1946) may have been dealing with a somewhat similar protein complex in their studies on “chromosin”; both preparations contained phosphorus and tryptophan. However, the ‘(chromosin” was not an acid-insoluble residue, but rather the total protein extracted with 2 M NaC1, and contained histones and DNA, as well as the “residua1 protein,” named by Mirsky and Pollister “Tr.Pr.” because of the high content of tryptophan.

62

HARRIS BUSCH AND WILLIAM J. STEELE

Microscopic studies of the Tr.Pr. obtained after extraction of DNA and histone showed that it was a coiled thread that was part of the structure of their “interphase chromosomes.” The Tr.Pr. fraction comprised &-lo% of the “chromosomal” or deoxyribonucleoprotein mass and the content of tryptophan was actually quite low, i.e., about 1.36%. Mirsky and Ris (1950-1951) suggested that the “residual” or Tr.Pr. protein was the basic structural protein of the chromosome and that these proteins formed a thread around which the chromosomal structure was condensed. A similar conclusion was reached by Bernstein and Mazia (1957) who found that sea urchin sperm contain considerable nonliistone protein totaling 40% of the dry weight of the nuclei. I n preparations of the mitotic apparatus, Mazia (1954) and his colleagues (1952) found a group of alkali-soluble proteins that were precipitable by bringing the pH to 6. Studies on amino acid composition of these proteins have not indicated characteristic features, since glycine, serine, argininc, lysine, glutamic acid, and other amino acids were present, but quantitative data were not presented. Moreover, Mazia and his colleagues also found that RNA, but no DNA, was present in these proteins.

2. Amount in Tumors One interesting feature of these proteins was their relatively high concentration in tumors. Studies by Edgar and Ellen Stedman (1943, 1944) and later by Debov (1951, 1953) showed that the percentage of the dry weight of nuclear preparations composed of these proteins was in fact higher in both tumors and regenerating livers than in other tissues. The lack of purity of the nuclear preparations (Georgiev et al., 1960a,b) indicates the need for re-evaluation of such data on nuclei isolated by improved techniques. B. NUCLEAR LIPOPROTEINS The relationship of the “residual proteins,” Tr.Pr., “chromosoiiiin,” and the “chroniosins” to the other nuclear protein fractions has remained in doubt. There is some question a t the present time as to the relationship of these proteins to the nuclear lipoproteins and the nuclear globulins. Using the technique of Behrens (1932) for isolation of nuclei, Mayer and Gulick (1942) found a sulfur-rich nuclear protein that had an isoelectric point of about 5.3, a value that is similar to the isoelectric point of many soluble proteins. Chargaff (1949) and later Carver and Thomas (1952) found lipoproteins in nuclei of calf thymus. The isoelectric points of these proteins were 4.7 and 4.8 in the nuclear lipoprotein fractions obtained from calf thymus and liver, respectively. The possibility that these proteins were similar to the residual protein of the “chromosomin” lias

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

63

been suggested by Engbring and Laskowski (1953) , who noted that their alkali-soluble protein contained tryptophan. A protein fraction with a high lipid content, i.e., about 20-30%, was isolated from sperm heads. The protein comprised about 20% of the dry weight of the sperm head. Dallam (1955) noted that lipoproteins could be extracted from the nuclei of sperm heads in the course of fractionation by salt solutions; he found that the lipoprotein was alkali-soluble. The lipid content of the lipoprotein was 27%) of the total mass and a variety of lipids was present, including cholesterol and phospholipids. Wang et al. (1950, 1953) also found that lipoproteins could be extracted from nuclei and, like that of other authors, their protein had an isoelectric point of approximately 6.0. Their lipoprotein contained cholesterol and phospholipid and was composed of a variety of amino acids of which arginine comprised 5.7%. Small amounts of tryptophan and tyrosine, i.e., 2.9 and 3.20/0, respectively, were also found. The possibility that these proteins might be either related to or a part of the chromosomin complex was suggested, but in the light of present findings (cf. below) this possibility seems unlikely. It seems more likely that these authors may have been isolating lipoproteins of the ribosome or polysome precursors. Recently, Levin and Thomas (1961) have presented evidence that lipoproteins of the nuclei and mitochondria were similar, but differed from those of the microsomal fraction. VI. Nuclear Globulins

Much interest has been generated by reports that there were globulinlike proteins in the nucleus and recently by evidence that globulins in tumor nuclei are abnormal (Mantieva and Belousov, 1962). I n essence, globulins are defined as simple proteins, that is, they consist only of amino acids. Second, they are soluble in dilute salt solutions and are precipitated a t high salt concentration. I n addition, they are supposed to be coagulable by heat. I n studies on globulins, the dilute solutions of salt usually employed for extraction are 5 or 10% solutions of NaCl, i.e., about 1 or 2 M . Kirkham and Thomas (1953) extracted nuclei with saline solutions and obtained a protein fraction that was soluble in 0.14M NaCl and was precipitable by half-saturation with (NH,) ,SO,. Using varying concentrations of salt, Dallam (1955) found that there was a globulin fraction and an albumin-globulin fraction, as well as the nucleoprotein fractions and the lipoprotein fraction mentioned above. The proteins that were believed to be globulins and were originally found by Kirkham and Thomas (1953) have come to have much significance, inasmuch as they contain the nucTear ribonucleopfoteins (-Wang, 1960). Their fractions were probably different from the fractions obtained by Zbarsky and

64

HARRIS BUSCH AND WILLIAM J. STEELE

Debov (1948) whose proteins were more like those called “chromosomin” by the Stedmans; the fractions obtained by Zbarsky and Debov (1948) were soluble in alkali after the nuclei were treated with 1 M NaCl. This fraction was similar to the other fractions only in insolubility a t pH 5.3, like the corresponding proteins isolated by Mazia and Dan (1952), Mayer and Gulick (1942), Kirkham and Thomas (1953), and Wang et al. (1950, 1953). One interesting point with regard to the globulins is their solubility in dilute acid, a feature that results in their extraction from nuclei in combination with histones. The globulins are said to precipitate in 0.33A4 HgSO, and the histones do not. The precipitation of histones from dilute salt solutions is said to result in their association with the globulins in difficulty separable complexes (Dounce and Umana, 1962). A. AMINO ACID COMPOSITION In studies on the amino acid composition of the nuclear globulins of the Walker tumor and liver, the globulins were precipitated from the saline supernate by dialysis of the solution against distilled water overTABLE IV AMINOACID COMPOSITION O F NUCLEAR PROTEINS SOLUBLEIN 0.14 M NACL” Nuclear ribosomes Amino acid

Nuclear sap

Microsomes

Calf thymusb Liver Tumor Liver Tumor Liver Tumor Liver Tumor

Alanine Arginine Asparticacid Cystine Glutamic acid G1ycin e Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine a

Globulins

7.4 7.8 9.1 0.5 10.3 9.3 2.5 5.2 8.7 10.0 2.0 3.6 4.8 4.5 4.8 3.0 6.6

7.5 5.6 9.8 11.2 10.8 2.3 3.9 7.8 8.7 1.4 4.3 5.5 6.7 4.8 a.5 5.1

7.9 6.3 9.6

-

11.5 9.7 2.2 4.5

8.5 9.1 1.1 3.7 5.2 6.0 5.0 a.2 6.0

7.8 5.1 10.0 11.9 9.4 2.6 4.3 8.6 8.5 1.1 4.0 5.2 6.3 4.9 3.4

6.7

7.9 5.2 10.4 0.9 12.3 7.1 2.9 4.7 9.0 9.0 1.4 3.6 5.0 6.0 5.2 2.4 7.1

9.8 2.9 10.5 0.8 9.2 8.4 4.0 3.8 9.9 8.3 1.3 4.4 5.5 6.2 5.1 2.2 7.6

9.1 3.6 10.6 0.5 10.7 8.5 3.6 4.0 9.0 9.1 1.2 3.8 5.4 6.4 5.2 2.0 7.3

The values are per cent of total moles of amino acids recovered. (1963b).

* From Wan,

8.1 4.9 10.0 12.1 8.4 2.2 4.4 9.6 7.0 1.9 5.1 6.0 6.7 5.1 2.9 6.5

8.0 5.8 9.6 0.6 11.6 8.7 2.4 4.7 8.8 9.1 1.4 3.6 5.2 5.8 5.2 2.3 7.1

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

65

night after the nuclear ribonucleoproteins were sedimented a t 105,000 g. The globulins differed in amino acid composition from the ribosomal proteins only in that they had a higher concentration of arginine than did the ribosomal proteins (Table IV). The values for the globulins of the tumor were not significantly different from those of the liver. As is indicated in Table IV, the amino acid composition of the proteins of the microsomes of the liver and tumor were also very similar and the similarity of these values to those of the globulins was greater than their similarity to the amino acid composition of the nuclear ribosomes. The proteins of the saline extract that were neither sedimented a t 105,000 g nor precipitated by dialysis (“nuclear sap”) had a significantly different amino acid composition from the other fractions extracted in the dilute salt solution. The concentrations of alanine and histidine were higher than those found in the other fractions studied, but the concentrations of arginine and glutamic acid were significantly lower in these proteins than those of the other saline-soluble proteins. VII. The Nuclear Ribonucleoproteins

Following the report of Logan and Davidson (1957) that nuclear RNA was heterogeneous as determined by its extractability by 0.15 M NaCl and 2.OM NaC1, or in dilute phosphate buffer and Tris buffer followed by extraction with 1 or 2 M NaCl (Allfrey and Mirsky, 1957; Osawa et al., 1958), i t became clear that the possibility existed that there were ribonucleoproteins present in the dilute salt extract of the nuclei. Evidence has been presented (Smetana et al., 1963) that in the nuclei of cells of normal liver as well as the Walker tumor there is a well-developed ribonucleoprotein network of which the nucleolus is a large mass. Emanating from the nucleolus is a large number of radiating lines of ribonucleoproteins which, when cut thinly for studies with the electron microscope, break up into granular masses that have been referred to as both Swift and Watson granules, differentiated by size. It would appear that the granules of these masses as well as those of the nucleolus are the sources of the nuclear ribonucleoproteins, or “ribosomes,” that are extractable from nuclei of thymus cells with 0.14M NaCl. It seems likely that in calf thymus preparations, the particles obtained are typical or true ribosomes as indicated by their appearance under the electron microscope (Allfrey, 1963a). As with cytoplasmic ribosomes, the concentration of Mg++in the medium determines both the size of the ribosomes and their activity in uptake of labeled amino acids. As indicated previously, purification of the nuclear ribosomes was achieved by centrifugation of the preparation a t 105,000 g for 60-90 minutes after the initial extraction and low speed centrifugation to remove high-molecular-weight impurities.

66

HARRIS BUSCH AND WILLIAM J . STEELE

Further purification was accomplished by centrifugation in sucrose density gradients and then by treatment of the preparations with detergents to remove the lipids of the lipoproteins that comprise a very substantial weight of these particles. The same lipoproteins may also be present in the nuclear ribonucleoprotein network found in other cells.

PROTEINS A. RIBOSOMAL Although relatively little information is available on the proteins of the ribosomes, the finding that there are multiple species of the ribosomal particles (Wang, 1963a’b) has inevitably raised the question of whether there are many molecular species in the proteins present (Kit, 1960). The amino acid composition of the nuclear ribosomes of calf thymus has been determined by Wang (1963a,b) whose data has been translated to per cent total moles in Table IV. According to Wang’s data, the values are not particularly characteristic for any type of protein inasmuch as the ratios of acidic to basic amino acids are approximately one. Table IV also presents values obtained in this laboratory for the amino acid composition of the nuclear ribosomes obtained from the Walker tumor and the liver. Both of these groups of proteins were obtained in very low yields. Although there is considerable question whether these proteins arc identical either in structure or function with those obtained from the preparations of calf thymus, there was reasonably close agreement between the values obtained by Wang (1963a’b) and those obtained in this laboratory. The exceptions were that he found more of the basic amino acids and less glutamic acid in his preparation. It should be noted that the similarities are much greater than the differences, considering that the proteins originated from different species of animals. Determination of the amino acid composition is only one means for characterization of a given group of proteins or of a given protein species. Although the amino acid composition of some proteins is quite characteristic, that of mixtures such as are found in the saline extracts are frequently not too informative. Only the proteins that were neither sedimented a t 105,000g nor precipitated by dialysis (“nuclear sap”) had significantly different amino acid compositions from those of the other proteins extracted with dilute saline solution (Table IV). The notable differences were in alanine and arginine content. B. NH*-TERMINAL AMINOACIDSOF

THE

SALINE-SOLUBLE PROTEINS

The determination of the NHz-terminal amino acids of proteins lacks the remarkable refinement of the procedures for determination of the amino acid composition. Nonetheless useful results can be obtained as illustrated in Table V. The data present quantitative differences between

67

NUCLEAR PROTEINS O F NEOPLASTIC CELLS

TABLE V NUCLEAR PROTEIN N H 2 - T AMINO ~ ~ ACID ~ ~ ANALYSIS ~ ~ ~ OF~ SALINE-SOLUBLE FRACTION OF WALKER TUMOR" Amino acid Alanine Aspartic acid Glutamic acid Glycine Lysine Serine Threonine Leucine 1 Valine ,j 0

\

Nuclear ribosomes

Nuclear globulin

Nuclear sap

Microsomes

20.5 11.9

15.8 21.4

8.5 23.0

19.0 18.6

28.5 3.8 17.1 6.6 11.5

29.6 2.4 11.8 5.1 15.2

25.5

29.4 2.0 15.8 6.1 9.1

J

-

12.8 5.3 23.9

The values are per cent total moles recovered.

the NH,-terminal amino acids of the various fractions of the Walker tumor and the liver. The chief NH,-terminal amino acids were alanine, glycine, aspartic or glutamic acid, serine, and leucine or valine. I n the Walker tumor, the nuclear sap was about equally rich in proteins with either aspartic or glutamic acid, glycine, and leucine or valine as NH,terminal amino acids. I n the liver, on the other hand, the proteins with leucine or valine as the NH,-terminal amino acid were the main cornponents. I n each of the other fractions studied, including the nuclear globulins, nuclear ribosomes, and microsomes, glycine was the chief NH,terminal amino acid. The globulins differed quantitatively from the other protein fractions in that aspartic or glutamic acid was the second most prevalent group of NH,-terminal amino acids ; the other NH,-terminals were relatively of minor significance in the other fractions. The cytoplasmic microsomal fraction and the nuclear ribosomal fractions of the Walker tumor contained essentially equal amounts of the various NH,-terminal amino acids. I n both of these groups of proteins, alanine was the NH2-terminal found in second largest amount. On the basis of the two parameters of amino acid analysis and NH,terminal amino acid analysis, the greatest differences were found between the saline-soluble proteins that were not sedimented a t 105,OOOg or by dialysis (nuclear sap) and the other nuclear proteins. Although the amino acid analyses were essentially the same for the globulins and the nuclear ribosomes, the NH2-terminal amino acid analyses were somewhat different. However, in none of the analyses were significant differences found for the nuclear and cytoplasmic ribosomes. This latter finding may reflect the possibilities that nuclear ribosomes are precursors of cytoplasmic

68

HARRIS B U S C H AND WILLIAM J. STEELE

ribosomes, that some ribosonies shuttle between the nucleus and the cytoplasm, as was recently suggested by Potter (1963)) or that, in fact, there are no free nuclear ribosomes in the Walker tumor and the liver. Thc possibility certainly exists that the very sinall amount of the nuclear ribosomes reflects a minor degree of contamination of the nuclei with ergastoplasm of the cytoplasm. I n view of the proximity of the nucleus to the ergastoplasm i t is virtually impossible to eliniinate all contamination. Data on labeling of the proteins of the nuclear fractions of the liver and the Walker tumor that were extractable with 0.14 M NaCl solutions (Table VI) show that the saline-soluble proteins that were neither sediTABLE VI SPECIFICACTIVITIESOF SODIUMCHLORIDE-EXTRACTABLE NUCLEAR PROTEINS" Fractions

Tumor

Liver

509 417 258

225 261 192

Globulins Ribosomes Nuclear sap

a Expressed as c.p.m./mg. of nuclear proteins extracted from isolated nuclei with 0.14 M NaCI.

iiiented a t 105,OOOg nor precipitated by dialysis (nuclear sap) had considerably lower specific activities in both the Walker tumor and the liver than did the other proteins. I n the tumor, the labeling of the nuclear globulins exceeded the labeling of the other proteins, although the values were not markedly greater than the specific activities of the proteins of the nuclear ribosomes. The data on labeling of these fractions supports the concept that the greatest differences between these groups of nuclear proteins exist between the globulins and ribonucleoproteins on one hand and the more soluble nuclear proteins on the other.

C. ELECTROPHORETIC STUDIES Bakay e t al. (1963, in manuscript) have utilized free boundary electrophoresis t o study the nuclear proteins of the livers of normal rats and rats treated with 3'-methyl, 4-dimethylaminoazobenzene, as well as liver tumors. Saline-phosphate solutions were used to extract the nuclei and on boundary electrophoresis, in veronal buffer, the protein composition of the various nuclear extracts was found to be very similar. At least one positive and eight negatively charged classes of proteins were found in the normal and dye-treated liver nuclei, I n the tumors, there was less of

NlJCLEAH PROTEINS O F Ir;EOPLASTIC CELLS

69

a slower component (P) and more of faster components (negative) LMN and 0. The positive component was not found in the tumors. In their studies on the cytoplasm, they found an increase in acidic proteins and a loss of basic proteins. In contrast to nuclear extracts, the cytoplasm of tumors had less of the weakly negatively charged proteins than did the cytoplasmic extracts of normal liver. These weakly negatively charged proteins are “h-proteins,” deleted in hepatomas. I n livers of dye-treated animals these h-proteins contained the carcinogen. I n the cytoplasm of the tumors there was also a decrease in the neutral or less acidic components and an increase in the more acidic components. However, the near-neutral components were essentially unchanged in the tumor nucleus. With regard to molecular size, the relative amounts of various classes were changed significantly, i.e., there was a marked increase in the tumors of proteins with a sedimentation constant of 4s.

D. CYTONUCLEOPROTEINS Although many studies have shown that nuclear proteins do not exchange either freely or completely with cytoplasmic proteins, recently Byers et al. (1963a,b, in manuscript) have found a group of proteins that apparently readily exchange between the nucleus and cytoplasm of amoebas. Using the technique of nuclear transplantation following incubation of the donor cells with radioactive amino acids, the most interesting fact noted was that label emigrated from the donor nucleus to the cytoplasm and shortly thereafter the label localized in the recipient nucleus. Prescott (1963, in manuscript) has found that during mitosis labeled proteins emerge from the nuclei of amoebas; after mitosis is completed, there is a kind of mass migration back into the nucleus. Horn (1962) has suggested that such a phenomenon occurs in frog embryos in the morula stage; using the fast green stain (Alfert and Geschwind, 1953), he found that prior to the morula stage the nucleus did not take the stain, but that after this point the nucleus was readily stained. Conversely, prior to the morula stage, the cytoplasm stained positively with the fast green stain and did not take the stain after the morula stage. From the studies on labeling of the cytonucleoproteins in amoebas, the conclusion was drawn by Byers e t al. (1963a,b, in manuscript) that the cytonucleoproteins and the nuclear proteins are both synthesized in the cytoplasm. They suggested that the rapid transport of the cytonucleoproteins from nucleus to cytoplasm to nucleus might mean that these proteins could be involved in communication of signals to and from these cellular compartments. The amount of protein of this type is apparently large, inasmuch as 30% of the label in the donor nuclei was transferred

70

HARRIS BUSCH AND WILLIAM J . STEELE

to the recipient nucleus. Another 10 to 40% of the label was transferred to the cytoplasm. VIII. Acidic Proteins of the Deoxyribonucleoprotein Complex

The extract of the residue obtained with 2 M NaCl after extraction of the proteins soluble in 0.14 M NaCl contains the deoxyribonucleoproteins as the major component. I n the liver and in the Walker tumor approximately 64 and 57%, respectively, of the total lipid-free dry weight of the nucleus were found in these fractions (Table 11). The deoxyribonucleoprotein complex was found to consist of nucleic acids that comprised about one third of the dry weight, histones that comprised about 40% of the dry weight, and acid-insoluble proteins that comprised about 25% of the dry weight of this complex. It is outside the scope of this review to discuss the nucleic acids of this complex (Busch, 1962) ; the histones will be reviewed in Section IX. OF ANTITUMOR AGENTSON ACIDICNUCLEAR PROTEINS A. EFFECTS

Interest in this group of acidic nuclear proteins has been much increased in recent years, in view of the findings that these proteins were shown to be involved in the effects of alkylating agents. I n studies from this laboratory, it was found that the rate of labeling of the acidic nuclear proteins was markedly diminished in animals treated with aminouracil mustard at earlier times and a t lower doses of the drug than were found to suppress the labeling of other nuclear proteins or cytoplasmic proteins (Busch e t al., 1959a, 1961 ; Strozier and Nyhan, 1962). Whether the label used to determine biosynthesis of proteins was lysine or arginine, the same result was found and was later extended to a variety of antitumor agents including Myleran, 6-mercaptopurine, leukeran, 5-fluorouracil, and HN,. Although in a number of instances the suppression of labeling of the acid-insoluble nuclear proteins was not in excess of that of other nuclear fractions, i t was the only constant feature of the effects of antitumor agents on protein labeling. I n experiments on the localization of action of mustards in Ehrlich ascites cells in uitro, Steele and Price (1961) and Steele (1962) found that the proteins cross-linked to DNA and RNA were not the histones or acid-soluble nuclear proteins, but rather were the acidic nuclear proteins. I n experiments carried out with therapeutically effective levels of nitrogen mustards, i t was found that the amount of protein remaining linked to the DNA in the interphase between phenol and water (Kirby, 1957; Sibatani, 1963) increased proportionately to the level of the mustard in nuclei of cells treated with difunctional alkylating agents. No increase was found in similar fractions of cells treated with monofunctional

TABLE VII

AMINO ACID COMPOSITION OF VARIOUSNUCLEAR PROTEIN FRACTIONS OF LIVERAND WALKER TUMOR" Liver Fraction Alanine Arginine Aspartic acid 1/2 Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

Tris

Acidinsol.

Walker tumor

Phenol- Alkaliinsol. sol.

Residual

Tris

Acid- Phenol- Alkaliinsol. insol. sol.

Ehrlich aacites crossResidual linked proteins

6.7 7.1 8.8 12.8 7.3 2.3 3.7 7.9 7.0 2.0

7.4 5.7 9.3 1.3 12.1 8.0 2.3 4.6 9.4 6.3 2.6

9.5 5.4 7.0 0.4 10.0 25.7 1.3 2.8 5.1 4.7 1.o

7.6 6.2 9.3 1.8

7.5 7.0 9.7 0.3

7.4 6.3 9.9 0.3

7.9 6.2 9.2 0.7

8.4 5.7 8.4 0.2

7.7 5.3 10.5 -

11.1 10.9 2.2 3.6 7.5 8.4 1.7

6.7 7.7 9.1 0.4 12.4 9.0 2.3 4.4 8.3 7.3 2.2

12.2 6.6 2.0 4.4 8.7 8.1 2.0

13.5 8.1 2.4 4.8 8.8 7.3 2.3

14.7 7.3 2.4 4.9 9.2 7.6 2.2

13.0 7.4 2.2 4.6 10.1 6.5 2.0

10.5 23.8 1.5 3.2 5.7 4.5 1.1

15.3 7.0 1.8 4.4 8.3 7.9 1.1

3.8 5.9 7.5 4.7 3.2 4.9

3.8 6.4 6.9 5.0 3.2 5.1

3.1 7.0 10.4 5.1 2.4 6.4

4.0 5.5 7.4 5.7 2.8 6.1

2.7 9.0 5.8 3.7 1.3 4.0

3.5 5.6 6.6 5.4 2.6 7.6

3.7 5.0 6.6 5.6 3.2 5.6

3.5 4.3 6.8 5.8 2.3 5.2

3.8 4.6 7.0 5.7 2.7 6.6

2.4 8.8 6.3 4.4 1.4 3.9

4.1 5.9 7.1 5.2 1.8 5.8

7.5 7.4 9.8

-

a The table presents the percentages of total moles of amino acids recovered by chromatography of protein hydrolyzates on a Beckman automatic amino acid analyzer. The values are averages of two to five analyses.

$ E!

a

s cd

H

2 0

r

3

0

cd

r

%

8 0

E

u,

72

HARRIS BUSCH AND WILLIAM J. STEELE

agents. The cross-linked proteins were characterized by a high content of glutamic acid and a low content of basic amino acids (Table V I I ) .

B. LABELINGOF ACIDICNUCLEAR PROTEINS Another evidence of the importance of these proteins was derived from studies on the labeling of nuclear proteins. In studies on fractions reported to be derived from “nuclear preparations,” Daly e t al. (1952) noted that although the cytoplasmic fractions had the highest specific activities in terms of labeling of the proteins, there was actually a higher labeling of the residual “chromosomal proteins” than there was of the other nuclear proteins, including the histones (see also Allfrey et al., 1954). Despite the fact that their results cast some doubts on the usefulness of isotopes for determination of the metabolic turnover of various nuclear proteins in vitro, Brunish and Luck (1952a,b) also found that, of the nuclear proteins, those with the highest turnover in livers in vivo were the acidic nuclear proteins, i.e., the “residual proteins” of Nlirsky and Ris (1947-1948a, 1950-1951). Smellie and his colleagues (1953) also noted that the alkali-soluble nuclear proteins had a high rate of labeling in rat liver and indeed, even higher than proteins in a number of cytoplasmic fractions including the mitochondria, microeomes, and proteins of the cytoplasmic sap. Their data on this point, however, are not in general agreement with those found by other workers. Significantly, this is not the case in tumors in which the labeling of the acidic nuclear proteins was consistently less than that of the histones when the data were compared on the usual basis of counts per minute per milligram of protein. I n studies carried out in this laboratory, the previous reports of the high rate of labeling of the acidic proteins of the deoxyribonucleic protein complex of the liver have been confirmed (Table VIII). Both the acidic proteins of this complex and the acidic proteins of the liver that were extractable with alkali (the NaOH-soluble proteins) were highly labeled 1 hour after intraperitoneal injection of L-lysine-U-C14 into tumor-bearing rats (Steele and Busch, 1963a). Following extraction of the previously acid-treated deoxyribonucleoprotein residue with phenol, a fraction remained that was insoluble in phenol. The protein of this fraction had the highest specific activity of any of the fractions of the nucleus of the liver. About 42% of the total isotope in the nuclear proteins of the liver was in the acidic proteins a t one hour. I n the Walker tumor and other tumors, there was marked labeling of the acidic proteins linked to DNA at 1 hour after the injection of the labeled lysine into the rat (Steele and Busch, 1963a). I n the Walker tumor, however, the specific activity of the Tris-extractable fraction was almost as high as that of the proteins of the phenol-insoluble fraction

73

NUCLEAR PROTEINS O F NEOPLASTIC CELLS

TABLE VIII SPECIFIC ACTIVITIES OF VARIOUSNUCLEAR PROTEINFRACTIONS OF LIVER AND WALKER 256 CARCINOSARCOMA~~~ Liver

__ Fraction Isolated nuclei 0 . 1 4 M NaCl 0.10 M Tris 2 . 0 M NaCl

Acid-soluble Acid-insoluble Phenol-insoluble 0.05 N NaOH Residual

Walker tumor

C.p.m./mg. protein

C.p.m./pmole lysine

C.p.m./mg. protein

C.p.m./pmole lysine

210 160 2-10

260 370

450 360 540

580 860

220 300 440 270 130

180 530 810 530 320

680 550 590 420 160

640 9so 990 840 410

-

One hour after administration of 5 pc. Lysine-U-C14. Specific activities are corrected on the basis of the sum of the total amino acids recovered by chromatography of acid hydrolyzates.

and the acidic proteins of the deoxyribonucleoprotein complex. Interestingly, only 26% of the total isotope in the nuclear proteins of the tumor was found in the acidic proteins of the deoxyribonucleoproteins. Unlike the liver, however, the highest specific activity of the nuclear proteins of the tumor was found in the histone fraction which contained approximately 42% of the total isotope in the nuclear proteins of the tumor at 1 hour after injection of the labeled lysine into the tumor-bearing rat. From studies of this type it would appear, as has been stated a number of times by different authors (Busch, 1962; Butler and Laurence, 1960; Busch e t al., 1958; McGillivray and Greenwood, 1962), that a main function of the neoplastic cell is the biosynthesis of the histones. In nontumor tissues, the biosynthesis of the acidic nuclear proteins would appear to be the predominant process. At the present time, data have not been accumulated for a variety of other tissues that might be important to include in such a study, such as the regenerating liver and the liver of thioacetamide-treated animals that is essentially in a preneoplastic state. Perhaps the relative percentages of isotope and the specific activities of the proteins reflect differences in the rates of synthesis of ribosomes for cytoplasmic protein synthesis.

C. FRACTIONATION OF ACIDICNUCLEAR PROTEINS At the present time, there are no satisfactory methods for further fractionation of the acidic nuclear proteins following the extractions with salt solutions and the phenol-extraction procedures indicated above. Al-

74

HARRIS BUSCH AND WILLIAM J . STEELE

though further fractionation procedures with salt solutions have been attempted, as well as chromatography of the fractions on starch gel and columns with various types of absorbents, none of these procedures have permitted separation of the components of these proteins. The analytical data on these fractions are the resultant of the values for various components of the mixtures of proteins present and much analytical information remains to be obtained following fractionation of the proteins. As indicated elsewhere (Busch e t al., 1963d), the methods for extraction of histones with acids are certainly subject to criticism and some soulsearching on the part of the protein chemists utilizing such procedures but at least (Zubay and Wilkins, 1962) there is some justification for such procedures. There is no clear logic in the employment of alkaline solutions to dissolve the acidic proteins, other than the fact that they have thus far defied being dissolved in other solvents.

D. AMINOACIDANALYSIS The amino acid analysis of these fractions (Table VII) reveals that the concentration of glutamic acid and aspartic acid is high and that these amino acids comprise approximately 20-25% of the total amino acid residues in these proteins. The basic amino acids, such as arginine, lysine, and histidine, comprise 16% of the total amino acids of these proteins. It should be noted that these values are quite constant for the entire group of acidic proteins throughout the nucleus (see Table I V ) , but they do not serve to provide reasons for the extreme insolubility of these proteins, particularly those linked to the deoxyribonucleoproteins and those of the “residual” group. One possibility is that the acidic proteins really are not so acidic as may seem from the amino acid analysis, and that the glutamyl and aspartyl residues are present as the amides, glutamine and asparagine. Slow hydrolysis of covalent linkages such as those of esters or anhydrides in alkali could result in the slow entry of these proteins into solution. Significant differences between the amino acid composition of the acidic proteins of the Walker tumor and the liver were found in only a few instances (Table VII). The glutamic acid content of proteins of the Tris extract was lower than that of the acid-insoluble, phenol-insoluble, and alkali-soluble proteins in both tumor and liver. I n each group of proteins, however, the glutamic acid content was higher in the tumor than in the liver. The glycine content of the proteins of the Tris extract was higher in the liver than in the tumor. The leucine content of the alkalisoluble proteins was higher than that of the proteins of the Tris-extract and acid-insoluble proteins in both the tumor and the liver. The lysine

NUCLEAR PROTEINS O F NEOPLASTIC CELLS

75

content of the proteins of the Tris extract was higher than that of the other nonhistone proteins, although the lysine content of the histones was significantly greater than that of the other groups of proteins. The serine content of the phenol-insoluble proteins of the liver was much higher than that of any of the other proteins studied. The valine content of the proteins of the Tris extract of the Walker tumor was higher than that of the corresponding proteins of the liver.

E. NH,-TERMINALAMINOACIDS Until studies were made of the amino terminals present in the acidic proteins, no indication of their heterogeneity had been available since there have been no successful attempts to chromatograph or otherwise separate the acidic proteins of the nucleus. The analytical data obtained for NH,-terminals of acidic proteins and the various fractions isolated from nuclei of the liver and the Walker tumor are presented in Table IX. Evidence for considerable heterogeneity of these fractions was obtained from the finding that 10 or more NH,-terminal amino acids were present in each of the fractions studied, However, it was notable that serine, alanine, and glycine were the major NH,-terminal amino acids and together they accounted for about 55% of the total. The data are qualitatively similar to those presented in Table V for the proteins of the nuclear sap extracted with 0.14M NaCl. At the moment, there is no certainty that the same or very similar proteins were not present in all of the fractions. However, the quantitative differences in the NH,-terminal amino acids as well as the different rates of labeling suggest that some differences may exist. One of the differences in the NH,-terminal amino acids was that of the higher proline and lower alanine and serine of the phenol-insoluble proteins and the acid-insoluble proteins. Both of these fractions were treated with hot TCA prior to the determination of the NH,-terminal amino acids in order to remove nucleic acids. The possibility exists that this procedure has produced some of the similarities of the NH,-terminal amino acids. The present status of comprehension of the numbers and functions of the acidic proteins of the nucleus is most unsatisfactory. Perhaps the chief stumbling block in the studies on these proteins is the development of methods for their isolation and purification. The procedures employing alkaline extraction are obviously likely to denature and possibly hydrolyze the acidic nuclear proteins and their limited solubility in other solvents has made them difficult to study with our present methods. The need for further techniques is apparent since these proteins have high turnover rates in both normal and tumor tissues. Whether they are re-

TABLE IX N H 2 - T AMINO ~ ~ ACIDS ~ ~ I N~ NUCLEAR ~ ~ PROTEIN FRACTIONS O F LIVERA N D WALKER 256 CARCINOSARCOMA" Liver

Walker tumor

_____

____

~

____---

__

Acidinsol.

Histone

Alkalisol.

Residual

Acidinsol.

TCAtreated, acidinsol.

17

35

18

20

18

11

35

9

17

24

Aspartic acid

8

1

10

9

8

8

1

9

8

8

Glutamic acid

7

1

9

6

8

6

1

4

7

8

Glycine

15

4

15

14

12

13

3

15

16

15

Leucine

5

1

7

6

6

6

2

6

5

5

Lycine

8

3

10

6

8

8

8

10

7

8

Phenylalanine

2

2

3

4

2

3

1

2

3

1

Fraction Alanine

Proline

2

49

4

Serine

26

5

17

5

1

5

Threonine Valine

3

3

The values are per cent t,otal moles recovered.

Histone

Phenolinsol.

Alkalisol.

Residual

5

21

42

19

8

-

20

26

13

6

15

21

16

8

5

6

1

P

6

8

4

-

5

3

J

4

4

~

77

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

lated to the enzymes involved in synthesis of the nucleic acids or serve to form structural components of nuclear products, such as the cytoplasmic polysomes, is simply not clear a t the moment. IX. The Histones

A very extensive literature has developed with regard to the histones of both nontumor tissues (Tazawa, 1953; Phillips, 1961, Busch, 1962), and tumors (Busch and Davis, 1958; Busch, 1962). One of the problems encountered in reviewing the literature on histones has been the recent complication of the accepted definition of histones as basic proteins of the chromosomes and interphase chromatin. Evidence has accumulated that basic proteins are components of cytoplasmic ribosomes (Crampton and Peterman, 1959; Butler e t al., 1960) as well as in globulins of the plasma (Porter, 1959) and inucinous secretions (Spicer, 1962). Moreover, evidence has been provided by studies in this laboratory that some of the proteins of the nuclear sap (Busch e t al., 1963d) are basic proteins and may represent entities that are of the group generally referred t o as “arginine-rich” histones (Table X) . Thus, the concept of localization of the histones to the chromatin or the nucleus has been subjected to some uncertainty and is less, rather than more, restrictive as time has gone by. AND DEFINITIONS A. NOMENCLATURE

One of the points of confusion in the literature on the histones is the nomenclature. I n order to achieve clarity in their own laboratories, a number of workers have used the different symbols indicated in Table X for classifying their products. As an increasing number of studies have been made, it has become clear that various fractions from different laboratories are essentially the same and hence the variable nomenclature should now be abandoned (Mauritzen and Stedman, 1959, 1960). TABLE X HISTONE FRACTION NOMENCLATURE Fraction Lysine-rich Slightly lysine-rich Arginine-rich

Designation (Y

Y

B

A B

-

I

I1 111, IV

Lys/Arg Fl F2 F3

>3 1-3 <1

Another problem in the definition of histones has been that of their basicity. The early studies on histones (Kossel, 1921; Kossel and Kutscher, 1900; Felix et al., 1952, 1956) indicated that these proteins were strongly basic and had isoelectric points that were high, i.e., pH 10-11.

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No doubt this basicity was the reflection of the presence of specific proteins of the groups that were very rich in lysine. As indicated below, some of the “lysine-rich” or “very lysine-rich” proteins (the literature is confused in terminology) have as many as 35% of all the residues accountable as lysine. Recently, Johns (1963) has ascribed these high lysine values to very lysine-rich peptides in partially degraded histone fractions. There is some kind of a relationship of lysine content to alanine content in these proteins, because they are almost equally “alanine-rich” (Busch and Davis, 1958, 1961). This enormous lysine content would make any protein very basic, but these proteins are all the more so because of their very low content of glutamic and aspartic acids (Greenstein, 1944). Interestingly, the content of amide nitrogen in histones has not been reported. Recently, a newer group of histones has been studied that has been described as the “slightly lysine-rich” group ; some of the proteins of this group have almost a 1:l ratio of basic:acidic amino acids. Thus, they are significantly less rich in basic amino acids than the over-all histone fractions and those containing much more lysine ; hence, the relevance of the older concepts of extreme basicity for the histones needs also to be reconsidered. These exceptions to the definition presented above are not yet assessed for their over-all significance but the original components of the definition of histones, a t least temporarily, are subject to some reconsideration. Although many studies have been made on the truly arginine-rich protamines, they are of less significance to the problem of neoplasia since they are found only in sperm cells (Felix e t al., 1952, 1956; Ando e t al., 1953, 1959; Callanan et al., 1957; Scanes and Toaer, 1956).

B. PHYSICAL RELATIONSHIP OF DNA

AND

HISTONES

The amount of histone associated with DNA is not completely established. Most workers have found that the amount of histone is approximately equal to that of DNA in nucleoprotein preparations. On the basis of amino nitrogen to phosphoric acid groups, the ratio has been variously estimated to be approximately 0.8 to 0.9 in such preparations (Perugnini e t al., 1956a,b,c, 1957a-e; Peacocke, 1960; Phillips, 1961; Laurence et al., 1963; Steele and Busch, 1963a). However, Dounce has reported that the ratio of histone to DNA in normal liver approaches 2, although in regenerating liver and in tumors the ratio is closer to 1 (Dounce, 1963). Although a variety of models has been proposed for the interrelationship between DNA and histone (Busch, 1962), there is in fact no completely satisfactory physical model a t the present time. One of the reasons for this is that the physical methods used for analysis of structure of macromolecules, including X-ray (Wilkins et al., 1959a,b; Zubay and

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Wilkins, 1962) and infrared analysis (de Loze, 1958a,b, 1959; Klamerth, 1957; Lenormant and de Loze, 1959; Bradbury e t al., 1962a,b) do not provide evidence for a highly ordered structure. I n the case of DNA, a structure with regular order has been found and it has been relatively uncomplicated to work out a structure with a high degree of regularity. The addition of histone to DNA diminishes the order of the structure. Thus, there are reflections in nucleohistone preparations a t 34,20,37,25,60, and 55A.; from such data, models have been drawn consisting of several linear strands of DNA with a crossing-over of linear histone strands at an angle of 55-65' to form a sort of lattice in which the lines do not intersect a t right angles. However, the arrangement is apparently more complex than this lattice-like arrangement that would suggest that histones interlink DNA strands and thereby promote coiling. Infrared analysis has suggested that there may be no regular arrangement of histones with DNA (Bradbury et al., 1962a,b), although the lattice-like structure is not ruled out by this technique. One of the points of agreement of the physical methods is that there is an a-helical structure cf a t least 65% of the histones linked to DNA. Moreover, treatment with ethanol denatures some of the histones so that they form /&structures. Thus far, there is no evidence from such physicochemical studies that the histones are either uniformly or irregularly spread on the surfaces of DNA molecules, although the data are in agreement with the concept proposed by Swift (1959, 1963) that the chromosomes consist of a series of interwound rope-like structures.

C. FUNCTIONS OF THE HISTONES The close relationship between DNA and the histones has been brought out by many studies utilizing both biochemical and cytochemical techniques. Although RNA is present in the chromosomes and, at least in Drosophila, becomes very heavily labeled with nucleic acid precursors a t some stages of development, the amount is relatively small and the RNA-linked proteins have not been intensively studied. The development of a relatively specific stain for the histones by Alfert and Geschwind (1953) has permitted many studies on the intracellular localization of the histones and simultaneous studies on the localization of DNA have been made with the aid of the Feulgen reaction. The technique of Alfert and Geschwind (1953) is quite simple, consisting of extraction of fixed sections with trichloracetic acid (TCA) or treatment of the sections with DNase to remove the nucleic acids and DNA. If DNA is not removed, the histones do not pick up the stain. Fast green is used to stain the histones in 0.1% aqueous solutions adjusted to p H 8. The pH is quite critical, since below this pH many other proteins besides

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the histones take the stain. The greatest staining was found with protamine, although methylated serum albumin also takes the stain a t pH 8. I n these studies a constancy of ratio of histone to DNA was found for different species and different organs from the same species (Alfert, 1956, 1957, 1958; Bloch and Godman, 1955). A number of reports have emphasized the very close relationships between the histones and the DNA in the chromosomal bands of Drosophila. The studies of Horn and Ward (1957) have clearly shown that wherever there is a DNA band, there is a concomitant histone band as revealed by staining with fast green a t alkaline pH. The various methods for isolation of deoxyribonucleoprotein complexes from which histones may be extracted (Kossel, 1921; Crampton et al., 1954a,b; Daly and Mirsky, 1954-1955; Daly et al., 1950-1951, 1952) also serve to emphasize the close relationships of the histones and the DNA. Since so many studies with viral and bacterial nucleic acids (FraenkelConrat and Williams, 1955; Gierer and Schramm, 1956; Hershey and Chase, 1952; Griffith, 1928; Avery et al., 1944) have now established that the nucleic acids serve as the bearers of genetic information, intracellular devices for carrying the genetic messages from nuclei to the cytoplasm and the essential components of the templates on which proteins are formed, the question that has logically arisen is: What is the function of the histones and the other nuclear proteins that are intimately associated with the genetic apparatus? A list of functions proposed for the histones is presented in Table XI. PROPOSED

TABLE XI FUNCTIONS FOR THE HISTONES

Stabilizers of DNA Stabilizers of RNA Backbones of the rhromosomes Ends of telomeres Unfolding of DNA Coiling of the chromosomes Enhancement of genetic functions Information transfer to cytoplasm Information transfer to nucleus

Regulators of genes Regulators of differentia tion Neutralizing agents for acidic macromolerrilrs Ribonucleases Releasing agents for RNA from DNA Suppressors of RNA synthesis Accelerators of RNA synthesis Suppressors of DNA synthesis

1. Histones and Genetic Control

It is somewhat puzzling t o read the earlier literature on the histones and to attempt to comprehend the results of the experiments or the conclusions. Stedman and Stedman (1943, 1944, 1950) noted that, in all probability, the chromosomes were related to the chemical basis of hered-

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ity and that they were composed of a mysterious “chromosomin,” an acidic residue that was largely or entirely protein and was slowly soluble in dilute alkali. They assigned to the nucleic acids the role of formation of the spindle during mitosis and in combination with chromosomin, the synthesis of protein during the resting stage. I n studies on the nuclei of a variety of cells, including the Walker tumor, a mouse carcinoma (2146), and embryonic tissue as well as spleen, erythrocytes, thymus glands, and sperm of a variety of species, they noted that the nucleic acids comprised about one third of the dry weight of the nucleus. No criteria of purity were presented for the nuclei. Using the technique of acid extraction, the histones were found to comprise 1.6 to 24% of the total dry weight of the nucleus. The chromosomin comprised 33 to 72.4% of the total dry weight of the nucleus. I n the tumors and in the chick embryonic cells, the histones comprised 1.6 to 3% of the total dry weight of the nuclei. On the basis of these values, Stedman and Stedman (1943) concluded that the high histone content of some cells, i.e., the nondividing cells, inhibited the processes that lead to mitosis. The low level of histones in embryonic cells and in tumors presumably would not be sufficient to prevent the nucleic acids and the chromosomin from forming the chromosomal structures and/or the structures of the mitotic apparatus. Hence, these cells would go on to cell division, These experiments suggested the possibility that histones prevent mitosis. This suggestion was based upon data that to the present time have not been confirmed. If anything, the content of the histones in the tumors studied in this laboratory and others is a t least as great, if not greater, than that of the other tissues studied. However, the possibility exists that the concentrations or structures of the individual species of histones in tumors are different from those of the other tissues. Another of the recent suggestions for the functions of the histones was that of Leslie (1961) who found high concentrations of ribonucleases in histone preparations. He also found that on chromatographic analysis of the histones, there was a peak of ribonuclease activity in each peak of the histones. I n recognition of the fact that messenger RNA would necessarily be of a variety of sizes and of base ratios, Leslie reasoned that perhaps the histones might be important in stabilizing RNA templates, in hydrolyzing complementary or nonsense strands of RNA and in releasing RNA from the DNA templates. Subsequent studies by Leslie (1963) and other workers (Irvin et al., 1963) have not supported the concept that histones function as ribonucleases since histones could be isolated that had no apparent ribonuclease activity. Moreover, it should be noted that ribonucleases are basic pro-

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teins and as such, they may have been extracted along with histones in Leslie’s procedure. Since complete details of the methods employed have not yet become available, this possibility cannot be evaluated. The possibility that histones might serve as some sort of vehicle for transmitting nuclear information to the cytoplasm (Busch, 1962) has also been suggested but, a t the moment, evidence is lacking for the presence of significant amounts of histones in either the cytoplasm or the nucleolus and hence it is not clear a t what cellular site the histones might transmit information. As indicated previously, there is some evidence accumulating that basic proteins are not limited in their localization to the nucleus, inasmuch as in the gamma globulins, protein components with gn over-all basic character have been found. Since it is obvious that the genome is essentially stable for the life of a mammalian cell and yet possesses the capacity to respond to both environmental and biochemical changes, a kind of corollary concept would be that the histones may bring information to the genome from outside the nucleus. The attractiveness of this hypothesis seems to be limited by the fact that histones are found to only a limited extent, if a t all, outside the nucleus and any message would have to come a t least as far as the nuclear membrane to cause the formation of histones. 2. Histones and Ribonucleic Acid Syn.thesis From all of the concepts presented (Table XI) and the paucity of evidence for the support of any one, it is clear that the over-all functions of the histones remain a mystery. Recent attention has focussed on the possibility that the histones serve to suppress formation of RNA in DNAprimed systems. I n studies on the biosynthesis of ribonucleic acids of pea seedling preparations, Huang and Bonner (1962) employed polymerase systems in which ribonucleotide triphosphates were substrates and DNA was the primer for RNA synthesis. I n such systems, “histone” preparations were found to inhibit the biosynthetic reactions by combination with the DNA template. The interaction of DNA and histone was apparently stoichiometric, and was also reversible, as was shown by the fact that DNA reisolated from the DNA-histone complex was again active as a primer. The methods used for preparation of the histones were modifications of the procedure of Crampton e t al. (1954a,b) in which the DNA was separated from the histones with the aid of high concentrations of ethanol, a procedure that has been shown by Zubay and Wilkins (1962) to modify the structure of the histones as determined by X-ray diffraction analysis. However, the studies of the latter workers were carried out on deoxyribonucleoproteins of calf thymus and Huang and Bonner (1962) employed

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pea seedling preparations. Since there is little or no evidence that the histones of the pea seedling are the same as those of the calf thymus, the same experiment should be attempted with DNA, histone, and RNA polymerase from other sources. I n the studies carried out by Huang and Bonner (1962) the DNAhistone complex was a soluble preparation. The histones and DNA were mixed in 1-2.5M NaCl, and the solution was dialyzed to a low ionic strength against dilute sodium citrate that functioned as a buffer. This preparation was centrifuged and the supernatant fraction derived from soluble histone and soluble DNA was used for further studies. These workers (Bonner and Huang, 1963) found that histones influenced the temperature of strand separation of DNA ( T m )to differing extents. The T , of DNA alone was 70°C. and that of the deoxyribonucleoprotein complex was 84°C. DNP prepared by mixing DNA with lysine-rich histones had a T , of 83°C. and DNP prepared by mixing DNA with other histones had the same T , as free DNA. They found that the complex of DNA and lysine-rich histones had the least primer activity of any of the complexes formed, i.e., the primer activity could be reduced to a negligible level compared to that of free DNA. The primer activity of “native” nucleohistone was found to be 10% of that of free DNA. A partial confirmation of the experiment of Huang and Bonner (1962) has been reported by Allfrey et al. (1963), who found that in calf thymus nuclear preparations, histones inhibited the activity of RNA polymerase. However, their results with purified histone preparations were the obverse of those obtained by Bonner and Huang (1963). Allfrey and his colleagues found that the lysine-rich histones either did not inhibit RNA synthesis or actually stimulated i t ; they found that the arginine-rich histones were inhibitory. I n experiments with trypsinized thymus nuclear preparations, the incorporation of labeled precursors into RNA was much increased, but the reasons for such an increase may be multiple. Interestingly, Weiss (1963) has found that a variety of polyamines, such as spermidine, enhance the activity of free RNA polymerase. When histones were added to such preparations a t low concentrations, they enhanced, rather than suppressed, the activity of the isolated enzyme system.

3. Histones and D N A Synthesis Billen and Hnilica (1963) employed the DNA polymerase or nucleotidy1 transferase derived from Escherichia coli as a catalyst for formation of new DNA in the presence of calf thymus DNA as primer and the appropriate deoxyribonucleotide triphosphates. I n their study, histones were added to the system and they noted that the activity of the DNA poly-

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merase was suppressed. However, the suppression was apparently due to the formation of the deoxyribonucleoprotein precipitates in salt-loaded systems. Whether this precipitation of deoxyribonucleoproteins was the whole basis of the inhibition or whether there was any greater specificity to these suppressions is not clear a t the moment. Although the evidence obtained (Huang and Bonner, 1962; Allfrey et al., 1963; Billen and Hnilica, 1963) serves to suggest that the histones may function to suppress nucleic acid templates by decreasing their availability to the appropriate enzymes, the critical question is whether the cell utilizes the histones selectively for such purposes. There are now known to be many DNA molecules in each cell and, depending upon the calculation of DNA concentration and molecular weight, the number is approximately 1 million in mammalian cells. In a liver cell containing 12 picogram (pg) DNA per nucleus, there would be 740,000 molecules of DNA, assuming a molecular weight of approximately loF for the DNA. Since each DNA contains about 5000 coding units, there are sufficient coding units to produce about 7 million proteins in each mammalian cell, assuming a molecular weight of about 50,000 for proteins and about 400 amino acids per protein. The total DNA coding units approximate 4 x lo9 in the mammalian cell and the total histone molecules, assuming a molecular weight of about 20,000, approximate 2.5 x lo8. These values not only reflect the enormous number of possible coding complexes that are available to mammalian cells but also indicate that the histones could block one in 15 coding units in DNA, if one histone blocked one coding unit. If the histones were strung along the DNA in the form of strands on the surface or fit as strands in grooves, the histones would be more than sufficient in amount to block all of the protein codes, assuming that a block of one in three phosphates of a codon would be sufficient to block the codon. As indicated previously, there are enough amino groups in histones to combine with 80-90% of the phosphate groups of DNA. Presumably, messenger-RNA synthesis could be prevented by blocks of key segments of DNA. Assuming that histones do function to suppress the biosynthesis of RNA on DNA templates, the question that arises is whether histones possess sufficient “information” to control genetic loci, or whether they simply serve to function as some sorts of “stops” that are released under appropriate conditions. There is one school of thought that suggests there are hundreds of histones and that, indeed, they may possess genetic information. However, as indicated in the sections to follow, there would appear to be only 10-20 types of histones and of these, perhaps 8-10 constitute the great bulk of the histones. According to Phillips (1961), one histone type is probably linked to many DNA phosphates, but the association may not be random. Thus, it is very difficult indeed to consider that the histones have genetic infor-

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mation, or information from the cytoplasm that could control the function of the genome. From all of these considerations, it is clear that the role of histones is by no means elucidated thus far and that many pieces need to be fitted into the complex questions raised by structural, enzymatic, degradative, biological, and metabolic experiments before one can even begin to satisfy the question of the intranuclear role of these structures.2 Perhaps they do function to block all the genomes and in this way resemble the various types of stops used on musical instruments. The question would then be: What functions to unstop the stops? One suggestion made for this was that acidic proteins may be specifically combining with histones to produce the type of equilibria illustrated below (Busch et al., 1963d) : ki

+ (DNA)ks (histone)+ + (acidic protein)Histone-acidic protein Histone-DNA

kz

(histone)+

k4

D. ORIGINSOF

THE

HISTONES 1. Gene Control

Bloch (1962a,b; Bloch and Hew, 1960) has raised the question of the number of sites in the nucleus that are responsible for formation of the histones. He has argued, inductively rather than on the basis of evidence, that an individual DNA is unlikely t o code both for protein and RNA, and hence, that histones are not likely to be synthesized on DNA templates. Bloch (1962a,b) indicates that there is a 1: 1 ratio of histone to DNA on a weight basis; this means that for each nucleotide of DNA there are approximately two to three amino acids. Since two or three nucleotides are required to code for a single amino acid, it is impossible that each DNA codes for its own histone on any kind of a unit for unit basis. There is simply not enough information in DNA to permit this type of coding. Accordingly, there must be a number of sites that code for more than one histone molecule and from this concept, the question was raised as to whether or not there are merely several genomes or perhaps up to one hundred genomes that code for histone synthesis. Apparently, even these genomes are functional in different dosages, since there are different amounts of the individual histones (see Section IX,H,2). The question may be raised whether some of these genomes code for repression or depression of RNA synthesis and others for repression of DNA synthesis. *Other functions, such as control of respiration and mitochondria1 activity, h a w been suggested for histones (Hanson and Swanson, 1962; Rivenbark and Hanson, 1962; Wolfe and McIlwain, 1961; McIlwain et al., 1961).

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The intranuclear sites of synthesis of histones are not a t all clear a t the present time. There is evidence that direct synthesis of proteins occurs on the chromosomes, a t least in Chironomous and in Drosophila (Sirlin and Knight, 1960) but the nature of the proteins synthesized is not known. From biological evidence, it is established that in periods of increased histone synthesis there is an increased size of the nucleoli, i.e., in Walker tumor, regenerating liver, thioacetamide-treated liver, etc. However, i t is not possible to relate specifically the timing of the biosynthesis of the histones to the increments in the size of the nucleoli; consequently, it is not clear whether these are simultaneous events, or causal in any way for the other. 2. Studies on Regenerating Liver There are many statements in the literature that there is a simultaneous synthesis of histones and DNA. Most of these statements (Alfert, 1957; Prescott, 1960; Bloch, 1962a,b) are based upon cytochemical studies or autoradiograms since there is no biochemical evidence to support these concepts (Lindner, 1960; Baserga, 1962; Mattingly, 1962; Holbrook et al., 1962; Butler and Cohn, 1963). Certainly, there is no evidence of a relationship of the biosynthesis of specific histone fractions to the specialized biosynthetic reactions within specific cells. Recently, Holbrook et al. (1962) and Irvin et al. (1963) have examined the question of relationship of DNA synthesis to that of histone synthesis in the regenerating liver. The initial wave of cell division in their studies was found at 31 hours after hepatectomy; this time was the initial maximum of mitosis in these liver cells. The event that occurred most rapidly was a marked increase in the synthesis of nuclear RNA that became maximal a t 15 hours after hepatectomy. The next intranuclear event to occur was the increased synthesis of histones and this reached a maximum a t about 18 hours after hepatectomy (Muramatsu and Busch, 1962). Although these workers divided their histone fractions into a number of subfractions by salt extractions, the labeling of all fractions reached a maximum a t the same time. The maximum labeling of DNA was reached a t about 26 hours after hepatectomy and the rise in labeling occurred after the maximum had been reached for the labeling of the histones. These data indicate that biosynthesis of the histones actually precedes that of the DNA, a t least in this system, and may actually stimulate it (Holoubek, 1962). These results, which do not support the earlier cytochemical suggestion that histone synthesis is simultaneous with that of DNA, have recently been confirmed (Butler and Cohn, 1963).

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3. R N A Dependence of Histone Synthesis The data obtained by Holbrook et al. (1962) suggest that the synthesis of the histones is RNA-dependent in the sense that the DNA that might synthesize the histones has always been present, but apparently it is not active until derepressed. The increased biosynthesis of RNA that precedes histone synthesis (and probably that of many other cellular proteins in the regenerating liver) may result in availability of templates for the synthesis of the histones. If the histones are synthesized on RNA, it remains to be shown which RNA is involved. Since the role of the nucleoli would appear to be that of synthesis of masses of GC-type RNA (Muramatsu et al., 1963a) that are the basic building blocks of the ribosomes (Busch et al., 1963b), the question necessarily arises as to the locus of the RNA involved in these synthetic reactions. As indicated previously, there is no good evidence for the synthesis of histones in the cytoplasm, particularly since these proteins are not found in the cytoplasm in the amount or the variety found in the nucleus. Accordingly, it would appear that the RNA templates involved in histone synthesis must be present in the nucleus or adjacent to the nuclear membrane. The nuclear ribonucleoprotein (RNP) network has been described previously (Smetana et al., 1963) as a network in the nucleus which contains the nucleolus and is composed of ribonucleoproteins. I n further studies with the aid of the electron microscope, it became apparent that the particles referred to as the “Swift” and “Watson” R N P particles were in effect the cross-sections of this network and appeared to be very electron-dense. Using improved techniques, Smetana and Busch ( 1963) were able to demonstrate that these R N P particles were part of a tubular network in which the small tubules of the network apparently were derived from the nucleolus. From the electron micrographs prepared, it would appear that these highly coiled microtubules formed in the nucleolus migrate toward the nuclear membrane and that a layer of RNA or ribonucleoprotein is added to the periphery of the tubule as it moves through the nucleolus-associated chromatin toward the cytoplasm. This added layer may be the so-called messenger RNA and may account for the denser appearance of the cut tubules (or dense particles) as the tubules move toward the cytoplasm; the particles in the periphery of the RNP network were even larger than those in the nucleolus. The possibility exists that the larger R N P particles are precursors of ribosomes or polysomes (Wettstein et al., 1963; Gierer, 1963) formed in the nucleolus, to which ribonucleoproteins have been added in the outward course of the ribosomes toward the nuclear membrane. The presence of large amounts

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of this network in the nucleus of the tumor cell raises the question whether this nuclear substructural component may play a part in the biosynthesis of the histones and other nuclear proteins. Another possibility is that nuclear ribosomes may serve as sites of synthesis of these proteins. Thus far, studies in this and other laboratories have not permitted a conclusion of the types of proteins biosynthesized by the nuclear ribosomes since net synthesis of protein has not been obtained employing these preparations. Another possible candidate for biosynthesis of histones is the ribonucleoprotein complex that passes through the nuclear membrane and apparently is very closely attached to it. As indicated previously, in tumors there is such a large amount of these ribonucleoproteins that i t is almost impossible to isolate defined nuclei from tumors in the presence of calcium ions in the medium in appreciable quantities. The presence of such a large amount of ribonucleoprotein a t the periphery of the nucleus suggests that the rate of biosynthesis of tubules has not kept pace with the rate of breakup of the tubules and combination of the released dense particles with the product of the lumenal forming system of the ergastoplasm. However, it is not known whether this third possible histone template subserves any active function in protein synthesis until it is actively a portion of the ergastoplasm. Since some histones are so rich in lysine, it would seem that the RNA involved in their synthesis would necessarily be rich in adenine, since the code for lysine is AAA.

E. CHEMISTRY OF THE HISTONES Only snatches of the information ultimately required for characterization of the histones are available at the present t h e . To understand fully the chemistry of the histones, the following information is required: ( 1 ) the number of the histones; (2) their primary or linear amino acid sequence; ( 3 ) their spatial relationships to DNA; ( 4 ) their physicochemical properties including their “native” tertiary structure. For the complete development of this information, it is clear that much work remains to be done, Although, for the most part, the problem of histone chemistry has not moved forward rapidly, the pressing requirement that has been raised by the findings of differences in rates of histone synthesis in tumors and other tissues is whether there are differences in the structures of histones of tumors and other tissues. Partial answers to this question are inadequate. 1. Distribution of the Histones Ilistones are found in a variety of mammalian cells and in cells of other species, phyla, and classes including many chordates such as birds,

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fish, amphibia, as well as plants, including pea seedlings (Setterfield et al., 1960) and some lower forms such as starfish and sea urchins (Phillips, 1961). I n some specialized cell forms, such as the spermatozoa, the histones are replaced by protamines, a group of basic proteins that are limited in their occurrence and obscure in their function. The histones are found in very large amounts in calf thymus and other thymus tissues and hence the extracts of these tissues are frequently studied (Phillips, 1961). Whether there is any relationship between the high concentration of histones in these tissues and the increasingly evident role of the thymus as a tissue important in the production of antibodies is not yet understood, but in view of the finding of Porter (1959) that basic proteins form an important part of the structure of 7-globulins, the possibility may not be so far-fetched. 2. Sources of Histones

I n general, two procedures are commonly used for isolation of nucleoproteins. I n the first (Butler et al., 1954; Lucy and Butler, 1956; Butler and Davison, 1957; Shooter and Butler, 1957; Shooter e t al., 1954; Johns e t al., 1960, 1961) calf thymus or other tissue is homogenized in 0.14M NaCl with or without a pretreatment with a ball mill to more or less purify a “nuclear preparation.” TO the residue obtained after a number of extractions may be added DFP (diisopropylfluorophosphate) or citrate buffer to control proteolysis and the pH. The product is a whitish solid that is referred to as “crude DNP,” deoxyribonucleoprotein, or simply nucleoprotein, since it is likely that 5-10% of the preparation is composed of ribonucleoprotein. The second procedure involves the initial extraction of the deoxyribonucleoprotein from nuclei or from whole cells and is based upon the high solubility of nucleoproteins in distilled water or aqueous media of very low ionic strength and/or salt solutions 2 M or more with respect to NaC1. Regardless of the method employed for extraction of the nucleoprotein, the extract is finally diluted or enriched with NaCl to make a final concentration of 0.1-0.2 M ; the deoxyribonucleoproteins are precipitated out of such dilute salt solutions, as noted previously. The finding that proteins of cytoplasmic ribosomes were similar to histones (Butler e t al., 1960) has suggested that, for isolation of the histones from nuclei, the optimal starting material should be isolated nuclei. I n studies on calf thymus, the procedure employed for isolation of nuclei is frequently inadequate since the cells contain little cytoplasm in the first place, and really satisfactory methods for isolating nuclei from the very large amounts of calf thymus usually used are not available a t the present time (Roof and Aub, 1960). The procedure employed is not sufficiently discriminating to wash out the relatively insoluble ribonucleopro-

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tein fraction recently found by Steele and Okamura (1963, unpublished) in this laboratory to be partially extracted by Tris buffer after washes have been made with dilute salt solutions. It goes without saying that the product extracted by prolonged treatment with Tris should be examined for the types or presence of histones in the extract. The problems of the procedures for isolation of the nuclei have been discussed previously, and the procedures for isolation of the “nucleoproteins” also have difficulties. To the question that has been raised previously whether the histones are present in such tight linkage to DNA that they will not come off during extraction procedures must be added the question whether the histones are generally distributed throughout the nucleus under normal circumstances. I n other words, does direct extraction of the nucleus with acid, as carried out by Kossel (1921), Stedman and Stedman (1943), and by other workers (Davis and Busch, 1959, 1960), give a histone product that is more representative of the nuclear histones, or does the isolation of the nucleoprotein fraction provide a product that is more representative? At the moment, an answer to the question cannot be provided. There is evidence that the products obtained apparently differ when these different methods are utilized.

F. EXTRACTION OF THE HISTONES Since the time of Kossel (1921) , it has been standard practice to extract histones with dilute mineral acids of which HC1 is by far the most commonly employed (Busch and Davis, 1958). Although different workers have had their individual preferences, it has been the custom to utilize 0.2-0.3N HCl (Table XII). Some preference has been expressed for extracting histones with dilute H,SO,, but this acid is not removable by lyophilization and hence is a little less satisfactory than HCl. There are a few indications that the product obtained is different from the product obtained with HCl in terms of migration on starch gel electrophoresis; for example, the f3 zone has been found to be decreased when H,SO, has been used for the extraction of histones. There had been no justification for the use of the apparently brutal method of acid extraction of biological materials other than the fact that it was used by Kossel and most other workers. Recently, Zubay and Wilkins (1962) studied the X-ray diffraction patterns of histones both in complexes with DNA and as isolated by the use of acids. Their study showed that undenatured histones produced diffuse rings and a reflection a t 10 and 4.5A. These diffuse patterns are compatible with the presence of a-helices. They also noted a reflection of 4.7A. that was apparently compatible with the presence of a /I-type structure produced by denaturation, Dried preparations of the nucleohistones produced patterns similar to those of the proteins extracted with acid.

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

91

Zubay and Wilkins (1962) also extracted histones by the procedure of Crampton e t al. (1954a,b) which uses ethanol to separate the DNA strands from the histones left in ethanol-water solution. I n essence, the steps in this procedure consist of solubilization of the deoxyribonucleoprotein complex in saline solutions followed by addition of 1, 2, or 4 volumes of ethanol and stirring of the mixture to remove the DNA that comes out of the solution. Although the samples prepared by acid extraction did not have a pronounced ring a t 4.7A., the samples prepared by the procedure employing alcohol gave a strong ring a t 4.7A. and also did not reconstitute satisfactory deoxyribonucleoprotein preparations when added to DNA. The studies of Zubay and Wilkins (1962) thus give some comfort to those who have been extracting histones with the aid of acids from either nuclei or nucleoproteins and raise some questions about experiments such as those of Huang and Bonner (1962) in which the procedures of Crampton e t al. (1954a,b) have been employed. Of course, the data obtained by Huang and Bonner (1962) were obtained with histones from pea seedlings and the results of Zubay and Wilkins do not necessarily apply to such preparations.

G. SEPARATION OF THE HISTONES Some degree of fractionation of the histones has been achieved by electrophoresis (Cruft et al., 1954, 1957a,b; de Mende e t al., 1960; Nooij and Niemejer, 1962) or with the aid of chemical fractionations such as salting-out techniques, low-temperature alcohol precipitation, and other procedures. Separation of the very lysine-rich histones (Phillips, 1961) from the other histones can be achieved by precipitation of these proteins a t low temperatures by ethanol in concentrations of 20-33% a t p H 6.3 (Smillie et al., 1955; Cruft et aE., 1957a,b; Bijvoet, 1957; Ui, 1956, 1957a-e). Precipitation of the mixture of the arginine-rich and slightly lysine-rich histones from the other proteins can be achieved by addition of ammonia to the solution to p H 11. A t this pH, the very lysine-rich histones can be precipitated by addition of 2-3 volumes of either acetone or ethanol or by decreasing the pH to 6. One of the problems in working with the histones a t the high pH ranges is the rapid formation of aggregates (Cruft e t al., 1958) ; hence in most recent studies, efforts have been made by almost all workers to keep the p H below 4.0. There is considerable evidence that the fractions obtained by these different salting-out procedures are mixtures of a number of components, and regardless of the techniques employed, amino acid analysis and NH,terminal amino acid analysis show the presence of a variety of components. One of the possible explanations for the presence of a number of components in these fractions is the hydrolytic action of the proteases

TABLE XI1 FRACTIONATION OF CALFTHYMUS HISTONES

Methods of fractionation

Number of fractions

Differential precipitation with Ethanol

2

Ethanol

2

Ethanol

2

Ethanol

2

Alkali and ethanol

2

Ammonia and acetone

2

NaCl (saturation)

2

NaCl (successive extraction)

6

Electrophoresis

2

Ultracentrifugation

2 2 2 2

Characteristics of fractions

Starting material

Histone extraction

References

Arginine- and lysine-rich Arginine- and lysine-rich Arginine- and lysine-rich Arginine- and lysine-rich Arginine- and lysine-rich Arginine- and lysine-rich Arginine- and lysine-rich Inverse proportionality between arginine and lysine Arginine- and lysine-rich Heavy and light Heavy and light Heavy and light Heavy and light

Isolated nuclei

0.1 N HzSO,

Stedman and Stedman (1943)

Nucleohistone

0 . 2 N HCl

Bijvoet (1957)

Isolated nuclei

0 . 5 N HzSO,

Ui (1957a-)

Nucleoprotein

10% NaCl

Isolated nuclei

0 . 5 N HzSO4

Gregoire et al. (1953); Gregoire and Limozin (1954) Daly and Mirsky (1954-1955)

Nucleoprotein

0 . 2 N HCI

Davison and Butler (1954)

Nucleoprotein

0 . 3 N HCl

Smillie et al. (1958) Lucy and But!er (1955)

Nucleoprotein

Nucleoprotein

0 . 2 N HCl

Nucleoprotein Isolated nuclei Nucleoprotein Nucleoprotein

0 . 2 N HCI 0 . 2 5 N HCl HCl pH 2 . 5 0 . 1 N HCI

Butler et al. (1954); Davison and Butler (1954) Butler et al. (1954) Luck et al. (1956) Bakay et al. (1955) Hamer (1951h)

Electrophoresis and ultracentrifugation

Column chromatography Ba IRG50 column eluted with Ra(AC)2

Ba IRC-50 column eluted with Ba(AC)2and guanidinium chloride Amberlite IRC-50 column eluted with guanidinium chloride Carboxymethylcellulose column eluted with NaCl

Diethylaminoethylcellulose column eluted with NaCl

6

Two fractions rich in arginine and 4 fractions rich in lysinp

Isolated nuclei

0 . 1 N HzSO4

Cruft et al. (1957a,b)

3

One fraction rich in arginine and 2 fractions rich in lysine

Nucleoprotein

2 . 6 M NaCIethanol

Crampton et al. (1955)

Nucleoprotein

2.6 M NaClethanol

Luck et al. (1956)

6

2

Arginine- and lysine-rich

Nucleoprotein

Dil. H2S04

Luck et al. (1958)

3

One fraction rich in dicarboxylic amino acids and 2 fractions rich in lysine Arginine- and lysine-rich

Nucleoprotein

0.2 N HCI

Davison (1957a,b)

2

Nucleoprotin

Bakay and Kirschner (1958)

tc

GJ

94

HARRIS BUSCH AND WILLIAM J. STEELE

studied by Dounce and Umana (1962). To minimize protease activity these workers have carried out their initial extractions a t pH 6 ; others have preferred to add aitrate buffers or DFP to their preparations. Despite the fact that the variety of precipitation methods have provided fractions that were mixtures in varying degrees of all of the histones, as indicated by Table XII, there was some separation of the histone fractions by these procedures. Of course, their use permitted the separation of the very lysine-rich and arginine-rich fractions initially (Tables XIIXIV) . Recently, the methods for fractionating the histones have been aided by the development of extraction procedures by Johns et al. (1960, 1961 ; Johns and Butler, 1962), who initially extracted histones from deoxyribonucleoproteins with a mixture of 80% ethanol and 20% 1.25N HCI. This procedure permitted the extraction of small amounts of the very lysinerich histones of fraction 1, and considerable amounts of fractions 2a and 3 from a residue; fraction 2a is said to be a mixture of slightly or moderately lysine- and glycine-rich histones and fraction 3 is said to be a mixture of arginine-rich histones, although, indeed, i t is no more argininerich than the proteins of fraction 2a (Hnilica and Busch, 1963). When this extraction was completed, the precipitate was extracted with 0.25 hr HC1. This procedure solubilizes more of the proteins of very lysine-rich histones, or fraction 1 and the fraction coded as 2b, a moderately or slightly lysine-rich fraction, and some more of fraction 3. The differential extraction with acidified ethanol solutions and dilute HCI aided in the further isolation of individual components of the histones. 1. Precipitation of the Histones Further purification of the histones was achieved by dialysis and precipitation. I n the procedure employed in this laboratory, which is a modification of the procedure employed by Johns et al. (1960, 1961; Johns and Butler, 1962), the proteins of fractions 2a and 3 were separated by dialysis of the mixture against ethanol, or by direct precipitation with TCA and then by chromatography on carboxymethylcellulose columns. Precipitation of the fraction coded as 2b was effected with 570 TCA after the fraction was dialyzed against water and precipitation of fraction 1 was effected with 2070 TCA. 2. Chromatography The first attempts a t chromatography of the histones were made by Crampton et al. (1955, 1957), who employed a column of Ba IRC-50 and eluted the histones with barium acetate. The recoveries were very low, approximating one third of the total amount of histones added to the

TABLE XI11 AMINO ACID COMPOSITION OF CALFTHYMUS HISTONESPRIOR Starting material: Histone extraction: Hietone precipitation: Arginine Lysine Histidine Alanine Glutamic acid Aspartic acid Methionine Leucine Isoleucine Phenylalanine Valine Tyrosine Proline Threonine Serine Glycine Cystine Tryptophan

Nucleohistone Isolated nuclei (Daly et al., (Kossel, 1921) 1950-1951) dil. H2S04 0.2 A' HCl Ammonia NaOH to pH 10.0 9.6 6.1 0.9 5.4 0.8

18.0 3.4 8.0 2.3

0.8

8.5 7.9 1.7 11.2 9.2 6.1 0.8 8.5 4.4 2.4 5.9 2.1 4.5 6.0 5.1 9.0 0.4

Nucleoprotein (Hamer, 1951a,b) 0.1 N H C I Ammonia 11.0 8.0 2.0 9.8 ? .8

5.5 0.9 14.8 3.0 8.7 2.7

3.8 6.0 4.3 9.3 0.03 0.21

TO

FRACTIONATION'

Chromosomal Nucleoprotein material (Morris (Crampton et al., and Harper, 1953) 1955) 0.2 N HC1 2.6 M NaCI-ethanol Lyophilization NaOH to pH 10.4 9.3 11.6 2.5 14.2 9.4 6.0 0.6 7.3 6.3 1.9 4.7 2.8 4.4 6.6 4.8 7.8 0.0

8.3 14.7 2.0 13.5 8.2 4.8 0.9 7.7 4.3 2.2 6.2 2.5 4.9 5.7 5.9 8.3 0.0 0.0

a Values are per cent of total moles of amino acids recovered in the particular amino acid. The data are recalculated from the original papers.

0

m F

6

96

HARRIS BPSCH A N D WILLIAiM UJ. STEELE

column. Three fractions were isolated: fraction A, a lysine-rich fraction or very lysine-rich fraction corresponding to fraction 2a of later workers; fraction B, a fraction corresponding to fraction 2a of later experiments; and fraction C, a fraction that is distinctly different from any fraction obtained by other workers (Table XIV). Fraction A accounted for about 30% of the total histones recovered, and fraction B accounted for about two thirds of the total histones recovered. Experiments with a similar system, employing guanidinium chloride as an eluting agent, were carried forward by Luck et al. (1956, 1958), who noted that three additional peaks could be recovered and, moreover, that the recovery could be increased to essentially quantitative recovery of the histones. Later studies of a similar type employing IRC-50 columns were also reported by Neelin and Butler (1959, 1961), Rasmussen et al. (1962), and Satake et al. (1960). a . Chromatography on Carboxymethylcellulose. The first use of carboxymethylcellulose columns was reported by Davison (1957a,b), who noted that total recovery of the histones could be obtained. Three fractions were obtained by elution of the histones from the columns with NaCl solutions. Attempts to utilize the system employed by Davison in this laboratory did not succeed, either because of differences in preparation of the histones or differences in the carboxymethylcellulose. Thus far, a verification of the results of Davison has not been reported in view of the fact that workers in Butler’s laboratory have turned to elution of the histones with acid and with buffers. The reason given by Phillips and Johns (1959a,b) for the change in eluting agents was that the products obtained by the procedure of Davison were mixtures, as indicated by NH,-terminal analysis. Accordingly, they attempted to obtain a fractionation of the histones by elution of the carboxymethylcellulose columns with 0.01 N HCI and 0.02N HCI. Interestingly, both of these elution steps have been carried out on histones in contact with the adsorbent and a t room temperature. Reruns of the fractions obtained demonstrated that the protein was again absorbed on the columns and readily eluted. The fractions were apparently homogeneous, as indicated by their position on elution from the columns. Evidence for increased purity of the fractions obtained by elution of histones from carboxymethylcellulose columns with strong acid was the increased concentration of proline and alanine as NH,-terminal amino acids in the fractions referred to as 2 and 3, respectively. In 1960, Johns et al. reported a modification in which an acetate buffer a t pH 4.1 was used as an eluting agent to precede the 0.01N HC1 previously employed initially. This modification provided an initial elution of fraction 1 in higher yield and the isolation of a purer preparation of

AMINOACID COMPOSITION

Daly and Mirsky (1954-1955) Name of fraction: I I1 Amino acid Arginine Lysine Histidine Alanine Glutamic acid Aspartic acid Methionine Leucine Isoleucine Phenylalanine Valine Tyrosine Proline Threonine Serine Gly cine Cystine Tryptophan

11.3 11.5 1.9 12.1 9.9 6.1 1.1 8.1 3.4 2.3 4.3 2.5 4.0 6.1 5.0 9.8 0.4

3.0 33.7 0.0 19.1 4.0 2.4 0.2 4.9 1.5 0.8 4.5 0.8 7.7 4.4 6.0 7.0 0.0

Davison and Butler (1954) Fast Slow 11.6 8.4 3.2 11.6 8.5 4.9 1.7 9.3 6.5 2.8 6.9 4.5 2.3 6.4 2.7 8.7

2.7 26.2 0.0 27.4 2.3 2.7 0.0 3.1 1.3 0.0 5.0 0.7 8.9 5.8 5.9 7.9

OF

TABLE XIV VARIOUSCALFTHYMUS HISTONEF R I C T I O N S a

Gregoire et al. (1953); Gregoire and Limozin (1954)

P

S

10.4 10.5 1.4 9.5 7.7 6.5 0.7 17.0

5.5 15.2 1.3

2.1 6.9 3.4 3.4 5.1 5.4 9.3 0.4 0.2

2.0

Crampton et al. (1955) A B C

( ~ 1

Cruft et al. (1957a,b) ( ~ 3 @ 0.8s

02

1.6s

2.4 7.3 4.7 0 . 0 3.3 4.3 11.4 4.8 8.1 26.0 12.6 18.8 38.0 26.0 20.4 8 . 1 17.7 11.3 0.0 2.4 1.1 0.0 0.8 1.7 3 . 3 1 . 6 3.2 24.5 11.3 19.6 30.4 22.8 19.5 11.4 17.7 11.3 1.1 4.1 5.1 9.8 6.5 8.9 3.6 8.2 5.8 4.9 5.6 6.5 2 . 1 5.2 3 . 3 0.8 2.4 4.2 0.0 0 . 9 0 . 5 0 . 0 0.0 0.0 1.6 0.8 1.6 4.7 8.0 5.9 0 . 8 4.1 6.8 9 . 8 7 . 3 8.9 1.1 4 . 5 2.4 0.0 1.6 2.5 4.9 2.4 4.9 8.5 2.4 1.6 1.3 0.7 1 . 3 1.4 0.0 0.8 5.3 6.4 4.7 3 . 8 4.9 5.1 5.7 5.6 6.5 0.7 3.0 2.0 0 . 0 0.8 2.2 3.2 1.6 3.2 9.2 4.4 7.6 13.7 9.8 6.8 4.9 4.8 4.0 5.7 4.9 5 . 8 4.6 4.9 4.2 6 . 5 4.8 5.7 7.3 6.5 7.1 7.0 6.8 3.0 4.9 6 . 8 4.1 7.1 8.0 7.4 3 . 8 9.0 11.0 8.1 9.7 8 . 1 0.0 0.0 0 . 0

= Starting materials and extraction procedures are the same as in Table XIII. Values are per cent of total moles of amino acids recovered in the particular amino acid. The data are recalculated from the original papers.

98

HARRIS BUSCH AND WILLIAM J. STEELE

fractions 2 and 3. The amino acid analyses of these fractions are presented in Table XV. TABLE XV AMINOACID ANALYSES OF VARIOUS HISTONEFRACTIONP Extraction methods f2(a)

f2(b)

Starch gel fl

Amino acid Aspartic acid Glutamic acid Glycine Alanine Valine Leucine +isoleucine Phenylalanine Tyrosine Berine Threonine Proline Histidine Lysine Arginine

6.4 9.0 11.8 10.3

6.5 14.9 1.7 3.0 3.2 5.7 3.2 1.9 11.4

10.9

{ 16.9 7.8 11.4 7.2 10.3 1.8 2.7 7.6 5.9 5.2 3.0 15.8 4.5

2.7 7.0 5.6 22.9 4.3 6.4 1.3

-

5.5 4.2 7.6 0.7 29.7 2.1

7.3 9.2 10.7 12.1 6.9 12.8 1.4 2.0 4.3 4.9 4.3 3.0 11.6 10.3

7.4 5.9 8.9 11.o 6.9 10.6 1.9 2.5 7.2 6.6 4.2 3.0 14.8 9.2

-

-

NHz-Terminal amino acids Alanine Proline Glycine Others Wt. (g./moIe of NHzterminal amino acids)

54.0 12.0 11 .o 23.0 112 000

11.0 77.0 3.0 8.0 17 000

9.0 8.0 34.0 49.0 52 000

-

-

The amino acids are expressed as moles/lOO moIes of all amino acids found and no correction has been made for the hydrolytic losses of amino acids. The proportions of NHz-terminal amino acids are molar percentages of all such groups found (Johns and Butler, 1962).

b. Chromotography of Histones of Tumors. Following the findings of the group in England that showed that the histones could be adsorbed by and eluted from the carboxymethylcellulose columns, an effort was made in this laboratory to achieve improved resolution of histones of tumors by the use of formic acid to elute the proteins from carboxymethylcellulose columns (Davis and Busch, 1959). The experiments were designed to determine whether the greater uptake of labeled lysine into histones of tumors as compared with nontumor tissues (see Section IX,J) was due to the higher turnover of histones of tumors or was due to the formation

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

99

of different products in tumors. Since it was ne.cessary to work with the tissues of the rat, only small amounts of the proteins were available. The preparations utilized for chromatography were acid extracts of the whole nuclear preparations, which were not always as satisfactory as those of the liver and the tumor when the technique used for isolation of nuclei of the Walker tumor was employed (Busch et al., 1 9 5 9 ~ ) . The chromatographic patterns for the acidic nuclear proteins differed markedly in the various tissues studied. The pattern for each tissue contained a group of peaks ranging in number from five in the chromatogram for the liver to eight in the chromatogram for the intestine. Although the over-all chromatogram was similar in the Walker tumor, liver, kidney, and pancreas, two peaks were found in the B region of the spleen, four were found in the B region of the intestine and two were found in the E region of the thymus. Since the procedure employing formic acid elution was carried out on whole acid extracts, i t was apparent from the beginning that complete fractionation of the histones could not be anticipated, and this was found to be the case by starch gel electrophoresis (Busch et al., 1962). The presence of proteins other than histones may have produced some complexing of histones such that the histones were eluted somewhat differently in the different tissues studied, as shown particularly by the presence of RP2-1, in tumors (see Section IX,K). From the standpoint of over-all protein distribution, significant differences were not found for the tumors and a number of the other tissues studied (Davis and Busch, 1959). 3. Recent Developments in Fractionation of the Histones Each development of new chromatographic adsorbents has been followed by an endeavor to utilize them in fractionation of histones (Kent et al., 1958). Cruft (1961) has recently found that improved separations of histones can be achieved on Sephadex. He employs a column 1.5 meters in length and elutes the fractions with 0.02N HC1. The recovery of the protein is essentially quantitative. The degree of purification depends upon the concentration of HC1 employed and 0.02N HCI has been found to be optimal; lanthanum salts are being used to aid in the purification. Johns (1963, in manuscript) has recently separated the histones of the F1 fraction into three subfractions, differing in solubility in trichloroacetic acid. The fractionation was achieved on carboxymethylcellulose eluted at pH 9 with varying concentrations of NaCl. Interestingly, Bome of the fractions aggregated a t low pH. I n one of the fractions, glycine was the NH,-terminal amino acid ; in another, the NH,-terminal was blocked by an acetyl group. None of the subfractions had as high a lysine content as the a-1 fraction described by Cruft et al. (1958) but a very lysine-

100

HARRIS BUSCH A N D WILLIAM J. STEELE

rich fraction, equivalent in amino acid composition to that described by Cruft e t al., was obtained by hydrolysis of the F1 fraction for 30 seconds in 3 N HC1 a t 100°C. At least one fraction had a large number of glutamic and aspartic acid residues, but it is not known whether these were present as the amides, glutamine and asparagine. Many attempts have been made to utilize the starch gel electrophoretic procedure introduced by Neelin and his colleagues (Neelin and Butler, 1959, 1961; Neelin and Connell, 1959; Neelin and Neelin, 1960) for separation of large amounts of histones; thus far, however, these have not been successful and neither has the procedure employing polyacrylamide gels (Murray, 1962; McAllister e t al., 1963, in press).

H. PURIFICATION OF HISTONES OF TUMORS Using the procedures previously described by Johns e t al. (1960, 1961; Johns and Butler, 1962) the histones of the Walker tumor were separated into fractions 1, 2a, 2b, and 3 by means of chemical and chromatographic fractionations (Hnilica and Busch, 1963). A number of fractions isolated from the tumors were actually of higher purity than those obtained from the calf thymus. Subsequently, interest in this laboratory has centered around the fractions 2a and 2b that were parts of the RP2-L complex (see Section IX,K). The amino acid analyses of the proteins in fractions 2a and 2b are shown in Table XVI. Fraction 2a was actually richer in glycine and arginine than in other amino acids and may be referred to as a glycinerich histone. Fraction 2b actually contained more lysine than any other fraction with the exception of fraction 1 and in amino acid composition strongly resembled the composition of the purified fraction RP2-L (Busch e t al., 1962). Although fraction 3 is supposed to be an arginine-rich fraction, it had little more arginine than the other fractions, particularly 2a. From the electrophoretograms obtained from the histone fractions it appeared that fraction 3 was the least pure, and that fractions 2b and 2a were much more purified. The components of fraction 1 were separated into a number of bands by means of starch gel electrophoresis following treatment with TCA. The electrophoretic patterns on the gels showed that the purity of the proteins was considerably enhanced by the procedures employed for their isolation. Moreover, the NH,-terminal analysis (Biserte and SautiBre, 1958; Phillips, 1958) showed that the protein with the proline NH,-terminal was the predominant one in fraction 2b (Table XVII). If there was only one protein containing proline in this fraction, the purity of the protein would be about 81%. I n further studies on isolation of these proteins, the percentage of the proline end group increased to almost 90% (Hnilica e t al., 1963). The number and types

TABLE XVI AMINOACID COMPOSITION OF NUCLEAR PROTEINS FROM WALKERTUMOR^ Amino acid

Fraction

Fraction 2a

Fraction 2b

2.8 6.1 5.9 7.6 7.9 6.8 17.8

5.1

2.9 5.9 6.3 8.2 8.9 7.5 20.4 5.1

0.1 0.1 1.6 4.2 0.7 0.9

1.7 4.5 0.5 0.7

5.3 8.5 5.6 3.2 3.1 12.5 10.4 7.2 0.4 0.1 4.8 9.9 2.8 1.6 0.1

5.0 9.0 6.4 9.5 4.6 6.9 10.4 6.9 0.7 0.2 5.1 6.0 3.1 1.8

26.7 0.5 3.8 7.0 3.5

23.8 0.3 3.4 7.0 3.1

9.9

14.3 2.3 7.1 2.0 1.7

-

1c

-

-

2.4 11.7 0.8 1.7

-

Fraction Fraction NaOH3C 3(NH,OH) soluble 6.8 14.6 5.6 4.9 3.7 5.7 11.5 5.1 1.4 0.3 4.1 10.6 1.9 2.6 0.2 9.1 1.9 10.2 0.9 1.o

5.5 10.2 6.8 4.6 4.3 8.5 10.7 6.0 0.7 0.2 5.2 9.0 2.3 2.9 0.1 9.8 2.1 11.8 0.8 1.5

8.9 11.9 5.4 6.7 5.0 7.0 7.3 5.4 2.0 0.3 5.0 9.0 2.7 3.9 5.2 6.2 2.5 5.2 1.2 0.7

NaOHinsoluble 7.6 11.5 5.9 6.8 4.4 6.7 7.8 6.1 2.0 0.4 3.5 9.7 2.5 3.8 6.0 6.8 2.1 6.2 1.1 0.7

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

Aspartic acid Glutamic acid Threonine Serine Proline Glycine Alanine Valine Methionine Half-cystine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Ratio of lysine t o arginine Ratio of basic to acidic amino acids

Fraction 1T

~~

101

0 The amino acids are expressed as moles per 100 moles of all amino acids found. A correction was made for the 10% losses of t h r e e nine and serine during hydrolysis. The average standard errors for the values for fractions 2a, 2b, and 1C were 0.1 for histidine, halfcystine, methionine, arginine, and phenylalanine; 0.2 for lysine, threonine, isoleucine, leucine, tyrosine, and aspartic acid; 0.3 for serine, proline, glycine, and alanine; and 0.4 for glutamic acid and valine. Fractions 2a, 3C, and 3(NH,OH) contained 0.5 t o 0.8% of eN-methyllysine (Hnilica and Eusch, 1963).

102

HARRlS BUSCH A N D WILLIAM J. STEELE

N

H

Amino acid Alanine Aspartic glutamic acids Glycine Leucine isoleucine Lysine Proline Serine Threonine Valine

2

TABLE XVII - AMINO ~ ACIDS ~ ~ OF ~HISTONES ~ ~ FROM ~ WALKERTUMOR"

Fraction 1 Fraction (trichloro1 acetic (chroma- Fraction acid) tography) 2a

Fraction 3 Fmrtion Fraction (chroma3 2b tography) (NH,OH)

+

10.6 3.9

21. 1 5.6

66.5 1.2

6.2 2.7

51.2 6.5

75.0 1.5

+

23.5 2.2

18.4 2.0

19.5

4.8 0.6

3.9 1.6

-

17. 5 14.6 18.2 6.5 3.0

18.4

4.9 6.7 0.9

3.3 80.8 0.3 1.5 1 .o

5.8 21.6 4.4 3.9 1.2

-

17.5 14.9 2.1

-

-

-

7.3

4.2 9.3

-

2.2 -

a The values for NH2-terminal amino acids are molar percentages of the total found. The standard error for the value for NH2-terminal proline in fraction 2b was 3.9. The standard errors for the values for NHz-terminal alanine and glycine in fraction 2a were 4.4 and 1.0, respectively. Five determinations were carried out for each fraction (Hnilica and Busch, 1963).

of the end groups in the other protein fractions were greater than those in fraction 2b. Because all of the subsequent evidence has shown that the protein of fraction 2b containing proline as the NH,-terminal amino acid was a single molecular species, the protein has been referred to as the N-proline histone. Another indication of the purification of the N-proline histone was the ultracentrifugation pattern that showed a single peak (Busch et al., 1963d). This fraction had a single peak on ultracentrifugation and on diffusion analysis ; from the constants obtained, the molecular weight was determined to be 22,000, a value that was in close agreement with the value of the average molecular weight of RP2-L. By contrast fraction 2a was resolved into two peaks on ultracentrifugation analysis, one of which had a shoulder; hence, at least three components were present in this fraction.

1. Comparative Studies on the Amino Acid Composition and NH,-Terminal Analysis of Histones of Tumors and Other Tissues I n a series of studies from Butler's laboratory, the general conclusion has emerged that if there are differences between histones of tumors and other tissues, they are not detectable by methods available. This conclu-

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

103

sion was drawn by Davison (1957b) based upon his chromatographic procedure discussed previously. Later, employing the analytical methods developed by Johns et al. (1960, 1961; Johns and Butler, 1962), Hnilica et al. (1962) drew the conclusion that the compositions of histones of thymus, spleen, and livers of calves and livers and spleens of normal and leukemic rats were essentially identical. Employing the starch gel fractionation scheme, no differences were noted, although the mobility of the fractions was not identical for all of the tissues studied. The only problem was that of the Ehrlich ascites tumor for which a complete resolution of the fractions was not obtained. I n the Walker tumor (Hnilica and Busch, 1963), fraction 1 corresponded to the fraction A of Crampton et al. (1957) in many respects, i.e., i t contained very low amounts of methionine or histidine and resembled the lysine-rich histones of a number of authors. The ratio of lysine to alanine was higher than that found in other tissues because of the relatively low amount of alanine. It is fortunate that methods for the fractionation of these histones are now becoming available so that the subcomponents can be compared for tumors and other tissues. Although many similarities were found for amino acid composition of other subfractions, 2b and B, significant differences in amino acid analyses were found. The over-all amino acid analyses for histones are clearly much less meaningful. Recently, Laurence et al. (1963) have compared the electrophoretic patterns for histones of two mouse tumors, rat liver, and calf thymus, and have reported that they were essentially the same. The lysine contents of the whole histone fractions were compared and although numerical differences were noted, the authors apparently drew the conclusion that the results were not significantly different. The NH,-terminal amino acids of subfractions of the histones were studied and although there was marked variation in the results, no significant differences were found. One of the peculiar features of the study, from which the conclusion was drawn that there were no differences in the histones of the tissues studied, was the arginine content of fraction 3 of the Crocker tumor and thymus. Differences of as much as 3% were found for the arginine content of this fraction between the tumor and the thymus and the difference was consistent for the different methods used to prepare the fraction. This difference, which suggests that there is a 20% difference in the arginine content of the histones of this fraction in the tumor and the thymus was not regarded as significant, and was not commented upon by the authors. 2. Special Features of Histones and Histone Subfractions

In the Walker tumor, the percentages of the whole histone recovered in fractions 1, 2a, 2b, and 3 were 10, 30-40, 30, and 20-30% of the total,

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HARRlS BUSCH AND WILLIAM J. STEELE

respectively. The content of fraction 1 approximates 2076 of the total histones in calf thymus, and the over-all content of this fraction in Walker tumors may be low, The proline content of this fraction approximates 8% of the total amino acid residue; there may be a relationship between this high proline concentration and the inhibitory effects of this histone fraction on RNA biosynthesis noted previously. The other histone fractions contained only 3-57% of proline. The content of c-N-methyl lysine (Murray and Luck, 1962) and acetylated amino terminals (Phillips, 1963) in these proteins has not yet been determined. One of the interesting findings of workers studying plant histones (Setterfield e t al., 1960; Huang and Bonner, 1962) is that there is a low content of coniponents of fraction 3 in plants (Rasch and Woodard, 1959; Johns, 1962). I n electrophoretic studies on histones of chicken red blood cells, Neelin and his colleagues have reported the presence of a different histone from those found in other tissues (Neelin and Butler, 1959, 1961; Neelin and Connell, 1959; Neelin and Neelin, 1960). One question that remains open is whether bacteria have any histones (Cruft and Leaver, 1961; Palmade e t al., 1958; Zubay and Watson, 1959).

I. STRUCTURAL ANALYSISOF

THE

N-PROLINE HISTONE(FRACTION 2b)

The purification of the N-proline histone of the Walker tumor suggested that a t least this one histone might be compared for a variety of tissues. The amino acid analysis of the 2b fraction of the Walker tumor, rat spleen, calf thymus and rat thymus was essentially the same, although minor quantitative differences were found (Hnilica et al., 1963).However, it would not have been possible to distinguish these fractions, from one tissue to another, on the basis of this analysis. The purity of the A'proline histones from the various tissues ranged from 86-90%, as measured by the percentage of proline as the NH,-terminal amino acid. The most critical studies carried out to compare the structures of these proteins involved fingerprinting of the proteins, in which the peptides obtained by tryptic hydrolysis of the proteins were initially chromatographed in a modification of Phillips' system (Hnilica et al., 1963; Phillips and Simson, 1962) and then were subjected to electrophoresis in a Gilson high-voltage electrophorator. Essentially the same number of spots moving in the same positions was found for calf thymus and for the Walker tumor. The patterns for the other N-proline histones were not significantly different. The peptides ranged in size from dipeptides to dodecapeptides. 1. Analysis of Peptides of Histones

The only satisfactory methods available a t the present, time for the determination of the linear sequence of amino acids is the technique

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105

initially developed by Sanger (1945) in his studies on the structure of insulin. I n essence, this technique depends upon the determination of the NH,-terminal amino acid of the whole protein and a number of its cleavage products obtained by hydrolysis with a number of proteolytic enzymes (Hirs et d.,1954). Since the products overlap with one another, the over-all structure of the protein can be determined through the use of information on the structure of the peptides and their overlapping units. At present, three sets of studies have been carried out on the fractions obtained by the procedure employed by Johns e t al. (1960-1961). In these, peptides have been derived by tryptic hydrolysis of fractions 2a and the N-proline histone of the Walker tumor and calf thymus (Hnilica e t al., 1963; Busch et al., 1963d) and from fraction 3 of calf thymus (Phillips and Simson, 1962). A comparison of the amino acid analyses of the peptides of fraction 2a shows that differences exist in the composition of the peptides of this fraction in the Walker tumor and the calf thymus, as reflected in these early studies (Table XVIII). Of the fourteen peptides for which complete elementary amino acid composition was available, differences of one or more amino acids were found in seven. TABLE XVIII DIFFERENCES IN ELEMENTARY AMINOACID COMPOSITION OF PEPTIDES OF FRACTION 2a Peptide No. 2 3 5 6 7 9 10

Tumor

Calf thymus

Asp, gb, thr(?) Ser Ala, arg Lysr Ala,, argz, gly Leu

I n studies on the N-proline histone, about thirty peptides have been analyzed. Of these, none corresponded to the peptides obtained from fraction 2a. Moreover, differences were found for the number and kinds of amino acids in the peptides of calf thymus and of the Walker tumor (Table XIX). I n peptides 5 and 10, there were differences in the amounts of leucine and tyrosine. These studies on the peptide composition of histone fractions of the Walker tumor and calf thymus suggest that there are specific differences in the proteins of these two tissues and, as may have been evident from the amino acid composition, there are marked differences in the over-all peptides of the different protein fractions. The latter difference was so

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TABLE XIX SOMEPEPTIDES OF FRACTION 2b (N-PROLINEHISTONE) Peptide No.

Source

Formula

1

Calf thymus Walker tumor

Lys (glu, gly, sen, tyr, Val) Lys (glu, gly?, sera, tyr, Val)

2

Calf thymus Walker tumor

Lys (asp, glu, gly, his, ile, pro, ser2, thr, v:d) Lys (asp, glu, gly, his, ile, pro, ser2, thr, Val)

3

Calf thymus Walker tumor

LYS (asp, d Y ) LYS (asp, gly)

4

Calf thymus Walker tumor

Arg (ala, glu, ile, thr, Val) Arg (ala, gluz, ile, thr, Val)

5

Calf thymus Walker tumor

Lys (ala, glu, gly, leu, pro) Lys (ala, glu, gly, leu, proj

10

Calf thymus Walker tumor

His (ala, leu, tyr) His (ala, leu)

14

Calf thymus Walker tumor

Lys (ala, thr, Val) Lys (ala, thr, Val)

15

Calf thymus Walker tumor

Lys (tyr, Val) Lys (tyr, Val)

16

Calf thymus Walker tumor

Lys (leu, Val) Lys (leu, Val)

marked that different chromatographic procedures are necessary for the fingerprinting of fractions 2a and 2b. The procedure utilized by Phillips and Simson (1962) was quite adequate for fractionation of peptides of fraction 2a but a rather different procedure is required for the N-proline histone (Hnilica et al., 1963). 2. Methods f o r Fractionating Peptides One point that is worth mentioning has to do with the limits of accuracy of the procedure utilizing paper for fingerprinting. Some alterations of the peptides are possible during the procedure of heating the sheets for visualization of the peptides with ultraviolet light and, more significantly, total resolution of the peptides was not possible. Further efforts to separate the peptides have been made with the aid of the amino acid analyzer and the Aminex types of cation exchange resins (Spackman e t al., 1958). From the early results obtained, it seems that a more satisfactory separation of peptides will be possible with the aid of these columns and with the aid of the stream divider system for isolation of the peptides.

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NUCLEAR PROTEINS OF NEOPLASTIC CELLS

It should be noted that there is a great need for determination of the linear sequence of amino acids in individual histones. All of the theories regarding function (Table X I ) and structural relationships of histones to individual nucleotide sequences in DNA are subject to the revisions that will accompany the working out of the structures of these proteins. Obviously, the purer the proteins, the more likely satisfactory resolution of the peptides will be and, more importantly, it would be possible to determine the over-all primary structure. However, a t the moment, none of the fractions is completely pure. The N-proline histone approaches the necessary level of purity for determination of the peptide structure, but i t would be of great value to purify it to the point where there would be no question that the peptides in the fingerprint actually originated from the N-proline histone.

J. METABOLISM OF HISTONES 1. In Vitro Studies

As part of a series of studies in the authors' laboratory on the effects of conditions on metabolism of tumor slices, a series of experiments was undertaken to ascertain the intracellular distribution of labeled amino acids. A number of such studies has previously been made with nontumor tissues (Daly e t al., 1952; Lang e t al., 1953; Smellie e t al., 1953) and extensive incorporation was found of labeled aspartic acid into the nuclear proteins of slices of the Walker tumor as compared to its incorporation into the nuclear proteins of liver slices (Davis and Busch, 1958). TABLE XX ISOTOPE IN HISTONESAS PERCENTAGE OF TOTAL ISOTOPE IN CELLUL-IR PROTEINS" Source Walker 256 carcinosarcoma Ehrlich ascites tumor (mouse) Rat liver Mouse liver Regenerating liver (rat) Rat kidney Rat spleen Rat brain Mouse pancreas

Per cent 22.0 24.0 8.0 7.1

7.7 7.3 6.5 7.7 1 .O

The values in the table represent the percentage in the histones of the total isotope incorporated into proteins of the cell suspensions or slices in uitro. The values for the ~-alanine-U-C14and ~-1ysine-U-Cl4were not significantly different and hence the data are pooled. Cell suspensions or slices were incubated for 1 hour in Krebs-Ringer bicarbonate buffer (Rusch et al., 1960).

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I n studies with labeled lysine, more than 20% of the isotope incorporated into the tissue proteins was in the histones in the tumor (Table XX), while less than 10% of the total isotope was in the histones in the other tissues studied (Busch et al., 1960). Evidence from this and other laboratories has indicated that the kinetics of labeling of the histones were essentially linear for approximately 1 hour of incubation of the slices in the Krebs-Ringer bicarbonate medium selected for these studies (Busch e t al., 1960; Campbell et al., 1957; Starbuck and Busch, 1960). The specific activities of the histones exceeded those of the proteins of the whole homogenate or most cytoplasmic fractions of the tumors (Busch et al., 1960; Starbuck and Busch, 1960). However, in the other tissues studied, the specific activities of the proteins of the microsomes and other cytoplasmic fractions were markedly in excess of those of the tumors. I n the pancreas, there was a very large incorporation of isotope into the proteins of the zymogen granules (Busch e t al., 1958, 1959b; Starbuck and Buscli, 1960). One of the interesting points of the in vitro studies was the finding that the specific activities (or isotope incorporation into the proteins) of the whole homogenate or the microsomes of nontumor tissues, particularly the spleen, did not differ markedly from the values obtained with the tumors. However, the specific activities of the histones of the neoplastic tissues were markedly greater than those of the nontumor tissues studied (Starbuck and Busch, 1960). 2. In Vivo Studies Some experiments on neoplastic tissues performed in vitro have provided rather striking results which tended to differentiate the neoplastic tissues from the other tissues studied. However, the history of metabolic studies on neoplastic tissues is replete with findings which are different in vivo from those found in vitro. Very early in this series of experiments, the in vivo labeling of nuclear proteins of tumors and other tissues was determined (Busch et al., 1958). The labeling of the histones of the Walker tumor exceeded the labeling of the other proteins of the tumors in the time period of 30-60 minutes after intraperitoneal injection of thc tracer. I n the other tissues studied, there was a considerable difference in the pattern found in that in each case the specific activities of the proteins of cytoplasmic fractions far exceeded those of the nuclear fractions studied. The pattern for distribution of the isotope into the proteins of the tumor was different from that of the distribution of the isotope in other tissues in that a greater percentage of the total amino acids entering the proteins of the tumor entered the nuclear proteins (Busch et al., 1958; Butler and Laurence, 1960). Since the initial findings were obtained with lysine labeled with C",

NUCLEAH PROTEINS OF NEOPLASTIC CELLS

109

it seemed necessary to determine the universality of these findings with other amino acids. Accordingly, similar studies were carried out with seventeen radioactive amino acids (Busch et al., 195913; Konikova et al., 1963). With twelve of the seventeen amino acids as tracers, the specific activity of the histones exceeded the specific activity of other tumor proteins. I n other tissues, specific activities of nuclear proteins were considerably lower than those of other tissue proteins. In view of these data and corroborative findings obtained, the fate of the amino acid pool in tumors was diagrammatically compared to other tissues as shown in Fig 1.

.

Albumin. other plasma pmteins resorotion

FANCREAS

AMINO ACID POOL SPLEEN

ZymogQ, grander.

* digestive enzymes

gmnules Lymphocyfer. mzymes fa

' pmtedyws of red cells

FIG.1. Fate of amino acid pool of Walker tumor diagrammatically compared with those of other tissues.

Other evidence indicating that the histones of a hepatoma had a higher specific activity following the administration of glycine to tumor-bearing rats than the corresponding liver was obtained by Rotherham et al. (1957). However, this finding was modified in later studies from the same laboratory (Holbrook et al., 1960). Butler and Laurence (1960),Samarina (1961), and Zbarsky and Samarina (1962) also reported a high nucleus to cytoplasmic ratio of labeling of proteins in tumors in vitro.

K. RP2-L To determine whether the higher labeling of histones of the tumor was due to differences in the composition of the histones of tumors (Black et al., 1960; Cruft et al., 1954) or simply due to higher over-all rates of synthesis, tumor-bearing rats were injected with 10 pc. of L-1y~ine-U-C~~ and the acid extracts of nuclear preparations were chromatographed using formic acid to elute the proteins from carboxymethylcellulose. The localization of radioactivity differed in the tissues studied. I n the heart and skeletal muscle, little radioactivity was found in any portion of the elution pattern other than in the breakthrough peak. In the chromatogram of the liver, spleen, intestine, thymus, and the kidney, the greatest proportion of the isotope was found in the region under peak E and smaller

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HARRIS BUSCH AND WILLIAM J . STEELE

amounts were found in the region between peaks A and B. The former radioactive peak was coded as peak 3 and the latter was coded as peak 1 (Davis and Busch, 1959). I n the Walker tumor, a very large and highly radioactive peak emerged between peaks B and C. Since it was the second radioactive peak to emerge, it was coded as peak 2, or RP2-L, meaning the second radioactive peak with labeled lysine as a precursor. The fact that this peak appeared in the Walker tumor and not in the other tissues studied suggested that the peak might be related to the neoplastic character of the Walker tumor and subsequent experiments were carried out on a variety of tumors. Regardless of the tumor studied, there was a peak, corresponding in position to RPP-L of the Walker tumor in the chromatogram when the animals had been injected with labeled lysine. However, in growing nontumor tissues, such as embryonic tissue and regenerating rat liver, this peak was not found. Accordingly, the possibility that the presence of RP2-L was related to neoplastic disease was enhanced by these studies. 1. Confirmatory Evidence for the Presence of RP2-L in Tumors

A number of reports has served to verify the findings mentioned above. McGillivray and Greenwood (1962) have reported that in human leukemic cells, RP2-L is formed in the presence of labeled lysine. The peak was not present in normal marrow cells incubated with labeled lysine. When the marrow of patients treated with alkylating agents was examined for the presence of RP2-L, after it was initially found to be present in the marrow of the same patients prior to treatment, the amount of RP2-L was found to be diminished. Studies were carried out by other workers (Hidvegi et al., 1963) on the presence of RP2-L in Walker tumors and these have verified the finding of the peak; moreover, studies on tumors treated with a variety of analog inhibitors showed that the peak disappeared in the presence of the analogs. Experiments on the effects of antitumor agents on the nuclear proteins have been discussed in previous sections, but it should be noted that selective inhibition of the biosynthesis of RP2-L did not occur following the administration of nitrogen mustards (Honig et al., 1961; Davis et al., 1961). As indicated previously, one of the main reasons that studies were initiated in this laboratory on the acidic nuclear proteins was that the biosynthesis of these proteins was markedly suppressed by the administration of mustards to tumor-bearing animals. 2. Composition of RP2-L

A logical consequence of the finding of RP2-L in tumors of murine and human origin was an endeavor to ascertain the number and types of

NUCLEAR PROTEINS OF NEOPLASTIC CELLS

111

proteins present in this peak. Because purification of proteins results in marked losses, purification of substantial amounts of a mixture of proteins is required for the achievement of any degree of purity of any one constituent from a mixture. The problem of obtaining mass amounts of RP2-L was partially solved by injecting as many as 100 rats with Walker tumor implants at as many as 12 sites per rat. From these rats, as much as 1 kg. of tumor tissue could be obtained in a period of 2 to 4 weeks. Either every other rat or every fourth rat was injected with labeled lysine to provide the radioactivity in the peak. The acid extracts of the nuclear preparations were chromatographed on carboxymethylcellulose columns 9 cm. in diameter and 45 cm. in height. For purification of RP2-L in amounts up to 100 mg., the procedure was scaled down and some impurities could be removed, but i t was apparent from the appearance of a shoulder on the chromatogram that the protein was not pure. Evidence that the proteins in the peak were rapidly biosynthesized was shown by the high specific activities of the proteins. The amino acid composition of the proteins in the RP2-L peak showed that the proteins in this peak were largely the slightly lysine-rich proteins, inasmuch as lysine, alanine and arginine comprised 14, 11, and 876,respectively, of the total amino acid residues. Studies on the NH,-terminal amino acids revealed that in the purified RP2-L fraction almost half of the NH,-terminal residues were proline residues. This value was higher than that of the other fractions obtained. Starch gel electrophoresis showed that the slowest moving fractions, the 1 and 3 fractions of Johns e t al. (19611, were not present in the RP2-L peak in appreciable amounts. This peak contained the fast-moving bands referred to as the 2a and 2b bands (Busch e t al., 1962) that were subj ected to the more precise analyses described previously. As indicated previously, the proteins in this group are being purified and analyzed. Thus far, it is not possible to account for the difference in the chromatographic behavior of the acid-soluble nuclear proteins of the tumors and other tissues in terms of a specific difference in proteins present in this peak. However, it is hoped that structural analysis, as well as further quantitative and qualitative analysis of the components of the acid-soluble proteins of the tumors, will permit interpretation of this interesting difference between tumors and other tissues. X. Discussion

The field of nuclear protein chemistry is in its infancy. Progress has been made in defining some of the conditions necessary for studies of the nuclear enzymes and for isolation of the histones. However, there is

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virtually no satisfactory information available on the very important acidic nuclear proteins from the point of view of isolation and purification. The multiplicity of theories regarding the function of nuclear proteins serves to indicate that clear evidence for the function of these proteins in living cells is not yet available. The mechanism of chromosome reduplication is of obvious significance in the cancer problem, and the recent findings of Cairns (1963) suggest that a number of enzymes must be involved. The alteration in structure of the chromatin spireme to the defined chromosome of metaphase must require the breakage of a number of chemical bonds. If one assumes that the DNA remains intact, the possibility exists that a number of histones or acidic proteins may form the lengthy chains of the spireme. Evidence that has been presented by chromosome morphologists that the telomeres of chromosomes are particularly prone to become closed in normal cells, presumably by proteins and possibly by specific histones. It is conceivable that the closure of the telomere prevents chromosome reduplication in interphase cells. The lack of specific histones or acidic proteins for blocking chromosome reduplication could be a key feature of the neoplastic cell. If the histones do not function as blocking agents and if they are removed from their normal equilibrium with DNA by virtue of the presence of acidic proteins in excessive amounts, DNA synthesis could bc unimpeded in neoplastic cells. One possible function of viruses either free in the cytoplasm or lysogenixed to DNA could be the excessive production of such acidic proteins. Evidence has been provided by Franklin and Baltimore (1962) that viral proteins can produce marked effects on nuclear metabolism in cells infected with the Mengo virus. Powerful suppressors of RNA synthesis have been shown to be produced by the virus. Similar substances might be influential in altering cellular metabolism to produce neoplastic disease. REFERENCES Alfert, M. 1956. J . Biophys. Biochem. Cytol. 2, 109-114. Alfert, M. 1957. I n “The Chemical Basis of Heredity” (W. D. McElroy and B. Glass, eds.), pp. 187-194. Johns Hopkins Press, Baltimore, Maryland. Alfert, M. 1958. Exptl. Cell Res. Suppl. 6, 227-235. Alfert, M., and Geschwind, I. I. 1953. Proc. Natl. Acad. Sci. U . S. 39, 991-999. Allfrey, V. G. 1963a. Exptl. Cell Res. Suppl. 9, 183-212. Allfrey, V. G. 196313. Exptl. Cell Res. Suppl. 9, 418-429. Allfrey, V. G., and Mirsky, A, E. 1957. Proc. Natl. Acad. Sci. U . S. 43, 821-826. Allfrey, V. G., Stern, H., Mirsky, A. E., and Saetren, H. 1952. J. Gen. Physiol. 35, 529-554. Allfrey, V. G., Daly, M. M., and Mirsky, A. E. 1954. J. Gen. Physiol. 38, 415-424. Allfrey, V. G., Mirsky, A. E., and Osawa, S. 1955a. Nature 176, 1042-1049.

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Webster, G . W. 1960. Biochem. Biophys. Res. Commun. 2,5648. Weiss, S.1963. I n manuscript. Wettstein, F. O., Staehelin, T., and Noll, H. 1963. Nature 197, 430-435. Wilbur, K. M., and Anderson, N. G. 1951. Exptl. Cell Res. 2, 47-57. Wilkins, M. H. F., Zubay, G. L., and Wilson, H. R. 1959a. Trans. Faraday Soc. 55, 497. Wilkins, M. H. F., Zubay, G. L., and Wilson, H. R. 1959b. J . Mol. Biol. 1, 179-185. Wolfe, L. S.,and McIlwain, H. 1961. Biochem. J . 78, 33-40. Yanagisawa, K. 1962. Biochem. Biophys. Res. Commun. 9, 88-93. Zbarsky, I. B., and Debov, S. S. 1948. Doklady Akad. Nauk SSSR 63, 795-798. Zbarsky, I. B., and Georgiev, G. P. 1959. Biochim. Biophys. Acta 32, 301402. Zbarsky, I. B., and Perevoschchikova, K. A. 1951a. Biokhimiya 16,112-124. Zbarsky, I. B., and Perevoschchikova, K. A. 195lb. Biokhimiya 16, 347-355. Zbarsky, I. B., and Samarina, 0. P. 1962. Biokhimiya 27, 557-564. Zbarsky, I. B., Dmitrieva, N. P., and Yerniolayeva, L. P. 1962. Exptl. Cell Res. 27, 573-576. Zubay, G., and Doty, P. 1959. J. Mol. Biol. I, 1-20. Zubay, G., and Wilkins, M. H. F. 1962. J. Mol. Biol. 4, 444-450. Zubay, G., and Watson, M. R. 1959. J . Biophys. Biochem. Cytol. 5, 51-54.

NUCLEOLAR CHROMOSOMES: STRUCTURES. INTERACTIONS. AND PERSPECTIVES M. J . Kopac and Gladys M. Mateyko All-University Deportment of Biology. Graduate School of Arts and Science. N e w York University. N e w York. N e w York

I . Introduction . . . . . . . . . . . . . . . I1. Nucleolar Bodies . . . . . . . . . . . . . . A . Nucleolar Structure . . . . . . . . . . . . B . Extrusion of Nucleoli . . . . . . . . . . . . C . Variations in Nucleoli in Different Tissues . . . . . . . D. Prenucleolar Bodies . . . . . . . . . . . . I11. Nucleolar Chromosomes . . . . . . . . . . . . A . The Nucleolar-Chromosomal Complex . . . . . . . . B. Nature and Properties of Nucleolar Organizers . . . . . . C . The Secondary Constriction as the Site for Nucleolar Organizers . . D . Number of Nucleoli vs . Number of Nucleolar Organizers . . . E . Nucleolar and Non-Nucleolar Chromosomes during Interphase . . F. Human Nucleolar Chromosomes . . . . . . . . . IV . Experimental Studies on Nucleoli . . . . . . . . . . A . Modification of Nucleoli by Chemical Agents . . . . . . B . Action of Microbeam Ultraviolet Irradiation on Nucleoli . . . C . Modifications in Nucleoli by Genetics . . . . . . . . D. Transplantation of Nucleoli . . . . . . . . . . V . Lampbrush Chromosomes . . . . . . . . . . . VI . Polytene Chromosomes . . . . . . . . . . . . A . Variations of Puffing Patterns in Different Tissues and Cells . . B . Variations of Puffing Patterns with Stages of Development . . . VII . Experimental Modification of Puffing Patterns in Salivary Gland Chromosomes . . . . . . . . . . . . . . A . Action of Nonspecific Environmental Factors . . . . . . B. Action of Hormones on Puffing Patterns . . . . . . . C . Action of Hormone Imitators on Puffing Patterns . . . . . D. Production of Deficient Karyotypes . . . . . . . . E . Transplantation of Salivary Gland Nuclei . . . . . . . VIII . Perspectives Involving Nucleolar and Non-Nucleolar Chromosomes . A . The Fate of Anucleolate Genomes . . . . . . . . B. mRNA Production and Transfer from Nucleus to Cytoplasm . . C . Where Are the Ribosomes Formed7 . . . . . . . . D. Mechanisms for Activating Gene Loci . . . . . . . . E . Serial Activation of Gene Loci . . . . . . . . . . F . Polytene Chromosomes vs . Conventional Interphase Chromosomes . G . The Reversal and Recapitulation of Chromosomal Activation . . References . . . . . . . . . . . . . . 121

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I. Introduction

“Observations have begun to accumulate in favor of the conclusion that the true nucleoli may be concerned in the secretory processes of the cell” (Wilson, 1924). In the forty years that have passed since this statement was written, data slowly and steadily have been accumulating to support this view. In all probability, the first published observation of the nucleus and nucleolus ( m e tache) in adult animal cells was made by Fontana (1781). It is interesting to note that prior to the discovery of mitosis, Schleiden (1842), who formulated the theory of “free cell formation,” thought that the nucleolus was the first organelle to show itself by separating out of formative fluid. Granules from the formative fluid then condensed around the nucleolus to be followed by the condensation of a membrane. This became the nucleus which was then followed by a further condensation of material enveloped by a membrane. Thus, a new cell wab born ! Although the nucleolus in the interphasic nucleus seems to be a reasonably simple structure, there is as yet no universally accepted interpretation of its structure or function. The earlier studies of nucleolar morphology have been reviewed by Montgomery (1898), Gardiner (19351, and Gates (l942), and bear thoughtful rereading periodically. Pianese (1896) was probably the first investigator to note an increased nucleolar size in neoplastic cells. Impetus to the study of the nucleolus then came from the provocative work of Caspersson (1941), Caspersson and Schultz (1940), Brachet (1941), and a decade later by the concept of nucleolar architecture composed of pars amorpha and nucleolonema by Estable and Sotel0 (1951). Since then several reviews on nucleoli have been published including those by Vincent (1955), Hertl (1957), and Stich (1956). Various examples of atypical nucleoli in human and amphibian neoplastic cells were described by Kopac and Mateyko (1958). Other reviews have appeared, for example, by Bolognari (1959a,c, 1960) emphasizing nucleolar structures and physicochemical properties, and by Swift (1959) eniphasizing nucleolar functions. More recently we have the reviews by Sirlin (1960a,b, 1961, 1962a,b) which dwell on various structural and biochemical aspects of nucleoli. I n addition, Busch et al. (1963) published an extensive review of the nucleolus in the cancer cell. Although many of the studies of nucleoli have been directed toward the nucleolar body, per se, i t is increasingly evident that there is much more to the nucleolar problem. To date, there are no clear-cut differences between normal and malignant nucleoli that can be described as distinctive or consistent, either on morphological or biochemical grounds. As Oberling and Bernhard (1961) have so well stated, the nucleolus of the

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cancer cell shows no specific properties that can be associated with the neoplastic transformation-at least by the present methods of analysis. And since we can explain so very little about a normal cell-in fact, we cannot offer any irrefutable explanation why a zygote goes on to divide so precisely and produce a functional organism-we must admit that we do not know what property of the nucleolus we should investigate nor where to look for i t in the nucleus of a normal cell, let alone in a cancer cell. Therefore, there is no question that some attention, perhaps primary attention, should be directed toward the nucleolar-chromosomal complex, which was first elucidated by McClintock (1934). Without the nucleolar organizer, a specific locus on a chromosome, the nucleolar body will not be formed. Accordingly, this review is directed toward a consideration of nucleolar chromosomes, their nucleolar organizers, and the origin of nucleolar substance. The role of the nucleolus in transmitting genetic information from the gene locus in the chromosome to the cytoplasm is also considered. In addition, there is also the role of nucleolar chromosomes not only in normal cellular differentiation but also possibly in the cancer process. Thus, if we err in omitting many excellent papers, it is only because papers particularly relevant to these topics have been selected. II. Nucleolar Bodies

I n conventional cells, during interphase, only the nucleolar bodies are clearly visible as nuclear structures. I n most instances, the chromosomes are invisible. Occasional allocycly may provide a conspicuous chromatin mass or masses that may be associated with the nucleolar bodies. To what extent are nucleolar chromosomes different in the differentiated cells of the organism? This can be answered, a t present, only in terms of structures that can be seen. Thus during interphase, the differences have to be confined to observable differences in the nucleolar bodies. Such differences are: size, shape, number, vacuolization, chromophobic coronas, etc. The major differences between nucleolar chromosomes and their associated nucleolar bodies would probably be reflected in the specific content of the nucleolar body. If the assumption is correct that nucleoli are the centers for ribosome assembly, among other things, then there should be differences among the various differentiated cells (Kopac, 1962). There is absolutely no reason to be satisfied that liver cells or spermatocytes have exactly the same nucleolar cytogenetics as pancreatic cells or neurons. On the other hand, there is no adequate evidence to the contrary. We simply do not know. Nor do we know what precisely might be the differences in nucleolar content in differentiated cells. Simple cyto-

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chemistry will not solve the problem. One would need to know the nucleotide sequence in the RNA (ribonucleic acid) present in these nucleoli. Differences in nucleoli, such as size, shape, number, composition, vacuoles, or other inclusions, could impIy possible cytogenetic differences in the cells. But even if two types of differentiated cells had identical nucleoli (based on currently available criteria), there would be no assurance that the nucleoli are indeed identical.

A. NUCLEOLAR STRUCTURE One of the more illuminating concepts of nucleolar morphology is that elaborated by Estable and Sotelo (1951, 1955). These investigators postulated that the nucleolus consists of two principal components, that is, a filamentous structure called the “nucleolonema,” and the other more bulky and homogeneous structure called the “pars amorpha.” When Estable and Sotelo (1955) tempered and modified their earlier (1951) statements, there appeared to be no doubt that the concept of a pars amorpha and nucleolonema was displaced from the status of an acceptable biological law. Serra (1958) also writes: “It is not a permanent structure in the nucleus nor does i t necessarily exist in every nucleolus at all times.” He does, however, admit that the nucleolus does show internal inhomogeneity which may be related to nucleolar activity. Thus, while the status of the nucleolonema as a permanent ultrastructural component of the nucleolus-and of the cell-is uncertain, it is generally a useful and realistic morphological concept of nucleolar structure. The recent electron microscope studies of Davis (1960, 1963) on the ultrastructural relationships of the nucleolus and chromosomes during mitosis are quite illuminating, but despite these and many other investigations in this field, the ultrastructural nuclear relationships are not particularly clear. One such extensive electron microscope study is that of Yazuzumi e t al. (1958). They concluded, based upon the morphology of various cell types, including Ehrlich ascites cells, that as a rule nucleolar morphology is constant and the nucleolonema is a fairly uniform structure being composed of filaments, 50A. in diameter which are helically coiled and make their appearance in the premitotic stage. But Bolognari (1960) reports that the Walker carcinoma cells had nucleoli composed of 200 A. granules which in another phase were only 50 A. in diameter. Thus, a t one extreme nucleolar substructure may be described as having the traditional nucleolonema and pars amorpha pattern (Cotte, 1959), and a t the other, as being composed of a granular mass (Serra, 1958). To cite an example of the former situation, amphibian oocyte nucleoli have tubules 100-150 A. in diameter which are surrounded by amorphous matter (Brown and Ris, 1959). Supporting the latter view is the work of Lafontaine and Chouinard (1961, 1963), who observed nucleolar struc-

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ture to be composed of particles 150A. in diameter. Bolognari (1959bj reports that, a t least in the oocytes of Patella coerulea, there are two types of nucleoli, one with and the other without the filamentous component. I n addition, Kutosumi and Akiyama (1958) describe a third ultrastructural component, in both vertebrate and invertebrate material, the pars spheroidea, which is composed of an aggregation of granules. The pars spheroidea, which may represent an inactive state of material, may show continuity with the nucleolonema and, in turn, the nucleolonema may be extended to the nuclear membrane. I n some plant cells, according to Rodkiewicz (1959), a typical thread is not recognized, but there may be a densely granular network with occasional threads in which a varying number of vacuoles are interspersed. Bolognari (1956-1957) indicated that this component could be impregnated, particularly by heavy metals as silver and platinum. H e (1957) also identified Feulgen-positive granules in the nucleoli of oocytes of invertebrate material, which he felt could be related to the nucleolonemal concept. Bernhard (1958) indicated that the threadlike component is characteristic of some tumors as the Shope fibroma and the Yoshida sarcoma, while in other types the filamentous network may be obscured by the matrix. Dmochowski’s (1960) observations agree with these views. According to Gonzalez-Ramirez (1959) a complex nucleolar reticulum characteristic of young, normal, and pathological cells of hematopoietic tissue becomes more dense in mature cells. Other reports in the literature that provide evidence along these lines abound. For example, Journey and Goldstein (1961) observed a threadlike element in HeLa cell nucleoli. Also hepatic tumors induced by butter yellow showed a filamentous component in the enlarged nucleolus (Heine et al., 1957). Ashworth e t al. (1960) described in human adenocarcinomata irregular enlarged nucleoli with the appearance of being “uncoiled” and suggest the presence of significant amounts of deoxyribonucleoprotein. The identity of the internal filament, however, is not fully clarified. LettrB and Siebs (1955), based upon their studies in a number of human tumors as well as normal cells, state that the internal structural component is chromosomal. O’Donnell (1961) clearly demonstrated that the internal filaments visible in the large nucleoli of Spirogyra grassa and S. lamellata were susceptible to digestion by DNase (deoxyribonuclease) . I n the interphasic nucleus of renal tumor cells a residual component exists. It is argyrophilic, evenly dispersed, but unlike the nucleolonema, not removed by RNase or DNase or lipid extraction, and is probably a protein rich in basic amino acids by virtue of its residual acidophilia (Mateyko and Kopac, 1957, unpublished). During mitosis the pars amorpha disappears, as postulated by Estable

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and Sotelo ( 1955) but, during mitosis, the nucleolonema, the nuclcolar organizer, and its chromosome must be replicated. During reconstitution of the nucleus, following the early telophase, the nucleolonema becomes condensed and entangled to form a tuft that then represents a substrate on which the pars amorpha accumulates. If the nucleolar-chromosoma1 complex is correctly interpreted, then it could be that this complex not only determines the number of nucleoli, but the number of nucleolar chromosomes as well. I n addition, the onset of replication must be mutually controlled in that one cannot replicate without the other. Where persistent nucleoli occur normally, for example, in Fritillaria (Frankel, 1937), this may be indicative that the demands on nucleolar substance are different.

B. EXTRUSION OF NUCLEOLI At present, the fate of nucleolar substance dispersed during prophasc is not fully clarified, although we know that a great deal of it enters into the ribosomaI material. The extrusion of nucleolar material probabIy falls into three categories: ( I ) the extrusion of macromolecular aggregates or even sizable fragments of nucleoli readily visible with conventional optics ; (a) the extrusion of RNA or ribonucleoprotein as submicroscopic, subvisible particulates; and ( 3 ) the extrusion of a low density liquid substance from vacuoles or interstices in the nucleolus, The extrusion of pieces, even entire nucleoli, has long been known. Hsu and Lou (1959) reported in the Cloudman melanoma the formation of nuclear pockets which expanded and shrank with a piece of nucleolar material moving back and forth from the center to the pocket. By this mechanism, presumably, massive doses of ribonucleoprotein and other nucleolar constituents are delivered to the cytoplasm. As has been reported by earlier investigators, the nucleolus frequently may be found close to the nuclear membrane (Frajola e t al., 1958). Extrusion of entire nucleoli is not unknown, for example, in rabbit anterior pituitaries as many as 21.8% of the cells showed extrusion, mostly to chromophobes (Ma and Hou, 1959). Kaushiva (1961) observed nucleolar buds given off in ophidian oocytes which eventually were extruded t o the cytoplasm, He claimed that the nucleolar organizer did not seem to enter visibly into the formation of these derivatives. The evidence for the release of minute particulates of RNA from the nucleolus to the cytoplasm is incontrovertible and well documented by many isotopic experiments which have shown that the nucleolar RNA contributes to cytoplasmic RNA (review by Zalokar, 1961). The intranucleolar products condense in vacuoles. Some intranucleolar vacuoles which have an extremely low density may break or deliver their product to the nucleus. Johansen and Flint (1959) observed that in some

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HeLa cells, nucleoli attracted to the nuclear membrane exhibited a fading of the dark reticular substance, and the appearance of a darker zone in the contiguous cytoplasmic area. The nucleoIar vacuoles contain less than 5% dry matter whereas the dense matter contains 85% (Vincent and Huxley, 1954). Gonzalez-Ramirez e t al. (1959) studied in HeLa cells the emergence of materials from the nucleolus. They reported that nucleolar vacuoles approached the surface to empty their contents and thereby left a halo. These studies confirm the observations of Kopac and Mateyko (1958) and Mateyko and Kopac (1958), who reported the occurrence of low density nucleolar vacuoles, chromophobic coronas, and nuclear canaliculi to the cytoplasm in tumor cells. The nature of the highly dilute material is not known. And, in fact, there is no critical method a t present for studying the contents of intranucleolar vacuoles or coronas. The above data are not consonant with Sirlin’s (1961) statement: “It has been frequently claimed that oocytes and cancer cells produce nucleolar substance that is visible under the microscope, but the significance, and even general occurrence of this is still doubtful, especially as it is rarely observed with the electron microscope.” On the contrary, i t would be remarkable for a low density, highly fluid substance such as has been observed in nucleolar vacuoles (Vincent and Huxley, 1954; Mateyko and Kopac, 1958) to be preserved by electron microscopic techniques. The extrusion of liquid materials from the nucleolus probably falls into two categories: ( 1 ) the elimination of a product necessary for metabolism as a highly dilute aqueous system and ( 2 ) the elimination of water, per se, which results in the great concentration of solids, thus making the nucleolus one of the most dense of cellular organelles, C. VARIATIONS IN NUCLEOLI IN DIFFERENT TISSUES Nucleoli in different somatic tissues of an organism are generally structurally similar but those in neurons and tissues subjected to unusual demands for protein and RNA synthesis are larger and, in developing oocytes, may be quite numerous as well as unusual in structure and composition. During cellular differentiation nucleoli grow ; when specialization is achieved, both nuclear and nucleolar growth is decelerated. The exception is, of course, in protein-secreting cells; this was noted by Caspersson (1941) and most recently by Wallace (1963). Accordingly, the nucleolus, in many ways a plastic body, undergoes active alterations in size, shape, and chemical make-up during cellular life, and probably more so in malignant cells. Lett& and Siebs (1954) reported that the nucleolus changes when cells are cultured in vitro. Attempts have been made to study nucleolar

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structure on nucleoli isolated in bulk; for example, Finamore (1961) reported on their physical properties when isolated from amphibian eggs. Such nucleoli appeared to be refractile and vacuolate spheres. The physical properties of nucleoli and nuclei are not the same in all cell types. To illustrate, Chambers and Fell (1931) transected nucleoli in fibroblast cells in tissue culture without damage to the nucleus. I n other cells, for example, immature oocytes of Asterias, puncture of the nucleus brings about not only nuclear dissolution, preceded by disappearance of nucleolus, but widespread cytolysis as well (Chambers, 1924). Although isolated nucleoli appear to be discrete and, as such, do not fuse even though in contact with each other (unpublished data), nucleolar fusion in viwo or in vitro cultures is common. Auerbach (1890) stated that the number of nucleoli is more or less constant for all the cells of a given species, and in view of the constancy of chromosome number this seems a likely premise. However, there are many reports in the literature which indicate that there may be a variability in number of nucleoli not only in different tissues of an organism but even in the same tissue, Montgomery (1898) indicated that the mollusc, Doto, had from l to 13 nucleoli in ganglion cells, the medium-sized ones containing 1 to 4, generally 2 to 3. I n blood cells, there was only one nucleolus per nucleus while giant cells which were larger than ova, had often as many as 40 nucleoli. I n the polychaete annelid, Piscicola, one nucleolus was characteristic of the ovum and ganglion cells, but in muscle cells and subcuticular gland cells, nucleolar fragmentation occurred so that there could be as many as 12 in mature muscle cells or 400 in the subcuticular gland cells. Dearing (1934) observed that larval tissues of Ambystoma tigrinum generally contained 2 nucleoli per nucleus but 1 and 3 were also common. Recently, Inone (1959) indicated that there is a discrepancy in number of nucleoli in the nucleus of different organs of rodents. And, of course, i t is commonly known by all cytologists that the number of nucleoli in developing oocytes may be extraordinarily high. This may be brought about by the budding of nucleoli and the enhanced activity of the chromosomes of the germinal vesicle stage, Olah (1962) interpreted the budding of nucleoli during meiosis of Corypha elata as being a means of increasing the surface of nucleoli. During interphase, the number of nucleoli which corresponds to the number of haploid nuclear chromosome sets may be visible. However, nucleolar fusion may occur when nucleolar chromosomes are close to each other (Kopac and Mateyko, 1958). I n fact, a criterion of nuclear differentiation is nucleolar number, a diminution usually being attributed to fusion. Swift (1959) makes the interpretation that the number of nucleoli

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as well as the size is dependent to a degree on the “competition” between metabolites in the nucleus and the nucleolus-forming sites. Many reports on nucleolar fusion have appeared. Swift (1959) reported that the diminution in nucleolar numbers in Vicia faba was caused by nucleolar fusion. In an interesting analysis of different tissue types in Xenopus, Wallace (1963) points out that the frequency of nucleolar fusion is rather similar in tissues which go on to differentiate without additional mitosis. On the other hand, in other tissues where cell division does occur during differentiation, the frequency of fusion shows variability. It is important to distinguish between nucleolar fragmentation and multiple organization of nucleoli, both of which can account for the presence of more than one nucleolus per nucleus. Nucleolar fusion, of course, reduced the number of nucleoli and frequently gives an idea as to the number of chromosomes that participate in the formation of nucleoli. Nucleolar fusion and fragmentation may occur in the same tissue so that it is difficult to say whether multiple nucleoli are the original products or are fragments budded off secondarily. To illustrate, Amenta (1961) observed that heart cells in tissue culture (Triturus viridescens) exhibited from 1 to 4 nucleoli. Where fusion occurred a reduction in number was present, yet fragmentation of these nucleolar bodies often restored the original number. Fusion occurred with greater frequency after telophase, probably due to the physical properties.

D. PRENUCLEOLAR BODIES A considerable amount of work is required to identify the chemical make-up and destiny of the nucleolar filaments and granules during the mitotic cycle. I n fact, a definition of a nucleolar cycle was attempted by Carleton (1920). Most recently, we have the precise work of Lin (1955), Love (1957), Lafontaine (1958b), La Cour and Chayen (1958),Bolognari ( 1 9 5 9 ~ Lafontaine )~ and Chouinard (1961, 1963), Stevens (1961), Olah (1962), Das (1963), and Davis (1963), among others. However, a clarification of what is meant by pars amorpha, nucleolonema, nucleolini, nucleolar vacuoles, and matrix in terms of nucleolar chemistry and ultrastructure and relationships to chromosomes and what happens to each during the mitotic cycle of a specific cell is sorely needed before one can sensibly ascribe a function to any of them. The activity of a cell nucleus may be so heightened that the “middleman” stage, that of the organized nucleolar body, does not appear. Material is rapidly synthesized and rapidly consumed, as during the active division in amphibian embryos prior to gastrulation where defined nucleoli are rarely visible with the optical microscope (Brachet, 1952). It is pos-

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sible, therefore, to postulate that an organized nucleolus is an evolutionary step in the development and differentiation of the nucleus. Prophase nucleoli as viewed with electron microscopy are generally irregular in shape and have many zones of lesser density (Lafontaine, 1958b). The meiotic prophase nucleolus as described by Bopp-Hassenkamp (1959) is a most informative stage since it is in a state of incipient dissolution. It appears to be composed of fibrillar elements, but similarly dimensioned (120 A.) fibrils are also characteristic of the nucleus. Short fibrils may also penetrate into vacuoles and elementary fibrils of the nucleus penetrate partly and extend into the nucleolus. Horstmann and Knoop (1957) suggested that fibrillar structures of the karyoplasm that continued into the nucleoli might be of chromosomal origin. According to Zenkteler (1959) the nucleolonema spreads during prophase to the nucleoloplasm and appears in short bands on the metaphase and anaphase Chromosomes. These thicken during telophase, thus forming the nucleoli. The original of nucleolar material on various loci of telophasic chromosomes has been frequently reported (Heitz, 1931; Austin, 1953; Vincent, 1955). The definitive experiments of McClintock (1934), however, demonstrated that the formation of the nucleolus was under the influence of a particular region of the chromosome. Moreover, she described that during late prophase the nucleolus contributed to the formation of the matrix. Many papers have appeared describing the elaboration of prenucleolar substance by the chromosomes. Stevens (1961) described “pronucleoli” as small granules arising a t the beginning of telophase as an uneven layer on the chromosomes. At mid-telophase, these detach from the chromosomes as bodies 0.3 to 0.5 p in diameter. Harris (1961) likewise concluded that the telophasic reappearance of nucleoli was dependent on the aggregation of proteins and presumably other materials that were synthesized before mitosis. According to Lafontaine and Chouinard (1961) , the prenucleolar material appears as early as late anaphase in the form of tightly packed granules in a filament. Once compact nucleoli are apparent a t late telophase the prenucleolar material is not detectable. These data, however, do not negate the fact that one chromosomal locus, the nucleolar organizer region, may be the most active metabolic center for prenucleolar material. Evidently, the reorganization of nucleoli a t telophase must involve a true synthesis of new material as well as a reassembly of dispersed nucleolar material. For example, Lafontaine and Chouinard (1963) have observed both from light and electron microscopy (Vicia faba) that the bulk of prenucleolar substance comes from the synthesis of material by early and mid-telophase chromosomes. This material

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is resolved a s fibrillogranular, threadlike material, 0.1 p in diameter, which appears in the interchromosomal spaces. Lafontaine (1958a,b) and Das and Alfert (1959) presented convincing evidence with the electron microscope that prenucleolar bodies derive in different degrees from the chromosomes and coalesce to form the organized body. Loose granules, 140A. in diameter, first appear around anaphase chromosomes and agglomerate and finally fuse during telophase. Tandler’s (1959) observations confirm this pattern of a particulate organization of the nucleolus. Das (1962) in a variety of cell types, observed a t telophase non-RNA granules around all chromosomes. These were identified as protein or lipoprotein unaffected by the removal of nucleic acids, and also not the nucleolonema. A direct relationship between the size of the nucleolus and the number of nucleolonemal granules was found by Albanese and Bolognari (1960). These data, then, give a clue that the elaboration of RNA and other products may be dependent on, or controlled by, chromosomal material. I n an electron microscope analysis of mitosis in regenerating rat liver, Davis (1963) reported that fibrous inclusions of the order of 1000A. during telophase probably represented stages in the reorganization of the nucleolus. I n an attempt to get a more precise electron microscope picture of nucleolar ultrastructure, Davis (1963) followed buffered osmium fixation with phosphotungstic acid. His studies revealed that both the nucleolonema and the pars amorpha consisted of fibers of about 100A. in diameter and, moreover, maintained the same ultrastructure despite changes in the over-all density of the nucleolus. The steps in the reorganization of nucleolar material are by no means certain, but Davis’ (1963) evidence shows that a t mid-telophase new structures appear that are embedded in chromosomal masses. These are spherical masses, 1000 to 1500A. in diameter, with a fibrous ultrastructure. The presumed nucleolonemal fibers have a diameter ranging from 50A. for thinner ones and 100A. for thicker ones. Unfortunately, the reforming nucleolonemal-like fibers do not show any clear-cut structural relationship to the chromosomal fibers. Davis (1963) concludes that: ( 1 ) either prenucleolar bodies arise from parts of the chromosomes and the nucleolonemata are formed in them, de nova, or ( 2 ) nucleolonemata are “first formed among the chromosome masses in mid-telophase” and subsequently unite to form the prenucleolar structures. The substances elaborated a t the site of the nucleolar organizer or nucleoli may be described as “matrix” in the sense that McClintock (1934) and Olah (1956, 1962) describe nucleolar phenomena. Preferably, the material should be identified by its biochemical make-up and structural modifications. On the whole, the term “matrix” may perhaps best

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be avoided since it brings in the concept of a chromosomal matrix, a point of view which is disputed. Swift’s (1959) rejection of the possible organization of nucleoli from matrical substance during telophase, such as the aggregation of “droplets of material” (prenucleolar substance), seeme untenable in view of the clear-cut evidence that such aggregation of prenucleolar bodies does occur (Lafontaine, 195813; Das and Alfert, 1959; Tandler, 1959; Lafontaine and Chouinard, 1961 ; Stevens, 1961). Moreover, the evidence that where the nucleolar organizer is lacking, no typical nucleoli develop. On the other hand, small bodies-“nucleolar substance in search of an organizer”-do appear (McClintock, 1934; Beermann, 1960; Fischberg and Wallace, 1960; Wallace, 1962a,b). Swift (1959) has also cautioned that apparent prenucleolar bodies might be in reality spindle remnants, and emphasizes the difficulty in distinguishing and interpreting nucleolar materials in electron microscope pictures. Ill. Nucleolar Chromosomes

The nucleolar chromosome consists of three integrated components: ( I ) the nucleolar body, usually most prominent during interphase, and referred to as the nucleolus; (2) the nucleolar organizer, a specialized region on a chromosome, probably heterochromatin-rich, and visible usually as a secondary constriction from late prophase to early telophase; and (3) the chromosome itself, with perhaps no other characteristics that would distinguish it from other chromosomes in the genome except that it carries the nucleolar organizer. I n some genomes, the nucleolar chromosome may carry considerable heterochromatin, especially if i t happens t o be a sex chromosome. COMPLEX A. THENUCLEOLAR-CHROMOSOMAL The relationship of certain chromosomes to nucleoli by way of the nucleolar organizer has been extensively studied by McClintock (19341, Kaufmann (1934), and by Matsuura (1938), and has been reviewed by Kaufmann (1948). McClintock (1934) described the nucIeolar organizer of the 6th chromosome in Zea as specific organizing bodies located next to the point of origin of the nucleolus and the secondary constriction. The fact that nucleoli are formed a t specific loci on specific chromosomes was pointed out by Heitz (1935). Using larval stages of the fly, Bibio, Heitz and Bauer (1933) demonstrated a nucleolus a t a fixed locus in one pair of chromosomes in the cells of Malpighian tubules. McClintock (1934) likewise demonstrated that if the heteropycnotic knob in the nucleolar organizer of Zea is broken by X-rays into unequal

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halves, both portions are capable of forming nucleoli. If a cell has only one portion of the knob, a full-sized nucleolus is formed, indicating that one nucleolar organizer or even a portion of i t is capable of assembling all the nucleolar material. With two nucleolar organizers, however, a competition is established. The intact nucleolar organizer must be a compound structure whose parts have differential rates of organizing ability. Kaufmann (1938) later showed that in Drosophila melanogaster various inversions and translocations occurred in which nucleolar organizer regions were broken into two parts. Since each part retained its specific property of forming nucleolar material, such stocks showed two or Inore independent nucleoli. The position of the nucleolar organizer in chromosome X is not coincident with the centromere, but lies some distance away from it. Darlington’s (1942) view was that the organizing bodies were chromomeres capable of being fractured by X-rays and present in any region in a chromosome. By the study of inversions and translocations in both Zen and Drosophila in which nucleolar organizers were divided, i t is clear that each portion is capable of assembling the nucleolar material into a body. Ohno and Kinosita (1955) found that the nucleoli in rat lymphoblasts are organized by a few autosomes. Subsequently, in mesothelial cells of the rat omentum, Ohno and Kinosita (1956) showed an attachment of certain chromosomes at their subterminal regions t o the nucleolus, which resembled nucleolar chromosomes with SAT zones (sine acido thymonucleinico) as described in other organisms. On the other hand, in the mouse, only the X and Y chromosomes are found associated with the nucleolus, the nucleolar organizer being located near the centromeres (Ohno et aE., 1957). Tobias (1956) described chromosomes in the gerbil (Tatera).The two largest chromosomes have a characteristic pattern of constrictions. One constriction, median in position and always present, is the centromeric or primary constriction. I n addition, each arm is divided into a longer central segment and a shorter distal segment by a secondary constriction. I n one arm, the distal segment is shorter than in the other. These two chromosomes are probably the sex chromosomes, the largest being the presumptive XI and the shorter, the presumptive Y. Both members of the second largest pair possess a submedian centromeric constriction and a subterminal constriction in one arm. The karyotype of R a m pipiens was studied during embryonic differentiation by D i Berardino (1962). Chromosome No. 10 has been designated the nucleolar chromosome and is characterized by possessing a prominent secondary constriction in the long arm. Interestingly, the sec-

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ondary constriction in No. 10 is much longer in gastrula chromosomes than in tail chromosomes. Also, the secondary constriction length does not maintain a constant relationship to the rest of the chromosome, when gastrula nuclei and tail cell nuclei are compared. Moreover, the nucleolus is larger in early gastrula nuclei than in tail cell nuclei, and this may perhaps be a factor in differences in length of the secondary constriction. One larva with 3 nucleoli in interphase turned out to be trisomic with regard to chromosome No. 10. Briggs and Humphrey (1962) reported on certain cytological effects in axolotl larvae. Nucleoli become apparent in cells of the late blastula, but not during cleavage or early blastula stages. I n arrested blastulae, due to the presence of a lethal gene, v, cells may contain from 3 to 14 nucleoli per cell. Thus, there is a suggestion that chromosomal replication without nuclear division is responsible for this increase in nucleolar number. I n other stages, especially if developmentally arrested, the cells may show more than two nucleoli. The diploid axolotl cell normally has two nucleolar chromosomes and thus one or two nucleoli may be seen during interphase (Fankhauser and Humphrey, 1943). The karyotype of Neurospora crassa as studied by McClintock (1945) discloses that the chromosome, designated No. 2, has a nucleolar organizer close to the end of the short arm. Also a minute satellite is present on this chromosome during mitosis. The nucleolar organizer develops a nucleolus in each telophase nucleus. Navaschin (1934) worked with Crepis hybrids. The satellite on the chromosome of one parent frequently disappears in F, hybrids. The competitive activity of the nucleolar organizer of various species apparently differs in strength-the stronger nucleolar organizer then takes over the entire nucleolar activity of the cell. Thus a chromosome carrying a nonfunctional element consequently develops no constriction in absence of the nucleolus and thus its metaphase morphology is changed. C. capillaris has a strong nucleolar organizer and in F, hybrids, i t suppresses the activity of the organizers derived from several other species. The C. capillaris nucleolar organizer in turn is suppressed by that of C. pariflora. I n hybrids of C. pariflora and C. setosa, both have nucleolar organizers of the same length in F, hybrids and both organizers form nucleoli. The resulting nucleolar bodies, however, are reduced in size. According to Kahn (1962), the nucleolar mutation of X e n o p s has been recognized a t mitosis as the absence of a secondary constriction, with the nucleolar organizer either deleted or inactivated in this mutation. However, bodies or blobs with a cytochemical resemblance to nucleoli are found in the nucleoli of the On mutant, which develop through stages normally associated with striking nucleolar activity.

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The field of avian chromosomal cytology has not been as fully explored as the mammalian, partly because of the multiplicity of microchromosomes. Yet, these microchromosomes have been implicated in the formation of nucleoli. For example, Ohno e t al. (1962) observed that a t least 12 of about 60 microchromosomes of the domestic fowl (embryonic liver) were in association with the nucleolus. B. NATUREAND PROPERTIES OF NUCLEOLAR ORGANIZERS I n conventional cells, during interphase, only the products of the nucleolar organizer are visible. In most instances, both nucleolar and nonnucleolar chromosomes are invisible. But occasional allocycly may provide a conspicuous chromatin mass or masses associated with the nucleolar bodies. Beyond prophase stages and extending through early telophase, the nucleolar body is not evident, but the sites of the nucleolar organizer are. These sites are the secondary constrictions. In addition, if the secondary constriction is near the terminus of the chromosome, there may be evident small satellited bodies. Heitz (1931) discovered that during telophase each nucleolus had its origin in a chromosome with a satellite. Nucleolar organizers, as are secondary constrictions, are frequently poor in chromatin and are therefore similar to heterochromatic segments, Usually, nucleolar development is associated with a particular site on a specific chromosome. Moreover, in every cell in an organism a nucleolar organizer is present, unless a specific mutation has occurred. Crosby (1957) suggested that nucleolar and non-nucleolar chromosomes as well, might contribute to the elaboration of nucleolar material with each type of chromosome being the factor that limited nucleolar size. One point of view that may be advanced is that the nucleolar organizer is a highly specialized, active genetic site on a chromosome and may be considered as the main, but temporary assembly point for the ribonucleoproteins which are then distributed to the cytoplasm. The early work of Poulson and Metz (1938) describing the assembly of achromatic substance at the Balbiani rings and puffs of dipteran salivary gland chromosomes (similar to the activity of the nucleolar organizers) may be considered as a transition point in a n interpretation of a relationship of active sites such as puffs and nucleolar organizers. In fact, more and more evidence indicates that chromosomal and nucleolar specialization is probably concomitant of cellular differentiation. For example, in the salivary gland of Bradysia the nucleolar organizer is not functional, but is functional in other larval tissues (Jacob and Sirlin, 1963). These investigators likewise speculate that there may be an “evolutionary acquisition of organizer.”

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The formation of Feulgen-positive material a t the nucleolar organizer region has been clearly demonstrated by Olah (1962). The earlier observations made by Olah (1956) on the DNA-containing material formed by the nucleolar organizer participating in matrix formation deserves emphasis since i t appears to provide evidence for Caspersson’s assertion that the nucleolus-associated chromatin is an active site of cellular metabolism. Olah used meiotic tissues of Corypha umbraculifera and noted that a semiliquid, Feulgen-positive material was formed a t the nucleolar organizer region and was “drained off” by the SAT threads and distributed along the chromonemata. At early pachynema the substance disappeared among the chromosomes. Interestingly enough, there is a distribution and dissolution of the material coincident with the beginning of the condensation of the chromosomes, that is, the assembly of the matrix. The relationship of the nucleolus to the chromosome and to its cycle during mitosis would be more intelligible if the nucleolonema were continuous with the nucleolar organizer or were part of it. Thus, the nucleolonema, the nucleolar organizer, and the nucleolar chromosomes would be self-replicating structures. As might be expected, considerable variability in the nucleolonema may be found on comparing one cell type with another. I n some instances, the nucleolonema resembles a tuft; in others, a meandering filament. In each instance, however, there is a structural relationship between the nucleolonema and the nucleolar organizer. Perhaps, the simplest relationship between nucleolar organizer, nucleolonema, and the nucleolar chromosome is that found in Spirogyra, as first described by Godward (1950). The points established were : ( I ) two nucleolar organizing chromosomes are present; (2) two nucleoli are organized which may fuse as one; and (3) nucleoli contain coiled structures through which the elongated nucleolar organizing regions of the chromosomes pass. Fine chromonemata, or structures representing the chromonema, must extend through the organizer track. This portion of the chromonema is the nucleolar organizer. During interphase, the nucleolar organizer region is coiled up inside the nucleolus; and the fine chromonema is embedded in a thick coating of deeply staining material, the organizer track. O’Donnell (1961) studied the effects of RNase and DNase on Spirogyra nucleoli. DNase creates an empty tubule within the nucleolar mass probably due to removal of the chromonema; the latter when present is clearly Feulgen-positive. The use of RNase left the inside of the nucleolus something like a continuous cave. The spaces in between those occupied by the depolymerized material showed a Feulgen-positive filamentous structure. The interesting feature in Spirogyra is that the nucleolar organizer

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is clearly a part of the chromosome and i t appears as a meandering structure inside the nucleolar body. There are two nucleolar chromosomeseach one capable of forming a nucleolar body, albeit of different sizes, with some relationship of nucleolar size to the nucleolar organizer. The importance of the nucleolar organizer in enabling a cell to undergo mitosis has been emphasized in the investigations of plant micronuclei ; in order for mitosis to ensue at least one nucleolar organizer is necessary (La Cour, 1953; McLeish, 1954; Darlington and Haque, 1955).

C. THESECONDARY CONSTRICTION AS THE SITEFOR NUCLEOLAR ORGANIZERS Secondary constrictions, if associated with nucleolar organizers, are the sites for nucleolar formation. Such specific regions are referred to as nucleolar zones, nucleolar organizers, or simply SAT zones. The nucleolar organizer, as a secondary constriction on the chromosome, is not readily distinguished from other secondary constrictions. As a rule, the secondary constrictions are due to the negative heteropycnosis (possibly a lesser degree of folding or coiling of the chromonema) at the site. Resende (1946) designated secondary constrictions as olistherozonesto describe the weak chromatin staining of these regions. The portion of the chromosome distal to the secondary constriction is the satellite or trabant. Usually the satellite is connected to the main body of the chromosome by a delicate chromatin filament. White (1954) pointed out that the size of the nucleolus is a property of the nucleolar organizer. Nucleolar organizers are divisible and may extend over a moderate length of the chromosome and, thus, are not to be regarded as a single gene. This helps to explain the great variation in size and number of nucleoli that are seen in Chironomidae. Most chironomids have definite nucleolar chromosomes in salivary gland cells. A repeated fragmentation and redistribution of portions of chromosomes by inversions or other structural rearrangements could lead to an increase in nucleolar number as well as a change in location. There may be a considerable variability between the length of thc secondary constriction and the size of the nucleolus. This probably dcpends on where the nucleolar organizer is precisely localized in reference to the constriction. I n some instances, as in Zea, the nucleolar organizer may have a secondary constriction developed within i t (that is, blocks of heterochromatin are on either end of the constriction). I n other instances, the secondary constriction and nucleolar organizer may be coincident. Still in others, the nucleolar organizer may be localized within the secondary constriction and, in fact, Darlington (1937) has shown that certain small nucleoli do not exhibit a secondary constriction at metaphase. Clearly,

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here the nucleolar organizer may be too small to disclose a recognizable constriction with the optical microscope. Secondary constrictions need not always elaborate a nucleolus. The lack of nucleolar organization may be due to: ( 1 ) the absence of a nucleolar organizer in the constriction, or ( 2 ) a competition between nucleolar organizers in the karyotype with the “strongest” competitor being successful in organizing a nucleolus.

D. NUMBER OF NUCLEOLI vs. NUMBER OF NUCLEOLAR ORGANIZERS

A problem that bears investigation is the specialization of the nucleus and also the specialization of the nucleolus during the differentiation of cells. Despite the fact that the number of nucleolar organizers is as constant as the chromosome number, the number of organized nucleoli per any nucleus in a particular cell type may be variable, for example, liuinan cells may show a range of 1 to 6 nucleoli. A reversible fragmentation and fusion of nucleoli during the telophase have been reported in Trillium by Matsuura (1938) and in Tmesipteris by Yeates (1925). Tobias (1956) proposed the term Lisynergisni”for the “cooperation of more than one organizer in forming a plasmosome” (nucleolus). He further pointed out the synergism of homologous nucleolar organizers (homologous synergism) and the synergism of nonhomologous nucleolar organizers (nonhomologous synergism). Examples of synergism are found in Fritillaria, Crepis, and Zea. Whether the tendency toward single nucleolar bodies (in cells with multiple nucleolar organizers) is due to synergism or fusion cannot always be judged. If two nucleolar organizers are in close proximity and if a nucleolar body is formed around one organizer and not the other, this is probably true synergism. On the other hand, where several nucleolar chromosomes may be associated with a single nucleolus, this might he synergism or i t might be simple fusion. Such fusion definitely occurs in human cells (Ohno e t at., 1961a). There is apparently no definite pattern of fusion, which implies that there is no special attraction between the nucleoli of homologous chromosomes (Chen, 1936). Zirkle (1931) had observed that there were six nucleoli in the interphasic nucleus of Pinus strobus, each of which became associated with the spireme chromosomes a t the inception of mitosis. Zirkle’s “plastin,” which may be interpreted in modern terms, was distributed to the chromosomes, and a t telophasic reconstruction this “plastin” was consolidated into typical nucleoli. Lin (1955), using microsporocytes of maize, produced cells with as many as five extra chromosomes with nucleolar organizers, and also cells with an increase in number of non-nucleolar chromosomes. In all cells,

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there was a single nucleolus. Also the amount of RNA in the nucleolus was proportional to the number of nucleolar organizers present in the nucleus. Lin considered the entire nucleolar chromosome to be involved in the synthesis of nucleolar RNA-so that capacity for RNA synthesis can be enhanced by the addition of an extra segment of chromatin to the nucleolar chromosome. Krivshenko (1959) induced mutants in Drosophila busckii by X-rays. Six lines of such mutants were found to have chromosomal aberrations with breaks in the nucleolar organizer. Breaks which occurred in the nucleolar organizer were made apparent by the new, permanent connections between the nucleolus and regions of the chromosomes which otherwise were never found attached to the nucleolus. The nucleolar organizer is, therefore, divisible at many points along its length. Each fragment of the nucleolar organizer continues to function independently of its size, but its effectiveness depends directly upon the size of the organizer. Accordingly, in some cells there may be as many nucleoli as there arc nucleolar organizers. In such instances, synergistic or fusion effects are not operating (examples: hyacinth, salamander, axolotl larvae, mouse, gerbil). I n other cells, the number of nucleoli may be less than the number of nucleolar organizers. Here, synergistic or fusion forces are operatin: (examples : Pisum, Drosophila, meiosis of many species, spermatogonia and Sertoli cells in Tatera, man).

E. NUCLEOLAR AND NON-NUCLEOLAR CHROMOSOMES DURING INTERPHASE Crosby (1957) suggested that both nucleolar and non-nucleolar chromosomes might play a role in the formation of nucleolar material with each type of chromosome being the factor that limited nucleolar sizes. Crosby Longwell and Svihla (1960) pinpoint the close relationship between the nucleolus and its chromosome. I n the microsporocyte of wheat, the synthesis of nucleolar and cytoplasmic protein and RNA is under the control of the nucleolar chromosome. Knowing that in the absence of nucleoli, blobs of presumptive nucleolar material have been noticed on chromosomes (Wallace, 1962a), we may ask the question: Is the nucleolar organizer a system for bringing together the products generated a t specific loci on all chromosomes? There is no question that i t does in many instances. On the other hand, material elaborated a t chromosomal loci may leave the chromosome and pass directly into the cytoplasm. The entire nucleolar chromosome is probably involved in the formation of the nucleolus since changes in the content of RNA were found in plants carrying a translocation involving a nucleolar chromosome (Lin, 1955). Genes in other chromosomes as well have an effect on the ribonucleoprotein of the nucleolus.

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Both nucleolar and non-nucleolar chromosomes are fully extended linearly during interphase with the exception of those chromosomes undergoing allocycly. I n some cells, the X chromosomes may show this tendency. I n mouse and man, one of the two X chromosomes in the female karyotype will undergo allocycly and appear as a chromocentric mass during interphase. If the same general plan persists throughout as i t occurs in the salivary glands of dipteran larvae, then only a relatively small number of loci will be active a t any time. If certain operons are functional, then the immediate reaction a t such loci would be the production of mRNA (messenger RNA). The products would then appear in the nucleolar bodies where ( I ) 'further replication of mRNA could occur, or ( 2 ) various components could be assembled to form specific ribosomes. Since most of the active loci might be on non-nucleolar chromosomes, then the question might be asked: What is the position of such loci relative to the nucleolar body? For this question we have no immediate answer. For all we know, the active loci might be fairly close to the nucleolar bodies; indeed, they might even be partly embedded in the nucleolus. Thus, each differentiated cell, with a different spectrum of active loci, might show a reasonable intimacy of such loci with the nucleolus. Unfortunately, chromosomes during interphase are so elongated and attenuated that they cannot be seen properly. Present pictures of interphasic nuclei can only yield information that a t best indicates a random distribution of the chromosomes in a nucleus. Perhaps this degree of randomness may be more apparent than real. Not even the electron microscope, a t present, can clear up this question.

F. HUMAN NUCLEOLAR CHROMOSOMES Schultz and St. Lawrence (1949) have produced cytological maps of the autosomal nucleolar chromosome in man following a study of the pachytene male chromosomes during spermatogenesis. They were able to show that the nucleolar organizer in the human autosome is associated with the tenth and eleventh chromomeres of a chromosome that has 22 recognizable chromomeres. This position seems to be fairly constant and, furthermore, the chromomeres to which the nucleolus is attached are unusually large and become densely stained with orcein. Schultz and St. Lawrence stated that in many instances the nucleolus appeared to be medial, flanked on either side by a chromosomal arm, and in almost an equal number of cases the nucleolus was either terminal or subterminal. Two interpretations were considered : ( 1 ) the nucleolar chromosome was frequently broken by smearing, or (2) there were two chromosomes concerned, whose separate nucleoli frequently fused. These

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authors favored the first interpretation since, a t that time, the location of secondary constrictions in metaphase chromosomes was not known. They also favored the possibility that no consistent differences in nucleolar sizes were found on comparing medial or terminal located nucleoli. I n view of the present information (Ohno e t al., 1961a) that acrocentric chromosomes in the human karyotype are satellited and also considering the location of the secondary constriction, i t would seem that the second interpretation is probably the correct one. Schultz and St. Lawrence also pointed out that the nucleolus in man is formed in association with a group of large chromomeres essentially similar to the type of chromosomal region called heterochromatin by Heitz (1935). They also considered that the X chromosome can form a nucleolus. I n addition, the nucleolus formed on the sex chromosomes differs from that seen on the autosomal chromosomes, in staining less intensely with fast green and in the pattern of its association with the chromomeres of its chromosome, the X. Thus, a t least two separate nucleolar organizers were identified in the human chromosomes. Kodani (1954) described the chromomere pattern of the %econd nucleolar chromosome” in human cells. Yerganian (1957) also studied human pachytene bivalent chromosomes in acetocarmine squashes of human testicular tissue. He observed bivalents with medially placed nucleoli and dense chromomeres on either side, similar to those described earlier by Schultz and St. Lawrence (1949). Also noted were separate arms with attached nucleoli. Thus, two types of bivalent, human nucleolar chromosomes have been observed, i.e., chromosomes with median nucleoli and chromosomes with terminal nucleoli. Tobias (1956) made a study of human diplotene chromosomes. I n a drawing of a squash preparation, he depicts an autosomal pair associated with a prominent trabant-like chromomere and a nucleolus. This is similar to the several nucleolar chromosomes with terminal nucleoli as illustrated by Schultz and St. Lawrence and by Yerganian. I n view of the information that the acrocentric chromosomes in the human karyotype are satellited (metaphase chromosomes), it would seem that those pachytene chromosomes with a terminal nucleolus may be examples of the acrocentric chromosomes. I n those chromosomes showing a median nucleolus (Schultz and St. Lawrence, 1949; Yerganian, 1957), we may be dealing with a situation where two chromosomes are sharing the same nucleolus. This situation was postulated by Ohno e t al., (1961a). Ferguson-Smith and Handmaker (1961) mentioned that all ten acrocentric chromosomes, in the human karyotype, may be satellited. This includes chromosomes 13, 14, 15, 21 and 22 (Denver classification). To this list may be added chromosomes 1, 4 or 5 , 9, 16, and Y (Moorhead,

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cited by Chu, 1963). Ohno e t al. (1961a) pointed out that either some of the satellited chromosomes are not true nucleolar chromosomes or else not all nucleolar organizers are functional, a t the same time. Ohno et nl. (1961b) reported that, in one chromosome pair of guinea pig cells, only one chromosome is active as a nucleolar organizer, the other being inactive. Only one member of the largest autosomal pair showed a SAT zone in any given metaphase; moreover, a t prophase only one member of this pair was found in association with a nucleolus. The corresponding region of the other chromosome was positively heteropyknotic, with no visible satellite. Moreover, Ohno et al. (1961a) pointed out that when two chromosomes of the 21-22 group are satellited, they may not be a pair of chromosomes 21, but rather one member of each pair. They have shown a late prophase from liver cells of a 4-month-old normal male fetus showing six chromosomes with nucleolar organizing regions. These might be as follows: a pair of chromosomes 13 (the largest); the two medium-sized chromosomes might be either pair 14 or pair 15 or one of each of these pairs; and the smallest two may be pair 21 or pair 22, or one of each. As many as six chromosomes may be attached to a single nucleolus. On the other hand, as many as six nucleoli were found in fetal liver cellscoincident with the number of observed nucleolar chromosomes. Most frequently, fewer nucleoli are seen during interphase, generally only one. Thus, human nucleoli show a strong tendency to fuse and thereby tend to bring together the nucleolar organizers. I n 20 prophase figures, 16 cells showed a nucleolus associated with 6 chromosomes; 3 cells showed 4 ; and 1 cell showed a nucleolus associated with only 3 chromosomes. From the frequent association, during interphase, of several nucleolar chromosomes, a number of interesting changes may then occur in such chromosomes. Some of these changes result in reciprocal translocations. Among the possibilities considered by Ohno et al. (1961a) are: ( 1 ) The translocation of a prominent satellite on chromosome 13 to chromosome 21, and vice versa. As a result, chromosome 21 would now have a large satellite, and chromosome 13 would have the small satellite. ( 2 ) The translocation of the long arm of chromosome 21 to the short arm of chromosomes 14 or 15. The result would be a metacentric chromosome without a satellite and chromosome 21 would be even smaller than the so-called “Philadelphia” chromosome. (3) Two small acrocentrics (21 and 22) could produce a mediocentric chromosome representing the long arm of one, and the centromere and long arm of the other. The other product would be a greatly reduced metacentric and satellited chromosome. ( 4 ) If one break occurs in the middle of long arm of chromosome 21 and a subsequent reciprocal translocation t o the short arm of chromosomes 14

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or 15 occurs, then chromosome 21 would now be similar to the “Philadelphia” chromosome. It is of interest to point out that functfonal trisomics have been reported in patients with Down’s syndrome. Here most of chromosome 21 is translocated to the short arm of chromosome 15 (presumably). It is not known whether these composite 21/15 chromosomes are satellited or functionally dicentric. Thus, by translocation, chromosomes other than the acrocentrics could acquire satellites, and this may account for the reported satellites on chromosomes 1, 4 or 5, 9, 16, and Y. Unquestionably, the frequent attachment of nucleolar chromosomes to one nucleolar body during interphase could lead to chromosomal aberrations as postulated by Ohno e t al. (1961a). Information is sadly lacking on the status of satellited chromosomes in other cells of the body. Most of the current information is based on cells from fetal liver or during spermatogenesis. Are the same chromosomes satellited in the pancreas, erythroblasts, and pituitary, for example? Is there the same tendency for the nucleolar chromosomes to be brought together by nucleolar fusion? Until such information becomes available, it will be impossible to evaluate the cytogenetics of human cell differentiation. Up to now, the best information along these lines is available from embryonal-albeit differentiated cells-of dipteran larvae. IV. Experimental Studies on Nucleoli

The nucleolus has long been known to be a sensitive indicator to alterations in the cellular milieu; for example, cells exposed to a pH greater than 9.6 show an irrcversiblc dissolution of the nucleolus which is subscquently followed by death of the cell. Even exposure to normal cytoplasm brings about changes in nucleoli. To illustrate, when nuclei of immature starfish eggs are punctured by microneedles, the nucleolus undergoes rapid dissolution even though no extraneous medium has been introduced (Chambers, 1924) . Recent studies have emphasized nucleolar changes brought about by the action of chemical agents, of microbeam ultraviolet irradiation, genetic modifications of nucleoli, and by transplantation of nucleoli.

A. MODIFICATION OF NUCLEOLI BY CHEMICAL AGENTS Kleinfeld (1957), Kleinfeld and Koulish (1957), and Kleinfeld and von Haam (1959) studied the effects of thioacetimide on the nucleoli of rat kidney and liver cells. I n most instances, the more obvious effect of thioacetimide was to produce nucleolar enlargements. The enlargement of nucleoli was reversible if the thioacetimide treatments were suspended early enough. The nucleolar enlargements may be due mainly to an in-

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crease in ploidy (by endomitotic replication) thus producing more nucleolar-organizer chromosomes which, if fusion of their products occurred, would lead to larger nucleolar bodies. The metabolic inhibitors, actinomycin C and D, bring about a decrease in the RNA of nucleoli and cytoplasm (Kobayashi, 1960; Pomerat, 1961). Cobb and Walker (1958) used actinomycin D to decrease nuclcolar size in leukemic cells. HeLa cells treated with actinomycin D responded selectively based on their sensitivity to the antibiotic (Journey and Goldstein, 1961). The resistant cells persisted as dense bodies with coiled nucleolonema, while the sensitive cells, with a loss of RNA and protein, showed fragmented and budding nucleoli with a hyaline, less dense interior surrounded by a granular osmiophilic ring. Goldstein et d. (1961) also observed with fluorescence microscopy that HeLa cells sensitive to the antibiotic exhibited a similar disappearance of nucleolar RNA. Recently, the suggestion was made by Goldstein et al. (1961) as to the mechanism of operation of actinomycin D. Cultures of HeLa cells exposed for 5 to 6 hours prior to addition of tritiated cytidine did not incorporate the label into any component of RNA but did, on the other hand, incorporate i t to a slight degree in DNA. These investigators suggested that actinomycin D appeared to interfere with nuclear RNA and thus alteration in nucleolar morphology and RNA content occurred. Reich e t al. (1961) reported that actinomycin D inhibited the synthesis of cellular RNA and viral DNA, but not the converse. Specifically, actinomycin D seems to inhibit the synthesis of DNA-primed RNA, presumably mRNA. Reynolds e t al. (1963) studied the morphological changes in nucleoli produced by the action of 4-nitroquinoline-N-oxide on Chang liver cells in culture. The principal changes reported include: ( 1 ) nucleolar exhaustion manifested by a progressive decrease in the size of the nucleoli, ( 2 ) fusion of the nucleoli, and ( 3 ) separation of the pars amorpha and nucleoloneme to produce two types of nucleolar “caps.” The condensation of dense particles produced “dark nucleolar caps.” In other instances, nucleolar material is redistributed to produce an aggregate of less dense particles which forms the “light nucleolar cap.” Vacuoles within the nucleolar body may coalesce and migrate to one side of the nucleolus where they form light nucleolar caps. Thus, it is believed that the nucleolar vacuoles are distinct components of the nucleolus and not defects or holes. More recently, Reynolds and Montgomery (1963) have observed identical nucleolar cap formation through the action of actinomycin D.

B. ACTIONOF MICROBEAM ULTRAVIOLET IRRADIATION ON NUCLEOLI It i s assumed that the nucleolus plays a central role in cell division

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and RNA-protein synthesis since destruction of the nucleolus in a tissue cell by microbeam ultraviolet (UV) blocks mitosis (Gaulden and Perry, 1958). Moreover, irradiation of nucleolus inhibits RNA and protein synthesis (Perry et aE., 1961). Montgomery and Hundley (1961), using UV microbeam irradiation on living cells, obtained a loss of absorbing material from nucleoli which was ascribed to an alteration of the protein and nucleic acid composition. The independence and sensitivity of the nucleolus was demonstrated by Perry (1960) using radioautography involving tritiated cytidine with the inactivation of localized areas of nucleoplasm and nucleolus by microbeam UV irradiation. He concluded that the incorporation of cytidine into RNA was suppressed only by irradiation of the nucleolus. The action of UV here may be to suppress the RNA-primed RNA synthesis, believed to occur in nucleoli. Gaulden and Perry (1958) applying a microbeam technique after Uretz and Perry (1957) provided a beam less than 3 p in diameter and with an energy flux (polychromatic ultraviolet) of 0.01 erg p? sec.3. Neuroblast cells from grasshopper (Chortophaga) embryos were cultured in vitro. Irradiation of one (of two) nucleoli between late telophase through mid-prophase for 3 sec. prevented further division. Similar irradiation of one or both nucleoli during late prophase generally did not prevent division. These observations suggest that nucleoli are involved in mitosis within the period from late telophase through mid-prophase (includes interphase). During later stages, and especially during late prophase, the nucleolus is no longer involved in mitosis. Irradiated nucleoli may become smaller in size, however, they disappear in the usual manner a t late prophase. The nucleolus appears to be metabolically active during the sensitive period as indicated by the incorporation of cytidine into RNA. The assumption is that neuroblast nucleoli are damaged by UV, but apparently the nucleolar organizers are not. Gaulden (1960) indicated that a polychromatic UV microbeam (3 p in diameter) irradiation for 3 sec. (0.03 erg P - ~ ) almost immediately arrested mitotic activity, and permanently, if nucleoli were irradiated a t stages from late telophase to the middle of mid-prophase. Cells with nucleoli irradiated a t very late prophase divide. When nucleoli were irradiated a t 2650 or 2804A., the wavelength of 2650 was more effective in retarding mitosis. This is the wavelength absorbed by the purines and pyrimidines of nucleic acids. At mid-prophase, however, either wavelength was equally effective. I n these cells, nuclcoli form and grow a t mid-telophase. During this time, the nucleolus is most sensitive to UV irradiation. Perry and Errera (1960) state that with microbeam UV irradiation a

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cell can be effectively enucleolated and the capacity of the nucleolus to incorporate cytidine into RNA can be reduced by more than 90%. If one nucleolus in the cell is inactivated, the other nucleolus continues to incorporate cytidine. Perry et al. (1961) continued their studies on the effects of microbeam UV irradiation on the metabolic properties of nucleoli using HeLa cells. Again, the cells were effectively enucleolated with the microbeam. B y measuring the incorporation of tritiated cytidine into nucleolar RNA, the authors concluded that approximately two thirds of cytoplasmic RNA is nucleolar dependent, while one third of the incorporation into extra nucleolar parts of the nucleus is nucleolar dependent. The same system was used by Errera et al. (1961) for an evaluation of amino acid incorporation. Their results indicate that an irradiated nucleolus that has nearly no capacity to incorporate cytidine, will have its capacity to incorporate amino acids reduced by approximately 30%. This partial inhibition becomes effective approximately 6 hours after irradiation. There is also a 30% drop in amino acid incorporation into the cytoplasm, indicating that the nucleolus participates in controlling cytoplasmic protein metabolism, a t least in part.

C. MODIFICATIONS IN NUCLEOLI BY GENETICS Fischberg and Wallace (1960) have identified a mutant form of Xenopus laevis that is anucleolate. I n the normal genome, there is one pair of chromosomes with nucleolar organizers. Accordingly, the cells may have one or two nucleoli, normally. Heterozygous hybrids have one nucleolus and are otherwise normal, since sperm cells lacking a nucleolus are competent for fertilization. Cells of anucleolate embryos show no true nucleoli. Some cells contain several small spherical bodies which vary in size and number and have essentially the same optical features as nucleoli. These bodies show Brownian movement (as if detached), while true nucleoli do not show Brownian movement. These bodies, called “blobs,” contain RNA. Wallace (1962a,b) discussed the cytological aspects of anucleolate Xenopus larvae. H e considered the possibility that chromosomal loci besides nucleolar organizers act in a nucleolar collection system, but to a limited degree. I n a way, this might be considered analogous to the puffs seen in dipteran larvae and which will be discussed later. Signoret et al. (1962) in their studies on nuclear transplantation in the axolotl, referred t o a number of mutants seen in this amphibian. One of these involves the nucleolus. The nucleolus in the mutant is considerably smaller than the normal nucleolus. There is a possibility that the mutation involves the nucleolar organizer where, indeed, part of nucleolar organizer may be deleted. I n the tail biopsies of animals homozygous for

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the nucleolar mutation, two small nucleoli may be seen. These larvae are nonpigmented and tend to be much less viable. Von Borstel and Rekemeyer (1958) described a mutation in Drosophila in which the nucleolus was missing. Development proceeds, however, only through 10 to 12 mitotic divisions. According to Beermann (1960), Chironomus tentans has nucleolar organizers on chromosomes I1 and 111, while C. pallidivittatus has only one nucleolar organizer on chromosome 11. The organizers on chromosome I1 are not homologous since they appear in different loci on the chromosome. Hybrids between these sibling species are fertile and various recombinant genotypes are obtained to show any combination from three nucleolar organizers to none. Hybrids with any one of the three nucleolar organizers will survive, but those hybrids without a nucleolar organizer are not viable. That all nucleoli within one cell type may not be functionally alike is clearly demonstrated in Beermann’s (1961) work on the accessory nucleolar organizer that occurs in the mutant chromosome IV in both C. tentans and C. pallidivittatus. The accessory nucleolus is different in structure and, particularly in function, in that i t does not fuse with the normal nucleoli that develop on chromosomes I1 and 111. Moreover, the accessory nucleolar organizer on chromosome IV is incapable of sustaining development in otherwise anucleolate embryos. It is quite interesting that the nucleolar organizer may be disrupted into fragments of different length which, when combined to the other chromosomal breaks, are capable of organizing a nucleolus. Apparently, each fragment of the nucleolar organizer is capable of organizing a nucleolus. This is similar to the nucleolar organizer in Zea (McClintock, 1934). Thus, if one of the partial nucleolar organizers is introduced into an otherwise anucleolate genome, normal development of the embryo is sustained. This, however, does not hold true for the accessory organizer on chromosome IV.

D. TRANSPLANTATION OF NUCLEOLI Studies on the transplantation of nucleoli especially from cells of the Luck6 adenocarcinoma of the frog kidney have been reported by Kopac (1956, 1957, 1959-1962) and by Kopac and Mateyko (1958). The nucleolus can be placed into the cytoplasm with minimal damage to the cell. The more difficult procedure is the removal of the nucleolus from its normal nuclear site. Even though chromosomes cannot be seen distinctly during interphase because of their thinness, the nucleolar bodies attain their most prominent configuration. Since the nucleolar body and its

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chromosome (or chromosomes) are firmly integrated, the transplantation of the nucleolus also means the transplantation of the nucleolar chromosome (s) . The transplantation of nucleoli into the nucleus presents special problems. I n most instances, the nucleus will be severely damaged if it is punctured with a micropipette-essential to removal or implantation of the nucleolus. A procedure was devised which accomplishes the inclusion of a nucleolus into the nucleus without inflicting mechanical injury to the nucleus. I n this procedure, a nucleolus is placed behind one or two anaphase sets of chromosomes in a cell undergoing mitosis, The transplanted nucleolus may subsequently become incorporated by the nucleus (surrounded by nuclear membrane and other nuclear structures formed during telophase) . Not every cell survives this delicate operation. I n the cells that did recover, cytokinesis did not occur. Therefore, the two or more nuclei resulting from karyokinesis remained close together, permitting easy comparison between the nuclei and their nucleolar complement, namely, between transplanted and in situ nucleoli. Quite frequently, the transplanted nucleoli are recognizable because of size or shape and sometimes by the extent of formation of a chromophobic corona about them. I n most instances, the degree of staining of both transplanted and in situ nucleoli is approximately the same. On the basis of a limited number of successful nucleolar transplants into nuclei, Kopac (1960) indicated the following trends: (1) Nucleoli, if placed in the right position relative to the spindle and chromosomes of a cell in anaphase, will be incorporated by the nucleus reconstituted during telophase. ( 2 ) The staining capacities of the transplanted nucleoli with hematoxylin and eosin or with pyronine are essentially the same as those of the in situ nucleoli. ( 3 ) Nucleoli, even if fragmented or deformed during transplantation, become incorporated within the nucleus and still retain their structural modifications as well as their typical staining capacities. ( 4 ) The chromophobic corona may be less prominent around transplanted nucleoli than around the in situ nucleoli. ( 5 ) If the spindle is damaged or the chromosomes are drastically deranged during implantation of the nucleolus, either the cell will be irreversibly damaged and die, or else the nuclei may re-form as pinched-off fragments during telophase. As pointed out by Kopac and Mateyko (1958),if the nucleolar-chromosomal complex is intact, then the propagation of the nucleolus should be through normal mitotic cycles. The transplantation of an atypical nucleolus along with its chromosome(s) into a normal nucleus makes possible the testing of hypotheses concerning nucleolar cycles. Such transplanted nucleoli are in the most favorable position to replicate and

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participate in mitotic cycles along with those already present in the nucleus. The nucleolar chromosomes, the nucleolar organizers, and, in all probability, the nucleolonemata are structures that replicate and, therefore, can be passed on to the next generation of cells. I n other words, the nucleolus, if transplanted so that i t becomes part of the new nuclear complex, can serve as a special marker to be followed in successive generations of cells. Whether the pars amorpha can be similarly transmitted is uncertain. Accordingly, those nucleoli in which the pars amorpha alone has been especially modified might not show the abnormal characteristics in future cell generations, especially if the pars amorpha is elaborated de novo a t the end of each mitotic cycle. After transplantation, the appearance of atypical nucleoli in successive cell divisions or changes in the appearance of in situ nucleoli might add important information concerning the function of nucleoli and their associated structures. Hoskins and Montgomery (1962) have performed another type of nucleolar transplant using HeLa or Chang liver cells in culture. The nucleolus was removed from the donor cell by rupturing the nucleus with a microneedle and teasing the nucleolus free. The nucleolus was then placed within 1 p of the recipient cell’s surface with a microneedle. If the surface of the recipient cell came in contact with the nucleolus spontaneously, the cytoplasm actively enveloped the nucleolus and pulled i t rapidly toward the center of the cell where i t generally came to rest near the nucleus. The average time for pliagocytosis of the nucleolus was about 35 minutes. Nucleolar size decreased and in some instances the phagocytized nucleoli became fragmented. The effects observed on phagocytized nucleoli were quite similar to those observed by Kopac (1960) on nucleoli placed into the cytoplasm by a micropipette. Although, as Hoskins and Montgomery point out, the technique of transplantation via phagocytosis eliminates the problem of physical injury to the recipient cell, the method does not offer much protection to the nucleolus once i t is removed from a nucleus. Up to now, there is no known medium that will duplicate the normal environment of a subcellular structure. The use of fluorocarbon liquids, as described by Kopac (1955) and Kroeger (1960), could minimize the effects on the isolated nucleolus. On the other hand, the effects of inserting a micropipette into the cytoplasm are minimal, if done properly. There is no question that nucleoli can be temporarily protected during transfer from one cell to another, if done with a micropipette. Similar instances, involving the transfer of viable nuclei, have been reported (Briggs and King, 1952, for example).

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The nucleolar mutant in the axolotl may turn out to be a more useful system than the Xenopus mutant. I n the axolotl, the nucleolar chromosome carries a marker-a reduced size nucleolus-during the interphase. Quite possibly, the deletion within the nucleolar organizer, if this is correct, may involve other markers-in particular, pigmentation. It would seem that the axolotl nucleolar mutant could be one of the more useful organisms for the transplantation of nucleolar chromosomes. By transplanting a nucleolar chromosome (either mutant or normal) behind one set of anaphase chromosomes, following incorporation of the implant, a new karyotype could be established (Kopac, 1961). Indeed, experiments along these lines are now in progress (Kopac, 1963, unpublished). V. Lampbrush Chromosomes

The lampbrush chromosome is an architectural variant that occurs in certain vertebrate oocytes prior to maturation. The loops which provide the lampbrush configuration are lateral extensions of the principal linear configuration of the chromosome (Gall, 1958, 1963). These lampbrush loops tend to accumulate a substance rich in RNA and are presumably concerned with the synthesis of RNA or possibly even ribonucleoprotein ( R N P ) . Only certain sites on the chromosome, however, appear to be active a t any one time (Callan and Lloyd, 1960). There are other sites that are nonfunctional. Such sites appear merely as chromomeres with no lateral loops. It is the loop that is the apparent functional component. The lampbrush chromosome seems to be primarily designed for the purpose of generating what appear to be copious amounts of RNA-rich substances. It may be assumed, therefore, that the active sites of the chromosomes are transmitting genetic information. Although there may be an analogy between the puff on polytene chromosomes (see Section VI) and the loop in lampbrush chromosomes, i t has not yet been possible to correlate a given loop pair with a known genetic locus (Gall, 1963). I n the first place, a single chromosome of average length in Triturus may have roughly 1000 loops. Although the loop pairs differ from one another in terms of size, shape, and amount of RNA substance (Duryee, 1950), i t is still too complex a system for analyzing chromosomal loci and genetic effects. Perhaps one outstanding point may be mentioned. Becker (1959) has catalogued some 70 puffs in salivary gland cells of Drosophila melanogaster, indicating that during development ranging from the 2nd instar t o the pupa, a t least 70 gene sites become unusually active in the salivary glands. On the other hand, the number of loop pairs is generally of the order of thousands. I n the developing oocyte, apparently many more gene loci must become active. Meyer (1963) described paired and polarized functional structures in

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Y chromosomes in the spermatocytes of Drosophila hydei and D . neohydei. These paired structures originate from the nucleolar region which is situated a t one pole of the nucleus in the spermatocyte stage. All nuclei contain four different components: ( 1 ) the “threads,” ( 2 ) the clublike structures, ( 3 ) the “pseudonucleolus,” and (4j the tubular strands. These four components are interpreted as paired loops of a lampbrush type Y chromosome. All these structures with exception of the threads are strongly basophilic. The results of treatment with RNase show that a substantial component of all these structures may be RNA. The functional structures are Feulgen-negative a t the completion of the growth stage. DNA can be shown by the dispersion of weak Feulgen-positive chromocenters near the nucleolus directly before the functional structures are formed. The pseudonucleolus in D . hydei looks like a sponge while the possible counterpart in D. neohydei is a loosely coiled and tubular strand in the corresponding nuclear region. VI. Polytene Chromosomes

Another type of chromosome found in the salivary glands, intestinal cells, and Malpighian tubules of larval Diptera is the polytene, so-called giant chromosome. These chromosomes are several hundred times larger than conventional somatic chromosomes. They are characterized by prominent transverse bands. The giant salivary gland chromosomes of Drosophila are composites of several hundred, or indeed thousands, of individual chromosomal strands of chromonemata cabled together. Somatic synapsis also occurs. Microsurgery on these chromosomes has been performed by several investigators (Kopac, 1961). One of them, D’Angelo (1950), may be singled out. A band in Chironomus chromosomes could be resolved, by transverse stretching, into a series of dots which represent chromomeres of the individual chromonemata. Furthermore, by delicate teasing with a microneedle, a single strand could be pulled out. This information, together with other studies, clearly demonstrates that these chromosomes are a composite cable of hundreds or thousands of individual chromonemata. The band is the concerted effect of the chromomeres which, depending on their size and shape, will form bands of various sizes, patterns, and staining intensities. It is now believed that the polytene chromosomes are formed by endomitotic replication, but without separation of the individual chromonenia (Gall, 1958). Since the chromonema is in essentially an early leptotene stage, its length would be considerable. I n addition, the leptotene stage has chromomeres to produce a fairly constant architecture within the

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chromonema. The polytene chromosome, therefore, represents the length of a cable of chromonemata, with chromomeres in the same position for each and thus providing the banding patterns. The width of the polytene chromosome will depend on the number of chromonemata in the cable and this would depend on the number of endomitotic cycles experienced during the genesis of the chromosome. As in lampbrush chromosomes, the giant polytene chromosomes also show signs of localized intense activity. In Drosophila, such activity is manifested by the formation of puffs, or swellings, which appear to be centers of intense synthetic activity and produce RNA and possibly proteins. I n other Diptera, in addition to puffs, the Balbiani ring (BR) configurations are formed. The development of a puff or Balbiani ring represents a dramatic change in the structure of the chromosome. In the puff, as seen in Drosophila, for example, two things happen. First, there is the localized separation of the chromonemata from one another. Then, the chromomeres in this segment undergo uncoiling, with the result that much, if not all, the preexisting band architecture is disrupted (Swift, 1962). This now represents a complex of separated, fully uncoiled chromonemata, ideally suited morphologicalIy for the task of transferring genetic information. Present thinking along these lines suggests that the DNA code present in the chromosome is transcribed through the synthesis of mRNA which carries the functional code needed to synthesis a polypeptide, for example. The Balbiani ring is a more elaborate manifestation of a similar localized structural change in the chromosome. Beermann and Bahr (1954) studied the submicroscopic structure of the Balbiani ring. They state that individual chromonemata give up their lateral union for a short part of their length, with each forming a loop before uniting again with other elements to resume the cable-like configuration. In many ways, the Balbiani ring resembles the nucleoli (Beermann, 1956) as seen in chironomid salivary gland chromosomes. Indeed, these nucleoli might be considered as permanent Balbiani rings. Also, the Balbiani ring resembles the lampbrush configuration, except that there are several hundred or thousands of loops a t a given locus on the chromosome. Puffs are large enough so that the uptake of labeled nucleosides can be demonstrated by radioautography. For example, tritiated thymidine is incorporated during DNA synthesis as during the endomitotic replication of chromonemata. I n some instances, there will be an uptake of tritiated thymidine in certain puffs (Rudkin and Corlette, 1957). Tritiated uridine likewise is incorporated in puffs. Longer incubation in the presence of labeled nucleosides will show the presence of labeled RNA in the cytoplasm. The pathway from puff to cytoplasm may be direct, in some in-

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stances, or indirect, from puff to nucleolus to cytoplasm, in other instances. The non-nucleolar transfer of RNA to cytoplasm can be clearly inferred only from a study of embryos of lethal mutants lacking the nucleolar organizer regions (Beermann, 1960). Tritiated leucine is also incorporated in puffs, suggesting that proteins are synthesized in these loci, as well. Rudkin and Coriette (1957) reported that in Rhyncosciara and various chironomids certain regions in the salivary gland chromosomes undergo structural changes-puff s-in which a disproportionate DNA synthesis occurs. They found that the DNA was a t least twice the amount in a puff as compared with an unexpanded site. It is not yet determined whether this is replication or a “metabolic” DNA. I n some chromosomal regions there appears to be a disproportionate synthesis of DNA a t the early stages. Not all puffs show a disproportionate DNA synthesis. I n the chironomids, Stich and Naylor (1958) reported a building-up of DNA of 8 to 10 times the amount when compared with the unpuffed regions. I n Drosophila, the formation of a puff was not accompanied by any appreciable DNA synthesis. Cytidine-C14 was incorporated into chromosomal RNA, especially in the puffed regions. Thus, the puff is a center of RNA synthesis and not simply the incorporation of cytidine into previously existing material. The point is that these experiments indicate a synthesis of RNA on a chromosomal site in addition to the nucleolus. I n Drosophila, all polytene chromosomes are joined together in one place-the chromocenter-to which the nucleolus is also attached. This chromocenter is apparently a fusion of the regions of the centromeres of each one of the chromosomes and it appears to be an inert region composed of heterochromatin. The heterochromatic region of the X chromosome is attached to the nucleolar body by a delicate filament. This filament may be one or a few chromonemic strands of the chromosome. Kaufmann (1938) described two principal nucleolar organizers in D. melanogaster. One nucleolar organizer is situated in the proximal heterochromatic segment of the X chromosome, and one on the short limb of the Y chromosome. I n the salivary gland nuclei, these nucleolar organizers behave as if homologous, being fused together and buried in the chromocenter. I n Chironomus, Simulium, Bibio, and Sn’ara no chromocenters are present, and the homologous chromosomes are more or less separated. In chironomids, the most intimate association of the nucleolus and chromosome is a t the center of the nucleolus. The nucleolar region of the chromosome bearing the nucleolus is a single hypertrophied band or interband (Beermann, 1959). I n Drosophila the nucleolus is externally attached to the chromocenter while in chironomids, the nucleolus is internally attached to one of the chromosomes. In Cecidomyia, on the other hand, the

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nucleolus arises from a chromosomal region composed of loose heterochromatin. There is no chromocenter. White (1946) described a supergiant cell in each salivary gland of Lestodiplosis, with the nucleus being both polytenic and 32-ploid. T h e other cells in the gland are polytene but not polyploid. I n the polytenepolyploid nucleus, the pairing of homologous chromosomes is restricted to pairs-unlike the triploid Drosophila larvae. It is not known whether endopolyploidy precedes polyteny or not. There is a possibility, however, that the polyploid situation resulted from the fusion of a number of nuclci which were polytenic. Both the puff and Balbiani ring are specialized regions of activity within chromosomes. The polytene chromosomes might, therefore, represent a self-amplified system in which local gene action can be seen clcarly. Thus, the specific locus of a specific genetic “factory” elaborating a specific salivary protein can be clearly identified. The puffing or gene activation not only shows different degrees of amplification but is also reversible (Beermann, 1956). It is this aspect of independent puffing that Beermann (1956) has interpreted as a reprcsentative of activated genetic loci. DetaiIs of such activated sites hare been presented by Ficq and Pavan (1957), Stich and Naylor (1958), and Rudkin (1959). The expanded surface provided by the puff (which is here used as ti generic term) presents a greater area for enzymatic reactions by which genes can put into the cellular pool their synthesized materials. Mechelke (1958) and Swift (1962) view the puffing as an enlargement of the surfacc brought about by an unfolding of a tightly coiled submicroscopic structure. Rudkin and Corlette (1957) indicate that with this expansion a dilution of DNA occurs which is more active since the flow of metabolic materials is thereby increased. The formation of puffs and Balbiani rings represents a distinct physiological timetable of activity and is a functional catalytic site and, in this way, is analogous to the interphasic structure of typical mitotic chromosome. If the puffs or Balbiani rings are sites of local gene activity, then it should not be too surprising if different patterns of puffs or Balbiani rings could be found in different cells o r tissues a t the same time, or a t different times in the same cells or tissues. Such situations indeed have been described.

A. VARIATIONS OF PUFFING PATTERNS IN DIFFERENT TISSUES AND CELLS Pavan and Breuer (1955) were among the first to make a comparative analysis of the polytene chromosomes in different tissues of the same organism. Using Rhyncosciara angelae, they investigated the banding

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patterns in chromosomes of salivary glands, Malpighian tubules, and vesiculae seminalae and concluded that the band patterns were the same in cells of different tissues. They studied the behavior of various patterns during different phases of the organism’s physiology and found that these certain banding patterns were characteristic of only a special stage of the larval life (Breuer and Pavan, 1953, 1955). The cyclic behavior of puffing or bulb formations present in the salivary glands were not evident in homologous sections of Malpighian cell chromosomes. These studies provided morphological evidence of gene activities correlated with a specific time of larval development-by comparing hyperactivity of certain regions in salivary gland chromosomes to similar regions in the chromosomes of different tissues. Mechelke (1958) followed the activity of 12 loci of Acricotopus lucidus from the 4th larval instar through the prepupal to the young pupal stage and observed that each locus was not activated in each of the three lobes and that the duration of puffs and Balbiani rings was not simultaneous for all loci studied. There are three chromosomes in each salivary gland nucleus. One of these has a nucleolar organizer and nucleolar body almost adjacent to the centromere. A number of puffs are formed and some of these reach the status of a Balbiani ring. Chromosomes, in terms of number and location of the puffs, are different in different positions of the salivary glands. For example, chromosomes in the posterior and lateral lobes of the salivary gland have a Balbiani ring on each of the non-nucleolar chromosomes. I n cells of the anterior lobe, each non-nucleolar chromosome has a Balbiani ring, but in different loci. There is a puff located near the nucleolar body. As i t turns out, the cells in the anterior lobe secrete a brown substance during metamorphosis; those in the lateral and posterior lobes do not. It might be inferred, therefore, that the differences in puff (or Balbiani ring) patterns reflect a difference in function and, accordingly, gene activity. Studies on a Chinese species of Chironomus by Hsu and Liu (1948) have shown that a segment of chromosome I1 may be normally banded (loci inactive), puffed (loci active), or heterozygous (one synaptic member only is puffed, the other member is not). As Hsu and Liu point out, the heterozygous puff represents two expressions of a pair of allelomorphs which differ in activity. This has been referred to as an example of a “mutation” detected by cytological means. I n Chironomus pallidivittatus, four cells in each salivary gland produce special secretion granules-the SZ granules (Beermann, 1961). Such a situation does not occur in C . tentans. I n both species, three Balbiani rings are shared by all cells in the gland on chromosome IV. In the lobe specific cells, in C . pallidivittatus, a fourth Balbiani ring occurs near the

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centromeric end, but does not appear in chromosomes of the other cells of the gland. I n C. tentans, this additional Balbiani ring is not found. I n both C . pallidivittatus and C. tentans, the fine structural details within the bands of the chromosomes are maintained. Thus, the site for the fourth Balbiani ring is present in all cells of the salivary glands in both species. However, only those cells that produce the fourth Balbiani ring produce the SZ granules. I n hybrids, the additional Balbiani ring appears in the C. pallidivittatus chromosome, but not in the C. tentans chromosome. Secretion specificity is evident in those hybrids that show the fourth Balbiani ring. The hypothesis is that lobe-specific puffs or Balbiani rings would furnish the genetic information needed to produce a lobe-specific component of the secretion. Any change in the lobe-specific Balbiani ring would lead to loss of the specific component, especially if this change should prevent the development of the lobe-specific Balbiani ring. Becker (1959) has noted a difference between the anterior and posterior portions of the salivary gland in Drosophila melanogaster. For example, a puff on the X chromosome (section X-l5BC) appears in the cells of the anterior region, but does not appear in cells of the posterior region. Thus, we have two instances in which puff patterns in the same organ may show differences a t the same time, owing to differences in function of the various cells of the gland. IN PUFFING PATTERNS WITH STAGES OF DEVELOPMENT B. VARIATIONS

Perhaps some of the more useful data are the observations that, while the over-all pattern is usually constant in polytenic tissues, the secondary banding pattern may vary not only in the different tissues, but even more interestingly, in one tissue a t different larval stages (Beermann, 1952, 1956; Pavan and Breuer, 1955; Pavan, 1958). Becker (1959) extensively studied the puffing patterns in the salivary gland chromosomes of Drosophila melanogaster a t two stages in the life cycle of the larva. The first stage covered the last 24 hours of the 2-day 3rd instar larva; the second was following the 12-hour prepupal period. These represent the puffing periods. Each one extends over not more than 6 hours, mainly before, but also during and possibly slightly after, entering the next developmental stage. Puffs are formed in specific chromosomal sections and their formation and disappearance follows a characteristic order. Both puffing periods differ in that (1) some puffs appear in only one of the two periods, and (2) the other puffs appear in both periods but in a different order. There is some evidence for a puffing period a t the end of the second instar. Puffing in the salivary gland chromosomes is also

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closely connected to molting from one developmental stage to another. Over 70 puffs were registered in D. melanogaster, and these were divided fairly evenly over the chromosomes X, 2L, 2R, 3L, and 3R. Perhaps the most conspicuous puff patterns occur a t loci 3L-74EF and 3L-75B. These appear during the third larval stage, then disappear to reappear again during the postprepupal period. Both these puffs were prominently figured in Bridges’ (1935) maps of these salivary glands. Beermann (1963), in a review, summarized the currently available information on the visible differentiation of giant polytene chromosomes. Puffing, according to Beermann, is an expression of, or the actual mechanism, which causes enhanced genic activity. Differentiation with respect to puffing would be equivalent to differentiation with respect to genic activity. This would be expected if differential gene activation occurred during embryonic development. Moreover, there is a correlation between puffing and cellular differentiation. The mechanism of puffing probably involves the unfolding of the chromomeres in the many chromonemata in concert a t a specific locus. At this site there is the elaboration of RNA and the accumulation of nonhistone proteins. Loci that are puffed in any tissue (space), or stage in development (time), contain genetic information which is of importance to the cells under consideration (Beermann, 1963). Examples of both situations are known, however. Beermann (1956,1959) has shown different puff patterns, occurring a t the same time, in such tissues as salivary gland, Malpighian tubules, rectum, and mid-gut. Becker (1959) has also demonstrated that the puff patterns exhibited by salivary glands of D. melanogaster change with stages of development. Becker (1959) , among others, has shown that puffs in Drosophita may develop and then recede. Some of these puffs may repeat the process later. The present interpretation is that a puff is produced through the activation of an operational unit on the chromosomal locus. Probably, through a feed-back mechanism, the puff may be “turned off” and will thus recede. Subsequent events may reactivate it or not, depending on the specific genetic system involved. VII. Experimental Modification of Puffing Patterns in Salivary Gland Chromosomes

A. ACTIONBY NONSPECIFIC ENVIRONMENTAL FACTORS Recent experiments support the assumption that puffing is indicative of increased local activity of the genome. This reaction can be induced in a number of ways. Droplets that are Feulgen-negative and give positive reactions for RNA and proteins accumulate a t most sites of salivary

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gland chromosomes where puffing normally occurs when Glyptotendipes or Chi-ronomzcs larvae are subjected first to low temperatures (5-10°C.) for a t least an hour and subsequently transferred to 20°C. water (Stich and Naylor, 1958; Beermann, 1956, 1959). These droplets are of unknown composition, except as already indicated, and are probably produced by a temporary upzet of the metabolic balance of chromosonies, nuclear sap, and cytoplasm. Further confirmation of Beermann’s concept of differential gene activation was provided by Vogt-Kohne and Carlson (1963). The second Balbiani ring in chromosome IV of ChironomzLs tentans during the 4th instar was studied by UV and interference microscopy. This Balbiani ring can be activated by exposing the larvae to a temperature of 4°C. for 6 hours. The dry weight of the second Balbiani ring was 2 times higher and the nucleic acid content was 3 times higher when compared with controls (4th instar larvae maintained a t room temperature). Ritossa (1962, 1963) described the development of new puffing patterns in the salivary glands of Drosophila that were induced by teinperature shock and by immersing salivary glands into media containing dinitrophenol or salicylate. On the other hand, Schultz et al. (1955) found out that various pyrimidine analogs (in mixture) were ineffective in changing the frequency with which puffs are formed, although the concentrations of the mixtures were sufficiently high to impare further larval development. It is known that specific changes in development can be elicited by changes in temperature or ionic strength of the medium. These unspecific physical or chemical factors can elicit changes in puff patterns (Becker, 1959, 1962). Better clues to the understanding of puffing mechanisms can be derived from the application of specific biological agents, such as hormones. B. ACTIONOF HORMONES ON PUFFING PATTERNS Becker (1962) , Clever (1961) , Clever and Karlson (1960) and Kroeger (1963) have reported studies in which specific activities of chromosomal puffs were elicited by the application of molting and rejuvenation hormones, and by hormone imitators. The specific hormone concerned with molting is ecdysone which has been highly purified (Karlson, 1956) and there are indications that i t is a cholesterol derivative. Burdette and Bullock (1963) have recently identified five separate fractions in ecdysone extracted from Bombyx pupae. Prior to this, Butenandt and Karlson (1954) had produced two fractions. All five fractions isolated by Burdette and Bullock showed biological activity when assayed by pupation induction in Calliphora.

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Clever (1961) injected ecdysone into Chiranomus larvae. Within 30 minutes after injection, a new puff appeared in region I-18-C and then disappeared 2 hours after injection. Within 30 to 60 minutes after injection, another puff in region IV-2-B began to enlarge and maximum puffing was achieved in 2 hours after injection. This puff does not react to ecdysone unless puff I-18-C is activated. From 48 to 72 hours after injection, two additional loci became extremely active (I-1-A and 11-14-A) and, when compared to the noninjected control group, would seem to be specific puffs for prepupal development. Again, these latter two puffs would only be present if loci I-18-C and IV-2-B are active. It seems then that these specific and reproducible puffing reactions. occur in a definite sequence: I-18-C, IV-2-B, I-1-A, 11-14-A. The first two puffs probably emit gene products which trigger off other gene products culminating in the protein syntheses of the required substances for pupation. Puff I-18-C may be located nearer to the primary action of the hormone than puff IV-2-B. I n Chironomus, puffs I-17-B and I-19-A may be present in larvae during the whole last stage, although they may vary in size, sometimes small and sometimes large. After the onset of molting, the puffs always reach maximuin size and, a t the end of the prepupal period, these puffs regress. Puffs I-18-C and IV-2-B are either inactive or only slightly active before initiation of the molt. After experimental induction, these puffs are activated within less than one hour and their size depends on the concentration of the hormone applied. I n normal development, the appearance of puff I-18-C in the larva is the first known sign of the beginning of molting. There is a progressive increase in puff size and maximum size is reached near the end of the process. I n a later study, Clever (1963) established the reaction threshold for gene locus I-18-C a t a concentration of lo-? pg., and for locus IV-2-C a t pg. of ecdysone per mg. of larval weight. Both puffs increased gradually with increasing hormonal doses, reaching their maximal sizes a t concentrations of 10 (I-18-C) and (IV-2-B) pg. of ecdysone per mg. of larval weight. Accordingly, the size of puff I-18-C depends on a hormone concentration in a range of about 5 and puff IV-2-B in a range of 2 orders of magnitude. These hormones are quite active: lo-? pg./mg. larval weight is needed to activate a puff on chromosome 1; while 10 times this amount will activate the puff on chromosome IV. As Beermann (1963) has pointed out, these amounts are equivalent to a few hundred ecdysone molecules per single chromatid in each nucleus. Clever (1963) showed that puff I-18-C disappeared 8 hours after the pg. ecdysone. Up to a dose of 2 X injection of 2 x to 2 x

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pg., a reduction in gene activity was recognizable by a decrease in puff size. Puff IV-2-B disappeared 8 hours after the injection of 2 x pg. ecdysone. It is clear that this locus has a lower sensitivity to ecdysone. It was further concluded that both loci are under the permanent control of ecdysone. Ecdysone is constantly present in the hemolymph during the molting period, due to induction of hormone production in the animals themselves. During normal pupal molting, puff IV-2-B disappears 1 to 2 days later than puff I-18-C. From this and other considerations, Clever concluded that the titer of ecdysone during molting rises gradually and the activity of puffing in these loci is adjusted, depending upon the altering hormone titer. Also based on the injection experiments and puffing behavior, the concentration of ecdysone in young prepupae is estimated a t lo-? to pg. per mg. of larval weight and reaches a peak level of pg. in old prepupae. Becker (1962) provided additional information on the dependency of puff patterns in Drosophila melanogaster on the presence of metamorphosis hormones, The source of molting hormone in Drosophila is the ring gland. By ligating late 3rd instar larvae in or near the fourth larval segment, the ring gland lies in the anterior portion t o the ligature and the passage of hormones into the posterior portion (where the salivary glands are located) is effectively prevented, providing that the ligation is done before hormonaI production starts. If the anterior portion formed a puparium in less than 3% hours after ligating, the posterior portion also formed a puparium. On the other hand, if puparium formation in the anterior end set in more than 5 hours after ligating, the posterior portion remained larval, in all cases. From this, it was concluded that the ring gland produces hormones from 3% to 5 hours before puparium formation. At between 4 t o 5 hours before puparium formation, there appears to be a critical period for initiating puff formation. Since there is this close correlation between puparium formation and puffing, Becker concludes that the puffing period in larval salivary gland chromosomes is triggered by the hormones in the ring gland. The puffing reaction in chromosomes immediately follows the production of hormones. The ring gland hormones activate the complete gene pool provided for temporary salivary gland function, since the whole set of puffs was affected by the ligating experiment. Another set of experiments involved the transplantation of the salivary gland into the abdomen of other larvae. If the gland from an early 3rd instar larva is transplanted into a late 3rd instar larva, both the implanted and host salivary glands start with puff formation simultaneously. Thus, the implant reacts prematurely due to the influence exerted by the host’s milieu.

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A reversal in puffing pattern can also be demonstrated by transplanting the salivary gland from an early prepupa into the abdomen of a 3rd instar larva. The puffing pattern now shows the characteristics of the host. I n particular, section 3L-78D of the implant chromosomes forms a puff. This puff normally does not appear in prepupal chromosomes and is, therefore, a characteristic of the larva. From these observations, Becker (1962) concludes that the specificity of the puffing pattern depends, in part, on the milieu which surrounds the gland and this would be due to a change in relative amounts of the molting and juvenile hormones. Thus, both acceleration of development and retardation of development (rejuvenation) can be demonstrated in the salivary gland chromosomes of Drosophila. On the basis of ligation and transplantation experiments, Becker (1962) concludes that there may be two attributes of the function of metamorphosis hormones with regard to gene locus activation: (1) They can trigger the activation of the gene pool, which a cell holds in preparation according to its state of differentiation, and ( 2 ) the milieu (juvenile hormone-molting hormone) can select t o a certain degree from the offered gene pool, possibly by means of changed relative amounts of the hormones.

IMITATORS ON PUFFING PATTERNS C. ACTION OF HORMONE Kroeger (1963) discovered that the action of ecdysone on larvae of Chironomus thummi could be imitated by injecting ZnC1, into the hemolymph to make the concentration 0.023 M . The same effect can be elicited by explanting salivary glands into oil containing a droplet of hemolymph and ZnC1,. The reaction of puffing pattern to ecdysone is called: “acceleration of development.” Normal larval response occurs only a t a certain stage of development. At the proper stage, the larvae are competent. When larvae a t this phase of development do not respond, they are called incompetent. Heavy metals or narcotics apparently do not imitate ecdysone by increasing the sensitivity of cells to the hormone. This was shown by explanting matched salivary glands into hemolymph differing in ecdysone content. There is no difference in reaction when ZnC1, is added in a standard, or effective concentration. Puffing in competent larvae is not accelerated by KCN; indeed, KCN causes the puffs to disappear altogether. Juvenile hormone blocks the action of ecdysone. For example, if active corpora allata (the source of juvenile hormones) are transplanted into larvae competent to respond to ecdysone, the larvae are rendered incompetent for 5 days. The effects of the juvenile hormone can be imitated by inducing a “wounding metabolism” in the cells. Wounding metabolism is induced by mechanical injury, explantation, X-rays, oxygen poisoning, etc. Following the induction of wounding metabolism, the nuclei return to

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a puffing pattern characteristic of a younger stage, and become refractory to the action of ecdysone (Kroeger, 1963). Thus, the nuclei are rendered incompetent through this effect. This process is called rejuvenation. In the rejuvenated stage, the puffs are oversized as compared to those puffs that appear during normal development. Kroeger (1963) points out that ecdysone and juvenile hormones act in an antagonistic fashion on a system, depending on their levels in the nuclear sap. The genetic loci, in turn, respond a t various levels of these hormones by being activated or remaining dormant according to their individual sensitivities. Clearly, in metamorphosis it is the level of the controlling system which changes, not the sensitivity of the loci. Therefore, only specific combinations of activities can appear in response to a given hormonal situation. The occurrence of rejuvenation a t all stages shows the same control system to be operative along the whole developmental sequence. Rejuvenation can be halted a t all stages by ecdysone. It is a t first blocked by juvenile hormones, but as the juvenile hormone levels fall, the titer of ecdysone rises. This is the progression from one stage of metamorphosis to the next. Usually, juvenile hormone levels may fall before ecdysone is produced, a probably normal occurrence. Then the larva becomes conipetent. One can conclude that incompetent larvae must have a high titer of juvenile hormone.

PRODVCTION OF DEFICIENT KARYOTYPES Kroeger (1963) developed a technique for the production of highly deficient karyotypes by cutting away parts of chromosome IV (salivary gland nuclei of Chironomus thummi). By a process of elimination, i t was demonstrated that for the appearance of two puffs and for the enlargement of a third puff, during rejuvenation no other part of the genome is necessary. Only short stretches of chromosomal material neighboring the respective loci, on either side, were not tested for indispensability because they could not be removed by the procedure used. These experiments so far have been available only for rejuvenation effects since the excessive wounding metabolism induced by microsurgery blocks acceleration. If gene activities are regulated by other parts of the genome, then removal of certain parts of the chromosome set should interrupt this function. Accepting this premise, i t should be possible to localize such parts of the genome that are indispensable for the functioning of others. The control system of gene activities may involve an interplay of parts of the genome or may be an independent response of single loci (or groups of closely linked loci) to a specific biochemical content of the nuclear sap. It cannot be concluded, on the basis of present evidence, that closely D.

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linked chromosomal loci interact in a similar way as an operon in bacteria. However, activation of the whole functional unit cannot depend on sites (or loci) farther removed in the chromosome set.

E. TRANSPLANTATION OF SALIVARY GLANDNUCLEUS A beautifully designed test of the possible environmental effect on gene loci was executed by Kroeger (1960) when he induced new puffing patterns by the transplantation of prepuparium salivary gland nuclei of Drosophila busckii into the preblastoderm or blastoderm cytoplasm derived from D . melanogaster eggs. After 3 hours of incubation of the nuclei in preblastoderm cytoplasm, there is a change in certain puff patterns. Some of the puffs persist as they normally do through pupation; Iikewise some puffs disappear as they would normally. On the other hand, one puff a t locus 2R/22 was highly activated in preblastoderm but not in the blastoderm cytoplasm. A puff a t this locus has not been seen in polytene chromosomes under customary methods and times of observation (1st instar through prepupal stages). It would seem, as Kroeger pointed out, that the puff a t 2R/22 becomes active a t an early stage of development, and not later. Also, these gene loci involved in the utilization of yolk, for example, would not be active in salivary gland nuclei. Such an event would not be evident prior to polyteny. Here again, the polytene chromosome by functioning as an amplifying system discloses events which heretofore could never be seen in conventional chromosomes. VIII. Perspectives Involving Nucleolar and Non-Nucleolar Chromosomes

For the present, the polytene chromosomes are the best available systems for demonstrating functional activity of chromosomes and especially for obtaining an insight into the mechanisms of transferring genetic information from chromosomes to cytoplasm. Since these intriguing structures also have nucleoli (of various types, to be sure), one has a better opportunity to study the relationship between chromosome and nucleolus as well as between nucleolar and non-nucleolar chromosomes. For these reasons, this section is intended to summarize our knowledge as well as speculations on the relationships of chromosomes, nucleoli, nuclei, and other subcellular structures on the basic problems of cellular differentiation. A. THEFATEOF ANUCLEOLATE GENOMES Anucleolate cells, especially if the genome is anucleolate, do not fare well. This suggests that the sojourn of some of the RNA derived from chromosomal loci in nucleoli is essential. It may be that certain ribosomes can be assembled only in the nucleoli. On the other hand, not all RNA

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derived from chromosomal sites needs to be brought to the nucleolus to be functional. Perhaps some of the RNA derived from chromosomal loci may be tRNA (transfer RNA) , while the other obviously must be niRNA. Perhaps the latter needs to migrate to the nucleolus before it becomes functional. On the other hand, certain amounts of RNA-primed RNA synthesis occur in nucleoli and perhaps may not occur elsewhere. If such RNA is essential to the cell, then obviously this supply would be curtailed in the absence of a normal nucleolar system. I n mitotic divisions from the oocyte stage to the blastula, these embryonic nuclei divide rapidly, even though the oocyte nucleoli have been genetically removed in amphibian mutants (Elsdale et al., 1958), Drosophila (von Borstel and Rekemeyer, 1958), or Chironomus (Beerniann, 1960). Wallace (1962b) reported that anucleolate Xenopus embryos always hatched into abnormal animals. Cytologically, the nerve cells and some endodermal derivatives showed a large number of pycnotic nuclei. Mitosis did not occur. It must be pointed out, however, that while no circumscribed nucleoli were present, there were pyronin-staining intranuclear structures that were present in the nuclei, although smaller and more numerous than typical nucleoli. These bodies, which were called “blobs” were RNA-, arginine- , and alkaline phosphatase-positive, and Feulgennegative. Such anucleolate homozygous mutants developed into apparently normal elongated neurulae and tailbud stages with both morphogenesis and histogenesis normal up to hatching. Beyond this, development and growth were retarded. Severe pycnosis was evident in the central nervous system, eye, ear, and nose. Pycnosis was also seen in cells of the pharyngeal floor and some ectomesodermal organs, but not in other tissues. Because of the limited optical characteristics of ordinary chromosomes a t interphase, there is no way of knowing whether the blobs represent the accumulation of material generated a t the chromosomal sites, or whether these already are a partial accumulation of material generated in other parts of the chromosome, or even in other chromosomes. A possible interpretation is that the blobs in anucleolate cells are, in part, the material that would usually accumulate in the region of the nucleolar organizer, if it were present. McClintock (1934) noted that in Zea, cells which lacked nucleolar organizers, nucleolar-like bodies appeared, but of course failed to collect in a central nucleolar body. Beermann’s (1961) work on anucleolate hybrid embryos of Chironomus tentans x C. pallidivittatus indicates the physiological importance of the nucleolus. Anucleolate zygotes die as embryos. Development following the blastoderm stage is irregular. There is a failure t o gastrulate and

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endodenn is not formed. However, some mitotic divisions and differentiation of a few cells which contain numerous small granules of spherical shape instead of the typical, larger nucleoli do occur. The conclusion is that those hybrids which lack a nucleolar organizer fail to synthesize a special type of ribonucleoprotein which would be a depot for a protein important for growth. Moreover, the nucleolar organizer is, to a certain degree, involved in assembling the small disorganized nucleolar-like bodies which are found scattered within the nucleus (if the nucleolar organizer is absent). Since in the nuclei of anucleolate embryonic cells one may find small spherical granules instead of the nucleolar body, it is possible that this substance might make up the nucleolar body when one or more nucleolar organizers are present. Latent organizers might be those segments of chromosomes which do not ordinarily produce nucleoli when functional nucleolar organizers are present. If the main nucleolar organizer is absent or nonfunctional, then the so-called latent organizers show a greater tendency to accumulate nucleolar material than other chromosomal loci. Thus, the abnormal development of naturally occurring anucleolate organisms (von Borstel and Rekemeyer, 1958 ; Beermann, 1960; Fischberg and Wallace, 1960) and anucleolate cells produced by microbeam UV irradiation (Gaulden, 1960; Perry and Errera, 1960) clearly demonstrates the necessity of assembled nucleolar material for the proper functioning of cells.

B. MRNA PRODUCTION AND TRANSFER FROM NUCLEUS TO CYTOPLASM Goldstein and Micou (1959) point out that RNA is synthesized initially a t the chromosomal locus and the material is rapidly transported to nucleoli. Pelling (1959) believed that nucleolar RNA is synthesized only a t t,he nucleolar organizer. Moreover, other active loci may also be concerned. Bonner’s (1959) views were that RNA is made in the chromosomes, then is transferred to the nucleolus where the protein component is manufactured and, the finished product, the ribosome, is transferred to the cytoplasm. Feinendegen et al. (1960) for the most part, concurred with this view based upon observations of HeLa cells in culture, as did Zalokar (1961) in his work on oocytes of Blatella germanica. Not only is nucleolar RNA apparently a precursor for cytoplasmic RNA (McMaster-Kaye, 1960) , but the nucleolar turnover rate is more rapid than the chromosomal (McMaster-Kaye, 1962). The nematode, Meloidogyne, which induces the formation of giant cells in tomato roots with enlarged nuclei and nucleoli (Bird, 1961), brought about an enhanced synthesis of protein. Some information on hepatic viral nucleolar effects was given by Bearcroft and Peachey (1962). The earliest and progressive change was an enlargement and basophilia

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of the nucleolus which was associated with an increase in ribonucleoprotein, later to be found in the cytoplasm. I n fatal cases, nucleoli were also vacuolated. The interpretation was made that the infection was characterized by increased synthesis of ribonucleoprotein which originated in the nucleolus. Georgiev et al. (1961) revealed that the nuclear supernatant fraction had low polymeric RNA while the nucleochromosomal complex produced high polymeric RNA. They interpreted these data as signifying that two types of macromolecular synthesis occurred in the nuclei of resting cells. I n the first instance, there appeared to be a formation of soluble proteins in the ribosomes, the nuclear sap, and principally in the microsornes; in the second instance, the synthesis of ribosomes and soluble RNA, associated with the nucleolus-chromosomal complex, occurred which, in turn, moved out into the cytoplasm. That more than one type of RNA or R N P may exist in the nucleolus or be transferred to the cytoplasm has been suggested (Goldstein and Micou, 1959; Perry, 1960; Taylor and Woods, 1959). Sibatani et al. (1960) fractioned two metabolically distinct classes of RNA in animal cells. Moreover, the activity of acid RNase appeared to be much lower in tumor cells. I n addition, Finamore (1961) identified two RNA fractions and indicated that nucleolar RNA is characterized by a major component, compound X, which resembles uridylic acid and it may be pseudouridylic acid. Le Blond and Amano (1962), using labeled cytidine, confirmed the previous observations that RNA synthesis occurs in the nucleus and nucleolus; nucleolar RNA then migrates to the cytoplasm. With labeled leucine they demonstrated that protein synthesis was rather localized in the nucleus and cytoplasm, and minimal in the nucleolus. Bolognari (1960) assessed the function of the nucleolus as the obvious one, related t o ribonucleoprotein, and the other to substances probably connected with the filamentous threads or components concerned in the functional developmental stages (Albanese and Bolognari, 1961). Edstrom et al. (1961) , using nucleoli isolated by microdissection from Asterins rubens oocytes, concluded that the particular role of nucleolar RNA was to form the stable part of ribosomal RNA while the karyoplasmic RNA was destined as unstable mRNA. Edstrom’s (1960) elegant work on the RNA content of spider oocytes revealed the following order of distribution: highest in nucleoli, next in the cytoplasm, and quite low in the nucleoplasm. Nucleolar and cytoplasmic RNA appeared to be similar. Sirlin (1960a), using labeled uridine as a precursor on the larvae of Smittia, described the order of labeling as: nucleolus, Ralbiani ring, and

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chromosomes. He argued the interpretation that the precise site of incorporation was the chromosomal-nucleolar junction, and that the nucleolus, per se, could be considered as an assembly station for various ribonucleic acids. The rate of nucleolar RNA synthesis in the root tips of Allium and NigeZZa were studied by Das (1963), using tritiated cytidine as a pulse label. The rate of nucleolar RNA synthesis remains normal as long as nucleoli are still present in prophase cells; only the condensing chromosomes during prophase show reduced RNA synthesis. I n mitotic cells lacking distinct nucleoli, there is practically no RNA synthesis. RNA synthesis resumes in the late telophase or early interphase cells when pronucleolar bodies are formed. Nucleoli are active primary centers of RNA synthesis so that the nucleolus is more than a center for the accumulation of chromosomal RNA. For various reasons, the polytene chromosomes in dipteran larvae may offer clues for suggesting mechanisms of action in ceIls of other organisms, including man. The variation in localized chromosomal activity has already been discussed. Because several hundred or even thousands of identical chromosomes comprising the polytene complex are acting in concert, one has here a self-amplified system. Presumably in conventional chromosomes consisting of a single chromonema during interphase, only certain sites elaborate RNA. This RNA could be mRNA if it represents a transcription of the genetic code reposited in the chromosomal DNA. Edstrom and Beermann ( 1962) have succeeded in characterizing the RNA in puffs by utilizing the Edstrom fiber electrophoresis procedure. For this work, the large Balbiani rings located on chromosome IV of Chironomus tentuns were studied. The short chromosome was isolated with glass needles from formalin-fixed nuclei. The isolated chromosomes were dried and cut into three pieces, each one containing a Balbiani ring. The RNA from a number of homologous pieces (pooled) was extracted by repeated RNase action. The hydrolysis occurred in a micropipette. The hydrolyzates were subjected to electrophoresis on a rayon fiber (25p in diameter) and the concentration of nucleotides separated on the fiber were estimated by UV microphotometry. The sensitivity of the method is 10-log. RNA per analysis. This is equivalent to the RNA content of 50 Balbiani rings, 5 nucleoli, or one half of the cytoplasm of a salivary gland cell. The base composition of the anaIyzed sample was expressed in per cent, from which base ratios may also be calculated. The results of such an analysis are shown in Table I (Beermann, 1963). Even though there is no certainty that each puff contains homogeneous

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TABLE I BASECOMPOSITION (IN yo) OF RNA EXTRACTED FROM THREE BALBIANI RINGS(BR) LOCATED ON CHROMOSOME I V IN SALIVARY GLANDS OF Chironomus tentansa Base

BR 1 (proximal)

BR 2 (median)

BR 3 (distal)

Nucleolus

Adenine (A) Guanine (G) Cytosine (C) Uracil (U) A/U

35.7 f 0.6 20.6 f 1 . 7 23.2 f 1 . 2 20.8 f 0 . 8 1.72

38.0 f 0 . 6 20.5 f 0 . 6 24.5 f 0 . 6 17.1 f 0 . 6 2.22

31.2 f 2.2 22.0 5 2 . 0 26.4 f 1 . 9 20.2 f 1 . 4 1.54

30.6 f 0 . 8 20.1 Itr 0 . 5 22.1 k O . 6 27.1 k 0 . 6 1.13

0

Cytoplasm 29.4 22.9 22.1 25.7

k 0.4 k 0.3 f 0.4 k 0.3

1.14

From Beermann (1963).

RNA, the differences in base composition in each Balbiani ring are of interest. Likewise, the A/U ratios are different. It will be noted that nucleolar RNA composition differs from those shown by the Balbiani rings, although there is greater similarity between the base composition of nucleolar and cytoplasmic RNA. This similarity between nucleolar and cytoplasmic RNA may be spurious, as Beermann (1963) states, even though it agrees with the idea that ribosomal RNA is of nucleolar origin. The data suggest that Balbiani ring RNA is not stored in the nucleolus or else it is stationary ribosomal RNA. It could represent short-lived mRNA or tRNA. The mechanism of transcribing base sequence in RNA from base-pair sequence in DNA is not understood. Beermann (1963) points out two possibilities: (1) Single-strand RNA is formed from the selection by base pairs in DNA. This is the triple helix concept. ( 2 ) Two RNA strands, complementary to one another, are formed, with one strand either destroyed or rapidly transported away from the Balbiani ring, If mRNA is a direct copy of DNA, then the specific way in which it differs in its base composition from other RNA fractions becomes significant. As Edstrom and Beermann’s data show, the Balbiani ring RNA is not symmetrical with respect to A/U ratios, as would be expected in the A/T ratios of double-stranded DNA. Balbiani ring A/U ratios of 2+ or large deviations from 1 are evident. A/U symmetry is found more closely in nucleolar and cytoplasmic RNA. The RNA analyses suggest that Balbiani ring RNA is either the messenger itself or its complementary counterpart-the antimessenger RNA. The information on the RNA composition in Balbiani rings as stated by Edstrom and Beermann (1962) provides the most useful data. The next step is t o establish base sequence in the Balbiani ring RNA. Only in this way can the identity of RNA be established since i t is possible for

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two different RNA samples to have the same base composition percentagewise, yet they may be different only in terms of base sequence. If the present conception of the genetic code is correct, then it will be the base sequence that is important-and not base composition per se. Another aspect of the transport of “genetic information” from the nucleus to cytoplasm is that provided by Gay (1955, 1956). The evidence for this was obtained through electron microscopy of salivary gland cells during the time the cells are producing a protein which, when secreted, functions as a glue. The function of the glue is to permit the attachment of the larva to a solid substrate prior to pupation. By electron microscopy, at the time the special secretion granules are being formed, it can be seen that blebs appear in the nuclear membrane. The blebs are apparently outpocketings of the nuclear membrane (as double-layered membranes which evolve as cytoplasmic membranes later). Within the bleb is material that is continuous with and part of one of the polytene chromosomes. Thus, the interpretation here is that active regions of the chromosomes (puffs) are releasing substances that pass into the cytoplasm by way of the double-layered nuclear membrane blebs. Once the blebs separate from the nuclear membrane, their architecture eventually resembles the typical double-layered cytoplasmic membranes. There is a possibility that the material coming off the chromosome is essentially ribosomal in nature, although these ribosomes, if so, do not become attached to the membranes, as is common in other instances, where ribosomes are attached t o cytoplasmic membranes. The transport of mRNA, either by itself or already incorporated into ribosomes, has been inferred from the results of labeling and other techniques. I n general, through pulse labeling and chasing techniques, RNA carrying a labeled nucleotide may be traced from the chromosomal locus to cytoplasm, or from chromosomal locus to nucleolus to cytoplasm.

C. WHEREARE THE RIBOSOMES FORMED? A question that must be answered a t this time is: When and where are the ribosomes formed? The status of the ribosome in terms of size, structure, protein content, RNA content, and role in protein synthesis was reviewed by Watson (1963). It seems to be fairly clear that ribosomes may exist in different sizes (in terms of sedimentation constants and by electron microscopy)-the result of aggregation of smaller units or the disaggregation of larger units. These entities contain many kinds of proteins (enzymes ?). Classes of sizes so far described have sedimentation constants of 30, 50, 70, and 100 Svedberg units ( S ) . By starch gel electrophoresis, some 20 different protein bands can be demonstrated. Protein

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band patterns in 3 0 s ribosomes are different from those found in 5 0 s ribosomes (Walter and Harris, 1961; Spahr, 1962) . Several types of RNA were also found. There is the ribosomal RNA consisting of a polymer ranging from 16 to 23 S. I n addition, there is the tRNA with attached, activated amino acids. Finally, there is the mRNA to which as many as 5 ribosomes of the 7 0 s class may be attached. This institutes the class of substances referred to as the polyribosomes, with a sedimentation constant of 170 S (Warner e t al., 1962). It is believed that each one of these ribosomes, in the polyribosome complex, is polymerizing polypeptides in accordance with the instructions furnished by the mRNA. Are the specific tRNA’s attached to specific amino acids in the ribosome? Where do ribosomes acquire their mRNA? The nucleolus probably plays a part in this step. The mRNA, as it comes off a particular gene locus, gets into the nucleolus where it is then integrated with other proteins and, also presumably, with certain specific polymerizing enzymes. The resulting particles are probably similar to or identical with the ribosomes which are generally found in the cytoplasm. The ribosome, after it is formed in the nucleus, passes through the nuclear membrane into the cytoplasm where, along with cytoplasmic membranes, it can then produce a specific polypeptide or protein. Thus, a ribosome coded especially for insulin, for example, must assemble the various adapted (attached to tRNA) and activated amino acids to produce the 21 amino acid glycine chain and the 30 amino acid phenylalanine chain. Then, the ribosome must link these two chains together with three properly positioned disulfide bridges. I n the islet cells of the pancreas, one may presume that the gene locus for insulin is capable of being activated and so produce mRNA specifically coded for insulin. The muscle cell, on the other hand, produces myoglobin which has more amino acids than insulin and is also integrated with heme. Furthermore, there is considerably more structural complexity in the folding of the polymerized amino acids, as has been suggested by Kendrew’s (1961) X-ray diffraction studies. The problem which arises is that of assembling some 150 amino acids in precise sequence as well as the folding of the long chain into the intricate pattern which, when coupled with heme, constitutes the myoglobin molecule. It may be presumed that in the chromosomes of muscle cells there is a gene locus, distinct from the insulin locus, with the information required for myoglobin synthesis. Here again, mRNA must be copied from the DNA template, and integrated with proteins and enzymes in the nucleolus, where a ribosome designed specifically for myoglobulin synthesis is produced. An interesting problem is the following: Do islet cells and muscle cells

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carry a t the same time the genetic information needed for both insulin and myoglobin synthesis? If it is true that these cells still bear both kinds of genetic information, then another question arises: Why does the islet cell produce insulin and not myoglobin, and the muscle cell produce myoglobin and not insulin? Are the nucleoli in islet cells different from those in muscle cells? These are major problems in differentiation (Kopac, 1962). A possible parallel situation includes the special four cells in the salivary glands of Ch'ironomus pallidivittatus which produce the SZ secretion granules. Other cells in the salivary glands do not. These four special cells show a Balbiani ring in the fourth chromosome-the other cells do not. Thus, these differentiated cells become activated and a specific chromosomal locus generates the mRNA (and presumably ribosomes) which has the necessary information to produce either the SZ substance or the enzymes which lead t o the production of SZ substance. What is it that activates the locus? Is the mechanism one requiring an inducer? If so, why are not equivalent loci in the other cells similarly activated? We have no answers to these questions, but the study of polytene chromosomes is pointing the way. The answers t o specific questions involving differences in nucleoli of differentiated cells can come only when it will be possible to test the nucleoli for differences in their RNA content. Such tests can lead to base composition ; but ultimate information will require base sequence as well.

D. MECHANISMS FOR ACTIVATING GENELOCI Jacob and Monod (1963) in a stimulating and thought provoking review have considered the problems of genetic repression and cellular differentiation. They pose two questions: ( 1 ) Does differentiation operate a t the genetic level by turning on or off gene activity, thereby selecting the genetic potentialities to be expressed? ( 2 ) I n the affirmative, can the basic elements of regulatory circuits found in bacteria-regulator genes, repressors, operators, structural genes, and operons-be organized into other types of circuits whose properties could account for the main features of differentiation in higher organisms? The synthesis of mRNA can be initiated only at certain regions, or operators, which may control one or more structural genes. The rate of transcription of operons is negatively controlled by regulator genes. A regulator gene forms a cytoplasmic product, or repressor. The repressor tends to associate reversibly with the operator gene. The combination blocks the synthesis of mRNA by structural genes within the operon. The repressor (R) can react with small molecules (F),called effectors. Reactions are specific with respect to both repressor and effector, to pro-

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duce the RF complex. I n one class, the RF complex inactivates the repressor and blocks the action of the operator. Accordingly, regulator genes are transmitters of cytoplasmic chemical signals, the repressor which can be inactivated (induction) or activated (repression) by specific metabolites. The repressor is a product of the regulator gene and acts on the operator gene to prevent synthesis of mRNA by the operon. The repressor may be activated by a metabolite (repression) or inactivated by a metabolite (induction). It must be capable of recognizing a given operator and a given metabolite. The regulator gene is also susceptible to mutation and may, thereby, exhibit different properties. Identification of the repressor is difficult. Some think the repressor is a polyribonucleotide (Pardee and Prestidge, 1959; Jacob and Campbell, 1959). At present, Jacob et al. (1962) believe the repressor is partially or totally a protein. The arguments against repressor-RNA being the repressor substance are: ( I ) Polyribonucleotides, as primary products of the regulator genes, and not being transcribed into proteins, would be of a different nature from the mRNA as the product of structural genes. ( 2 ) Recognition of metabolites by a polyribonucleotide without a protein is difficult to visualize. ( 3 ) Properties of different alleles of regulator genes are difficult to account for if the product is not a protein. Thus, the expression of a regulator gene involves : ( 1 ) the transcription of the regulator gene as specific RNA, and ( b ) the transcription of the repressor RNA into a protein or polypeptide. Accordingly, the regulator gene has as its final product a specific protein. Now, it is possible that the repressor may be synthesized in the nucleus-perhaps a t the locus of the regulator gene. The total number of repressor molecules is probably small, perhaps no more than 10 to 15 molecules per chromosome in each instance (Jacob e t al., 1962). It seems fairly certain that the regulator action within the genome need not be morphologically identified with the genetic loci in the Mendelian sense. To this end, many studies have implicated heterochromatin as being the center of gene control (Caspersson, 1950; Caspersson et al., 1960; Schultz, 1947, 1956, 1958, 1959). Indeed, the variegation in maize (Mcclintock, 1956) is believed to be heterochromatin-induced. Welshons (1963) considers that variegation can be envisaged as an “inhibiting or inactivating effect of heterochromatin upon the genetic loci carried on the transposed piece of euchromatin.” Variegation requires the transposition of euchromatic loci to a region adjacent to heterochromatin. One or two euchromatic loci can be inactivated thereby. The existence of regulatory circuits of cells in different organisms is

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known. h4cClintock (1956) and Brink (1960) described systems which control gene activity and are composed of two elements. One element is closely associated with the structural gene (analogy to operator-structural gene relationship). The other may be located in another region of the genome. The latter determines the conditions to which the gene-associated element responds and, therefore, the change in activity of the structural gene. The systems are specific : each gene-associated element (operon) responds only to a particular second element (operator) and it affects only the activity of the gene located in the same chromosome (cis-position) . McClintock (1961) pointed out that such a dual control in maize may, in many respects, be compared with the regulator-gene-operator control system in bacteria. Maize elements also operate by way of specific molecules acting as chemical signals like bacterial repressors. Where, in the genome, would be the expected site of the regulator genes? Now if the functional repressor substances turn out to be a protein, or possibly even a histone, then it is quite possibIe that these proteins could be synthesized in the heterochromatin. Whether they would be eventually fed into a nucleolus is an unanswered point. It is, therefore, not too surprising that there might be a parallel between the control systems in maize (McClintock, 1956) and in bacteria (Jacob and Monod, 1963), with the primary site for repressor production assigned to the heterochromatin. Indeed, Falk (1963) postulated a similar system in Drosophila. Steinberg (1942) reported that the transference of the sc+ (scute) locus from a position near Hw' (hairy wing) to the heterochromatin of the X chromosome produced variable expressions of bristle formation. I n this instance, scute would be analogous to the structural gene in bacterial chromosomes, and hairy wing would be the operator. The heterochromatin, by implication, could be comparable to the regulator in bacterial systems. Heterochromatin is generally found in both arms of a chromosome (if metacentric) adjacent to the centromere. Nucleolar organizers also show some of the characteristics of heterochromatin, especially allocycly or positive heteropycnosis (retention of high concentration of DNA during interphase). Various segments of X chromosomes also demonstrate allocy cly. I n Crepis, the replication of centromeres is later than found in other chromosomal segments (Taylor, 1958). I n other cells, the X chromosome may take longer to replicate than other chromosomes (Lima-de-Faria, 1959). Late replication might be a characteristic of heterochromatin, and this characteristic may be mutable or may change during differention (Lyon, 1961). By pulse labeling with tritiated uridine, a differential rate

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of RNA synthesis between euchromatin and heterochromatin in the H,, strain of mouse cells was demonstrated by Hsu (1962). Much more uridine was incorporated by euchromatic components than by heterochromatic centers. I n the salivary gland chromosomes of Drosophila, the heterochromatic regions proximal to the centromeres all synapse to form the chromocenter, as a single mass. Here, apparently, heterochromatin is less specific in synapsis than euchromatin, so that heterochromatic regions of non-homologous chromosomes may indeed synapse. Heterochromatic regions near the telomeres may also synapse with the chromocenter. Heterochromatin contains both DNA and RNA and is unusually rich in histones. Caspersson (1950) believed that the heterochromatin is a site for the production of histones and that the products collect into nucleoli. Accordingly, the nucleolar organizer regions are believed to be heterochromatic. Schultz (1947) reported that four types of substances are present in heterochromatin, namely, RNA, DNA, filler protein (histone ?), and elastic fibrous protein of the interchromomeres. There is apparently much less elastic interband protein in heterochromatin than in euchromatin. Heterochromatic regions may be more susceptible to radiation-induced structural changes than euchromatin. Could this susceptibility to breakage be caused by the lack of elastic type fibrillar structure as shown by Schultz (1947) ? Another characteristic of heterochromatin is its susceptibility t o breakage induced by 5-bromodeoxyuridine (5BUDR) (Hsu and Somers, 1961). It may be that heterochromatin, therefore, provides greater opportunity for translocation and inversion types of chromosomal aberrations and, in some instances, this result may lead to striking manifestations of the position effect. As reported by Hsu and Somers (1961), the vulnerable sites of action of 5BUDR are the regions rich in AT (adenine-thymine) pairs, which is also believed to be a characteristic of heterochromatin. 5BUDR is incorporated into replicating DNA in place of thymine. I n addition to some special loci, the telomeres are damaged by 5BUDR incorporation. With hydroxylamine, Somers and Hsu (1962) reported chromosomal damage in the region of the centromeres which should be rich in GC (guanine-cytosine) pairs. It is possible that heterochromatin may contain both types, namely, AT and GC pairs, but the feature here is that certain segments in the DNA complex may contain nothing but AT polymers, while others contain sequences of GC polymers. Thus, the centromeric region would be susceptible to agents which may replace 5BUDR for T during replication or by agents that modify C (hydroxylamine) . The operon is a complex consisting of an operator plus structural genes. If a structural gene were physically disconnected from the operon and

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located elsewhere, the displaced structural gene would escape control of the original operator and, thereby, become insensitive to the normal system of regulation. The operator may undergo a mutation, to be designated as an operator-negative mutation. Jacob and Monod (1963) commented on the SZ granule-producing cells in the salivary glands of Chironomus pullidivittutus. This is considered as an example reminiscent of the negative-operator type of mutation. Beennann (1961) points out that no difference in the bands of the chromosomal system can be detected. There may, obviously, be differences which cannot be detected by present means. An operator-negative mutation, according to Jacob and Monod (1963) results in a complete block of the transcriptive process along the structural genes in the operon; therefore, all enzymes that would ordinarily be produced if the structural genes in the operon were active, fail to be synthesized. Apparently, the operator-negative mutation is incapable of being activated by the usual RF induction. The operator may be the initiating point of the transcriptive process and the receiver of controlling signals. The operon, then, constitutes the unit of primary transcription, containing one or more structural genes (protein or enzyme producing). I n these situations, a distinction can be made between operational and informational mutants. Thus, the failure to form the fourth Balbiani ring in most cells of the salivary gland is an operational factor. Failure of the locus to form the fourth Balbiani ring in Chironomus tentans may also be an operational factor. The factor here may be something equivalent to the operator gene (Jacob and Monod, 1963). Thus, the Balbiani ring may be considered as an operon, representing more than one informational gene (structural gene of Jacob and Monod). If the operator gene were suppressed, then the development of a Balbiani ring would be suppressed, resulting in the failure of the cell to elaborate, in this instance, the SZ granules. If heterochromatin is the center of regulator genes, and if regulator genes are susceptible to mutation, one might consider the possibility that a change in regulator genes could lead to changes in the subsequent actions of an operon. Thus, a regulator gene whose repressor product ordinarily succeeds in maintaining an operon a t the inactive status could, following a change, either fail to produce the repressor or else its product would no longer control the operon. I n this way, a result equivalent to rejuvenation might alter the differentiation status of a cell. The cell then would most certainly be capable of doing something it ordinarily would not do.

E. SERIALACTIVATION OF

CHROMOSOMAL LOCI

Chromosomal loci can be stimulated by various non-nuclear agents or

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changes in subcellular environments. These extraneous stimuli and their effects are beautifully shown by the transplantation experiments of Kroeger (1960), the effects of hormones (Clever, 1963), or the effects of hormone imitators (Kroeger, 1963). The fact that the normal sequence of puffing may be reversed is also of significance. Juvenile hormones, or their imitators, can reverse the puffing patterns-at least in part. Thus a locus plus activator lead to RNA synthesis (and possibly protein synthesis or accumulation a t the site). This synthesis is continued until it is turned off. If the event occurs on a single chromonema, it would be too small to be seen; however, in polytene chromosomes a puff is elicited. The important point is that in any one cell, a t any time, relatively few loci in chromosomes are functional. An activated locus may be a single gene (classical sense), or it may be a number of functionally related genes (operon sense). Assuming that the mechanism of gene activation in nonbacterial cells is similar to that proposed by Jacob and Monod (1961) for bacteria, then in their scheme, the operon consists of an operator gene and one or more structural genes. The structural genes will not lead to polypeptide synthesis unless the operator gene permits them to do so. Presumably, the operator gene permits the code as double-stranded DNA to be transcribed into a single strand, mRNA. There is some evidence to suggest that a product of the operator gene itself is a form of RNA. As Clever (1962, 1963) has shown, the activation of locus I-18-C in Chironomus salivary gland chromosomes by ecdysone leads to the activation of other loci. Thus, the product resulting from the activation of the first locus now functions as the inducer for activating the second locus. A serial sequence of such events can be diagrammatically represented as shown in Scheme I:

The numbers 1 to 3 represent chromosomal loci (on same or different chromosomes) ; the letters A to C represent the products of the structural genes comprising each locus. Substance X is the inducer for locus 1 (I-18-C) , corresponding to ecdysone, for example. This substance, by

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triggering locus 1, leads to the production of substance A. The appearance of A now induces locus 2 to go into action by forming a puff, and so on.

F. POLYTENE CHROMOSOMES vs. CONVENTIONAL INTERPHASIC CHROMOSOMES I n the non-leptotene interphasic chromosome, there is maximal linear extension and presumably this affords maximal opportunity for ( 1 ) replication, prior to mitosis, and (2) transcription of genetic code via mRNA synthesis (DNA-primed RNA synthesis). Thus, the activation of a gene or operon locus results in the production of mRNA. I n a diploid cell, if homoaygous, each operon locus is represented twice. The rate of mRNA production is not known a t present. It may or may not be constant for each operon locus. If the rate of mRNA production is insufficient a t the operon locus, then the output of the specific mRNA might be augmented by RNA-primed RNA synthesis and this site presumably is the nucleolus. I n the polytene chromosome, one has a system in which each operon is represented several hundred or thousand times in a single nucleus. If structure is favorable, then each operon may participate in specific mRNA synthesis. Since the polytene chromosome seems to be a cable consisting of many individual chromosomes in substantially the leptotene stage, chromomeres are therefore present. A chromomere, however, is not morphologically suited for genetic code transcription. To do so, the chromomere must uncoil. If such sites then uncoil in concert and if the rate of mRNA production (and protein accumulation) is greater than the rate of diffusion away from the sites, the effect will appear as a puff, bulb, or Balbiani ring. I n any event, the puff functionally resembles a nucleolus. Likewise, the lateral loops of lampbrush chromosomes also resemble nucleoli, in the sense that the loop is functionally identical to the nucleolar organizer. The potential output of mRNA from a given locus on the polytene chromosome may be several hundred or thousand times greater than from the same locus in an interphasic chromosome. The activity in a puff may be high enough to provide the necessary mRNA and protein needed a t that time. I n a conventional cell, for a given locus, perhaps only one or two chromonemata are functional and the only sources of mRNA for the eventual synthesis of a protein. If the demands for mRNA are greater than can be supplied by the chromosomal locus, then the balance will have to be supplied by another site. This could be the nucleolus where RNA-primed RNA synthesis is possible, and some evidence for such production of RNA is available from tracer studies (Sirlin e t d., 1962). A

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general scheme for the production of mRNA. is given in Scheme I1 (XRTP, pool of ribose triphosphates) :

DNA

DNA-primed RNA synthesis (chromosomal locus) n (XRTP)

RNA-primed RNA synthesis (nucleolus) mRNA (11)

It is likely that both situations exist in cells containing polytene chroiiiosomes as well as in cells containing conventional chromosomes. Thus, the mRNA may pass from the chromosomal locus directly to the cytoplasm, in some instances, while in others, it may pass into the nucleolus, be replicated there, and then pass into the cytoplasm. I n each instance, presumably the mRNA is incorporated with ribosomes before passage into the cytoplasm occurs. The puff or Balbiani ring might facilitate the assembly of mRNA, enzymes, proteins, and other substances which eventually become the ribosome. I n a conventional chromosome, because of its few chromonemata, such assembly might not be possible a t the site but i t could be facilitated within the nucleolus.

G. THE REVERSAL AND RECAPITULATION OF CHROMOSOMAL ACTIVATION Von Borstel (1963) suggested that embryogeny could be a series of events, in time, of cytoplasmic inducers affecting specific genes that produce substrates which in turn function as new, qualitatively different cytoplasmic inducers and, in turn, activate new sets of genes. As the substrates build and become compounded, the entire intracellular milieu changes. The action of rejuvenating hormones or their imitators illustrates that the usual sequence of gene action as demonstrated by puffs can be modified. As Kroeger (1963) points out, the implantation of coq.mra allata (a source of juvenile hormone) into Chironomus larvae may bring about a reversal of the normal course of nuclear development. I n such nuclei, the chromosomes return to a puffing pattern characteristic of a younger stage and even become refractory to the action of ecdysone (Becker, 1959). According to Becker (1962), the rejuvenated puffs are larger than normal. Also Lezzi (1961) has shown by the uptake of labeled RNA pre-

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cursors and radioautography that puffs in the rejuvenated state are biochemically more active. There is evidence, therefore, to suggest that the sequence of gene activation can, in a number of instances, be reversed. Kroeger (1960) in his nuclear transplantation studies has shown that a t least one new puff is elicited by exposure of salivary gland nuclei t o preblastoderm cytoplasm. This puff has never been seen in the polytene chromosomes (1st instar through prepupal stages). This may mean that the specific locus in question is required to initiate some biochemical event that occurs only in early stages of development. I n due time, its mission is accomplished and the same locus never goes into action again, a t least not in the salivary gland cells during larval stages. I n normal development, a t the time locus 2R/22 is activated (preblastoderm stage), polytene chromosomes do not exist. Accordingly, it would be impossible with our present skills to demonstrate activity a t that locus in a nonpolytene chromosome. Fortunately, the salivary gland chromosomes a t the prepuparium stage are highly polytenic, and the concerted activation of a locus can be clearly demonstrated through the formation of a large puff (locus 2R/22). This experiment might, therefore, be considered as another example of reactivation of gene loci. I n Kroeger’s language, also, the locus a t 2R/22 is still competent, but requires a specific activation factor, presumably present in preblastoderm cytoplasm. The next possibilities deal with the participation of an entire genome. These studies are based on the transplantation of a nucleus from an embryonic cell of a frog (Runa pipiens) into the cytoplasm of an enucleated frog egg. This environment, of course, is identical to the normal environment experienced by the zygote nucleus-actually the most primitive state for a developing system. These experiments are based on the fundamental work of Briggs and King. Briggs and King (1953) transplanted the nuclei of animal pole cells of blastulae and early gastrulae into the cytoplasm of activated, enucleated frog eggs. I n their summary, they state: “In both cases the successful nuclear transfers lead to normal cleavage and complete development of the recipient eggs. This shows that the nuclei in question are not differentiated, for when they are transferred back into enucleated eggs they may still participate in all types of differentiation-a result that is more or less expected in view of the fact that the animal pole cells a t these stages are themselves still undetermined.” Later, King and Briggs (1954) transferred living nuclei from determined zones of late gastrulae, the chorda-mesoderm, and presumptive medullary disk, into enucIeated egg cytoplasm ( R a m pipiens). Less than 10% of the attempted transfers resulted in normal cleavage of the egg.

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The rest of the eggs either failed to cleave or cleaved abnormally. The normally cleaved eggs developed into complete blastulae, but some of these were arrested in blastula or gastrula stages. Approximately one half of the eggs cleaving nornially developed to neurula and postneurula stages. I n these embryos, all three germ layers and their derivatives were evident, regardless whether their nuclei were derived from chorda-mesoderm or from the presumptive medullary plate. From these studies, one may conclude that nuclei from undetermined animal pole cells are capable of repeating the cleavage and differentiating processes when returned to the primitive environment represented by egg cytoplasm. One may presume, therefore, that all requisite gene loci involved in the stages of development from the very beginning are capable of being reactivated when the nucleus is brought into an environment that is capable of stimulating such gene activation. This entire process could be considered as analogous to the rejuvenation effects described in salivary gland chromosomes. If the nuclei come from a later stage, the successful recapitulation becomes somewhat limited. Some of these limitations may be technical, as King and Briggs suggest. But in those instances where cleavage occurs (indication that the transferred nuclei were viable), the system in its new environment is still capable of recapitulating development events to produce all three germ layers and achievement of a neurula or postneurula stage. The question then comes up: When does this potential of recapitulation become reduced or even cease? T o this end, Briggs and King (1957) and Briggs et al. (1960) have summarized their results on transplanting late gastrula endoderm nuclei into enucleated frog egg cytoplasm. The following is a score sheet of the results (Briggs et al., 1960) : “1) About 40% of these eggs develop into post-neurula with deficiencies and degenerative changes in ectodermal and mesodermal organsbut not in the endoderm. I n such cases the pattern of differentiation exhibited by the test eggs is consistent with the endodermal origin of the nuclei. “2) About 30% are arrested early in developmentin blastula or early gastrula stages. Since development is stopped before any overt cell differentiation occurs i t is impossible to determine from simple observations of these embryos what the specific properties of the transplanted nuclei might be. “3) About 10% develop to post-gastrula stages, but fail to display any consistent pattern of deficiencies. These embryos are variable, exhibit deficiencies in any or all germ layer derivatives and obviously do not conform to the ‘endodermal pattern’ mentioned under (1) above. “4) About 20% develop normally throughout.”

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As Briggs e t al. (1960) point out, the majority of endoderm nuclei have undergone changes “restricting their capacities for promoting the balanced array of cell differentiations required for normal development.” Furthermore, these restrictions are stabilized, for descendents of a single endoderm nucleus, when transplanted serially to test eggs, exhibit a uniform set of deficiencies which persist unchanged after 39 or more nuclear generations (King and Briggs, 1956). Similar serial transplantations of endoderm nuclei of X e n o p u s were conducted by Fischberg et al. (1959) with results substantially similar to those obtained with R. pipiens nuclei. T o continue, group 1 eggs exhibit the endoderm pattern of deficiencies, while groups 2 and 3 do not. As Briggs et al. point out, these variations in the behavior of test eggs have made it difficult to arrive a t a definite decision concerning the specificity of nuclear differentiation both in endoderm and in other cell types. Briggs e t al. (1960) then pursued the problem further by studying the chromosomal constitution of some of the cells derived from early gastrula stages. The nature of developmental deficiencies obtained from late gastrula donor nuclei transplanted into enucleated frog egg cytoplasm were correlated with chromosomal constitution. Their results are summarized as follows: “1) Embryos which were arrested early in development and did not conform to the ‘endoderm’ pattern were abnormal in karyotype (aneuploids with acentric fragments, etc) . Aneuploids, or course, do not provide a valid test of the properties of the nuclei as they exist in the donor cells prior to transplantation. “2) The valid cases (those in which the chromosome complement remained euploid a t least until early gastrula stage) gave a different result They either developed normally, indicating that some of the endoderm nuclei of the late gastrula donor have undergone no stable differentiation, or they became abnormal and exhibited a pattern of deficiencies consistent with the endodermal origin of the nuclei. This result strengthens the case for a characteristic nuclear differentiation in the case of the endoderm. Whether other cell types have their own characteristic nuclear differentiation (‘specificity’) remains to be determined.” There are many factors involved in the ultimate potential of a system consisting of a nucleus from a determined or differentiated cell and undifferentiated (theoretically nascent) cytoplasm. The fact remains that in many instances the new nucleus-cytoplasm system is capable of cleaving and recapitulating some of the development stages, albeit inadequately, It could be that part of the differentiating process may render certain genetic loci noncompetent t o reinduction by the more primitive egg cytoplasm. It is amazing that so much development can indeed occur.

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It is also significant that the structures least affected, as shown by Briggs et al. (1960), are endodermal in origin-the germ layer from which the donor nucleus was derived. The appearance of aneuploids and aberrant chromosomes in some of the cells obtained from the nucleus-cytoplasm developing system was noted. As one possibility, Briggs e t al. (1960) considered that chromosomal abnormalities may arise as a consequence of nuclear differentiation. Mitotic specialization, involving (‘specific requirements for normal chromosomal replication” and subsequent behavior of chromosomes during mitosis (linear condensation, loss of nucleolar bodies, centriolar actions, etc.) may also be a part of the differentiating process. There is no question that a differentiated nucleus, when brought into undifferentiated cytoplasm will, during its mitotic cycle, be subjected to a new biochemical experience. This is a striking change in subcellular ecology. Some of this new experience might be favorable toward the formation of atypical chromosomes and equally atypical mitotic aberrations. Perhaps as nuclei become more and more differentiated, the exposure to new environments may be increasingly drastic and will obviously expose gene loci to new inducing factors. The more advanced the stage of development (differentiation) of the nucleus, the more atypical would be the environment consisting of undifferentiated cytoplasm. What may also be a factor is that there is a progressive loss of competence of some of those Ioci that have already been activated in an earlier stage of development. There is very likely a broad range of variability in the capacity of a gene locus to become reactivated, once i t has been activated previously and its action subsequently blocked by whatever mechanism. Obviously, not enough of the required loci for early stages of development can be reactivated, so that under these circumstances typical development of the nucleus-cytoplasm system fails. On the other hand, i t is quite clear that some of the loci do become reactivated in the presence of the new environment. A differentiated cell might be considered to represent a system with a special programing of its genetic complement so that all required biochemical processes occur at the right time. Any disruption of this programing would lead to the introduction of new biochemical reactions or to the suppression or modification of regular biochemical processes. This would tend to remove the cell from its normal differentiated pattern. The propagation of this situation to successive cell generations could lead to neoplasia. Cancer cells, even though still largely differentiated, are doing things that their normal prototypes cannot do. The fact that activities of chromosomal loci may be stimulated by extrinsic agents has a direct bearing on the cancer problem. If the dif-

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ferentiating processes during larval development can be revised, then why cannot other differentiated cells be similarly modified? By the time a neoplastic condition appears, there has been almost all the major cell differentiation that would normally occur within the life history of the individual. The exposure of the genome as represented by the complement of chromosomes to a new inducing a g e n t w h a t e v e r it tnight be-could be enough to reactivate a dormant locus. This reactivated locus plus the normal complement of activated loci might lead to a state of differentiation that enables such a cell and its progeny to pursue an atypical existence. Such a situation might indeed be a cancer cell. For example, if a pancreatic cell should start synthesizing myoglobin instead of insulin, i t would for all practical purposes be no worse off than a cancer cell, especially if the effect could be propagated to succeeding generations. We would like to know to what extent have the nucleolar chromosomes been modified? It can be presumed, if the interpretation is correct, that ribosomes are generated mainly in the nucleolus. Obviously, a pancreatic cell, modified to synthesize myoglobin instead of insulin, would be generating different ribosomes. It is true that perhaps the only change in the ribosomes would be in the mRNA provided for them. The key point is that if a gene locus or loci may become reactivated, then the capacity to remain reactivated must be propagated to the next generation of cells. I n the present state of our knowledge, such a permanent change in activation capacity might ensue if: (1) a change in the locus, per se, to make i t competent, or ( 2 ) change in position within the genome to render such a locus no longer under the control of the operon complex. The latter could happen by translocation or inversion. If the translocation or inversion involves only a portion of an operon, for example, it is highly unlikely that i t could be recognized in conventional chromosomes by our currently available techniques. If cellular differentiation in an organism is the result of orderly and properly paced sequential activations of gene loci, then a question to be asked is: Can such a sequence be interrupted, or reversed? The answer is yes. If we admit that the neoplastic process permits a cell, or a population of cells derived from it, to do things its normal ancestral cell would not do, then the cancer problem hinges directly on to the problems of cell differentiation. Excellent reviews and discussions focused on genes and developmental pathways are by Lewis (1963), Baker (1963), who is concerned with the genetic control of pigment differentiation, Ursprung (1963), who discusses the genetics of patterns, and by von Borstel (1963), who summarizes the above indicated reviews. It is interesting to note how the concept of the operon is being brought into an interpretation of genetic phenomena of

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nonbacterial organisms. Along with the operon, perhaps the position effects are more sharply defined. One has the impression that considerable progress is being made in the direction of explaining not only gene action, but perhaps even more important, the possible mechanisms involved in regulating gene action. With the availability of polytene chromosomes, coupled with experimental and genetic controls of puffing, the biologist has, for the first time, the material that indeed seeins to pinpoint an operon going into action as well as controlling not only when the operon acts, but also of controlling the period of time the operon is permitted to act. Without question, the polytene chromosomes will shed even more light on this fundamental biological problem-the control of differentiating mechanisms.

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CARCINOGENESIS RELATED TO FOODS CONTAMINATED BY PROCESSING AND FUNGAL METABQLITES

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. B . Shirnkinf'

H. F Kraybill* and M

National Cancer Institute. Betherdo. Maryland

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I1. Processed Rations and Trout Hepatoma

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G. Carcinogenesis of Other Chemicals . . . . . H. Ionizing Radiation and Parasitic Infestation . . . I . Projected Research on Trout Hepatoma . . . . 111. Role of Fungal Metabolites in Diet and Cancer . . . A . Occurrence and Development of Mycotoxins . . . B. Metabolism of Aflatoxin-Species Susceptibility . . C . Pathology in Mycotoxicoses . . . . . . D. Nutritional Considerations in Mycotoxicoses . . . E. Isolation and Characterization of Aflatoxins . . . F. Other Fungal Metabolites and Associated Compounds G . World-Wide Health Implications of Mycotoxicoses . IV . General Discussion . . . . . . . . . References . . . . . . . . . . . .

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Concurrent outbreaks of disease in poultry and fish in different parts of the world during early 1960 focused major attention on the significance of diet contamination in the induction of cancer . The first epidemic was uncovered by a seizure of a large shipment of diseased Idaho hatcheryreared trout a t a California border station . Many of the fish were found to have hepatomas . This incident led to national studies that demonstrated the wide prevalence of hepatic carcinoma in this popular and commercially important species of fish . For thirty years (Haddow and Blake. 1933) sporadic cases of liver tumors in 'trout have been described but

* Present address: Bureau of Environmental Health. U.S. Public Health Service. Department of Health. Education and Welfare. Washington. D.C. f Present address : Temple University School of Medicine. Fels Research Institute. Philadelphia. Pennsylvania . 191

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within the past ten years the disease assumed epidemiological importance in the United States and Europe (Ghittino and Ceretto, 1962). The second situation relates t o a disease, presumed to have been a new entity, which killed over 100,OOO turkey poults in the south and east of England. The British named this disease “turkey X disease” (Blount, 1961). On post-mortem examination, the poults were found to have acute hepatic necrosis associated with generalized bile duct proliferation. The liver lesions were similar to those observed in fowl by Campbell (19551957) from Senecio alkaloid poisoning. A search for the toxic agent revealed no contamination from pesticides or plant alkaloids, but Blount (1961) quickly associated this disease with the use of Brazilian groundnuts (peanut meal) in the feed. Studies on weanling rats showed that the toxic peanut meal fed a t 20% level in the diet produced hepatic carcinoma by 30 weeks. (Lancaster e t al., 1961). These two events were related to the adoption of specially prepared commercial food rations. Trout feed is composed of rations put up in the form of dry pellets. The high prevalence of hepatomas in trout occurred following the wide-scale use of these commercial feeds. Essentially all trout populations in the United States have their origin in large federal, state, or commercial hatcheries. Consequently, in early months of trout rearing, prior to release into streams, there is exposure to commercially prepared rations. Practically no population of trout in the United States is now reared exclusively on a so-called wild or native diet. An epidemiological survey of state and federal hatcheries revealed the occurrence of the disease in most trout-rearing areas of the country. The prevalence was highest in healthy, fast-growing older fish, the highest rate of hepatomas being in those beyond 3 years of age. The frequency of hepatomas in some hatcheries reached levels of 50 to 75% of the population (Wood and Larson, 1961). There is a paucity of information on frequency of disease among wild trout populations of the United States; a world-wide mail survey for this information was not convincing as to accuracy. Dry pelleted rations for trout are composed of steam- or flame-dried fish meal, cottonseed meal, meat scraps, and dried milk as protein sources. Recently fish meal and cottonseed meal have been used almost exclusively, since meat scraps are in greater demand for livestock feeding. Prior to use of these components a wet diet, consisting largely of viscera from sheep, swine, cattle, and other fish, was used in addition to ground beef and horse meat, according to Wood and co-workers (1957). These diets of unprocessed products were not known to produce hepatomas. Elucidation of etiological agents in the trout feeding studies has provided a clearer understanding of mechanisms of carcinogenesis relevant to the role of dietary sources to the genesis of neoplastic disease.

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193

While the trout hepatoma problem has demonstrated the significance of ration components, the “turkey X disease” incident subsequently showed that toxic peanut meal also produced an hepatotoxic effect in other domestic animals such as cattle, ducklings, chickens, swine, and horses. Simultaneously with the outbreak of disease in England, highly toxic samples of peanut meal contaminated with mold were fed to ducklings in Uganda, resulting in many deaths. This provided an important clue as to the origin of the toxin. By growing some of the fungal species on a synthetic medium, or on heat-sterilized nontoxic peanut meal, a toxinproducing fungus was identified by Sargeant et al. (1961b) as Aspergillus flavus Link ex Fries. The toxin so produced was named aflatoxin by the British investigators and has been shown to be fluorescent in the ultraviolet. There appeared to be a linear relationship between toxicity and the amount of fluorescence. Indeed, the fluorescence test has become the standard procedure for screening peanut meal samples used in livestock feeding. The bioassay procedure is standardized on day-old ducklings, which are extremely sensitive to aflatoxin. Aspergillus flavus and its associated mycotoxin is but one of a series of fungal contaminants which may occur in cereal products or other foods. Other fungi and fungal toxins are now receiving intensive consideration as they apply to a broad spectrum of animal and human foods. The disastrous acute effects of moldy feeds in turkey poults and ducklings and hepatic carcinoma in rats dramatized the biological importance of mycotoxins. Earlier work in this country by Burnside et al. (1957), Carll and co-workers (1954), and Forgacs et al. (1954) concentrated on diseases in poultry, swine, and cattle which consumed moldy corn diets. In the Soviet Union successful studies on stachybotryotoxicoses, a disease primarily affecting horses, cattle, and man, resulted in the organization of a laboratory in Moscow in 1940 for study of toxic and pathogenic fungi (Sarkisov, 1947). The subject of mycotoxicoses appears to have received more attention in diseases of livestock in the Soviet Union than it has in the United States. I n addition, correlated works on the influence of fungal-contaminated rice and excellent studies on the carcinogenic effect of actinomycin by the Japanese, Miyake et al. (1959) and Kawamata et al. (1959), have given further emphasis to the role of fungal metabolites in cancer. Mycotoxicoses assume world-wide importance in relation to programs on the alleviation of kwashiorkor through provision of various food or protein sources. These products may undergo unfavorable storage conditions with subsequent fungal contamination. The possible effect of mycotoxins in cancer and other diseases is now being considered (Forgacs, 1962; Forgacs and Carll, 1962).

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H . F. KRAYBILL AND M. B. SHIMKIN

I I . Processed Rations and Trout Hepatoma

From an evaluation of gross manifestations or detailed histology in rainbow trout of eastern and western fish hatcheries, as reported by various investigators (Nigrelli and Jakowska, 1955; Wood and Larson, 1961 ; Hueper and Payne, 1961 ; and others), it is apparent that trout hepatoma is common t o all parts of the United States and is the same disease. Rcports from hatcheries in France and Italy suggest that there may be the same disease in Europe operating through identical causal factors in the induction of hepatomas (Cudkowicz and Scolari, 1955; Ghittino and Ceretto, 1962; Levaditi et al., 1960). Ghittino and Ceretto (1962) believe that the action of an imbalanced diet on hepatocytes produces a regenerative-hyperplastic reaction with tumor development. They claim that in the rainbow trout, susceptibility to hepatomas is a process separate from degenerative cirrhosis. This species seems to be affected by metabolic diseases, such as lipoid degeneration of the liver and disturbances in fat metabolism. Wood and Lareon (1961) claim neoplastic changes in fatty livers, but fatty livers are common to hatchery trout. Furthermore, ceroid, an acid-fast sudanophilic pigment, is commonly observed, but this is of limited meaning since it is observed in many fish diseased from other causes. I n an appraisal of possible etiological factors, investigators have considered the following ( 1 ) genetic predisposition and related factors, ( 2 ) bacterial or viral invasion, (3) nutritional factors, ( 4 ) processed components in commercial dry pelleted rations (influence of processing and storage), (6) environmental chemical carcinogens (pesticides, herbicides, fungicides, amebicides) , ( 6 ) parasitic infestation, and (7) radiation originating from fallout radionuclides and radioactive cations from nuclear reactor effluents. These factors will be discussed with respect to potential influence they have on induction of hepatomas.

A. PATHOLOGY Rainbow trout afflicted with hepatoma are not obviously sick. They usually appear as normal, healthy, fast-growing fish. Mortalities occur only when the disease is so advanced that the metabolic processes of the liver are compromised. Obvious tumors in older fish can be seen bulging the body wall. On gross examination, multicentric, small, oval, translucent lesions with smooth, rounded contours are observed in the liver. The lighter color of the lesion contrasts i t with the normal liver tissue. The larger tumor in the nodular form encompasses much of the liver, replacing the liver parenchyma, as seen in hepatic carcinoma of mammals. These nodular or hemorrhagic tumors develop into huge abdominal masses

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195

in the late stages of the disease. They may become attached to the abdominal wall and be implanted along the peritoneum. Metastases to muscle and other organs are rare but have been observed. For a detailed review of the gross and microscopic pathology the reader is referred to the reports of Wood and Larson (1961) and of Hueper and Payne (1961). There are some common elements in the pathology of hepatoma induced by diet or environmental rodent carcinogens as observed in this exploratory research (Ashley et al., 1962). The various investigators involved in collaborative studies in initial research lacked uniform description and criteria of the pathology. Accordingly, in 1961, a classification was developed for delineation of the observed pathology. This classification is represented in Table I. TABLE I TROUTLIVERPATHOLOGY CLASSIFICATION IN HEP~ T O M ASTUDIES Class

Characteristics

N I I1 111 IV V

Normal liver 1 to 5 spots, remainder of liver normal 5 to 20 spots, remainder of liver normal Many spots, abnormal swollen liver Discrete nodule-classical characteristics Advanced massive nodules or metastases

I n histopathological classification classes I and I1 represent small necrotic areas on liver with only rare, small, basophilic-staining, incipient nodules. Class I11 consists of lesions that appear to be hepatomas on gross examination, but are not consistently so confirmed microscopically. I n all cases of classes I V and V grossly visible discrete tumors are confirmed histopathologically as hepatocellular carcinoma (Ashley, 1962). Attempts have been made to transplant hepatomas to other trout. Individual fish were triple-matched with serum, prepared to identify donors and acceptors, prior to study of transplantation. Histopathologically confirmed hepatomatous tissue was transplanted into liver, stomach wall, pyloric caeca, testes, cardiac chamber, and peritoneal cavity. When individual fish were closely matched genetically, hepatoma tissue could be transplanted successfully. Approximately 10% of all transplants were successful, indicating the genetic heterogenicity of the species (Halver, 1962).

B. GENETICPREDISPOSITION AND ASSOCIATED FACTORS Epidemiological surveys and considerations thus far have suggested the importance of genetic factors in the genesis of hepatomas. Although

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H. F. KRAYBILL AND M. B. SHIMKIN

genetic predisposition plays a minor role when viewed in terms of other causal factors, it is indeed of interest that the rainbow trout (Salmo gairdnerii) suffer from an epidemic of hepatocarcinomas and not merely from nodular hyperplasias. According to Snieszko (1961), brook and brown trout, when kept in similar environmental conditions to those of the rainbow and maintained on the same ration, do not develop hepatomas. However, Nielson (1960) has observed that different strains of rainbow trout kept on the same diet showed widely different hepatoma rates. It is possible, of course, that some “refractory” strains observed in early life may develop liver cancer a t a later stage in life than the susceptible strains. The breeding methods used in propagation of rainbow trout certainly differ from those practices of obtaining inbred strains of mice for cancer research. Hence it is perhaps inaccurate to refer to strains of rainbow trout in the context of inbred strains of mice. Some selective breeding of rainbow trout has indicated that there are certain strains that are refractory to hepatoma or other diseases but these are currently isolated cases. Investigations are also proceeding to establish the relative susceptibility of other fish, especially those related to trout or salmon. I n this respect chromosomal patterns of susceptible or resistant strains of fish are being studied. Allegedly, from current evidence “wild trout” do not show hepatomas. To apply the nomenclature “wild trout” to fish that have been “cultured” in the early months of life in federal or state hatcheries does not seem particularly relevant. Under hatchery conditions there is close control over water temperature, parasites, and bacteria, and feeding is based largely on commercial rations. The term “wild trout,” however, is loosely applied in the United States to those fish stocked from hatcheries and maintained for a predominant portion of their life in natural waters. Hence any so-called wild trout observed with hepatomas in the United States in natural waters might have been descendants of trout raised under hatchery conditions. Further surveys from other countries where hatcheries and related practices are not prevalent might help to clarify the point of incidence of hepatomas in a truly wild population of rainbow trout. Despite these distinctions, the current state of knowledge on strain specificity to liver cancer is not enough t o warrant conclusive association with one strain or species of fish. Such a conclusion must await more extensive genetic studies. Although in some instances hepatoma was present in trout less than 1 year old it was found more frequently in more advanced stages in adult fish. The prevalence was somewhat higher in older females than in males. The sex relationship postulates some probable hormonal effect. A potential association in this direction was noted by Halver (1963) when he found

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197

that prednisolone and high levels of protein in diets administered to trout fed a carcinogenic lipid fraction appeared to reduce the rate of hepatoma frequency. Whereas the hepatoma frequency in younger fish may approach 25% of population, in older fish the rate may be 75% and in some cases even 100%. Associated with genetic factors are the environmental conditions under which the trout are maintained in hatcheries. Water quality and temperature of water are quite relevant to growth and metabolism of the trout. Consumption of natural foods and growth rates of trout usually increase with rising temperature up to 60°F. and then decrease with rising temperature. The rate of hepatoma induction could then be expected to be related to the water temperature factor. Accordingly, in most of this research conducted a t hatcheries there has been close control of the water temperature, quality of water, and also control of bacteria and parasites.

C. BACTERIAL OR VIRALINVASION While some salt-water fish do not appear to demonstrate specific immunological evidence for an array of viruses, trout and goldfish are classes of fresh-water fish that encounter disease through viral infections. The possibility of viral infectious hepatitis with cirrhotic aftereffects, leading to primary carcinomas of the liver, has not been overlooked. It would appear, however, that in trout there is no histological evidence of an inflammatory hepatic process (Rucker, 1960). According to Heuper and Payne (1961), brown trout and brook trout in the same hatchery troughs with hepatoma-bearing rainbow trout did not acquire hepatomas through infection from the latter, It is also pointed out that observed sick rainbow trout reared with healthy ones did not appear to transmit the disease, as evidenced by ultimate induction of hepatoma (Cudkowicz and Scolari, 1955). While it was earlier conceded that the evidence did not support a viral theory of hepatoma formation, there is lacking certain criteria to confirm fully this original viewpoint. Serological evidence on trout populations may provide information on specific exposure to a virus. The possibility of a hepatocarcinogenic agent in the diet, including tumors with and without a viral-infected trout, has not been explored sufficiently to rule out a possible promoter action of the agent. I n some of the initial viral studies, according to Rucker (1961), the toxic commercial pellet was not available for challenging trout with a fish hepatoma virus-if indeed, such a virus exists. This investigator has now inferred that the initial studies on viruses lacked certain detailed aspects relevant to trout hepatoma. For example, it would be useful to test material from early stages of the tumor against multiple tissue culture systems. No systematic char-

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H. F. KRAYBILL AND M. B. SHIMKIN

acterization of an array of viruses has been made, and no other studies have been performed in relation to bacteria and viruses with respect to this neoplastic disease.

D. INFLUENCE OF DIETARY FACTORS The marked increase in hepatomas in trout populations in the United States has appeared to coincide with the utilization of dry pelleted rations in place of wet production diets in hatchery operations. This practice has been more predominant in the last ten years. Typical composition of a wet production diet is given in Table 11, as reported by Wood et al. TABLE I1 TYPICAL CONSTITUENTS OF WET PRODUCTION DIET” Constituent Liver (beef, hog, lamb, horse) Tripe (beef, sheep) Spleen (beef, hog) Lungs (beef) Lip (beef) Horse meat Beef, meat Fishery products Viscera, eggs, carcass (salmon) sole, carp, halibut, tuna, hake

Approx. per cent 23.0 3.0 8.0 6.0 6.0 10.0 4.0 40.0

100.0

a

According to Wood et al. (1957).

(1957). Prior to this wide-scale use of commercial dry rations the wet production diet offered trout consisted of packing house by-products and viscera of certain fish. As meat scraps, a good source of animal protein, became competitively unavailable because of utilization in livestock feeds, dried milk, fish meal, and cottonseed meal were then used exclusively, particularly the latter two components. Typical composition of a dry, pelleted, commercial-type trout ration is given in Table 111, as reported by Wood et al. (1957). Not all commercial dry rations consisting of fish meal and cottonseed meal as major protein sources produced hepatomas. Apparently conditions of processing, storage, or source of ration component had something to do with those sporadically produced hepatomas that were observed. I n those cases where a particular carcinogenic ration was encountered they were referred to as suspect rations or “hot” diets. Studies were initiated by Dollar and Katz (1962) and Halver (1962) to establish the hepato-

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CARCINOGENESIS AND CONTAMINATED FOODS

TABLE I11 TYPICAL COMPONENTS OF DRY PELLETED TROUT RATION" Component

Approx. per cent

Meat scrap meal Yeast Fish mealb Cottonseed meal Wheat shorts Dry milk Distillers solubles Salt (NaC1)

25.0 6.5 25.0 12.5 12.5 12.5 5.0 1.0

100.0

b

From Wood et al. (1957). Predominantly herring meal.

carcinogenic agent in those commercial rations which produced trout liver cancer. This phase of research has provided some promising and definitive leads in the elucidation of the carcinogenic agents involved in trout hepatoma induction. The range and average composition of trout diets and body composition of fish maintained on these diets have been determined by Wood et al. (1957) and are shown in Table IV. TABLE IV BODYCOMPOSITION OF HATCHERY AND WILD TROUT AND RANGEAND AVERAGECOMPOSITION OF DIETS USED FOR MAINTENANCE OF T R O U T ~ Hatchery fish Range

Average

(%)

(%I

Protein Lipid Ash Carbohydrate

56-80 12-38 7-14 0-8

68.1 22.5 9.7 0.6

Diet Protein Lipid Ash Carbohydrate

20-77 10-80 1-24 0-36

62.4 23 .O 10.0 4.8

Composition

a

According to Wood et al. (1957).

Wild fish Range

(75)

64-81 7-25 8-16 0-3

Average

(%) 75.8 13.4 12.3 0.4

64.6 12.4 9.6 13.5

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H. F. KRAYBlLL AND M. B. SHIMKIN

Feeding practices in fish are different from livestock in that metabolically trout cannot effectively utilize high proportions of carbohydrate in the diet but do utilize effectively high levels of protein. It is to be noted (Table IV) that commercial production diets average higher in lipid content and slightly lower in protein content. The utilization of production diets in hatcheries influence markedly the body composition of trout, with hatchery fish being somewhat higher in the proportion of body fat and lower in the proportion of lean body mass (protein and bone). Commercial producers of diets have been able to increase the proportion of lipids in diets through use of cottonseed meal and fish meal. To what degree this modification of the diet and the quality of these components have influenced the increase in frequency of hepatomas has indeed become an interesting and important phase of this extensive research. The diet of the wild trout in streams is, of course, high in protein and lacking in commercially processed plant and animal protein components. The foregoing discussion implicates the use of commercial dry pelletcd rations in trout hepatoma induction. From this knowledge, investigators centered their attention on the development of information on source of ration components, particularly cottonseed meal and fish meal, and the influence of adverse conditions of processing and storage on its hepatocarcinogenic properties.

E. INFLUENCE OF PROCESSING Sporadic occurrences of hepatomas, in some cases to the extent of 80 to 100% of a trout population, have been attributed to use of dry pelleted rations. Some investigators have believed that the etiological agent resided in the cottonseed meal, others have incriminated fish meal and its processing degradation products (Dollar and Kate, 1962 ; Hueper and Payne, 1961 ; LaRoche et aE., 1962). Since polymerized polyunsaturated vegetable oils will produce sarcomas in rats a t site of injection, it has been deduced that fish oils (contained in fish meal) subjected to high temperature processing during drying (150" to 700°C. during flame drying) readily undergo oxidative polymerization and hence carcinogenic conversion products may be found. The work of Sugai et al. (1962) has attested to the fact that peroxidized corn oil acts synergistically with 2acetylaminofluorene in the induction of ear duct and liver tumors in rats. Variation in levels of vitamins, protein, fat, or carbohydrate influences the induction rate and growth of tumors in rodents, as shown by Tannenbaum (1959). These considerations led Halver (1962) to investigate the role of the micro- and macronutrients in trout hepatoma. Although the variation in levels of these nutrients per se did not affect the frequency of liver tumors, Halver et al. (1962) did find that by incorporating the

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CARCINOGENESIS AND CONTAMINATED FOODS

respective protein, lipid, and carbohydrate fractions of a suspect commercial or “hot” diet into a control test diet they could influence the hepatoma frequency in experimental trout populations. The constituents of the control test diet used are shown in Table V. TABLE V TYPICAL CONTROL TESTDIET-SALMON Component

AND

Per cent in diet

Casein, vitamin-free Gelatin, purified Corn oil, purified Dextrin, white Alpha cellulose flour Mineral mixture DL-Methionine L-Tryptophan Vitamin supplement (1

TROUT~

54 15 9

8 9 4 1

0.5 0.04

From Halver (1957).

The solvent extractable material or lipid fraction from a suspect commercial ration or “hot” diet has been shown according to LaRoche et al. (1962), to contain the carcinogenic agents. I n Table VI is shown the effect TABLE VI FEEDING PROTEIN, LIPID, AND CARBOHYDRATE FRACTIONS “HOT” COMMERCIAL RATIONINCORPORATED IN CONTROL TESTDIET (CTD) ON RATEOF TROUT HEPATOMA INDUCTION“

I N F L U E N C E OF OF

Hepatoma frequency “Hot” ration fraction CTD alone Lipid fraction alone (6%) Lipid fraction 70% protein (CTD) Carbohydrate fraction CTD Protein fraction CTD

+

+

+

Number

Per cent

0/300 5/11 5/15 6/68 2/41

0 45 33 9 5

From Halver (1962)

of feeding the respective lipid, protein, and carbohydrate fractions of this “hot” diet in a control test diet (CTD) on the rate of hepatoma induction. The lipid fraction from the suspect dry ration is obtained by use of Bloor’s solvent extraction, using diethyl ether, ethanol, and ethyl acetate, with separation into a neutral and phospholipid fraction, The final phos-

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H. F. KHAYBILL AND M. B. SHIMKIN

pholipid fraction is obtained by elution with methanol. It is evident from the data in Table VI that the control test diet does not induce hepatomas. The lipid fraction alone apparently contains the tumor-inducing compounds, perhaps polymerized lipids, epoxides, or other compounds resulting from adverse conditions of processing or drying, as is the case in flame-drying of fish meal. The frequency of hepatoma induced by the protein and carbohydrate fraction should have approached the zero incidence with the control test diet if the lipid fraction contains the carcinogen. It is quite likely that the Bloor solvent extraction of lipids does not completely remove occluded lipid polymers and other compounds from the protein and carbohydrate fractions which were fed. The insolubility of these fat degradation products in common fat solvents has been shown by Kraybill and Nilson (1947) and other investigators. By autoxidation of fats or using stripped herring oil which is highly oxidized, the effect of peroxidized marine oils in hepatoma induction could be clearly demonstrated. Halver (1963) also found that the incorporation of prednisolone in the diet (tumorigenic lipid fraction plus control test diet) produced a decrease in the rate of liver tumor induction in experimental trout populations (Table VII). Since these findings are of a preliminary nature, further TABLE VII EFFECTOF LEVELOF PROTEIN AND PREDNISOLONE ON DECREASE OF HEPATOMA FREQUENCY IN TROUTMAINTAINED ON CARCINOGENIC LIPIDFRACTIONS IN DIET^ Hepatoma frequency Ration or component of diet

Number

Per cent

0/300 2/18

11

~~

Control test diet (CTD) CTD lipid fraction prednisolone CTD (70y0protein) lipid fraction prednisolone CTD (low protein) lipid fraction

+

0

+ + +

+

0/11

5/11

0 0 45

From Halver (1962, 1963).

work will have to be done before firm conclusions can be reached concerning the effect of this hormone and the additive effect of the levels of protein and prednisolone. It is evident that a control test diet does not induce hepatoma in trout on repeated trials and only sporadic observations are made of hepatoma induced from feeding trout commercial dry pelleted rations. Some investigators have also found that feeding high-quality cottonseed meal or fish meal alone will not produce the liver tumors. Presumably, then, conditions

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203

of processing or storage of these components alone, or the formulated ration, must yield specific processing degradation products which play a role in the etiology of this disease. Flame-dried, overprocessed fish meal, for example, yields almost 2% of a polymerized resinous fraction of fish meal fat (Kraybill and Nilson, 1947). Good quality fish meals or unoxidized marine oils have a minimum amount of these processing byproducts which have been associated with toxicity and physiological aberrations in experimental animals. As previously stated, the rainbow trout is particularly susceptible to this disease. Some investigators believe that these trout are susceptible to the disease because the liver of this species of fish normally contains no reserve of fat and therefore does not have provisions for prevention of oxidation of fat. Hatchery-raised rainbow trout, under conditions of intensive culture, accumulate an abundant supply of fat, resulting in deposition in the liver in a varying degree. Abnormal accumulations of considerable amounts of unsaturated fatty acids creates favorable conditions for the process of autoxidation which may be triggered by any upset of metabolism from whatever cause. Unpublished reports in early 1963 implicated cottonseed meal as the agent involved in an incidence of 60% hepatomas in trout. Conversely, a similar dry ration with 39% cottonseed meal did not yield any hepatomas in trout in the same period of time. These contradictory findings merely serve t o focus attention not on cottonseed meal per se but more significantly on the influence of processing, deterioration through storage, and contamination of the cottonseed meal with toxic and carcinogenic agents. A source of highly purified cottonseed meal, with gossypol, cyclopropene acids, pesticides, defoliants, and fungal metabolites removed, has been made available for investigation. Extension of research in this direction should provide some delineation of the combined effect of the aforementioned parameters or the singular role of these vectors in induction of trout liver cancer.

F. ISOLATION OF DIETCARCINOGENS Current evidence has demonstrated that the lipid fraction from tumorigenic rations fed to trout plays a significant role in the etiology of the disease. Peroxidized fats, as previously stated, have been implicated in toxicity syndromes and physiological aberrations and may have promoter action in carcinogenesis. Halver (1962, 1963) and LaRoche e t al. (1962) have further fractionated the total lipids into the neutral and the phospholipid fraction. The fractionation procedure is essentially that listed in Scheme I, using Bloor’s solvent extraction.

204

H. F. KRAYBILL AND M. B. S H I M K I B

Suspect or carcinogenic trout ration Diethyl ether ethanol 1 Diethyl ether ethyl acetate Extracted total lipids I Pass through silicic acid powdered glass 5 column Elution with ether

+

+

I

I(

Phwpholipids (remain on column) Elute with absolute methanol, then 50% methanol HzO Phospholipid fraction SCHEME I

1

Eluate = neutral lipid

+

These fractions have been bioassayed for their relative carcinogenic properties. It would appear that the neutral lipid fraction, with 17% frequency, rather than the phospholipid fraction with 2% rate contains the etiological agent. However, further isolation and purification of these fractions are required before definitive data are available. A crystalline compound with a specific melting point and other physicochemical criteria on structure has not been obtained as yet to characterize the chemical nature of the carcinogen in the lipid or neutral lipid fraction. Research is proceeding rapidly in this direction.

G. CARCINOGENESIS OF OTHER CHEMICALS The wide-scale use of agricultural chemicals and pesticides, with the associated problem of plant residues and contamination of water, has prompted environmental health research and established certain requirements on residues. For example, Aramite used for control of mites on fruit at a level of 1 p.p.m. was tested on mice, rats, and dogs; a t a level of 400 p.p.m. was found to induce hepatomas in several species, according to Oser and Oser (1960) and Sternberg et al. (1960). Similarly, thioacetamide and thiourea, used in fungicidal treatment of fruits, produced hepatomas in rats (Fitzhugh and Nelson, 1948). The herbicide, aminotriazole, used to control weeds in cranberry bogs, produced thyroid adenomas in rats when fed a t level of 15 p.p.m. (Jukes and Shaffer, 1960). Other examples of carcinogenic action of pesticides, herbicides, and fungicides could be cited, but the fact that these compounds gain entry into food supplies has not been overlooked (Kraybill, 1963), particularly DDT, which has been found in beef carcasses. Recently, DDT a t a level of 2.21 p.p.m. has been reported in human body fat (Hunter et al., 1963). Trout and other fish show a toxic and fatal response to some insecticides a t levels of parts per billion, according to Katz (1961). Commercial livestock feeds have added medicinal agents such as carbarsone, an amebicide, antioxidants, such as butylated hydroxyanisole and bactericidal agents (sulfa compounds) which may be incorporated acci-

205

CARCINOGENESIS AND CONTAMINATED FOODS

dentally or otherwise in trout rations. An enumeration of these compounds suggests that a few have a wide spectrum of possibilities for chemical carcinogenesis. With these possibilities for contamination of the ration or the environment of the trout, Halver (1962, 1963) determined the carcinogenicity of a series of these compounds. The compounds were fed to trout in a control test diet (previously shown in Table V) a t levels capable of producing tumors in rodents or other test animals. After challenging groups of rainbow trout with these compounds for 20 months some of the trout were sacrificed or surgically examined grossly. Nodular lesions were found which were confirmed histopathologically as classical hepatoma. Class IV or V of an arbitrary classification indicated in Table I is applicable here, since there were discrete nodules, advanced massive nodules, or metastatic nodules confirmed histopathologically. A summation of the trout hepatoma frequencies from screening various compounds a t various challenge doses are shown in Table VIII. TABLE VIII SUMMATION OF TROUT HEPATOMA FREQUENCY FROM SCREENINQ OF COMPOUNDS IN DIETFED TO TROUT FOR 20 MONTHS' ~~

Dose in diet Chemical

mg./100 g.

Number

Per cent

-

0/300 38/46 22/43 3/49 4/11 9/45 7/38 6/46 6/48 5/55 4/44 5/50

0 82 52 6 36 20 18 13 13 11 10 10

Control test diet (CTD) Dimethylnitrosamine Aminoazotoluene Aminoazotoluene Dichlorodiphenyltrichloroethane 2-Acetylaminofluorene Thiourea p-Dimethylaminoazobenaene Tannic acid Urethane Carbon tetrachloride Carbarsone 9

Frequency

480 120 30

8 30 480 30 120 480 120 480

According to Halver (1962, 1963).

These preliminary findings did not encompass a wide range of doses for these chemical carcinogen induction studies. The specific hepatoma frequency could not be calculated accurately in all cases because of the small number of trout remaining as the result of those sacrificed for histopathological evaluation and lack of duplicate lots for adequate statistical analyses.

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H. F. KRAYBILL AND M. B. SEIIMKIN

The experiments indicated that rodent carcinogens, or certain chemicals when fed to rainbow trout, consistently yielded a high rate of hepatoma formation when effective levels of these compounds were administered. It is, of course, interesting, as shown in Table VIII, that the control diet did not yield any evidence of hepatoma in 300 trout examined. Of the chemical carcinogens, dimethylnitrosoamine and aminoazotoluene appeared to be the most active tumorigenic compounds in trout. Of equal interest is the fact that DDT showed a significant frequency and that the amebicide carbarsone, an arsenic compound, has provided some of the first direct evidence of the carcinogenic properties of this type of compound. The information on arsenicals in the literature relative to carcinogenicity has been equivocal. Undoubtedly, these initial findings on carbarsone will require further confirmation in additional tests and with other species. The carcinogenic response of trout to tannic acid and thiourea has significance in that these compounds could readily be found in the environment of trout in the hatcheries and streams. The associated studies on the chemical carcinogens revealed interesting observations other than hepatomas. For example, urethane produced skin lesions after 10 months’ feeding a t a level of 1.9g. per 1OOg. diet. Thioacetamide fed 6 months a t the same level in the diet produced eye cataracts, impaired vision, and depigmentation, whereas, a t a lower dose of 480 mg. per 100 g. diet, liver lesions observed were comparable to those noted in fatty acid deficiency in trout (Halver, 1962). Necrosis, fibrosis, and hemorrhage were observed in trout fed dimethylnitrosamine after feeding 1.9 g. per 100 g. diet for 15 months. Diethylstilbesterol fed for 15 months to trout a t l e v ~ l sof up to 1.9g. in 1OOg. diet produced enlarged hearts and growth retardation. Another interesting aspect of this screening of rodent carcinogens on trout is the fact that trout can now be recognized as another sensitive test animal for the bioassay of natural and synthetic materials.

H. IONIZING RADIATION AND PARASITIC INFESTATION The potential influence of environmental radiation has been postulated in view of the fact that ionizing radiation a t a certain dose level can induce tumors. The degree of low level radiation which would be encountered here has been difficult to assess as to magnitude of biological response. Programs of research are going forward in this direction in other fields and with other test species to ascertain the tumorigenic effect of low level radiation. In this instance, thus far the influence of ionizing radiation on induction or promotion of trout hepatoma has not been studied.

CARCINOGENESIS AND CONTAMINATED FOODS

207

Various parasites which infest fish in their natural habitat have been suggested as agents in induction of liver cancer. I n reference to the induction of hepatomas in rainbow trout this possibility likewise has not been studied so an assessment of their role a t this time cannot be made.

I. PROJECTED RESEARCH ON TROUT HEPATOMA The various aspects of research on trout hepatoma have provided an extension of our knowledge on the role of environmental factors in cancer and have elucidated the mechanisms involved in the etiology of the disease through specific agents. The association of diet, nutrient level, and degradation products arising from food processing is perhaps now more fully comprehended in terms of genesis of tumors through this research. Further exploitation of these preliminary findings will be emphasized in future work through certain directions of research. These may well include the following: ( 1 ) Definitive studies on lipid fractions of carcinogenic trout rations with identification of the chemical entity or molecular species responsible; (2) further exploration of the influence of nutrient level and hormonal effects; (3) effect of processing and extent of processing on formation of breakdown products and the effect of these by-products in toxicity and carcinogenesis; ( 4 ) continuation of studies to determine the susceptibility of various strains of fish and genetic predisposition to hepatoma induction; ( 5 ) continuation of the screening of various chemicals and other carcinogens on rainbow trout; and (6) adaptation of the trout for study of the metabolic fate of some of the classical carcinogens, such as 2-acetylarninofluorene, dimethylnitrosamine, and others, through identification of urinary metabolites using cannulated experimental trout. The utilization of trout for metabolic studies presents many intriguing possibilities. Halver (1962, 1963) has developed a technique in which cannulation of an older trout, maintained in a single aquarium, can be used to collect urine samples and provide for measurement of protein utilization, drug metabolism, and studies in depth on use of radioactivelabeled compounds for measurement of metabolic fate of such compounds. No arrangement has yet been made for collection of fecal samples for measurement of exogenous compounds by this route. The addition of trout or fish to our armamentarium of test species provides another means of obtaining important data in the fields of toxicology and carcinogenesis with environmental materials. From a practical standpoint, investigations are also continuing to &mess the effect of water temperature, other fish diseases, and parasites in hepatoma induction. With respect to the effect of processing, the various ingredients of the ration, such as cottonseed meal and fish meal, will be treated under favorable and adverse conditions (steam-drying, flame-

208

H. F. KRAYBILL AND M. B. SHIMKIN

drying, high temperature storage, autoxidation of lipid components, etc.) to establish the magnitude of these parameters in the etiology of liver cancer. As previously mentioned, the conflicting viewpoints on the implication of cottonseed meal, a component of commercial dry rations, as the etiological agent in trout hepatoma have extended the studies on the influence of processing, storage, and possible contamination of rations with pesticides, gossypol, herbicides, and fungal metabolites in relation to hepatomagenesis in trout, The role of viruses and the immunological aspects of this problem have not been adequately explored. Further efforts on the design of research in this direction are anticipated to provide more conclusive evidence on the potential role, if any, of viral agents in the genesis of neoplastic disease in trout. Il l . Role of Fungal Metabolites in Diet and Cancer

The extensive research on trout hepatoma has clearly established the role of certain diets in induction of hepatomas in rainbow trout. What specific carcinogen or promoter of carcinogenesis is involved can only now be categorized as a neutral lipid component, represented in widely used commercial dry rations. Evidence thus far suggests that the etiological agent is introduced through modern practices of trout feeding associated with unfavorable processing procedures utilized in the production of dietary components. To some extent this environmental condition resulting in the occurrence of neoplastic disease has evolved from our modern technology. Contamination of products such as peanut meal with fungi, and the development of fungal metabolites which produce liver cancer in rats, however, is somewhat different. This process is not accelerated by technology but arises from a natural sequence of events in the environment.

A. OCCURRENCE AND DEVELOPMENT OF MYCOTOXINS 1. Production of Aflatoxin

I n the British work a mixture of toxins was obtained from pure cultures of certain of the fungal species which were grown on sterilized peanuts inoculated with the toxic strain of Aspergillus flavus. Good growth of toxin could also be produced on Czapek’s Dox medium with added zinc sulfate on which the fungus had been grown for 5 to 7 days a t 27°C. A typical mixture for fungal growth proposed by Brian et al. (1961), utilizing glucose, ammonium nitrate, zinc sulfate, potassium dehydrogen phosphate, calcium chloride, and other salts supports good growth of fungi.

CARCINOGENESIS AND CONTAMINATED FOODS

209

To identify the fungal species and fungal metabolite responsible for toxicity of peanut meal, the production was first established on peanut meal. The peanut meal is a defatted product produced by petroleum ether extraction of whole peanuts for 4 hours or ground peanuts for 2 hours, using a continuous extractor. I n subsequent studies, as reported by Wogan et al. (1963), most of the toxin production was carried out in liquid media by growing mold spores from a stock culture on Caapek’s agar for 5 to 9 days a t 30°C. Two methods are used, either stationary growth or submerged growth in shake flasks. These methods may not initially give as high a yield of fluorescent toxin compared to that obtained by growth on infected peanuts. Interestingly enough, yeast extract or corn steep liquor, added in concentrations of 1g. per liter of medium, is superior for yield of toxin. Since protein or protein hydrolyaates will also cause marked increase in yield of toxin, Wogan et al. (1963) studied the influence of various amino acids on growth. Alanine, lysine, methionine, thyrosine, aspartic acid, histidine, arginine, cystine, glycine, isoleucine, hydroxyproline, ornithine, and phenylalanine had no effect. On the other hand, glutamic acid and proline stimulated production of the blue fluorescent compound, whereas leucine, threonine, and tyrosine had a less pronounced effect. Tryptophan stimulated production of an unidentified green fluorescent component. Pilot plant production of aflatoxin is now carried out continuously, with the fermentation of 100 liters of broth yielding log. of chloroformextractable material, of which 3 to 57% consists of fluorescent compounds. In such a production 300 mg. of toxin was obtained for bioassay in a period of 6 days (Wogan et al., 1963). Animal products, rice, and corn appear to support toxin production better than peanuts. Soybeans or soya protein support weak production of the toxin. Incaparina (a protein supplement promoted for treatment of kwashiorkor, containing 3376 cottonseed meal) produced only a weak fluorescent toxin. Other protein sources, such as wheat, oats, millet, egg solids, and skim milk powder, appeared to support good growth and yield of toxin. The importance of these findings to fungal contamination and production of fungal metabolites is readily appreciated in terms of a world-wide food supply and its implication to the health of animals and man. The work with A . fkwus has suggested the importance of studying the fungal metabolites or toxins from other mold species such as A . tamarii, A . oryzae, A . glaucus, A . niger, and others. Information is not yet available on the yield of toxins and the fluorescence or biological action of the products. Just as the amino acid composition is critical in growth of toxin on

21 0

H. F. KRAYBILL AND iM. B. SHIMKIN

various solid substrates, in liquid media such as Brian’s mixture, the ammonium compounds or ammonium nitrate appear to be critical. The importance of amino acid has its corollary in the influence of amino acids in correction of toxicological response of turkey poults fed moldy diets; a subject which will be discussed later. 2. Extraction of AfEatoxin from Contaminated Peanut Meal Fungal-contaminated peanut meal is first extracted continuously with methanol for 18 hours in a Soxhlet apparatus. Under reduced pressure the solvent is removed yielding a black oily residue. After drying the extracted meal, it is re-extracted for 18 hours with methanol, after which the meal is again air-dried and finally dried over phosphorus pentoxide. The oily residue, dispersed in water, is continuously extracted for 3 hours with chloroform. The removal of chloroform leaves another black residue which is then extracted several times with petroleum ether, methanol, and water, leaving the final residue after the removal of the solvent. A typical extraction procedure is shown in the scheme of Sargeant et al. (1961a) (Scheme 11). Toxic Brazilian peanut meal

1

I 1 Methanol-insoluble extracted meal B (nontoxic 88%)

Methanol-soluble extract A (toxic 12%)

1

1 Water-soluble extract D (nontoxic 10%)

Chloroform-soluble extract C (toxic 1.5%)

1 1 Lower layer from petroleum ether-methanol-water extract E (toxic 0.4%)

SCHEME I1

1 Upper layer from petroleum-methanol-water extract F (nontoxic 1%)

3. Occurrence and Development of AfEatoxin in Peanuts prior to or after Harvesting

The growth of fungi on peanuts, or for that matter on any appropriate substrate during harvesting, drying, and storage, is optimum a t a relative hnmidity of 80-857h. This is true for A. fumigatus, A. niger, A. tamarii, and Penicillium martensii. The A. glaucus group, according to Austwick and Ayers (1963), will grow rapidly a t lower moisture levels and Penidlium spp. will grow well a t a high moisture content but a t lower tempera-

CARCINOGENESIS AND CONTAMINATED FOODS

21 1

tures than 25 to 30°C. The influence of moisture content on fungal growth is shown in Fig. 1. Groundnuts

,

Brazil nuts

60

8

40

E n c 20

0

10

20

30

40 10 % Moisture content

2

30

FIG. 1. The effect of high moisture content on the rate of fungal invasion of groundnuts and Brazil nuts.

Webb e t al. (1959) demonstrated that they could inactivate molds in ground corn by means of gamma radiation. When the moisture content was below 15.50/0, corn was safe from growth of molds during storage after receiving a radiation dose of 0.25 megarads. Higher moisture levels (16.5%) required 0.5 Mrads, and corn with moisture levels of 18.5 to 22.0% required 0.75 to 1.00 Mrads to prevent mold growth. The molds which grew a t high moisture levels were, however, more resistant to gamma radiation. For example, the species of Penicillium and Verticillium survived a dose of 0.75 Mrads in corn containing 12.5 to 23% moisture. To what degree the metabolites or toxins from molds can be inactivated or destroyed by gamma radiation has not been fully explored. However, preliminary unpublished work of British workers indicates that a 2 Mrad dose of radiation did not destroy aflatoxin. Further work, however, should be done in this area. The British studies on Brazilian peanuts suspected of having the toxic agent revealed that 20% of the cotyledon tissue had 20% fungal hyphae. Nontoxic peanut meal had no hyphae. The peanuts are infected by fungi during development, and some may decay in the ground; others that are contaminated mature normally, and invasion of the kernels is apparently more rapid during curing than prior to harvest. After harvesting of the whole plant the drying is first done in windrows or later by stacking for a few days or weeks. Any damage to the peanut permits rapid contamination a t this point. After picking the peanuts or threshing the fruit from

212

H. F. KRAYBILL AND M. B. SHIMKIN

the plant, decortication follows for shipping or milling. Kernals that show yellow, orange, buff -brown, or black flesh are invariably infected and toxic (approximately 80%). Visual examination provides a basis for selection of peanuts and the fluorescent test (fluorescence of mycotoxin) provides a further basis for sorting out undesirable products. Indeed the British have experimented with a sorting machine for this selection. The progress of this development cannot be reported on a t this time. While attention has been focused primarily on aflatoxin produced by A . fluvus, Austwick and Ayerst (1963) identified the fungi isolated from infected peanuts (see Table I X ) . It would appear that A . flavus represented the highest number of isolates (91), followed in order by A . tamarii (62), Phoma sp. (62), and A . niger (35). Austwick and Ayerst (1963) reported that 29 of 59 A . flavus isolates were fluorescent; of these, 19 were tested for toxicity and 9 produced TABLE IX FUNGIISOLATED FROM MOLDYPEANUTS' Fungus Acremoniella sp. Aspergillus carneus Aspergillus jlavus Aspergillus fumigatus Aspergillus glaucus series Aspergillus ruber Aspergillus nidulans Aspergillus niger Aspergillus ochraceus Aspergillus tamarii Aspergillus versicolor Botryodiplodia Chaetomium erectum Curvularia maculans Fusarium oxysporum sensu Fusarium solani Fusarium sporotrichioides Fusarium spp. Mucor sp Penicillium martensii Penicillium steckii Penicillium spp. Phomu sp. Rhizopus arrhizus Rhizopus sp. Syncephulastrum racemosum Trichothecium roseum a

According to Austwick and Ayerst (1963).

Number of isolates 1 1 91 7

5 1 1 35 1 62 1 16 1 1

3 4 1 6 4 2 1 9 62 10

5 1 1

CARCINOGENESIS AND CONTAMINATED FOODS

213

toxins. Eight isolates which were fluorescent were not toxin producers. Nonfluorescent culture filtrates from two isolates of A . flavw were not toxic. Culture filtrates from some isolates of A . tamarii and A. fumigatus were only faintly fluorescent but not toxic. A filtrate from an isolate of Penicillium martensii produced some fatty necrosis in the livers of ducklings. It has been stated by the British workers that mechanical damage to the peanut kernal may occur during drying after removal from the soil. At this point resistance to infection is reduced and the parasitic fungi invade. On further drying moisture becomes a limiting factor and any further damage only occurs by the “storage” or xerophytic fungi. Less than 3% of toxin-containing kernals can be enough to render a sample toxic (Austick and Ayerst, 1963).

B. METABOLISM OF AFLATOXIN-SPECIES SUSCEPTIBILITY Following the outbreak of disease in England in 1960 much of the controlled feeding experiments was carried out in turkey poults, ducklings, and chickens. The selection of the duckling as the standard for bioassay was made on the basis of its extreme susceptibility (Allcroft and Carnaghan, 1963a). It is interesting to note that hepatitis or other lesions had been observed frequently in laboratory animals such as the guinea pig as early as 1954 by Paget when peanut meal was used in the diet. The general order of toxicity in poultry might be represented as follows: duckling > turkey > chicken. For larger farm animals the order of toxicity is: swine > cattle > horses > sheep. 1. Rats and Mice

The carcinogenic effect of aflatoxin was noted by Lancaster et al. (1961) and later by Schoental (1961) when peanut meal was fed to rats for 6 months. Mice appear to be resistant in short-term experimental feeding but chronic feeding tests have not been undertaken (Allcroft and Carnaghan, 1963a). 2. Ducklings and Chickens Chick and duck embryos are susceptible to aflatoxin, the relative susceptibility being comparable to that of the toxicity on day-old birds of each species. The nonspecificity of the lesions produced by aflatoxin in duck embryos precluded the use of this technique (Carnaghan, 1962). Aspiration of spores of A. flavus (aspergillosis) into the respiratory tract failed to produce a toxicity or lesions in ducks or chicks (Carnaghan and Allcroft, 1962). This again established the fact that not the fungus but the fungal metabolite is the potent toxin. The preceding discussion has emphasized the fact that day-old duck-

214

H. F. KRAYBILL AND M. B. SHIMKIN

lings or turkey poults are extremely sensitive to the mycotoxin. Five hundred micrograms of aflatoxin administered to l-day-old ducklings, in 5 daily doses of 100 pg. each, will produce liver lesions; 2000 pg. over 5 days will yield 100% frequency of bile duct proliferation. Carnaghan and Sargeant (1961) showed that rations containing 6.25% toxic Brazilian groundnuts killed all of a group of 6-day-old ducklings in 13 days. Nesbitt et at. (1962) stated that the LD,, for a potent fraction of aflatoxin is of the order of 15 to 20 pg. for day-old ducklings. Wogan and co-workers (1963) confirmed this finding.

3. Swine Of the larger farm animals the pig appears to be the most susceptible, a t least a t the age of 3 to 12 weeks. The pregnant sow is most commonly affected (International Working Party Progress Rept., 1962 ; Loosmore and Markson, 1961). Pigs are the only species that develop generalized clinical jaundice. With increase in age the pigs become more resistant, except the pregnant sow as heretofore mentioned. 4. Cattle

Calves are quite sensitive to aflatoxin, with exposure leading to ductile proliferation and severe fibrosis in the liver. From the age of 1 to 6 months the calves are most susceptible, but show increasing resistance with age. Chronic feeding trials at Central Veterinary Laboratory (Weybridge, England) indicate that 3- to 4-year-old heifers are clinically affected when continuously fed a ration of 20% toxic groundnut meal (Allcroft and Carnaghan, 1963a). Some isolated cases of unthriftiness in cows 10 years old have been reported, but in general, cows are not as adversely affected clinically as heifers. Cows fed toxic peanut meal excreted in the milk a toxic factor having a biological effect in ducklings (Allcroft and Carnaghan, 1963a). Pasteurization or drying of milk did not reduce the toxicity when bioassayed on ducklings. The rennet precipitation of milk demonstrated that the aflatoxin resided in the cheese or paracaseinate fraction rather than in the whey. Allcroft and Carnaghan (1963b) maintain that no market-milk samples contained enough aflatoxin to give an effect on bioassay with ducklings. This indicates considerable dilution. More work should be done to determine the actual levels of toxin in market milk by repeated concentration and chemical extraction of the toxic fraction. 5. Sheep Three-month-oid sheep fed rations containing 20% strongly toxic peanut meal for 19 months failed to demonstrate any obvious clinical effects

CARCINOGENESIS AND CONTAMINATED FOODS

215

other than a slight growth retardation (Carnaghan and Allcroft, 1962). Hence, sheep appear to be quite resistant. Biochemical studies are underway in the British laboratories (Central Veterinary Laboratory, Weybridge) to study the detoxification mechanism. For example, in calves the serum alkaline phosphatase levels increase and then fall to normal levels prior to death as a result of peanut meal toxicity. Vitamin A levels in the liver are reduced until absent. 6. Man

No information is available regarding mycotoxicoses in man caused by contaminated peanuts or other cereal products, or products made from these materials. A recent editorial by Shimkin and Kraybill (1963) indicates that peanut oil used in the manufacture of margarine in England did not contain aflatoxin since alkali treatment in oil refinement removed the toxin. More significant that the health of game or farm animals and the associated economic problem is the relevant importance of this problem to the development of plant protein sources for augmentation of milk proteins in alleviation of kwashiorkor in less developed countries of the world. Thus far, no evidence of human hazard associated with consumption of such products has been revealed. The epidemiological aspect of this problem will be discussed later. C. PATHOLOGY I N MYCOTOXICOSES

1. Toxicity Exudative hepatitis in guinea pigs poisoned by peanut meal toxin has been referred to previously. This observation is comparable in other species although the degree of involvement may vary. Within a few days of feeding toxic peanut meal in ducklings, there is destruction of the liver parenchymal cells and extensive bile duct proliferation. Indeed, the latter changes in pathology have become the basis for detection of the my cotoxin. The following description of pathology in various species resulting from the toxic action of fungal-contaminated peanuts reflects the similarities and dissimilarities of response by different species. a. Calves. Macroscopic lesions are cirrhosis and pallor of liver associated with ascites and edema of the mesentery. The bovine liver, according to Allcroft and Carnaghan (1963a), differs from other species in that the central hepatic vein shows chronic centrilobular endophlebitis. Other lesions are megalocytosis, severe fibrosis, and proliferation of the bile duct. As indicated before, these lesions are comparable to that from ragwort poisoning or pyrollizidine alkaloids (seneciosis) .

216

13. F. KRAYBILL AND M. B. SH IMK IN

b. Swine. The only large animal showing generalized jaundice is the pig. White to yellow coloration and subcutaneous hemorrhages appear in the liver. The other microscopic changes are comparable to those for calves. c. Ruts. Heart, spleen, adrenals, the intestinal tract, and urogenital tract appear normal. The kidneys are microscopically normal but some groups on toxic meal show glomerular and tubular changes. I n the lungs there are some macroscopic grayish lesions with zones of hemorrhage. The livers of rats become grossly abnormal after 30 weeks’ feeding of toxic meal. The aflatoxin group had livers twice the size of controls; particularly true in male rats. Macroscopically there were brownish-yellow, irregular nodular surfaces, red and greenish cysts, and numerous yellowish focal lesions (Lancaster et al., 1961). The rats failed to reproduce the acute liver damage observed in turkey poults. Hepatomas were observed after feeding 20% Brazilian peanut meal for 6 months. d. Ducklings, Chickens, and Turkeys. Avian livers do not develop fibrosis to the extent seen in mammals. After feeding 6 days on toxic peanut meal a t a level of 10% in the diet, degenerative changes appear in the liver parenchymal cells. The cells are swollen and vacuoles are present. Nuclei are enlarged and karyolysis occurs. There is extensive proliferation of the bile duct epithelium. After 3 weeks of feeding, only small islands of normal parenchymal cells remain and these are surrounded by dense masses of bile duct epithelial cells and then fibrous tissue. The kidneys show multiple and diffuse hemorrhages, but glomerular changes seen in turkeys are not observed in ducklings (Asplin and Carnaghan, 1961). Siller and Ostler (1961) have described the histopathological lesions in turkeys. Hydropericardium and ascites are frequently noted. In acute cases there are hepatic hemorrhages. As with ducklings, there are extensive parenchymal cell degeneration, bile duct proliferation, and fibrosis. The catarrhal enteritis and marked swelling and congestion of the kidneys are the most striking macroscopic lesions. Chickens are comparatively resistant to the toxin. After extended periods on the toxic meal striking hepatic changes are noted. The retrogressive and regenerative changes in the parenchymal cells are similar to that in turkeys. Analogous to the duckling, there are some diffuse areas of regeneration of the acinar cells in the pancreas. 2. Carcinogenesis

Reports by Lancaster et al. (1961) and Schoental (1961) describe the chronic effects of feeding toxic peanut meal to rats, leading to development of liver tumors. In this early work none of the crystalline aflatoxins

217

CARCINOGENESIS AND CONTAMINATED FOODS

(B or G compounds) had been isolated and purified for these carcinogenesis experiments. The crude extract, or moldy meal, however, obviously contained the carcinogen or a precursor of the carcinogen which has now been clearly defined. The effect of toxic peanut meaI feeding on liver weights and frequency of liver tumors in rats is shown in Table X. The rats used by Lancaster TABLE X EFFECTS OF VARIOUS PEANUT MEALSON LIVERWEIGHT AND TUMOR INCIDENCE OF RATS's~ Average liver weight (g.) Group

8

0

Purified control diet Control diet Brazilian peanut meal (20%)

+

13.6 28.9

Control diet control highquality Indian peanut meal Suspect diet of peanut meal (Schoental's studies) Stock diet with 10% peanut meal

+

(1

Liver tumors 3

0

8.2 16.6

0/7 5/6"

0/5

17.2

10.2

1/7

0/6

-

-

Sex unspecified, 2/5

-

-

0/243

4/5

1/257

All animals 6 t o 12 months old.

* Data from Lancaster et al. (1961) and Schoental (1961). c

One death a t 6 weeks.

et al. (1961) were from an undefined inbred colony. Some of the rats fed the toxic Brazilian peanut meal were sacrificed a t 9 weeks and were found to have numerous subcapsular yellow focal lesions in the liver. After 30 weeks, when the males had eaten 650 g. peanut meal, 9 of the 11 animals had grossly abnormal livers with the nodules identified histopathologically as solid hemorrhagic or cystic hepatomas. Two of the rats had pulmonary metastases. One hepatoma occurred in a male rat that was maintained on Indian peanut meal plus control diet, suggesting the possibility of a carcinogen in this peanut meal. I n a group of 243 male and 257 female rats fed 30 weeks on a control or stock diet, there was 1 hepatoma (Table X) The relative levels of peanut meal (Brazilian and Indian) and the fungal metabolite, aflatoxin, that are required to produce a toxicity or liver lesions and tumors are compared in Table XI with the concentration of p-dimethylaminoazobenzene (DAB), carbon tetrachloride, and quino-

.

TABLE XI RELATIVE LEVELSOF FUNGAL-CONTANINATED PEANUT MEAL,FUNGAL METABOLITES, AND OTHERCOMPOUNDS WHICHPRODUCE TOXICRESPONSES AND LIVERTUMORS Biological response Tumor data Toxin or carcinogenic material

Species tested

Level fed/day

Moldy peanut meal (Brazil). Moldy peanut meal (Brazil)'

cow Weanling rat

15% (Diet) 195 g.

Moldy peanut meal (Brazil). Control peanut meal extract" (India) Aflatoxin (Brazilian meal)b Aflatoxin (Brazilian meal)' p-Dimethylaminoazobenzene (DAB)d 3-(p-Dimethylaminoaphenylazo) quinolinee Carbon tetrachloride (CC14)f

Duckling (7 days) Duckling (4 days)

10% in Diet 200 g. meal extract

Duckling (1 day) Weanling rat Weanling rat

Days fed 53 63 15-24 5

Toxic action

Induction period (days)

Frequency

(%I

Reduced lactation Subcapsular yellow focal lesions (liver) Death None

1 180 105

Death Hepatoma Hepatoma

-

-

180 300

83 66

Weanling rat

1000 pg.

120

Hepatoma

120

100

Strain A mouse

1800 pg.

1 2 0 ~ Hepatoma

150

100

From Sargeant et al. (1961a) and Allcroft and Carnaghan (1963a). From Lancaster et al. (1961). c Communication from Dr. J. Barnes (Medical Research Council, England): 1 g. meal contains 6 pg. aflatoxin. From Miller and Miller (1955). a From Brown et al. (1961). f From Eschenbrenner and Miller (1946). 0 Fed in 4 d a y intervals over the 120-day period (30 doses).

CARCINOGENESIS AND CONTAMINATED FOODS

219

line analog to DAB that are carcinogenic to the liver. These comparative data demonstrate the potency of aflatoxin both from the standpoint of toxicity and carcinogenicity. The data for DAB is cited since its carcinogenic action is well documented (Miller and Miller, 1955) and because it was formerly used to color foodstuffs. Carbon tetrachloride is also cited (Eschenbrenner and Miller, 1946) because of the low concentration required to produce hepatomas and because i t is a compound which a t low doses induces hepatomas in mice in the absence of morphological evidence of liver necrosis. A more reactive group of compounds than DAB are the quinoline and quinoline-N-oxide analogs of DAB, one of which, shown as 3- (p-dimethylaminophenylazo) quinoline in Table XI, is highly reactive. Interchanging one of the phenyl rings in DAB by a quinoline or quinoline-N-oxide group, Brown e t al. (1961) found this series of compounds very potent hepatocarcinogens. As to their relative reactivity compared to DAB they are about 20 to 200 times more active than the original azo dyes or DAB. Interestingly enough, the fungal metabolites, represented by aflatoxin, are perhaps the most potent hepatocarcinogens; this is demonstrated on the basis of the extremely low dose rate required to produce liver lesions and induction of hepatomas in rats This type of compound, an unsaturated lactone or difurano analog of coumarin, which will be described later, presents a potential spectrum of compounds for further investigation. Some years ago Salmon and Copeland (1954) reported hepatomas in rats with cirrhosis on a choline-deficient diet. I n retrospect i t has been interesting to note that these hepatomas developed when commercial peanut meal was used as the protein source in the diet. Quite recently, Salmon e t al. (1963) found a decrease in frequency of hepatomas when peanut meal used in the diet was previously extracted with hot methanol. Supplementation of the diet with choline or methionine did not decrease the original frequency of hepatomas reported. Some of the original peanut meal used some years ago may have been solvent-extracted but present knowledge now demonstrates that the degree of removal of aflatoxin is dependent upon the solvent used in extraction, Chloroform apparently produces more complete removal than methanol, according to Wogan (1963). Further confirmation of this work on the toxicity and carcinogenicity of mold-contaminated peanut meals has been reported by Newberne and Carlton (1963), and establishes the potential role of peanut meal in the original findings of Salmon and Copeland (1954). Hence, the studies conducted on choline deficiency and hepatomas may have been clouded by the presence of a toxic fungal metabolite contained in the peanut meal.

220

H. F. KRAYBILL AND M. B. SHIMKIN

TABLE XI1 HEPATOMAS IN RATS ON DIETS WITH AND EXTRACTED PEANUTMEAL'

WITHOUT

Protein sources Peanut meal

-

-

Beef, dry

Not Casein Extracted extracted

(%)

(%)

(%Ib

(%I

7.9

7.9 7.9 -

-

33.3 33.3 33.3 -

-

6.0 7.9 7.9 7.9 a

t.

-

-

25.0 33.3 33.3 33.3

-

Fat source and per cent in diet Tallow, Tallow, Tallow, Tallow, Tallow, Lard, Crisco,

18.5 18.5 18.5 20.0 18.5 18.5

18.5

Average time on experiment Hepatoma (days) frequency 334 424 470 574

283 488 574

4/10 8/10 15/15 2/10 0/10 0/10 0/10

According to Salmon et al. (1963). Extracted with methanol, which will remove aflatoxin.

Table XI1 presents some of these findings and shows various fat and protein sources used in diets fed to rats, including extracted and nonextracted peanut meal. A sharp reduction in hepatoma frequency is noted here by using hot methanolic-extracted peanut meal, with only one case showing a 20% frequency. It would have been interesting to determine what response would be noted if an additional extraction of peanut meal were carried out using chloroform. Since molds are ubiquitous and adverse conditions of storage may be represented, the potential influence of the mycotoxins must be anticipated whenever this or similar feed protein sources are utilized.

D. NUTRITIONAL CONSIDERATIONS IN MYCOTOXICOSES The heavy losses in turkeys observed in England in 1960 were followed by a similar disease in ducklings. Commercial feeds appeared common to all outbreaks of disease. The common ingredients were fish meal and peanut meal. The disease was controlled by substituting soybean meal or dried milk. Reports have appeared in the United States since 1950,but these attracted little attention. Burnside et al. (1957)and Richardson et al. (1962)studied the effects of moldy diets, especially corn, administered to farm animals and poultry. The British incident dramatized the over-all significant biological observation on moldy diets. Representative diets in rat feeding studies which were used to demonstrate the hepatomas from toxic peanut meal are shown in Table XIII.

22 1

CARCINOGENESIS AND CONTAMINATED FOODS

TABLE XI11 COMPOSITION OF DIETS" OF BRITISHPEANUT MEAL TOXICITY STUDY) Control basal diet Component

+

Casein 0.75% L-cystine Peanut oil Starch Cellulose Jones Foster salts Wheat germ oil Vitamin mixture Peanut meal a

(%I

20.15 10.00 57.85 6.00 4.00 1.50 0.5 -

Suspect Brazilian meal

Control Indian meal

(%)

(%)

20.15 10.00 37.85 6.00 4.00 1.50 0.5 20.0

20.15 10.00 37.85 6.00 4.00 1.50 0.5 20.0

Vitamin A (200 I.U.) and Vitamin D (21 I.U.) fed once weekly in peanut oil. According to Lancaster et al. (1961).

Whereas turkeys and ducklings exhibited acute toxic effects, rats fed toxic peanut meals did not readily show the acute effects; however, the latent effect was expressed by development of liver cancer in approximately 6 months, If the estimation that 1g. of toxic meal produces about 6 pg. of aflatoxin is correct, the rats in the chronic feeding studies carried out by Lancaster et al. (1961) must have had a daily intake of 6 to 12 pg. of the active compound. There was also a growth retardation noted in rats fed toxic meal. More recently Richardson and co-workers (1962) reported on the effect of moldy soybean meal on the growth of turkey poults. Retarded growth occurred in poults that received a diet of moldy soybean meal. The soybean meal was experimentally contaminated with naturally occurring fungi by allowing flasks with meal (19% moisture) to mold for a 6-week period a t 31°C. and 78% relative humidity. The predominant fungi on the soybean meal were Penicillium sp. and the Aspergillus glaums group. Some of the moldy soybean meal containing the mycotoxins was extracted with ethanol. The residue from this extraction, the moldy residue, and the moldy extract were fed to 1-day-old turkey poultti. Comparison of growth rate of these birds was made with a control diet of nonmoldy soybean and the ethanol extract of control soybean meal. Typical composition of the diet used in these studies is shown in Table XIV and the biological response in terms of growth rate of poults on the extracted and nonextracted moldy soybean diets, moldy residues, and extracts is shown in Table XV. The level of moldy soybean diet fed a t 35% is somewhat higher than the level of moldy peanut meal a t 20%

222

H. F. KRAYBILL AND M. B. SHIMKIN

TABLE XIV OF TYPICALDIET COMPOSITION (MOLDYSOYBEAN MEAL FOR POULT STUDY)' Sorghum grain (milo) Soybean meal Steamed bone meal NaCl MnS04H20 Corn oil DbMethionine Choline chloride Vitamins/100 g. Vitamin A Vitamin D Menadione a-Tocopherol Thiamine . HCl Riboflavin Pyridoxine HCl Calcium pantotenate Niacin Biotin Folic acid Vitamin BIZ Inositol a

58.0 35.0 2.0 0.5 0.1 0.5 0.2 0.3

3000 I.U. 400 I.U. 0.75 2.00 1.00 1.oo 1 .oo

4.00 2.00 0.02 0.20 0.01 10.00

According to Richardson et al. (1962).

TABLE XV ON VARIOUS FRACTIONS GROWTHRATE OF POULTS FROM CONTROL AND MOLDYSOYBEAN MEALS(SBM)O Number of poults Group No.

b

Fractions of soybean meal

Initial*

Final

k.1

Control SBM Moldy SBM Control residue Moldy residue Moldy residue moldy extractc Control SBM control extractc Control SBM moldy extract"

20 20 10 10 8 9 19

20 19 I0 5 6 9 19

240 73 131 118 98 238 292

+ + +

4

Av. gain in 3 weeks

According to Richardson et al. (1962).

* Initial = Number surviving one week.

.Fed at equivalent of three times 35% soybean meal. Extract = Ethanol extracted material from meal; Residue = Meal remaining after extraction (CZHSOH).

223

CARCINOGENESIS AND CONTAMINATED FOODS

used by the British workers in previously mentioned studies. It is apparent that the moldy diet and the fungal metabolites in the diet extract have adverse effects on the poults. More extensive extraction with chloroform and petroleum ether might have effected complete removal of toxins, thus resulting in a different response for the moldy residue. The addition of 0.8% lysine to the diet, or 0.8% lysine and 1.0% arginine, produced a dramatic improvement in growth rate, as shown in Table XVI. Arginine is not as critical as lysine. Richardson et al. (1962) TABLE XVI INFLUENCE OF LYSINEAND ARGININESUPPLEMENTATION ON CONTROL AND M.OLDYSOYBEAN MEALSIN DIETS FOR POULTSWEEK FEEDING STUDY)' Control Amino acids (%) None Lysine 0 . 6 Lysine 0 . 8 Lysine 1 . 2 Arginine 1 .O Arginine 1 . 5 Lysine 0 . 8 + arginine 1 . 0 a

b

Moldy SBM

Series No.

No. of poults

Gain (g.1

No. of poults

Gain

1 2 3 4 5 6 7

10 8 9

510 535 50 1 501 519 515 50 1

9 8 10 10

223 41 1 518 46 1 238 399 543

9

8 8 5

7 8 9

k.)

According to Richardson et al. (1962). Optimum levels to correct lysine unavailability and correct balance of amino acids.

also demonstrated that moldy soybean diets were too low in lysine or arginine, particularly the former, which is a limiting factor. For example, control (nonmoldy) soybean meals had an arginine level of 38.3 mg./g. and a lysine level of 23.7 mg./g., as contrasted with a level in moldy soybean meal of 24.8 mg./g. arginine and 18.2 mg./g. lysine. This reduction in amino acids, particularly lysine, is critical with respect to the biological response. Previous reference was made to the fact that for growth and metabolism of fungi certain amino acids are essential in the production of the toxin or fungal metabolites. Apparently the utilization of these amino acids by the fungal agent depletes the dietary protein of these limiting amino acids. To what degree the reduction of the level of amino acids influences hepatic function or detoxification in the liver has not a t this moment been established. Further evidence on the significance of the lysine level in the diet and its reduction through fungal contamination is illustrated by the data in Table XVII. Here i t is noted that the level of lysine remaining in soybean

224

H. F. KRAYBILL AND M. B. SHIMXIN

TABLE XVII LYSINEREQUIREMENT FOR POULTS AND LEVELFURNISHED BY MOLDYDIETS" Component

Per cent of diet

Lysine requirement Lysine from sorghum g r a h Lysine from control SBM Lysine from moldy SBM Lysine supplementation for good growth on moldy SBM Lysine level deficient in control sorghum moldy SBM

+

0

i1:;:;""

0.830 0.637 0.80 0.289

1.004

According to Richardson et al. (1962). According to filmquist (1952).

meal after mold contamination is significantly below the lysine requirement for growth in poults (Almquist, 1952). The level of lysine in the control sorghum and moldy soybean meal was deficient by about 25% (Table XVII). Apparently the role of amino acids is important in the pharmacological activities of compounds formed during fungal contamination and metabolism. As will be described later, aflatoxin is a lactone. Haynes (1948) has stated that the unsaturated y- and &lactones have marked pharmacological activity. The activities of the lactone include inhibition of growth of animal tissues (as noted in turkey studies above), antibiotic activity, inhibition of germination of seeds, and inhibition of plant growth. Alanine and glutathione had a protective influence and cysteine was antagonistic to 6-hexenolactone (Haynes, 1948). Cysteine counteracted the biological effect of some lactones through its action on the essential thiol groups in the organism affected. These substances are believed to exert their effect on cellular proliferation through these thiol groups in the amino acids or proteins available in the biological system (Dickens and Jones, 1961). Further biochemical support on the role of amino acids in this protective action may be forthcoming through metabolic studies and work underway on liver enzyme measurements (Allcroft and Carnaghan, 1963a). The mechanisms of toxicity, carcinogenicity, and liver detoxification may be elicited through metabolic studies on labeled aflatoxin since the structure of this compound is now known (Asao et al., 1963). The preliminary work of Richardson et al. (1962) on lysine and arginine in reversal of growth inhibition has focused attention on the efficacy of the amino acids in these biological effects relating to fungal metabolites. Obviously, nutritional factors play more than a minor role in the pharmacological action of mold contaminants, and their metabolic activity with respect to mechanisms of carcinogenesis needs further exploration. By

CARCINOGENESIS AND CONTAMINATED FOODS

225

competitive inhibition, these amino acids, particularly those with thiol groups, could indeed be limiting the tumorigenic response. The prevalent fungi that contaminate food and produce their metabolites have not been extensively studied with respect to their concentration in man’s dietary. Undoubtedly in some conditions of processing and storage of foodstuffs for domestic animals, fungal contamination and growth are not carefully controlled. Selection and control of man’s food is obviously more rigid, but certainly fungal metabolites must develop in those foods in which molds and fungi have been identified. The fact that Allcroft and Carnaghan (1963a) noted that aflatoxin was present in milk of cows fed moldy peanut meal emphasizes the fact that certain food products are potential sources of mycotoxins. Therefore, an assessment of this hazard to man through epidemiological studies is desirable. Biocheimcal and nutritional factors assume an important role in the epidemiological assessment of the pharmacological and carcinogenic activity of these natural products encountered in man’s environment.

E. ISOLATION AND CHARACTERIZATION OF AFLATOXINS Crude crystalline aflatoxin can be obtained from synthetic culture (Czapek’s Dox medium plus zinc sulfate) by direct extraction, after permitting good fungal growth and toxin development. After extraction with chloroform the solvent-suspended material is chromatographed on thin layer silica gel. Wogan et al. (1963) used A . flavus (strain 3734/10) grown on sterilized crushed wheat as substrate and then extracted with chloroform and precipitated the toxins with petroleum ether. There are varying reports on the spectrum of compounds which fluoresce under ultraviolet light (UV) (365 mp). Smith and McKernan (1962), using thin layer silica gel chromatograms, report 12 distinct spots observed under UV light, while Wogan et al. (1963) and Asao et al. (1963) report 15 discernible fluorescent components; there are two strong bands, one of which is blue fluorescent and the other yellow-green fluorescent. The most intense bands have R f values of 0.7 and 0.6, according to Sargeant et al. (1963). For these major bands, Wogan et al. (1963) report R f values of 0.75 and 0.69 for the blue fluorescent compounds and refer to them as compounds B, and B,. The green fluorescent compounds with R f vaIues of 0.59 and 0.52 are reported as the GI and G2 compounds. Nesbitt et al. (1962) separated the aflatoxin B (B, and B,) and G (G, and G,) by countercurrent distribution in CHCl,/CCl,/H,O/CH,OH, 2:2.5:1:3 by 200 transfers. The British workers first determined the empirical formulas of the B and G compounds. These formulas, their fluorescence, and their LD,,

226

H. F. KRAYBILL AND M. B. SHIMKIN

values, based on duckling bioassay, are shown in Table XVIII (Sargeant et al., 1963; Asao et al., 1963). TABLE XVIII TYPEOF FLUORESCENCE, EMPIRICAL FORMULA, AND LDao VALUES(DUCKLINGS) FOR AFLATOXINS B AND G Toxin

Formula

Fluorescence

Aflatoxin B Aflatoxin G

C17Hiz06 CuHi207

Blue Green

a

LDso values (pg.) 20a 6oa

28. 2b 90.06

According to Sargeant et al. (1963). According to Asao et al. (1963).

In investigating the structure of aflatoxin B and G, the British investigators (Nesbitt et al., 1962; Sargeant et al., 1963) determined the empirical formulas for the B compound as C17H1206 and the G compound as CI&& ; they obtained other physicochemical data which suggested that the compounds had a methoxy group, some carbonyls, and a lactone function. I n April, 1963, Asao et al. (1963) at the Massachusetts Institute of Technology made the first breakthrough on the structure of these compounds. Based on data shown in Table XIX and comparison of the nuTABLE XIX CHEMICAL AND PHYSICAL PROPERTIES OF AFLATOX~NS B AND Ga Physicochemical valuesb

Compound B

Compound G

CtiHizOc 65.6 4.4 30.9 312

CnH1207 61.8 4.0 34.4 328

268°C. (dec.) - 558"

244-246°C. (dec.) -556"

223, 265, 362 mp 1760; 1665; 1630; 1600 cm.-' 25,600; 13,400; 21,800

243, 257, 264, 362 mp 1760; 1695; 1630; 1595 cm.-' 11,500; 9,900; 10,000; 16,100

Elementary analysis Carbon (%) Hydrogen (%I Oxygen (%) Mass spectrometric data (mol. wt.) Melting point Optical rotation [LYIDCHC'S

UV and infrared spectrum X!d,.EtOH VM,.CHC'*

Molar extinction coefficient

According to Nesbitt et a2. (1962), Sargeant et al. (1963), and Asao et a!. (1963). Values of British investigators (Sargeant et al., 1963) differslightly from more recent American work (Asao et at., 1963). b

CARCINOGENESIS AND CONTAMINATED FOODS

227

clear magnetic resonance spectrum of a reduction product of aflatoxin B with a synthetic analog of coumarin, they arrived a t the structure of aflatoxin B. They reasoned that this compound B contained a carbonyl group in a five-membered ring and was cross-conjugated with the a,Punsaturated lactone function. The principal difficulty arose in the nature of the sixth oxygen atom and its location in the ring structure. From the nuclear magnetic resonance spectrum they concluded that two furan rings must be present attached to a coumarin nucleus. Accordingly, aflatoxins B and G are similar with the exception that the G compound has on the extreme right a six-membered ring with oxygen in the ring, whereas the B compound has a five-membered ring with no oxygen inside the ring. The structures for aflatoxin B (I) and G (11),according to Asao et al. (1963), are presented in Fig. 2.

Aflatoxin

B

G FIG.2. Structures of aflatoxins B and G.

Aflatoxin

The yields of crystalline material, as reported in the literature, varies from one investigator to another, depending on the potency of the A .

228

H. F. KRAYBILL AND

M.

B. SHIMKIN

flavus strain, the culturing technique, the substrate on which grown, and the solvents used for extraction. Sargeant et al. (1963) obtained a yield of 20 mg. of aflatoxin from 30 kg. of toxic meal, with an unrecovered 40 mg. in the mother liquor. Wogan and co-workers (1963) reported a yield of 300 mg. of aflatoxin from 100 kg. of fermentation broth, or 40 mg. of active B and G compounds from growth of A . flavus on 250g. of ground wheat. From a bioassay of aflatoxin B (I), Asao et aE. (1963) calculated the LD,, value on ducklings to be 28.2 pg. and for the G (11) compound to be 90 pg. These differ slightly from values of 20 and 60 pg., respectively reported by Sargeant et al. (1963) , shown in Table XVIII. Reduction of compound B leads to removal of the carbonyl group in the five-membered ring of structure I. In a dosage of 50 pg. of this reduced compound to ducklings there were no mortalities, whereas the same dose of the original aflatoxin B resulted in 100% mortalities (Asao e t al., 1963). Extensive investigations have been conducted on the carcinogenicity of lactones; hence further studies on analogs of the aflatoxins B and G which have the lactone function are anticipated (Dickens and ,Jones, 1961). The biological response (toxicity and carcinogenicity) of a series of compounds having this basic coumarin or furocoumarin nucleus is likewise expected for further study. Whether these compounds, B and G, or other fungal metabolites closely related to aflatoxin are the primary carcinogens or, are, precursors of the liver carcinogens is not known a t this time. Clarification on this point will have to await additional metabolic studies utilizing labeled aflatoxin. F. OTHERFUNGAL METABOLITES AND ASSOCIATED COMPOUNDS Quite recently, Perone and co-workers (1963) reported on the isolation and bioassay of metabolites of a fungus that attacks celery, resulting in a disease called “pink rot.” The fungus causing the disease is Selerotinia sclerotiorum. On contact with skin of animals or man and subsequent exposure to UV light or sunlight, the fungal product produces a severe dermatitis. The compounds isolated and identified are 8-methoxypsoralen and 4,5’8-trimethylpsoralen. Normal celery extracts bioassayed on rabbits gave a negative dermatological response; hence these photoreactive furocoumarins were not present originally. These compounds are similar structurally to aflatoxin, since there are in common a furocoumarin nucleus, a lactone and carbonyl function, and also a methoxy group (Fig. 2). No evidence is available, to our knowledge, on the carcinogenic activity of these psoralen compounds, which are highly toxic and may be concentrated in foods. According to the criteria of Dickens and Jones (1961),

CARCINOGENESIS AND CONTAMINATED FOODS

229

these compounds have the lactone ring, with double bonds properly positioned to fulfill the chemical constitution requirement for carcinogenicity. Furthermore, the lactone portion of the molecule is a six-membered ring, and in this respect is similar in structure to aflatoxin, which exhibits carcinogenic properties. Another phytotoxic metabolite from a species of Penicillium (PeniciElium urticae Bainer) has been isolated from subsurface tilled plots showing reduced wheat growth (Norstadt and McCalla, 1963). From melting point data, infrared and ultraviolet spectra of the crystalline material, this substance was identified as patulin or clavacin. This antibiotic, whose structure is shown in Fig. 4,is also a lactone and is derived from the metabolism of a number of fungi, e.g., A . clavatus, A . claviforme, Penicillium patulum, P . expansurn, P. melenii, P . leucopus and G y m n o a s a s sp., according to Dickens and Jones (1961). Patulin, administered to male rats subcutaneously twice a week, in the right flank, at a dose of 2 mg. per injection, caused histologically malignant sarcomas a t site of injection. The minimum time required for development of tumor was 62 weeks from start of injection. One tumor grew when transplanted into young male rats. Patulin will develop under certain ecological conditions on plants during normal or above-normal rainfall and good organir substrate or soil conditions. Since this metabolite appears on cereal crops its significancp as an environmental contaminant is obvious. Although the concentration of patulin in field crops and the ultimate level in foods and animal feeds has not been determined, its biological significance as a health hazard is evident from the work cited above. Another compound which fluoresces a greenish blue under UV light has recently been reported by Stob et al. (1962) as a metabolite of Fusarium graminearurn or Gibberella zeae through culturing on corn. This fungal metabolite produces marked uterotrophic activity in swine. Its structure has not been elucidated and its carcinogenic activity has not been measured. One of the highly active carcinogenic and mutagenic agents is ,&propiolactone (3-hydroxypropionic acid lactone) which possesses antibacterial and fungistetic properties. It has been used for sterilizing plasma and arterial grafts (Dickens and Jones, 1961 ; Rains et aZ., 1956; Hartman et al., 1954). Roe and Salaman (1958) first suggested its possible hazard as a carcinogenic agent in such clinical uses. I n the sterilizing procedure none of the propiolactone remains unhydrolyzed and no hazard may prevail since the hydrolysis product is p-hydroxypropionic acid. Walpole et al. (1954) have suggested that the original lactone and not the hydrolysis or reaction product is the carcinogenic agent.

230

H. F. KRAYBILL AND M. B. SHIMKIN

The chemical reactivity of p-propiolactone is high, owing to the highly strained four-membered lactone ring (see Fig. 4). This configuration is responsible for its high activity as a carcinogenic lactone. This relatively simple cyclic aliphatic compound bH,CH,CO.b, has been shown to produce sarcomas on injection in rats, as shown in Table XX (adapted from Dickens and Jones, 1961). Using 1 mg. of this compound in arachis oil, Dickens et al. (1958) and Dickens and Jones (1961) showed that after 44 weeks’ treatment, 10 tumors developed in 10 survivors with the first tumor appearing in 29 weeks. Also shown in Table XX are the relative carcinogenic activities of patulin (which have been previously described), penicillic acid, penicillin G, 4-hex-2-enolactone, and the condensation product of cysteine and ppropiolactone, namely, S-2-carLoxyethyl-~-cysteine. Repeated injections of penicillic acid for about 50 to 64 weeks produced tumors in rats which are histologically similar to those observed for patulin and P-propiolactone (Dickens and Jones, 1961). Penicillin G gave rise to two local fibrosarcomas after continuous administration for 70 weeks, one of which grew well as a subcutaneous transplant in 6 female rats inoculated. The observation time was about 100 weeks. Midway in activity between the highly reactive patulin and penicillic acid (the antibacterial agents) and penicillin G are two other lactones, 4-hex-2-enolactone and 4-hex-4-enolactone. After 100 weeks’ observation following 54 to 64 weeks’ treatment with 1 to 2 mg. of compound in oil, they produced 2 out of 4 and 3 out of 5 tumors respectively (Dickens and Jones, 1961). Some investigations have been conducted on the interaction of lactones with proteins, utilizing plasma and egg albumin, and the amino acids, particularly cysteine (Dickens et al., 1956; Dickens and Jones, 1961). This interaction of the lactone with the thiol group counteracts the biological effect of the lactone group and indeed has significance, as previously mentioned, as a selective inhibitory effect (Hauschka e t al., 1945). The detoxification of these compounds and reduction in carcinogenicity is suggested on the basis of potential in vivo reactions. A schematic presentation of the reaction of a typical lactone P-propiolactone and cysteine in Fig. 3 shows that end products S-p-carboxyethyl cysteine or hydracrylic acid may be formed. The above named compound, a reaction product of highly active P-propiolactone and cysteine, is indeed only a very weak carcinogen (Table XX), thus showing the efficacy of sulfhydry1 compounds in reduction of carcinogenicity. The requirement for further research on these nutritional factors through study of reaction mechanisms involved in detoxification and inhibition of carcinogenesis of patural products is implied from these preliminary studies.

TABLE XX LACTONES AND RELATED SUBSTANCES WHICH HAVEBEENBIOASSAYED FOR CARCINOGENIC ACTIVITY IN THE RAT THROUGH REPEATED SUBCUTANEOUS INJECTIONS' Structure No. (Figs. 2, 3, 4)

V

M

VII VIII IX X XI XI1 IV I or I1 a

c3

ti G

Compound injected

Dose per injection (in oil)

Treatment time (weeks)

First tumor (weeks)

Tumors per survivors

Timeof observation (weeks)

Control arachis oil Control arachis oil &Propiolactone Patulin Penicillic acid Penicillin G (Sodium salt) 4-Hex-2enolactone PHex-4+ziolactone a-Angeliea lactone 7-Butyrolactone 8-2-Carboxye thyl-ccysteine Aflatoxin B or G

0 . 5 ml. 0 . 5 ml. 1 . 0 mg. 0 . 2 mg. 1 .O mg. 2.0 mg. 2 . 0 mg. 1 .O mg. 2 . 0 mg. 2 . 0 mg. 0 . 5 mg. 0.006 mg. (fed)

54 61 44 61 64 46 64 58 61 61 52 26

-

0/6 0/3 10/10 4/4 4/4 1 /4 2/4 3/5 0/5 0 /5 1/1 5/6

54 107 44 69 67 100 102 99 100 100 87 26

-

29 58 48 59 79 63 87

-

3 8 5

E

* 0

0

2

E

zw U

r

0 0 U

m

Data from Dickena and Jones (1961) and Lancaster et al. (1961).

N

c3

232

H . F. KRAYBILL AND M. B. SHIMKIN

GENERAL REACTION

R.S.

1 + 1

H

___

CHz-CH2

I

R.S.CHz-CH2

I

O-C=O

COOH

Protein or Amino Acid f B-propiolactone REACTION POSSIBILITIES

THZ

(A) HOOC.CHCH2.7

+

HOOC.~HCH,.S

FH2-YH2

I //o

__c

H (cysteine)

0-C=O

t

HO.CH2CH2.C

(lactone)

111

(thioester)

II

Hydrolysis

HOOC.CHCH2.SH t HO.CH2CH2.COOH

I

(Hydracrylic acid)

NH2 NH2

I

(B) HOOC.CHCH,.S

I H

t

CH2-FH2

I

O-C=O

-

NH2

I

HOOC.CHCH2

HOOC.CH2.CH2

\

s /

IV

S-B-carboxyethylcysteine

FIG.3. Mechanism of action of carcinogenic lactones with proteins and amino acids.

The presence of lactones in natural products through fungal contamination with aflatoxin, patulin, penicillic acid, and others in plant materials or other biological sources of food suggests the need for a search of other naturally occurring lactones. With reference to the problem of smoking and health, Dickens and Jones (1961), for example, havc suggested that a search for lactones in tobacco smoke and bioassay of these compounds might be further explored. Carcinogenic activity of the lactones is conditioned on chemical structure, according to Dickens and Jones (1961). From their studies they set forth some structure criteria to assist in such an evaluation. They found, for example, that the highly strained four-membered ring, as exemplified in P-propiolactone and the lactam ring of penicillin, is significant in carcinogenic activity. Similarly, double bonds a t the 2- or 4-position appear critical for carcinogenic activity of lactones. Lack of a double bond in the lactone (or double bonds not properly positioned) tends to render the compound noncarcinogenic. There is a lack of data in the literature on the activities of six-mem-

233

CARCINOGENESIS A N D CONTAMINATED FOODS

bered ring lactones, since the compounds studied thus far are of the fourand five-membered ring-type structure. However, the current data on aflatoxin, a six-membered ring lactone, a t least provides some information on these structures (Fig. 2 ) . The structural requirement and criteria for carcinogenic activity are illustrated in Fig. 4. (A) CARCINOGENIC

I f" 0-c=o

CH2-

2

VI

V

VII

(CHJ)2f-CH.CO0.Na.

HC-

\

c; ; "

=0

C2H5

CH

I \ -c ~ o / c O

CH,HC=

2cI

Cljo,Cn

Y 2

0

L

x

IX

VIII

(B) NONCARCINOGENIC

CH-

I1

CH2 \

C H 3 F o ~ cO = XI

CH2-

I

CH2

CH

\20/

\

C=O

XI1

FIG.4. Relation of structure to carcinogenicity.

Other groups of natural products that have been investigated extensively in Japan are the several actinomycins (C, D, S, and L) and the fungal metabolites from contaminated rice, essentially those developed by Penicillium islandicum Sopp. The exploration of the mycotoxic effect

234

H. F. KRAYBILL A N D M. B. SHIMKIN

of the latter substances on the liver assumes special importance in view of the significantly higher incidence of malignant hepatoma and liver cirrhosis among the Orientals and some natives of Central Africa. According to Kobayashi et al. (1959), tumor-like enlarged livers were observed as early as 1918 in pigeons fed a few months on rice contaminated with P. commune. Miyake et al. (1959) reported briefly on some work with yellowed rice, contaminated with P. islandicum; primary hepatic carcinoma developed in rats through long-term feeding of this material. I n addition, the influence of these fungal metabolites on dimethylaminoazobenzene (DAB) carcinogenesis in rats was also studied. More extensive studies were later pursued by Miyake et al. (1960) on the toxic liver injuries and liver cirrhosis induced in mice and rats through long-term feeding of rice contaminated with P. islandicum Sopp. Kobayashi e t al. (1959), in feeding about 8010g. of P. islandicumcontaminated rice to Wistar rats (Table XXI, group A ) for 600 days, TABLE XXI THE I N F L U E N C E O F Penicillium i S h d i C U m - C O N T A M I N A T E D RICE DIET ON TUMOR INDUCTION AND DEATH OF RATP Groups on feeding experiment ~~

~

A

Conditions

B (moldy diet (moldy diet) i0.06% DAB)

Approximate amount of material consumed 8010.0 Moldy diet (g.) DAB k.1 Control diet (g.)

+

C (control 0.06% DAB)

-

8010.0 4.5 -

4.5 9000.0

Initial number of Wistar rats

30

30

30

Mortality data and life span Deaths (12-89 days) Deaths (235-396 days) Deaths (532-622 days)

12 2 13

8 13 1

17 9 -

Survivors after 600 days

3

8

4

Animals with tumors

5

11

6

Q

Adapted from Kobayashi el al. (1959).

observed 2 rats with primary hepatic carcinoma confirmed histologically and 3 rats with sarcoma. Metastasis of tumor to the lung was noted. By the use of a moldy diet plus DAB (total intake 4.5 g.) these investigators noted an increase in malignant tumors to 11, all primary hepatic car-

CARCINOGENESIS AND CONTAMINATED FOODS

235

oinomas (Table XXI, group B) . In utilization of a control diet (unpolished sterile Japanese rice, casein, vitamins, and salts) plus 0.06% DAB, where 4.5 g. of this compound was consumed by rats in 600 days, the tumor rate (6 hepatomas per 30 rats) was about the same as when moldy rice diet alone was fed (Table XXI, group C) , The lifetime of rats dying from carcinoma in B and C groups was slightly longer than that usually observed for rats developing DAB carcinoma, The lifespan of the B group (Table XXI) which showed the highest tumor incidence was shortened by about 100 days, compared t o the C group; and by 300 days compared to the A group, in which only moldy diet was fed (Kobayashi et al., 1959). These data establish the toxic and carcinogenic properties of more fungal metabolites and their promoter effect when used in combination with DAB, in inducing the formation of carcinoma in rat liver. I n describing the liver injuries and cirrhosis in mice and rats produced by P. islandicum Sopp. strains Ea and u d , Miyake e t al. (1960) stated that histologically there is centrolobular necrosis with fatty metamorphosis of the remaining liver cells a t the beginning, with ultimate fibrosis and bile duct proliferation. They have associated the toxic effects on the liver to two compounds; one a chloride-containing peptide (white needles with melting point 251-252°C.) stable to proteases and peptidases, and the other a pigmented substituted anthraquinone compound [yellow needles, melting point 273°C. and [ = -880" (in acetone) 1. The structure of the substance, called luteoskyrin, is shown in Fig. 5 (formula X I I I ) . By growing P. islandicum Sopp. Ea and Ud strains in Czapek media or on rice grains identical chromatograms indicated the presence of the

LUTEOSKYRIN

FIG.5. Structure of luteoskyrin.

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H. F. KRAYBILL AND M. B. SHIMKIN

following fungal pigments (a) islandicin, ( b ) iridoskyrin, (c) rubroskyrin, ( d ) skyrin, (e) luteoskyrin, and (f) erythroskyrin. Since the predominant pigment is the yellow toxic component in culture media, Miyake et al. (1960) reasoned that luteoskyrin was the principal toxic agent, since 1 g. of this compound was recovered from 150 g. of moldy yellowed rice. Further work should be done to establish the specific carcinogen or carcinogens from this array of pigments and the chlorine-containing peptide. Actinomycin, an antibiotic first isolated by Waksman and Woodruff (1940) , and highly active against Gram-positive bacteria, has been shown to have carcinostatic properties (Umezawa, 1955; Nishibori, 1956). Indeed, the carcinostatic activity of actinomycin L is about one third that of actinomycin S, according to Kawamata et al. (1959). Since it is recognized that antitumor substances may also be tumorigenic, Kawamata et al. (1958, 1959) studied the carcinogenic activity of actinomycins A, S, and L on various strains of mice. It is evident that the amount of drug administered greatly influences tumor production, as shown in Table XXII. Actinomycin L proved less effective than actino-

TUMOR PRODUCTION

Stock or strain Btk Ctk Btk Btk Btk Btk Btk Btk Btk Swiss albino Swiss albino Ctk Ctk Btk Btk 0

Sex

BY

TABLE XXII ACTINOMYCINS INJECTED Actinomycin compound injected A A L L L S S S S S

S S S

Solvent Solvent

I N VARIOUS STR.41NS OF b'fICEa

Dose, twice weekly (ficg./k@;.) 22.5 22.5 9.0 22.5 67.5 7.5 7.5 15.0 30.0 7.5 7.5 7.5 7.5 0 . 1 ml. 0 . 1 ml.

Tumor appearance time (weeks)

Tumor incidence ratio

22 28

10/10 4/8

35 21 25 20 16 26 40 25 20

0/8 0/5 5/8 9/10 9/10 7/8 10/10 1 /10 1 /8 9/9 7/8 0/7 0/9

According to Kawamata et al. (1958, 1959).

nycin S in producing tumors in mice. As much as 67.5 pg./kg. body weight of mice tested was required for the L compound to produce sarcoma in

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5 of 8 mice (Table X X I I ) . However, with actinomycin S only 7.5 pg./kg. produced sarcomas in 9 out of 10 male and 9 out of 10 female Btk mice. The effect of S compound on Swiss albino mice was less marked, showing a strain difference. All lesions and subsequent sarcomas were localized at the site of injection. With an advanced stage the sarcoma appeared as a massive tumor with pronounced rim ranging from 0.8 to 3.0 cm. in diameter (Kawamata et al., 1959). The reason for sarcoma production with actinomycin is not clear. It is known, for example, that 3-hydroxyanthranilic acid and 3-hydroxykynurenine produce cancer of the bladder in mice (Allen e t al., 1957). It is conceivable, according to Kawamata et al. (1959) that these compounds might act as carcinogens either as such or as their condensation products, the phenoxazone derivatives. Phenoxazone is an important moiety of actinomycin. For visualization of the structures of the series of actinomycins the reviewer is referred to the comprehensive listing of Miller (1961). Another possibility advanced by Bergel (1958) is that the peptide chains of actinomycin might serve as carriers for the biological activity of the phenoxazone. The variation in carcinogenic activity of the actinomycins, therefore, may reside in the different peptide chains which constitute the wide spectrum of actinomycins. Finally, the work of Kawamata and co-workers (1959) has shown that actinomycin inhibits protein synthesis, at least in microorganisms, and actinomycin combines with DNA of bacteria and calf thymus, providing further evidence as to a possible mode of action of actinomycin.

G. WORLD-WIDEHEALTHIMPLICATIONS OF MYCOTOXICOSES The mycotoxicosis problem has a world-wide significance in terms of public health, agriculture, and economics. To what degree the various metabolites from fungi represent a toxicological and carcinogenic hazard to man has not been fully explored. Most of the work in this field, as is apparent from the literature reported herein, has been focused on domestic animals. According to Berman (1951), primary hepatic carcinoma is observed a t autopsy in 1% of all Orientals, or about 14% of all carcinomas in tropical areas of the Orient or in Central Africa. I n the United States and Europe, liver cancer is found in 0.3% of autopsies, or less than 2.5% of all carcinomas. I n Japan, according to Takeda and Aizawa (1956), hepatomas represent about 1.5% of all autopsies, or about 7.6% of all carcinomas. The Japanese surveys during the period 1930-1955 (Shikata, 1959) indicate that 79% of malignant hepatoma cases by autopsy are associated with liver cirrhosis. Moreover, 43% of the cases of annular

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H. F. KRAYBILL AND M. B. S H I M K I N

liver cirrhosis detected during 1931-1954 was associated with malignant hepatoma. The potentia1 association of the high concentration of moldy diets with liver injury and liver carcinoma in man would appear inescapable from the preceding discussion on the problem of yellowed rice in Japan. From the standpoint of geographical pathology, this higher incidence of liver cancer in the rice-eating Orient perhaps has parallels in other countries with respect to toxic factors or carcinogens in the diet of man. I n the past, much significance was attached to nutritional factors in abnormal pathology of the liver, but there now is added the question of the contribution of an array of contaminants in the diet induced by natural conditions or introduced by man. At the moment, there are no data available to indicate what ill effects on man the consumption of peanuts or peanut by-products may have as the result of ingestion of aflatoxin or other mycotoxins not yet identified. One lead in this direction is the British work (International Working Party, 1962), which established that peanut oil used in margarine manufacture did not contain aflatoxin, since i t was removed after extraction by alkali treatment of the peanut oil. Some assays have been made on a few samples of peanut butter. By fluorescence testing and bioassay in the duckling, Lammers and Linde (1962) found 0.2 to 0.3 p.p.m. of aflatoxin in a few samples of peanut butter in the Netherlands. Most peanut butter samples do not contain aflatoxin because of careful selection of peanuts for products designated for human consumption. Consequently, the nonselected peanuts would be utilized for livestock feed. Fungal contamination could, however, involve a wide spectrum of cereal products or protein sources consumed by man, such a s soybeans, corn, oats, wheat, milk powders, cheese, bread, etc. No information on the biological effects of metabolites from fungal species other than Aspergillus flavus is available. Allcroft and Carnaghan’s (1963a) studies on toxic peanut meal fed to cows, referred to previously, have clearly defined the level to which the toxic factor prevails in milk, a human food product, and its effect on the liver of ducklings when bioassayed on this test animal. Schoental (1959) has drawn attention to the possibility that various liver disorders in childhood may be the result of poisoning by milk. She cites in particular those infants breast fed by mothers using medicinal herbs containing pyrrolizidine alkaloids during lactation or those who are given milk from cows that have consumed rations containing these alkaloids. Allcroft and Carnaghan (1963b) state that i t is somewhat paradoxical that a hepatotoxic agent such as aflatoxin was found to be excreted in milk of cows fed toxic peanut meal, yet none was found in the liver or blood of the cow.

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CARCINOGENESIS AND CONTAMINATED FOODS

With regard to another human food product, Allcroft and Carnaghan (196313) were unable to demonstrate any toxicity to ducklings that received freeze-dried, extracted eggs collected from pullets maintained on 15% toxic peanut meal. Further confirmation of these negative findings is anticipated by these investigators. In the United Kingdom, vital statistics are being examined. Epidemiological studies may provide some lead regarding the role of mold-contaminated foods in human disease, including neoplasms. In conjunction with the program on development of plant protein sources, one of which is peanut meal, for augmentation of milk protein in alleviation of kwashiorkor and other protein deficiencies in man in the less-developed countries, such studies assume more significance in view of the untoward effects of fungal-contaminated products. For example, moldy corn provides an important ingredient of African native diets (Quass, 1959-1960), as shown in Table XXIII. In the male

TABLE XXIII DAILYMAIZERATION OF AFRICANNATIVES“ Constituent

Amount (lb.)

Per cent of diet

5.62 1.25 1.50

67.1 15.0 17.9

Bantu prisoners Porridge (1.12 lb. maize meal) ‘‘Whole” mealies (0.5 lb. meal) Supplementary foods

Laborers in gold mine lb./cap./yr. Cereal products Maize Wheat Kaffircorn Rice

438.7 101.6 41.2

38.9 9.0 3.7

Total cereals Other foods (meat, eggs, fruit, milk, etc.)

581.5 544.5

48.3

Total

1126.0

According t o Quass (1959-1960). examination of Bantu showed 14 lb. maize porridge in stomach. Average amounts removed: 5-7 lb. High intake figure represented largely by water.

* Post-mortem

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H. F. KRAYBILL AND M. B. SHIMKIN

Bantu, hepatomas represent 68% of carcinomas (Oettle, 1956). I n considering racial, genetic, and environmental factors, Berman (1941) concluded that cirrhosis promoters, or environmental carcinogens, might potentiate the precancerous or cancerous state of the liver. Accordingly, some speculation has been advanced on association of moldy diet components with high rate of Bantu hepatomas. The age-adjusted rate for primary hepatomas per 100,OOO population is only 0.18 for Danes, 1.7 for United States whites, 3.2 for United States Negroes, but 14 for Bantus. I n considering etiological factors in this high rate of hepatomas for the Bantus, malnutrition and diet, coupled perhaps with viral agents, may have an important association; some investigators have discounted any significant association of schistosomiasis, alcoholism, or the hazard of mining as an occupation (Higginson and Oettle, 1960). Plans are under way to ascertain the possible association of the moldy diet components with the incidence of hepatomas in the African native. As first lines of attack, corn or moldy maize samples will be assayed chemically and biologically for their content of aflatoxin. Following the identification of aflatoxin or comparable toxic and carcinogenic mycotoxins in moldy maize diets, a sequential approach to the problem will be to study their metabolism in man. Since the structure of aflatoxin is known, the metabolic fate of this lactone can now be investigated and should provide information as to whether this compound is indeed the primary carcinogen or a precursor of a carcinogenic metabolite. As more of these natural fungal metabolites and lactones are discovered, their mechanism of action can also be studied. As a first step, identification of aflatoxin or its derivatives in the blood or urine of experimental animals would be desirable. If aflatoxin or a metabolite of aflatoxin is excreted in the urine, this technique might become a useful index in screening of human populations. Such studies may provide important clues to the potential origin of liver cancer mediated through dietary carcinogenesis. IV. General Discussion

The genesis of tumors may be influenced to a lesser or greater degree by the nutrient environment furnished the host. Accordingly, considerable information has been recorded on the role of nutrition and dietary factors in carcinogenesis in rodents (Tannenbaum, 1959). Whereas much significance has been attached to the association of nutrition and cancer, more recently attention is turning to the influence of food additives, processing degradation products, and food contaminants. The development of modern food processing with an appreciation of some of its benefits has also brought attention to the potential carcinogenicity of degradation

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products and food additives. Moreover, modern agricultural practices, reflected in the utilization of soil fumigants, pesticides, and herbicides, which remain as residues on fruits, vegetables, and cereal products, present further problems of environmental contamination if not adequately removed. Many of these contaminants have not been previously studied or characterized as poisons, but recently their latent effects are being assessed with respect to their potential contribution to toxicological and carcinogenic responses (Kraybill, 1963). Food consumed by man or foodstuffs utilized by farm animals represent substrates or carriers for toxic and carcinogenic agents and therefore represent major environmental sources for mediation of cancer through diet, particularly under conditions of long-term exposure. In processing of food by heat or radiation sterilization, degradation products are produced. Heat dehydration and overprocessing, such as flame-drying of fish meal, produce toxic lipid polymers (Kraybill and Nilson, 1947). I n the processing of foods for improvement of texture, flavor, and color, chemical additives are used, many of which are innocuous and quite beneficial, but a few have demonstrated toxicity and carcinogenicity, as enumerated by Kraybill (1963) in a recent review on this subject. In livestock feeds certain medicinals are added such as amebicides, ascaricides, and other drugs. These are important for their clinical efficacy, but they may have adverse effects through long-term ingestion and exposure. Inadvertently, chemicals used for deinfestation of grains and control of mites on fruits (ascaricides) and the previously mentioned fungicides, herbicides, and pesticides in soils and plants find their way into the food supply. Aramite (2-[p-tertiary-butylphenoxy ] isopropyl-2-chloroethyl sulfite) with a small contamination of 2- (p-tertiary-butylphenoxy) isopropyl dulfite, when fed to several species, produced hepatomas a t a level of 400 p.p.m. Its level of application was 1 p.p.m. in control of mites on fruits (Oser and Oser, 1960). Forgacs e t al. (1963) have demonstrated the efficacy of 8-hydroxyquinoline as an antifungal and mycotoxic agent in treatment of foodstuffs. This compound has been shown by Clayson e t al. (1958) to be carcinogenic to mice. Many other examples could be cited, but these few emphasize the necessity for cognizance of these environmental hazards. Another source of contamination that cannot be overlooked is that from bacteria and/or bacterial metabolites, especially those which have clinical and pathological significance. Salmonellosis and brucellosis are perhaps the more commonly known examples. Associated with these natural bacterial contaminants are the universally occurring molds, fungi, and their metabolites.

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Certain parasites select as their host meat animals which are intermediates in their life cycle. Fortunately this parasitic infestation, particularly in meat products consumed by man, is not as predominant as other examples of contamination, but should be cited since some countries have considered public health measures for their control. For example, trichinainfected pork and tapeworm-infected beef have been considered for gamnia radiation treatment as a control measure in the United States and England (Kraybill and Brunton, 1960). Massive neoplasia epidemics through parasitic infestation have not been overlooked in cancer research. Shimkin (1963) has referred to the bladder cancer among Egyptians, which may be secondary to Schistosoma haemotobium infestation acquired by the farming population along the Nile. The distribution of these various neoplastic entities within hunian and animal populations may have been individually recognized, but it is now necessary to focus on these vectors in relation to food as a carrier. Rapid advances in our knowledge of biochemistry, pharmacology, and toxicology have helped improve our methodology of detection and bioassay for such environmental hazards. Further impetus to improvement in methods of detection for latent or long-term toxic and carcinogenic effeck of this array of food contaminants has been created in the United States through adoption of Federal legislation on September 6,1958 (Public Law 85-929), commonly referred to as the Delaney Additives Amendment to the Federal Food, Drug, and Cosmetics Act. I n 1954, the Miller Pesticide Amendment of this act also established a procedure for setting safe amounts for residues of pesticides on fruits and vegetables. I n 1960 the enactment of the Color Additives Amendment to this original legislation duly recognized the potential carcinogenicity of coal tar colors and prevented establishment of their harmlessness by the Food and Drug Administration. The localization of tumors in rats after ingestion of a carcinogen, mediated through the injection of a noncarcinogen a t a particular site, has important and interesting implications in terms of diet contamination and cancer. This principle has been described in the recent work of Huggins and Grand (1963). They never detected sarcomas which had arisen spontaneously in an untreated rat. However, when they fed 3-methyIcholanthrene (3-MC) and gave repeated injections of 0.15 M sodium chloride, sarcomas developed exclusively a t site of injection. Compressed pellets of several aromatic compounds and of cholesterol and progesterone did not focalize sarcoma development. The most reasonable explanation of the formation of sarcoma in rats fed 3-MC and given injections of sodium chloride would appear to be localization of the carcinogenic compound at the injection site. Undoubtedly this example has its analog in

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natural situations where an unidentified carcinogen in the diet (contaminant) is localized and cancer-mediated at certain sites through another agent to which one ascribes its function as the primary carcinogen. There is some parallelism, illustrated in cases reported elsewhere, on the role of the nutrient environment in the evolution of neoplastic disease. For example, Kensler e t al. (1941) demonstrated the protective effect of riboflavin and casein against dimethylaminoazobenzene (DAB) liver carcinogenesis. Moreover, earlier work of Sasaki and Yoshida (1935) showed that unpolished rice soaked with an olive oil solution of o-aminoazotoluene (AAT) produced hepatomas in 24% of a rat population with 25% mortalities. American workers, using another source of protein, wheat, could not duplicate these findings. Kinosita (1937) in DAB carcinogenesis, using rice as protein source, obtained similar results on induction of hepatomas in rats. comparable experiments with wheat failed to reproduce these results. The conclusion reached was that good sources of high quality protein and B vitamins (specifically, B,) provided a defensive mechanism for inhibition of the formation of the neoplasm. A reconsideration of these findings in terms of the recent Japanese work on moldy rice diets and cancer (Table XXI) suggests an alternate conclusion for these results. The report of Kobayashi e t al. (1959), previously mentioned, emphasizes the prevalence of fungal toxins in sources of rice used in Japan. The utilization of rice or a comparable natural product introduces the possibility of contaminant carcinogens. Therefore, this environmental variant may have accelerated or promoted the induction of hepatomas that were observed. Conversely, if perchance wheat proteins used by the American workers had been contaminated through unfavorable storage and randomly selected, their use might have reversed the findings on DAB and AAT carcinogenesis and hepatomas would have been observed. A more striking illustration of the role of contaminants in natural products which, although unrecognized a t the time, inadvertently played a role in nutritional carcinogenesis, is the earlier work of Engel et al. (1947) related to choline-deficient diets potentiating hepatomagenesis in rats. This well-known study and findings remained unchallenged until only recently. It is indeed serendipitous that the moIdy peanut meal incident forced a reappraisal of this initial work on choline. As stated previously, the recent work of Salmon e t al. (1963) with rats and Newberne and Carlton’s work on ducklings have now ascribed the contributing role in this hepatomagenesis to the fungal metabolites in the peanut meal, the protein source in the diet. Addition of choline to the diet did not decrease the incidence of hepatomas of rats raised on a diet where peanut meal was utilized.

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Certainly these important and recent revelations on the role of diet contaminants should now suggest a reassessment of analogous situations reported elsewhere. Although there is a clearer understanding of this problem, occasional reports appear in which certain natural products are incriminated per se as potentiating the induction of cancer through diet. The use of the label “carcinogen” to any component of the diet or single food is therefore incorrect until all entities associated with the product or substrate have been defined. The two episodes considered in this chapter, the first dealing with trout hepatoma and the second relevant to mycotoxicoses and cancer, may be distinguishable in certain respects but they also have common elements. The contamination of foodstuffs or rations with ubiquitous fungi appears to be a natural event. However, the development of carcinogens in a dry ration administered to trout would appear to be conditioned by man’s control of his modern technology. Cognizance of the universality of the fungi in nature and their potential for contamination of foodstuffs or foods is now fully appreciated from the moldy peanut meal epidemic. Conversely, the action of a mycotoxin present in food must not be considered only in terms of a singular role, but also from the probability that observed toxic and carcinogenic responses may be augmented by other environmental contaminants. Much emphasis was placed on the processing of commercial trout rations on the assumption that peroxidation and lipid polymers were involved in hepatoma induction. Some investigators, particularly Ghittino and Ceretto (1962), believe that the carcinogenic process is initiated through absence of vitamin E or antioxidants and presence of lipid peroxides or polymers which hastens lipoid degeneration of the liver and ultimate development of liver cancer. With equivocal evidence advanced implicating cottonseed meal as an etiological or source agent, an inescapable conclusion now prevails that processing degradation products (largely concentrated in fish meal) may not be solely responsible for the genesis of this neoplastic disease. Other factors have been considered. The presence and influence of pesticides, defoliants, or fungal metabolites, particularly as cottonseed meal contaminants, must therefore be evaluated. Distillers’ solubles also used in rations have contributed to some observed hepatomas and have shown some fluorescence, which would suggest fungal metabolites. What role and to what degree these vectors are responsible for initiation of liver cancer in trout is not clearly understood. Moreover, the role of the viruses or viral chemical action in the development of this neoplastic disease has not been adequately explored. The lipid or neutral lipid fraction of the trout ration may have a lipid degradation product as the etiological agent, but the possibility now re-

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mains that other carcinogenic compounds may be solubilized and carried through in this fraction. Some of the fungal metabolites are lipid and solvent soluble, as are some of the pesticides, and these compounds could appear in the lipid fraction. With respect to the role of mycotoxic effects, the synergistic action of other compounds must be similarly studied. The Japanese work cited herein, which showed the influence of moldy rice metabolites on acceleration of DAB carcinogenesis, is certainly an important phase of research in this direction. The antagonistic action or competitive inhibition of other compounds with mycotoxins must be similarly evaluated in terms of the total response. The influence of certain amino acids such as cysteine, lysine, and arginine in suppression of biological action of lactones, and reversal of effects of moldy diets on growth retardation and liver damage are illustrations which suggest an extension of research in this area. A clearer understanding of the mechanisms of action of the array of environmental contaminants in food will have to await further studies on isolation, identification, and assessment of their biological responses. A major advance has been made in the isolation and structural identification of aflatoxin; other compounds similarly characterized are anticipated for further study. Isolation of carcinogenic fractions from commercial trout rations has equal significance in explaining the mechanisms of hepatoma induction. Rapid progress is likewise expected in this area of research. Huge industrial enterprises are founded on fungus-based chemistry, yet the discovery of toxicity in moldy peanut meal was serendipitous in that i t revised our attitudes on the pathogenicity of the fungal metabolites. Admittedly, our knowledge of these nonphotosynthetic members of the plant kingdom is very incomplete, as is true for the pathogenic fungi and their metabolites which are clinically observed in diseases of man. Estimates of the number of fungal species in nature may vary from 40,000 to 250,000. While many fungi are presumably not harmful, the striking effect of one of these, aflatoxin, has stimulated some thoughts on the possible role of other mycotoxins which affect animal and human health through the induction of cancer. A wide field for exploration has evolved as a result of these developments on the role of man-made and naturally occurring agents in the diet of animals and man which initiate the neoplastic disease process. There is also little doubt that complex and unknown integrative effects are possible with respect to the action of the wide spectrum of toxins and carcinogens t o which animals and man are exposed. Therefore the chemistry, toxicology, and carcinogenicity of these many natural products must now be fully investigated to ascertain their contribution to the problem of environmental carcinogenesis confronting animals and man.

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EXPERIMENTAL TOBACCO CARCINOGENESIS

.

Ernest 1 Wynder and Dietrich Hoffmann Division of Environmental Carcinogenesis. Sloan-Kettering Institute for Cancer Research. N e w York. N e w York

We dedicate this communication to the American Cancer Society. whose support of our studies. as well as of other investigative groups. has contributed to much of the progress made in the field of experimental tobacco carcinogenesis . I . Historical Aspects . . . . . . . . . . . . I1. Objective of Laboratory Studies . . . . . . . . . I11. Some Characteristics of Tobacco and Tobacco Smoke . . . A . Tobacco Products . . . . . . . . . . . . B . Physical State of Tobacco Smoke . . . . . . . . C . Smoking Machines and Smoking Techniques . . . . . D . Preparation of Tobacco Smoke . . . . . . . . E . Tobacco Extracts . . . . . . . . . . . . F. Combustion and Smoke Temperatuoes . . . . . . IV . Biological Tests for Tumorigenic Activity . . . . . . . A . Tobacco Smoke Condensate . . . . . . . . . B . Tobacco Extracts . . . . . . . . . . . . C . Tobacco Smoke . . . . . . . . . . . . D . Tobacco Smoke Condensate Fractions . . . . . . E . Studies of Special Factors . . . . . . . . . . F. Cilia-Toxic Components . . . . . . . . . . V . Certain Constituents of Tobacco Products . . . . . . . A . Smoke Condensate Yields . . . . . . . . . . B . Polynuclear Aromatic Hydrocarbons (PAH) . . . . . C . Terpenes, Phthalates, and Certain Esters . . . . . . . . . . . . . D . Paraffinic Hydrocarbons (Alkanes) E . Heterocyclic Nitrogen Compounds . . . . . . . . . . . . F . Phenolic Compounds and Carboxylic Acids G . Aldehydes and Ketones . . . . . . . . . . H . Steroids . . . . . . . . . . . . . . I. Epoxides, Peroxy Compounds, and Lactones . . . . . J . Nitrosamines . . . . . . . . . . . . . K . Volatile Components in Tobacco Smoke . . . . . . L . Electric Charges, Radicals, and Radioisotopes in Tobacco Smoke M . Arsenic . . . . . . . . . . . . . . N . Metallic Constituents . . . . . . . . . . . 0. Summary . . . . . . . . . . . . . VI . Reduction of Tumorigenic Activity . . . . . . . . A . Reduction of Total Smoke Condensate . . . . . . 249

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B. Reduction of Tumorigenic Agents . C. Reduction of Cilia-Toxic Agents . VII. Interpretation of Experimental Findings A. “A Complete Carcinogen” . . . B. Inhalation Studies . . . . C. Statistical Considerations . . . D. Future Studies . . . . . E. Relation to Human Data . . . VIII. Postscript . . . . . . . References . . . . . . .

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I. Historical Aspects

Epidemiological observations have given much impetus to experimental studies in the field of carcinogenesis. I n the light of historical events it now seems surprising that the classical observations of Pott (1775) were confirmed by laboratory studies only after more than a century had elapsed. Similarly, Sommering’s (1795) notation of an apparent association between pipe smoking and lip cancer took more than 150 years before appropriate experimental studies were undertaken (Fig. 1).

Sic Iabii carcinoma ibi frCquentkisfin~irin, iibi Iion&ics hhilis tabacinis indulgent ; 1 , t I i i i i r i i inferius ehim solurnmado carcinornate corriljitur ,\ qutjzl‘iiinz inter fistulsm tabacinaxn et tlenir.: corn primi tur. FIG.1. “Carcinoma of the lips occurs most frequently where men indulge in pipe smoking; the lower lip is particularly affected by cancer, when it is compressed between the tobacco pipe and the teeth.” Sommering (1795), p. 109.

Brosch (1900) appeared to be the first investigator involved in experimental tobacco carcinogenesis. He applied tobacco “juices” to guinea pigs, observing epithelial proliferation. The clinical observations by Abbe (1915), linking the use of tobacco to cancer of the oral cavity, may be regarded as one of the first major stimuli for subsequent laboratory studies with different tobacco products. His description of a patient brushing snuff on her tongue is of interest in line with the subsequently used procedure of applying cigarette smoke condensate by brush to experimental animals. Abbe wrote, “One of the worst cancers of the tongue I have recently seen was in a woman. All the left half of the tongue and half of the right was cancerous. I asked her how it began. . . . ‘I have, all my life, taken a small toothbrush in my right band, dipped it in snuff, and rubbed it hard on my tongue, mostly on

EXPERIMENTAL TOBACCO CARCINOGENESIS

25 1

the left side, of course’.’’ The first squamous cell cancer in the experimental animal obtained with “tar” extracted from tobacco pipes was reported by Taki (1937). F. H. Miiller (1939) found a statistically significant correlation between cigarette smoking and lung cancer. I n 1950, the publication of several large-scale epidemiologic studies (Levin e t al., 1950; Wynder and Graham, 1950; Doll and Hill, 1950) suggested a link between smoking and lung cancer and led to a series of laboratory studies testing the possible tumorigenic activity of tobacco smoke condensate on several animal species and tissues. Wynder e t al. (1953) reported the first extensive production of skin cancer in mice upon application of cigarette smoke condensate. These were some of the early reports that have stimulated the very extensive research efforts in experimental tobacco carcinogenesis during the last decade. II. Objective of laboratory Studies

The primary purpose of laboratory investigations in the field of tobacco carcinogenesis, a t least as i t appears to us, has been to determine whether various tobacco and tobacco smoke products might prove to be carcinogenic to different animal species and tissues, and if so, which of the Components may be held primarily responsible for this activity. Once such components have been identified, attempts might then be made to investigate the possibility of their removal or reduction. Some oncologists feel that the induction of cancer in the experimental animal with a given material indicates that this material is also carcinogenic to man, particularly if cancer has been produced in a variety of different animal species and tissues (Hueper, 1963a). In respect to human lung cancer and the experimental production of this type of cancer, a statement by the Food Protection Committee (Food and Nutrition Board of the National Academy of Sciences, 1959) may be pertinent: “In the absence of data on lung tumors, the induction of skin tumors in mice and rabbits treated repeatedly with tars from cigarettes and soots from the atmosphere and the finding of trace amounts of compounds of known carcinogenic activity in these crude mixtures become important experimental observations in support of the epidemiological inferences.” We feel that the basic proof for the correlation between tobacco usage and cancer in man rests today primarily, if not solely, on human data. These data recently have been summarized and evaluated by a special committee of the Royal College of Physicians of London (1962). The Advisory Committee to the Surgeon General of the United States released on January 11, 1964, its findings on Smoking and Health indicting

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

smoking as a cause of lung cancer. It is stated “that the magnitude of the effect of cigarette smoking far outweighs all other factors related to this disease” (U.S. Health Serv. Publ. N o . 1103, 1964). Obviously, if experimental findings support the human data, then the iniportance of both sets of data is strengthened. It is only in the laboratory, however, that tumorigenic components, antitumorigenic agents, cilia-toxic substances, either singly or their interrelationship, can be investigated. We shall review the progress made in this field of tobacco carcinogenesis during the past decade. I n addition to reviewing biological and chemical studies involving tumorigenic and cilia-toxic activity of tobacco products, fractions and single components, we shall present some discussion of pertinent analytical techniques employed, of different biological procedures utilized, of precursors that may give rise to certain components, as well as of some fundamental aspects of tumorigenesis as they may apply to the subject under review. I l l . Some Characteristics of Tobacco and Tobacco Smoke

A. TOBACCO PRODUCTS 1. Production of Tobacco

Any review of experimental tobacco carcinogenesis must, by necessity, be preceded by some discussion of the nature of tobacco products and tobacco smoke as such. The constitution of tobacco leaf employed in the manufacture of cigarettes, cigars, and pipe tobaccos will, of course, influence the nature of smoke derived from it. W. W. Garner has reviewed these aspects well in “The Production of Tobacco” (1951). Unfortunately, no similar publication has appeared since that time, which would include such subjects as growing of low-nicotine tobacco, production of tobacco sheets, and the use of various materials for filter tips. Aspects of tobacco chemistry have been reviewed more recently by Johnstone and Plimmer (1959). Tobacco is grown in appreciable amounts as far north as south Sweden, and as far south as New Zealand-a fact which in itself suggests a great variety of tobacco types. This world-wide cultivation of Nicotiaria provides the tobacco industry with types and grades of leaf especially suitable for the manufacture of one or more specific tobacco products. The characteristics of a given type of tobacco are mainly a result of such combinations as planted seed, soil and climatic conditions, and the original position of the leaf on the tobacco stalk. While modern organic

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insecticides can sometimes be a source of off-flavor in tobacco (Entomology Faculty, North Carolina State College, 1958), they appear to be only of minor, if in fact any, interest in tobacco carcinogenesis. Formerly, inorganic insecticides, such as lead arsenate and cryolite, were used. Since lead arsenate can be regarded as one precursor for arsenic (arsenic (111)oxide) in tobacco smoke, many investigators have studied the arsenic content of cigarettes (as will be discussed in a subsequent chapter). For more than a decade, however, arsenical sprays have been banned or discarded as insecticides in most countries, and it appears that the arsenic content in tobacco leaf is further decreasing owing to the diminishing amounts of arsenic in the soil. The possible importance of this aspect in experimental tobacco carcinogenesis will be discussed in Sections I V and V. 2. Curing of Tobacco

Major chemical changes occur during the curing processes of tobacco leaf (Darkis and Hackney, 1952). The most commonly used processes are: air curing-drying in barns at ambient temperatures and humidities ; flue-curing-drying in barns which are gradually heated to about 80°C. ; and sun-curing-drying by sunlight with the leaves hanging vertically. During air-curing, e.g., Burley and Maryland tobacco, most of the sugar and starch is decomposed and protein, as well as nicotine, is reduced. Polyphenols are also significantly lowered compared to fluecured leaf (Stedman, 1957; Weaving, 1958; Penn and Weybrew, 1958). The mainstream smoke of cigarettes made from Burley or Maryland air-cured tobacco is weakly acidic. The whole process of flue-curing, e.g., Virginia, North Carolina, South Carolina, Georgia, and Florida tobaccos, requires only a short period and delivers leaves containing not more than 10-15% moisture. The outstanding effect of the flue method of curing is the transformation of starch into sugar. The loss of total dry matter during the heat-treatment amounts to about 15%. There is a relatively small loss of nicotine and polyphenols, while the content of cellulose, total nitrogen, and organic acids remains unchanged. Garner e t al. (1934) demonstrated the pronounced changes in the leaf (South Carolina, low nicotine) by comparison of analytical values of certain components before and after fluecuring (Table I). The smoke of flue-cured tobacco is acidic. I n this respect a recent publication on the fate of carbohydrates during thermal degradation of tobacco brings some insight as to chemical changes that take place during the curing of tobacco (de la Burde et al., 1962). The sun-cured types of tobacco resemble the flue-cured tobacco in chemical composition, but contain somewhat less sugar.

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Pipe and cigar tobaccos are mostly prepared by air-curing. For pipe tobacco the whole plant is cut down and dried, during which time a considerable translocation of mineral salts occurs. Cigar tobaccos are cured either as leaves (wrapper), or whole stalks (filler). TABLE I COMPARATIVE CHEMICAL COMPOSITION O F CIGARETTE

AFTER

TYPEO F LEAF BEFORE

AND

CURING"

~

Constituents Starch Fkducing sugars Sucrose Cellulose (crude fiber) Citric acid Malic acid Oxalic acid Protein Nicotine Ash Total nitrogen

Eefore curingb

After curing*

29.29 6.68 1.73 7.28 0.40 8.62 0.96 4.07 1.10 9.23 1.18

6.38 18.94 8.45 8.31 0.48 10.10 0.98 3.70 1.12 10.71 1.22

From Garner et al. (1934).

* Based on per cent total dry mat,ter. The nicotine content of the different types of cured tobacco ranges as follows: air-cured cigar filler 2.0-4.0%, Burley 1.0-5.0%, and Maryland 2.0-4.0% ; flue-cured tobacco 1.0-3.5% ; sun-cured Oriental leaf 0.75-2.0%. Certain volatile carbonyl compounds in the smoke appear to be of interest in tobacco carcinogenesis. Weybrew and Stephens (1962) found that the carbonyl content of tobacco is affected by various factors such as tobacco types, curing, drying, fermenting, and others.

3. Cigarettes The more popular present-day cigarette, especially the American, is a blended cigarette. The details of leaf blending for cigarettes are trade secrets. Manufacturers try to maintain uniform blends year in and year out. Garner (1951) approximated a blend for the average American cigarette to consist of 55% flue-cured, 33% Burley, 10% Oriental, and 4% Maryland leaf tobacco. It appears, however, that this approximation may be altered somewhat when reconstituted tobacco sheet is added (Atkinson, 1961). Reconstituted tobacco is made up of a mixture of tobacco fines or dust (a by-product of cigarette production), ground tobacco midribs,

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sometimes admixed with certain cellulose derivatives in a slurry. Humectants are added; the mixture is pressed into sheets, dried, and then added to the blend (Rosenberg and Bandel, 1959; Meissner and Richter, 1961). The tobacco blend is also enriched by “casing,” representing the addition of such agents as sugar, chocolate, and licorice, with glycerol and glycols added as humectants. The blend is finally passed through a cutting machine which shreds it into strands of less than 1 mm. in width (30-40 cuts per inch). These strands are then fed to a cigarette-making machine. Here the tobacco blend is wrapped in paper constituting about 5% of the total weight of a cigarette. Pastes, consisting of either starch or casein, are used to secure paper seams. Recently Blackmore et al. (1963) reviewed the latest modifications in the production of cigarettes and use of various filters. The cigarette paper used in most countries in the Western world consists of refined flax fiber. To regulate its porosity and burning characteristics and to increase its opacity, between 20 and 25% calcium carbonate is added. Titanium dioxide has been added in the past, but its use today is limited (Calkin and Witham, 1957). Additional substances that are often added to improve the color and cohesiveness of the ash include ammonium phosphate and sodium potassium salts of citric acid (Brookshire, 1945). Among types used in other parts of the world are rice paper and parchmentlike papers. The type of cigarette paper used would seem to have a definite influence on the burning characteristics of a cigarette, and thus on the constituents in its smoke (Ayres et al., 1963). The moisture content of the final product averages about 12.5%. This, as all the other figures given in this description of cigarette manufacturing, is an approximation, since considerable deviations exist which change with time and local customs. I n addition, it should be remembered that in some countries (e.g., Canada, England, and Germany) tobacco additives are not permitted or are quite limited in use. In this regard, it also needs to be emphasized that different types of tobacco are used in various countries. I n England, for instance, fluecured tobaccos are used almost exclusively, while air-cured tobaccos are predominantly used in the Soviet Union. The tobacco cut and the amount of tobacco used for each cigarette may also vary from country t o country. Since filter-tipped cigarettes were introduced about a decade ago, they have become popular in most countries. Of all cigarettes sold during 1962-1963, more than 75% in Germany, and more than 50% in the United States were filter-tipped, while in England they represented only about 25% (Tobacco, 1963). Crimped secondary cellulose acetate of between 2- and 6-denier fiber is the basic filter material most widely

256

ERNEST L. WYNDER AND DIETRICH HOFFMANN

used. Occasionally, however, one also finds cellulose or certain impregnated composite filters as filter tips. I n some countries paper filters are in use. There exists a considerable variation in the efficiency of different filter materials. A discussion on smoke fiItration properties will be presented in more detail subsequently (Section VI) . The approximate compositions given for American and western European cigarettes do not apply to cigarettes of eastern European countries. Since several studies in tobacco carcinogenesis have been reported from the U.S.S.R., a mention of some basic differences between Western and U.S.S.R. cigarettes appears in order. The vast majority of the Russian “Makhorka” is, as stated above, air-cured. The tobacco in the cigarette is rather tightly packed. Several Russian cigarettes have a long mouthpiece. For example, a popular Russian cigarette, LIBelomorkanal,” (83 mm., diameter 8-9 mm.) contains a 32-mm. tobacco portion containing about 660 mg. tobacco with 12.5% moisture. The tobacco is rolled in relatively nonporous parchmentlike paper. The other 51 mm. of the cigarette consists of white cardboard paper which can be easily dented or pinched as smokers may like it. A. A. Shmuk (“Chemistry and Technology of Tobacco,” 1953) presents a good review on Russian tobacco.

4.Cigars As with cigarettes, there is also a wide range for cigars with respect to the leaf used and manufacturing techniques. The air-cured cigar tobacco is often subjected to short aging followed by fermentation, The purpose of fermentation is the reduction of nicotine and other nitrogenous compounds, resulting in improved flavor and smoking quality. The fermentation is achieved by repeated packing of the leaves in tight staples. During this time enzymatic and/or bacterial reactions take place as evidenced by increased temperature (Frankenburg, 1946, 1950; Jensen and Parmele, 1950; Garner, 1951). During the fermentation a great reduction of dry matter, mainly proteins and alkaloids, occurs. I n the final stage cigars are made up of a combination of tobaccos used as filler, binder, and wrapper. The flavor of the cigar, as well as its burning quality and its weak alkaline smoke, are mainly dependent upon the quality of the components used. 5. Smoking Tobacco Many pipe tobaccos are blends of various Ieaf types, but the most popular are of straight Burley. The older type of pipe tobacco was llplug-cutl’; granulated and “cube-cut” types are now preferred. Smoking tobaccos are treated with “saucing material,” licorice, and other sweeten-

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257

ing agents. Burley tobacco, as principal constituent of the blend, requires the addition of considerable amounts of sugar to develop smoking qualities. Some pipe tobaccos may obtain as much as 30% saucing and casing material, a factor which appears to influence the concentration of some smoke components (Hoffmann e t al., 1963). 6. Chewing Tobacco

Since World War I, chewing tobacco consumption has steadily decreased in the United States and western Europe. Today, therefore, this tobacco product is of relatively minor importance. It consists of a “plug” and “wrapper.” The plug, moderately sweetened when made from Burley leaf, is pressed into the proper shape and covered with a wrapper of natural leaf. Sweetened and unsweetened cigar leaf scrap is another type of chewing tobacco highly favored in America. In India and many East Asian countries chewing of tobacco has remained popular. The chewer sometimes prepares his own “Khaini” by mixing tobacco with slaked lime in his hand (Khanolkar, 1959). More common, however, is the chewing of air- or sun-cured tobacco, along with betel nut and slaked lime.

B. PHYSICAL STATEOF

TOBACCO

SMOKE

1. Particle Size D ~ s t r ~ b ~ t i o ~

The smoking of tobacco products yields a mainstream and sidestream smoke. Since it is primarily the mainstream smoke that enters the respiratory tract of a smoker, the great majority of studies in experimental tobacco carcinogenesis have concerned themselves with this portion of the smoke. Tobacco smoke is a two-phase system consisting of a vapor phase (gas phase) and disperse phase (aerosol). The latter contains practically all nongaseous components (particulate matter) .l The measurements of the mean diameter of cigarette smoke particles reported in the literature vary considerably, since different dilution ratio and different time factors play a role in these studies (Warner e t al., 1952, Sano et al., 1953; Langer and Fisher, 1956; Holmes et al., 1959). During smoke formation the size of the particles varies between 0.1 and 2.0 p . Keith and Derrick (1960) have measured the particle-size distribution and concentration in cigarette smoke, as well as variables affecting the particle-size distribution, such as the effects of aging, filtration of the smoke, and other conditions (Fig. 2). The size of the particles entering the respiratory system is of particular importance, ‘We wish to stress that these terms apply arbitrarily t o the materials and have no strictly defined physicochemical meaning.

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

since this variable and the mode of inhalation determine, to a great extent, the retention, distribution, and elimination of the smoke particles. In this respect, “lung-damaging” particles appear to range from 0.25 to 3 p (Kotin, 1956; Crider and Spinar, 1955; Eisenbud, 1952).

* WITHOUT -C

FILTER

WITH FILTER

Fro. 2. Particle size distributions of the smoke from filter and nonfilter cigarettes (Keith and Derrick, 1960).

Farr and Revere (1958) studied under the light microscope and under the electron microscope settled and impinged smoke deposits from four types of United States nonfilter cigarettes and six types of United States filter cigarettes. This investigation indicates that color, form, and size of droplets as well as the types and amounts of particulate matter in the smoke can be correlated, to some extent, with the composition and construction of the original cigarette. 2. Retention of Tobacco Smoke upon Inhalation

Schmahl et al. (1954), as well as Wynder and Hoffmann (1960), reported a retention of up to 90% of the particulate matter of cigarette smoke in the lung and bronchi of a smoker upon deep inhalation. Kahler and Lloyd (1957) illustrated by electron micrographs of electrically precipitated cigarette and cigar smoke a variety of forms in which the smoke condenses and dries. Observed by this technique, cigarette smoke particles range from submicroscopic size of 0.018 mp to 16p, with a weighted average diameter of 0.5 p . The configurations and arrangements of particles exhaled appear

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extensively changed compared to noninhaled cigarette smoke. Mitchell (1962) observed 20 to 50% smoke-particle retention in the buccal cavity when none of the smoke was inhaled. The retention when the smoke was held in the lung for 5 seconds was about 82%, and about 97% when the smoke was held for 30 seconds. Mitchell reported further, that the longer the smoke was retained in the respiratory tract, the smaller were the exhaled particles. This result does not appear to be in full agreement with the study of Polydorova (1961). However, we feel that the Mitchell data are based upon techniques that appear quite sophisticated and which give reproducible results. Several groups studied the chemical composition of aerosol particles of different diameter by chemical and optical methods and lately also by X-ray diagrams (Trillat et aE., 1958). These investigations suggest that various particle sizes contain various concentrations of a t least one group of components. The inhomogeneity of the smoke particles could possibly lead to the design of filter material which might remove toxic and tumorigenic agents from cigarette smoke (see Section V I ) .

C. SMOKINGMACHINESAND SMOKINGTECHNIQUES 1. Single Smoker I n studying tobacco carcinogenesis, the preparation of tobacco smoke becomes a prime concern. The physical state, chemical composition, as well as the toxicity of tobacco smoke, can be influenced by numerous factors, particularly by the burning of tobacco itself. For experimental studies, the production of tobacco smoke can be accomplished by machines designed to simulate the conditions of human smoking as far as possible. Among the first principles applied, to fill this need, was that of continuous suction (Pontag, 1903; Kissling, 1905; Garner, 1906; and others). The next step was the development of machines with interrupted suction, first by manually controlled devices (Habermann, 1901; Garner, 1906; Pfyl and Schmitt, 1927; Wenusch, 1931; and others) and later with automatic timing devices to control the opening of the suction valves (Bogen, 1929; Kenyon, 1934). I n addition, Jensen and Haley (1935) finally employed a draft control. The first preparation of tobacco “tar”2 for a carcinogenicity test, however, was done by mere destructive distillation of tobacco (Roffo, 1939), SO that one has to exclude this work from the field under discussion (Kosak, 1955). 2Throughout this paper the term “tar” is only used as B descriptive noun, realizing that i t is not strictly correct from the chemical point of view; the term “smoke condensate” is actually more accurate.

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During the last decade smoking machines have been based on one of two principles : either on constant-puff volume or on constant-smoking time. While the frequency of puffs per minute can be readily controlled, the draw resistance is constantly changing during the smoking of a tobacco product. The alternatives are either a machine smoking with constant-puff volume and varying smoking time (regulated by descend-

FIG.3. Ethel Mark VI smoking machine (Cigarette Components, Ltd., London).

ing liquid columns), or a machine with constant smoking time and varying puff volume (controlled by a vacuum reservoir; see Fig. 3). During recent years most groups preferred, for their chemical-analytical studies, smoking machines with constant-puff volume, since these condensate determinations can be kept within smaller standard errors (O’Keeffe and Lieser, 1958; Waltz et al., 1961). A smoking machine for quantitative studies was designed by Keith and Newsome (1957), which, it is claimed, minimized the dependence of constant volume and time smoking ma-

EXPERIMENTAL TOBACCO CARCINOGENESIS

26 1

chines upon the pressure drop of the cigarettes being smoked. Schepartz (1959) developed a constant-puff volume-smoking machine for cigars, and Miller (1962) for pipes that appear to be most useful. Recently, Williamson and Clark (1962) reported an improved laboratory smoking machine able to operate under a great variety of smoking conditions. 2. Manifold Smoker

Studies requiring large amounts of smoke condensate necessitated the design of so-called “manifold machines” [Wynder et al., 1953 (Fig. 4) ; Engelbreth-Holm and Ahlmann, 1957 ; Service d’Exploitation Industrielle

FIG.4. Multiple unit-automatic smoking machine (Wynder and G. Wright, 1957)

des Tabacs et des Allumettes, 1958; Kosak e t al., 1956; Bock and Moore, 1959; Roe e t al., 19591 and “smoking robots” (Schur and Rickards, 1957). The manifold machines have been subject to some criticism, since they puff some cigarettes more strongly than others (O’Keeffe and Lieser, 1958). Our studies have shown, however, that the chemical

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

analysis of these “tars” varies only insignificantly from that of a single smoker, when otherwise standard experimental conditions are maintained (Wynder and Hoffmann, 1961a). The reported deviations are negligible considering the biological conditions in experimental carcinogenesis, in general, and in the testing of combustion products, in particular (Wynder, 1961). For comparative studies, one basic requirement lies in the testing on a great number of animals which necessitates large amounts of “tar” and thereby the use of “manifold machines.’’ This is true for a majority of experimental groups. 3. Smoking Variables While the majority of studies in tobacco carcinogenesis were accomplished with machines which allow the simulation of human smoking, a great number of smoking variables were applied for specific studies. Puff durations from 1 to 5 seconds, puff frequencies from 1 to 4 per minute, and puff volumes from 20 to 40 ml. were used (Bock and Moore, 1962). The tobacco products were mostly ignited by gas flame or by hot wire. Cigarettes were smoked down to butt lengths of between 25 and 20 mm. So far as reported, the influence of these variables on different factors in tobacco carcinogenesis will be discussed subsequently. Fortunately, during recent years, investigators in the United States, Germany, and Sweden have used equal standard smoking conditions for cigarettes. These are 1 puff per minute with a volume of 35 ml. and a puff duration of 2 seconds. When possibIe the cigarettes are smoked down to 23 mm. (Wartman et al., 1959; Staberg, 1959; Wiss. Forschungsstelle im Verband der Cigaretten-Industrie, 1961). I n Austria, the same smoking conditions are accepted with the exception that sometimes 2 puffs per minute are taken (Kuhn and Marek, 1961; Pailer et al., 1962). The Tobacco Manufacturers’ Standing Committee (Bentley and Burgan, 1961) recommended for the United Kingdom the following smoking parameters: 1 puff per minute, duration 2 seconds, puff volume 25 ml. and 20 mm. butt length for untipped cigarettes. This smoking technique has special bearing on tobacco carcinogenesis studies carried out in Great Britain since the English Committee supplies, voluntarily, interested academic institutions in the United Kingdom with tobacco smoke condensate for animal experiments (Tobacco Research Council, 1963).

D. PREPARATION OF TOBACCO SMOKE 1. Aerosol The first choice for simulating human smoking would be a direct (active) inhalation experiment. Several investigators tried such studies,

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263

but only P. R. Peacock (1955) succeeded in training fowl to voluntarily inhale daily the smoke of a few cigarettes. Since direct inhalation experiments appeared most difficult to carry out on any significant scale, a variety of animals have been exposed in cages to the indirect (passive) inhalation of freshly generated and diluted tobacco smoke, a subject which we shall discuss more fully in Sections IV and VII. Since the dose level is by necessity low in experiments involving indirect inhalation, the more promising choice to test the carcinogenicity of tobacco smoke appears to be the repeated cutaneous application of the aerosols to mice and rabbits (Food Protection Committee, 1959) ; and if certain conditions are fulfilled, perhaps the subcutaneous tissue of rats and the installation into the pouches of hamsters (see Sections IV and VII). 2. Collection Methods for Smoke Condensate The separation of aerosols from tobacco smoke presents certain difficulties. First, the aerosols are separated from the gas phase by mechanical filtration. This method is quite successfully applied for chemical analysis, especially for the determination of volatile components in the smoke (Touey, 1955; Irby and Harlow, 1959; and others). However, for large-scale biological testing, this collection system is impractical. During recent years some investigators have used electrostatic precipitation to obtain particulate matter for animal experiments (Druckrey et al., 1960). It is known, however, that organic radicals are formed in electrostatic fields. Some of the radicals are known precursors for the formation of polynuclear aromatic hydrocarbons (PAH) (Badger, 1962a,b) ; thus the electrostatic precipitation of aerosols from cigarette smoke for the purpose of testing its tumorigenicity seems improper. In fact, our data showed the benzo [ a ]pyrene (B [ a ]P) content of “tar” obtained in this manner to be elevated from 1.1 0.1 p.p.m. (standard) to 1.45 f 0.15 p.p.m., a rather significant increase. Since other reactions also might occur in an electrostatic field, caution should be exercised in interpreting biological results obtained with such material. The more often employed and thoroughly studied collection system is that of the precipitation of smoke aerosols in cold traps, with and without organic solvents. One disadvantage of this principle might be that the gas phase and particulate phase are not clearly separated. The trapped material contains not only aerosols, but also components which are volatile a t smoke temperature, but not a t the low temperatures of the traps. Material collected with this trapping method should be, therefore, called smoke condensate, rather than aerosols or “tar” (see footnote 2, p. 259).

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

I n the most widely used arrangement for the condensate preparation, tobacco smoke is led from the holder through a glass vessel immersed in ice water and then through a trap cooled by dry ice-acetone. Direct trapping in a system which is cooled by dry ice-acetone proved to be impractical, since the resistance to draw within the whole system can be changed by condensed material. For biological testing, the condensate is dried to constant weight in a desiccator and then dissolved in an appropriate solvent and concentration, and stored in the dark. The drying of the “tar” appears to be essential, since recent studies indicate that the moisture content might affect the tumorigenic activity of the material (Wynder and Hoffmann, 1 9 6 3 ~ )It . is, of course, realized that even during this procedure some loss of volatile components occurs. It is not advisable, however, to dry the condensate by heating a t 110°C. to constant weight, since in this way there is an even greater loss of volatile components. I n addition, oxidations and secondary reactions might occur.

E. TOBACCO EXTRACTS Several epidemiological studies have suggested an increased risk for cancer of the upper alimentary tract among tobacco chewers (see Section IV). These observations, as well as the basic question concerning the origin of carcinogens and cocarcinogens in tobacco smoke, initiated several studies with tobacco extracts. The most extensive of these were carried out a t the Indian Cancer Research Center in Parel-Bombay. Large-scale extractions of sun-cured south Indian tobacco leaves were made with water, benzene, and also successively with petroleum ether, benzene, chloroform, and alcohol. From these extracts the basic portions (nicotine and others) were removed by acid extraction before the test material was used for biological experiments (Khanolkar, 1957; RIody and Ranadive, 1959). Unfortunately, no experimental data are given on the chemical part of these studies. The fact that there are relatively few studies dealing with water extractions of tobacco seems surprising, since a water extract of tobacco appears to yield material which might be comparable to that extracted during chewing by man with saliva. Morris et al. (1961) reported the yields of water extractions from Bright, Burley, and Turkisli tobacco as 54.1, 46.7, and 58.5%, respectively. Two of the results of Morris et al. appear to be of special interest for studies in tobacco carcinogenesis. The petroleum-ether extractable portion decreases during aging, and the amounts of petroleum-ether ex-

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265

tractables vary, being for Bright 6.3%, Burley 5.2%, and Turkish tobacco 3.6%. The analysis of various tobacco leaves (Stedman and Rusaniwskyij, 195913) indicates that these differences in yield are not due to the concentrations of paraffins, but rather to neophytadiene (Bilinsky and Stedman, 1962a) and solanesol-like substances (Bilinsky and Stedman, 196213). The Eastern Regional Research Laboratory of the United States Department of Agriculture has made large-scale extractions of cured, unaged United States type of leaf with n-hexane; the yield was 6.37%. The composition of the n-hexane solubles was compared with that of the leaf before extraction (Dymicki and Stedman, 1959a; Swain et al., 1961; Stedman et al., 1962a). The n-hexane extract contains about 83% of the paraffins, 82% of neophytadiene, all higher fatty acids and all solanesol of the tobacco leaf. The n-hexane extraction is comparable with that made by Wynder et al. (1958) from United States cigarette tobacco, resulting in 5.4% extracted material. The extract was found to contain mainly paraffinic and polyenic hydrocarbons, glycerides, and other esters, solanesol, phytosterols, aliphatic acids, and nicotine. Mouron et al. (1960) extracted Maryland cigarette tobacco with petroleum ether a t temperatures between 15" and 20°C. and obtained only 1.3% material. The same tobacco extracted at room temperature with various halogenated hydrocarbons gave yields between 1.9 and 2.8% of extracted material. To determine whether tobacco itself may contain tuniorigenic agents, alcohol extractions were carried out by two groups. Wynder and Wright (1957) extracted with methanol 34% from the tobacco of a United States cigarette. The extract contained paraffins, high-molecular-weight saturated aliphatic esters, phytosterols, an aromatic acid, isoprenoid polyenes, geraniol, and glycerides. The extraction of tobacco with 70% ethanol by Druckrey et al. (1960) yielded 20% extract, a result appearing unusually low compared to studies of Stedman et al. (196213). These data are discussed further in Section IV. For chemical and analytical studies of essential oils in tobacco leaves, steam distillation is frequently employed (Onishi and Yamasaki, 1955; R. Muller, 1960; Burdick et al., 1963a). This method is of importance for chemical studies in tobacco carcinogenesis, as it can be useful for isolating terpenes and terpene-like components from tobacco and tobacco smoke. Furthermore, Onishi et al. (1958) employed steam distillation in a large-scale separation as the initial step for the isolation of polynuclear aromatic hydrocarbons (PAH) in crystalline form from tobacco leaves.

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F. COMBUSTION AND SMOKE TEMPERATURES 1. Cigarettes

The temperatures existing during combustion and yielding a certain "tar" determine the chemical nature of this "tar" inasmuch as higher temperature favors the generating of higher molecular aromatic compounds. These compounds are "largely responsible for the carcinogenic action" of coal tar (Clar, 1952). Combustion temperatures existing during the burning of tobacco products considerably influence the yield of carcinogenic hydrocarbons in smoke. Several investigators have studied combustion profiles of various tobacco products. Among the first were E. A. Cooper e t al., 1932; Briickner, 1936; Flory, 1941; Wynder e t at., 1953; Lam, 1955; Seelkopf, 1955; Greene, 1955; Harlow, 1956; Ermala and Holsti, 1956; Touey and Mumpower, 1957a; Pietzsch, 1959; Pyriki and Miiller, 1959; and Kobashi et al., 1959, all of whom determined combustion temperatures by thermocouple measurements. Average combustion temperatures for cigarettes ranged from 650" to 880°C. Lam (1955) , Harlow (1956), and especially Touey and Mumpower (1957a) were able to improve combustion zone measurements by the use of thin, precision thermocouple wires of 0.025 inch diameter. Touey and Mumpower found that variations in tobacco blend, cigarette size, and pressure drop (draw resistance) have little influence on the combustion zone temperature of cigarettes. The temperature profile, however, is largely dependent upon the amount and volume of puffs drawn (Fig. 5 ) . Pyriki and Miiller (1959) found peak combustion temperatures as high as 98&1050"C. when working with fine-precision thermocouples and observed a temperature drop of looo-150° between puffs. Kobashi et al. (1959) interpreted their combustion temperature measurements as changing only insignificantly with puff velocity, type of smoking (continuous versus intermittent puffs), and moisture. Dry cigarettes (6.6% moisture) a t 40-ml. puff velocity and intermittently puffed showed about the same peak temperature as cigarettes with 10.9% moisture, namely, 805 k 17.9"C. compared to 805 rt 10.8"C. At 15.4% moisture the readings averaged 814 k 25.3"C. With continuous puffing a t the rate of 40 ml. per second, values ranged from 815 f 13.2"C. for dry tobacco to 8 2 7 k 2 1 " C . for the normal moisture with values from 15.4% tobacco moisture condition being somewhat lower a t 800 17.6"C. Borowski and Seehofer (1962) used an electronic thermometer, as well as a thermocouple, to measure temperatures in the glowing zone

*

267

EXPERIMENTAL TOBACCO CARCINOGENESIS

and in the butt for extremely dry, normally conditioned, and extremely moist cigarettes. At 4.7% moisture they found 755"C., a t 13.1% moisture, 84OoC., and a t 27.2% moisture 898°C. Neurath and Horstmann (1963) made a detailed study on glowing-zone measurements in the area of the fifth puff of a cigarette with calibrated platinum-platinum/rhodium thermocouples of 0.03 mm. diameter. This investigation emphasizes the

Temp., "C.

looor

,

Cigars

Recorder Chart Travel, In.

FIG.5. Potentiometer graphs : Temperature profiles of different tobacco products (Touey and Mumpower, 1957a).

relationship of tobacco moisture to the peak combustion temperature, but without showing definite trends. At the average moisture content (13.5%) the temperature was 913 -t- 40"C., a t maximal dryness (3.1% moisture) 894 f22"C., and at maximum moisture (21.75%) 867 & 40°C. Cuzin and Guillard (1963) measured peak combustion temperatures as well as thermal conditions during puffing and progressive consumption of a cigarette by "2-point" thermocouple systems. The influence of cigarette paper in controlling the burning conditions of a cigarette is apparent from a study by Croninger e t al. (1958). Using tobacco leaf instead of cigarette paper as a wrapper, cigarettes burned unevenly, with peak temperatures varying between 650" and 1025"C., with an average temperature of 701°C. Another technique of measuring combustion temperatures in cigarettes was described by Dobrowsky (1962), who used a calibrated series of inorganic crystals of known melting points. He introduced these crystals into cigarettes and determined microscopically whether or not the crystals appeared melted in the ashes. This technique showed that within

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

puffs combustion temperatures are over 730°C. and below 8OO"C., and in puff intervals, the temperature remained about 730°C. A most accurate and detailed study of the mechanism of smouldering in cigarettes is the one by Egerton et at. (1963), using cine-radiationpyrometry, cine-X-ray photography, and Schlieren photography. The X-ray photography was carried out with metal alloy inclusions of different melting points. These investigators concluded that the outermost annulus of the cigarette has particle temperatures exceeding 900°C. with surface temperatures reaching 1200"C., whereas the inner strand cylinder "smoulders chiefly by natural convection." 2. Pipes and Cigars Relatively few publications deal with combustion temperature nieasurements in cigars and pipes. Even though techniques of measurement were not as standardized as they are today, earlier studies demonstrate clearly that the combustion zone in pipes reaches temperatures far below those observed in cigarettes (E. A. Cooper et al., 1932; Briickner, 1936; Touey and Mumpower, 1957b; Greene, 1955). Cigarette tobacco in pipes, according to Greene, burned a t peak temperatures of only 450°C. as compared to "aromatic" pipe tobacco a t 590°C. Values for cigar smoking initially were in the range of cigarette combustion temperatures or somewhat below those (Greene, 1955; Harlow, 1956; Ermala and Holsti, 1956). Touey and Mumpower (1957b), however, observed peak temperatures during cigar smoking a t 930°C. decreasing to 820°C. in smoke pauses. I n this case, the automatization and standardization of smoking conditions, as well as the more accurate methods of temperature measurement, provide more reliable values than the earlier studies. Pietzsch (1959) differentiates between aromatic and nonaromatic pipe tobaccos and reports average combustion temperatures of 578" and 439"C., respectively. For cigars Pietzsch found an average temperature of 629°C. These data, especially when standardized and refined to the greatest possible degree of perfection, are valuable to the chemist in suggesting the possibilities of chemical reactions involved in combustion of organic materials. They have led to investigations of precursors for certain smoke constituents, as well as determination of the fate of certain tobacco constituents. 3. Smoke Temperatures Most investigators agree that the temperature of cigarette smoke reaching a smoker's mouth will be around 30°C. until the butt reaches a length of 25 mm. Thereafter, the temperature is reported to increase

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rapidly to 40” and even 50°C. (McNally, 1932; Greene, 1955; Harlow, 1956; Smyth, 1959; Stephano, 1960; Ingelstedt and Wallenius, 1961). Borowski and Seehofer (1962) found that the temperature of the mainstream smoke measured in the cigarette butt is virtually uninfluenced by differences in brand, type, or position of the cigarette during smoking. They also agree that the average temperature hardly exceeds 30°C. provided that a 23-mm. butt is left. Radiant heat from the glowing zone can bring temperatures up to 75°C. within a range of 5 mm. One may conclude from these studies that the temperatures of smoke reaching the smoker’s mouth are not unusual, except when such smoking habits as the use of “chutta” by Indian women is considered. “Chutta” is a type of cigar smoked by women in Andrah Pradesh with the burning end inside the mouth (Reddy e t al., 1960). To our knowledge temperature measurements have not been reported under t,hese conditions even though one study points to heat as a factor in tobacco carcinogenesis (Reddy et al., 1960). With these background data on the nature of tobacco products and the conditions that influence the composition of tobacco smoke and tobacco extract, we now proceed to a presentation of biological studies designed to examine the possible tumorigenic activity of these products. IV. Biological Tests for Turnorigenic Activity

Biological studies designed to test tumorigenic activity of tobacco products have been quite extensive. The more pertinent of these studies have been summarized in Table I1 (pp. 398-434) which includes all those studies using mouse skin as test system since 1953 when they were last summarized (Wynder e t al., 1953) and those using rabbit epidermis since 1957 when they were summarized by Graham e t al. (1957b). Listed separately are the tobacco products utilized by each investigator, the smoking techniques, the portion and dose of the tobacco product used, the species and strain of animal used, and d-etails of the method of application. These details on methods could, of course, be given only as they were described, and i t is apparent that in many instances details of the experiments are relatively limited. I n but relatively few experiments were sufficient details given to permit an adequate comparison of different studies. The toxicity of tobacco smoke condensate, largley due to its nicotine content, has been a major dficulty in biological studies. This was particularly true in earlier studies, when cigarettes were made from higher nicotine-containing tobacco. The toxic effect of the tobacco smoke condensate, in addition to affecting the life span of animals, also affected

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the weights of animals, thus probably adversely affected tumor development (Tannenbaum and Silverstone, 1953). The toxic effects of tobacco smoke, although not of special consideration in the light of the present review, nevertheless need to be considered in the evaluation of results reporting on the tumorigenic activity of tobacco smoke and tobacco condensate. Details on methodology must obviously be available before one can attempt comparisons. Since such data are not available for many of the studies reported, the various experiments should be compared on a qualitative rather than on a quantitative basis. It is appropriate to list some of the main variables involved in experiments with tobacco smoke condensate, since these are far greater than in experiments conducted with a solution of a single carcinogen (Table 111). Although different laboratories cannot adequately control all of TABLE I11 VARIABLES IN TESTINGFOR THE CARCINOGENICITY OF TOBACCO SMOKECONDENSATE A. The Tobacco Product 1. Curing process 2. c u t 3. Additives used 4. Use of homogenized tobacco 5. Use of stems B. Type of Tobacco Smoke Condensate 1. Type of operating cycle of smoking machine 2. Manner of smoke collection 3. Manner of dissolving condensate 4. Method of storing the condensate C. Species and Strains of Animals D. Method of Condensate Application 1. Preparation of and size of area to which condensate is applied 2, Type and size of brush used 3. Amount of condensate applied (should be standardized) 4. Frequency, duration of application 5. Duration of experiment

these variables and an absolute comparison of the data summarized in Table I1 is not possible, i t is clear that despite these differences a variety of animal tumors in a variety of animal species have been induced with tobacco products. I n fact, since 1953, investigators from several different countries have succeeded in producing cancers in animals with tobacco products. Therefore, there can be no doubt that tobacco products, and in particular smoke condensate, are tumorigenic to a variety of experimental animals and tissues.

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A. TOBACCO SMOKE CONDENSATE 1. Epidermis

Since 1953, when the first large-scale production of epidermoid cancer in experimental animals was reported, many investigators have verified these findings. Some negative results (Shotadze, 1953; Gwynn, 1954; Passey et al., 1954; Kakhiani, 1955; Hamer and Woodhouse, 1956; Gwynn and Salaman, 1956) are largely, if not exclusively, a result of inadequate dosage. The subject of dose will be dealt with in more detail in a subsequent section (Section VI). Clearly, tobacco smoke condensate as applied to mouse skin, or as tobacco smoke that may come in contact with bronchial epithelium, has a threshold level as does any other carcinogenic agent. In this connection, it would seem obvious that the results of an experiment in which smoke condensate is applied by brush three times a week to mouse skin clipped free of hair cannot be compared to an experiment in which the condensate is applied twice a week by a glass rod to an unshaved area of the skin. For instance, in a relatively recent study by Gritsiute and Mironova (1960), 12 to 22 mg. of undiluted tobacco “tar” was applied by glass rod to unshaved mouse skin three times a week for 10 months. The authors calculate that the mice received from 1.4-2.6 g. of “tar.” The very weakly positive results can not be compared with experiments in which, let us say, 60 mg. of smoke condensate dissolved in acetone is applied to a shaved mouse skin three times a week for 15 months. In a somewhat less obvious setting, the statistically insignificant differences ( p > 0.05) between two separate “tars” (Orris et al., 1958) could be due to water content rather than any more complicated factors. For a strict comparison of different experiments, a t the very least the amount of condensate applied, the method of application, and the frequency of application and the duration of the experiment must be similar. I n addition to the manner of “tar” application, the animal species and the animal strain used may be of importance. Swiss (Millerton) mice, for instance, were found to be more susceptible to the tumorigenic activity of cigarette smoke condensate than C57 black mice (Wynder et al., 1955). The susceptibility for papillomas has been found to be the same for both CAF, and Swiss mice but the “conversion rate” to cancer was greater for the Swiss mice (Wynder e t al., 1956). The true malignancy of skin cancer induced by cigarette smoke condensate has been demonstrated by Croninger and Suntzeff (1959), who transplanted R squamous cancer for 102 generations without losing its characteristics.

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I n addition, the variables listed in Table I11 may also have an effect on the constituents of specific tobacco smoke products, and thus on their tumorigenic activity, as shown by both chemical analysis and biological findings reported in this communication. It is apparent, however, from the review of the literature, that when different investigators use a methodology which is as similar as possible from laboratory to laboratory, the biological results are also quite similar. This was shown particularly in experiments by Engelbreth-Holm and Ahlmann (1958), Bock and Moore (1959), Kensler (1962), and Homburger et al. (1963), who set out to duplicate the experimental setting of the initial studies by Wynder, Graham, and Croninger in 1953. Even though the experimental setting may be reasonably well controlled, the tobacco product used will, as any tobacco expert knows, vary gradually from year to year, so that even with the strictest control of methodology the experimental product may vary (as is discussed in a later section). It must be realized, therefore, that studies with tobacco smoke products can never be as well standardized as those with a pure chemical carcinogen. The fact nonetheless remains that cigarette smoke condensates obtained by a smoking technique which simulates human smoking as closely as possible, have proved to be carcinogenic to mouse epidermis (Fig. 6). The smoke condensate from filtered cigarettes, compared on a gram-

FIG. 6. Carcinoma of mouse skin induced with cigarette smoke condensate (Wynder et ul., 1953).

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273

to-gram basis, has the same tumorigenic activity within experimental deviation as that from unfiltered cigarettes (Wynder and Mann, 1957). These findings were recently confirmed (Wynder and Hoffmann, 1 9 6 3 ~ ) . In this experimental setting the lower ((tar” yield from filtered cigarettes was not considered, a s i t was in a study by Bock et al. (1962). Skin cancer in mice also has been produced with “tar” from the sidestream smoke of cigarettes. From the condensate that had settled on the metal funnel covering our multiple smoking unit (see Fig. 4 ) we prepared a 50% (‘tar”-acetone solution, which was applied to 30 Swiss (Millerton) female mice by standard method (Wynder and Hoff111511111, 1 9 6 3 ~ ) The . tumorigenic response was similar to that found for standard cigarette smoke condensate. At the end of 15 months, 14 mice P content had developed papillomas and 3 carcinomas. While the B [a] of the sidestream “tar” is relatively high (see Section V ) , its phenol content, owing primarily to the manner in which i t was collected, is low. Cigarette smoke condensate similarly was demonstrated to be tumorigenic to the epidermis of rabbits by Graham et al. (195713) for both the ear and the nape of the back. The malignancy of some of the tumors was demonstrated by their metastatic spread to regional lymph nodes (Fig, 7 ) . Papillomas of the back in rabbits were also produced by Wynder and Wright (1957) with cigarette smoke condensate and with cigarette “tars” obtained from puffing cigarettes with a high- as well as low-puff volume (Wynder et al., 1958). Negative experiments (Hamer and Woodhouse, 1956; Gritsiute and Mironova, 1960) may, as with mouse skin studies, be attributed to the low doses of condensate applied. Perhaps because of the longer latent period and associated greater expense, relatively few investigators have utilized the rabbit as a test object. We see no particular advantage of using the rabbit instead of the mouse in these studies, now that it has been established that tobacco smoke condensate can induce carcinoma in rabbit epidermis. The effect of cigarette smoke condensate on human epidermis has been reported only by Rhoads et al. (1954). A 50% solution of the smoke condensate in acetone was painted on the upper back of a volunteer on four consecutive days. No major changes were found upon biopsy but slight nucleolar enlargement was observed. This was to be expected in view of the relatively short time and small dose. Studies with cigar smoke also have been conducted, primarily with mice. These studies suggest a greater, though not significantly greater, tumorigenic activity of cigar, as well as pipe smoke condensates to the skin of mice than of standard cigarette smoke condensate (Croninger et al., 1958). These studies necessitated the use of nicotine-free condensates because of the relatively high toxicity observed for the cigar

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and pipe smoke condensates. Kensler (1962) showed a trend toward more tumors among mice that received cigar smoke condensate, although the difference was not statistically significant. Homburger e t al. (1963) compared the tumorigenic activity of smoke condensate obtained from cigarettes made up respectively of blends of cigarette tobacco, cigar tobacco, and a blend used in pipe mixtures. Any differences noted

FIG.7. Carcinoma of rabbit ear with metastasis to cervical gland induced with cigarette smoke condensate (Graham et al., 195713).

therefore had to be due to differences in the tobacco blend and not in the manner in which the “tars” were prepared. On the basis of time of latency and cumulative number of tumors a t any given time, cigarette “tars” were found to induce carcinomas less rapidly than either pipe or cigar “tars.” Present results indicate therefore that the type and cut of tobacco used (see Section V I ) , as well as additives such as “saucing material,’’ especially sugars, have an influence on chemical constituents and tumorigenic activity of the resulting “tar” that is independent of differences in temperature profiles during combustion. Long-term studies with water pipe smoke have not been carried out. These studies are of epidemiological interest because of the apparent infrequency of lung cancer among Asiatic and African-born immigrants

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to Israel (Rakower, 1955, 1957; and Kallner, 1961). Similar data are reported from Syria. Smokers of oriental pipes are further believed to be noninhalers (Central Bureau of Statistics, Israel, 1959). Hoffmann et al. (1963) have conducted a short-term experiment with a 33% water pipe smoke condensate-acetone mixture and found only a

FIG.7a. Thoracic organs at autopsy of rabbit (Fig. 7) at 59 months showing extensive metastasis of carcinoma to lungs and heart (Graham et at., 1957b).

minimal amount of hyperplasia and no destruction of the sebaceous glands, as compared to a significant amount of hyperplasia and sebaceous gland destruction when these tests are conducted with cigarette smoke condensate in the same concentration. The authors suggest that on the basis of this short-term study the tumorigenic activity to mouse skin will be negligible.

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2. Subcutaneous Tissue The infrequency with which subcutaneous tissue has been used in testing for the carcinogenicity of tobacco smoke products may seem surprising in view of the favor that this experimental approach has enjoyed. Perhaps the increasing concern over the lack of specificity of producing subcutaneous sarcoma contributed to this change. We shall discuss this issue more fully subsequently (Section VII) .

FIG.8. Sarcoma in rat induced by basic free portion of cigarette smoke condensate (Druckrey et al., 1960).

Published data using the subcutaneous injection techniques with tobacco products have been reported in studies by Druckrey e t al. (1960); Druckrey and Schildbach (1963) from Germany; and by Gritsiute and Mironova (1960) from the Soviet Union. The latter work-

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ers injected about 2 g. of cigarette smoke condensate into rats divided into 50 applications: Of 25 rats none developed a sarcoma. I n a similar experiment with cigar “tar” one out of 26 rats developed a sarcoma. A pyroIysate of tobacco produced 2 sarcomas among 12 rats. The dosage used by these workers is, of course, considerably lower than that used by Druckrey’s group. Druckrey et al. (1960) have produced sarcoma in rats upon injection of 3.2 g of nicotine-free cigarette smoke condensate subcutaneously during a 60-week period (Fig. 8). Twenty per cent of 75 rats so treated developed sarcoma. Most of the tumors appeared more than 10 months after stoppage of injection, that is, near the end of the rat’s life span. There can be no question, therefore, that cigarette smoke condensate is also tumorigenic to subcutaneous tissues of rats. The studies by Seelkopf et al. (1963), testing different smoke condensate fractions subcutaneously in rats, will be reported subsequently.

3. Oral Cavity and Bladder We are grouping endeavors to produce oral cavity and bladder cancer together, since the few attempts a t inducing cancer in the latter site have involved applying tobacco products t o the oral cavity. I n man, a correlation between tobacco smoking and bladder cancer has been demonstrated primarily for cigarette smokers (Denoix and Schwartz, 1956; Lockwood, 1961; Wynder et al., 1963b). This may indicate that the absorption taking place upon inhalation is more important in affecting the development of bladder cancer than the absorption and swallowing of cigar and pipe smoke retained in the oral cavity. Cancer of the oral cavity in man has been shown to be associated with the use of various tobacco products-cigar, pipe, cigarette, and chewing tobacco. Here, association to cigar and pipe smoking appears to be greater than . date, experiments that of cigarette smoking (Wynder et al., 1 9 5 7 ~ ) To designed to produce cancer in either the oral cavity or the bladder with tobacco products have been largely unsuccessful. The amount of tobacco smoke products that will reach the bladder is of necessity low. It is known that the normal mucosa of the oral cavity is relatively resistant to the absorption of “tar” components, and the risk is further reduced by the “washing effect” of saliva. Moreover, epidemiological data also indicate a relatively low correlation between tobacco products and oral cavity cancer in the absence of nutritional deficiencies and heavy alcohol intake (Wynder et al., 1957c; Schwartz e t al., 1962). Experiments by Kreshover (1952, 1955), Kreshover and Salley (1957), and Salley (1963) have attempted to consider the nutritional factors, in particular as they relate to vitamin B deficiencies, a subject which we shall discuss subse-

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quently. Goldhaber (1957) discussed the relative resistance of the oral mucosa of mice to topical application of carcinogens and has implied a protective influence of saliva. Shotadze (1953) applied tobacco pyrolysate to the lower lip of 80 mice three times a week for 11 months and observed no precancerous or cancerous process microscopically ; only a slight subacute inflammation of the oral mucous membranes. L. R. Holsti and Ermala (1955) applied about 30 mg. of pipe smoke condensate daily to the lip and oral cavity of 60 albino mice. After 12 months, the authors reported, 87.5% of the mice had developed benign papillomatosis and 10% had papillary carcinoma of the urinary bladder. No oral lesions were observed. Bonser (196213) has stated that because Holsti and Ermala did not distent the bladder before histological examination, “the evidence so far presented has not clearly shown that bladder tumors were in fact produced.” So far no one has been able to duplicate the findings of Holsti and Ermala. Koerbler et al. (1959) applied pipe smoke condensate dissolved in sputum behind the ears of mice. Although no ear lesions were observed, 2 of the animals developed scirrhous and planocellular cancer, respcctively, of the lower jaw, probably as a consequence of licking the ears of other mice. Koerbler (1963) states that these tumors arose “within the oral cavity from the outer surface of the mandible.” These findings demand repetition particularly since application of far greater amounts of smoke condensate did not result in tumorigenic response in the lip or oral cavity. In our own experience we have never seen cancers of the oral cavity among mice whose backs had been painted with cigarette smoke condensate even though frequent licking must have taken place in this experimental setting. The production of cancer of the oral cavity has been reported by GuQrin (1959). Five of 68 surviving rats placed in plastic smoke chambers developed tumors of the buccal mucosa. Of particular interest were 3 rats with malignant invasive lesions. One female developed an epithelioma infiltrating the musculature ; a male rat presented a keratinizing epithelioma infiltrating the jaw bone; a third rat had an undifferentiated epithelioma invasive to both salivary glands and bone, in addition to metastases to the cervical lymph nodes. I n this experiment the smoke constituents could only have entered the oral cavity as a result of the rats licking their own or each other’s fur, contaminated with the heavier smoke particles which had collected on the sides and floor of the cages. I n view of these positive findings, the negative results of L. R. Holsti and Ermala and DiPaolo and G. E. Moore appear surprising. DiPaolo and Moore (1959) applied 20-30 mg. of cigarette smoke

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condensate 5 times a week to 50 Swiss mice, and 60-90 mg. 5 times a week to 50 mice for 13 months. Application was to the lips and oral areas of the mice. One anaplastic sarcoma of the bladder occurred in the latter group, but they found no lesions in the oral cavity. Further studies in this field appear indicated. A number of investigators have used the hamster cheek pouch to test possible tumorigenic activity of tobacco products (C. Moore and Miller, 1958; E. E. Peacock, Jr., and Brawley, 1959; E. E. Peacock, Jr., e t aE., 1960; C. Moore and Christopherson, 1962). No tumors so far have been observed. E. E. Peacock, Jr., et al. (1960) instilled snuff and chewing tobacco. C. Moore and A. J. Miller (1958) used cotton and gauze wads impregnated with tobacco “tar.” They noted some hyperkeratinization and slight hyperplasia but no gross lesions. The relatively poor absorption of materials implanted in the hamster pouch can be seen from the observation (E. E. Peacock, Jr., e t al., 1960) that 30 mg. of powdered strychnine implanted in the pouch did not kill the animals (E. E. Peacock, Jr., 1964). Inadequate absorption of tobacco ‘%ar” by the mucosa of the hamster pouch therefore seems to be a major hindrance to the satisfactory testing for tumorigenic activity of tobacco products in this area. Kreshover and Salley (1957) made some interesting contributions to the effect of tobacco on epithelial tissue. including that of the oral cavity of mice and hamsters. Kreshover (1952) applied cigarette smoke on alternate days to the lips and ears of mice without producing malignant changes. In this experiment he applied ten 2-second puffs every other day for 76 days. It is doubtful whether in this experimental setting sufficient condensates are absorbed by the epithelium to expect such changes. I n a subsequent study Kreshover (1955) also applied tobacco smoke to the pouches of hamsters without producing any cutaneous changes. Salley and Kreshover (1959), as well as Akamatsu (1960a,b,c) demonstrated the susceptibility of hamster pouches to 7,12-dimethylbenz [alanthracene (DMBA) and B [ a l p , indicating that relatively high doses are needed in order for carcinomas to develop in this area. Combining the application of DMBA and tobacco smoke to the pouches of hamsters, both Akamatsu (1960a,b) and Salley (1963) showed that tobacco smoke together with DMBA caused cancer earlier than when DMBA was applied alone. In the Akamatsu study, the tumor yield was not significantly different, whereas in the Salley study slightly more cancers were produced in animals treated with both materials. I n studies dealing with tobacco smoke, the amount of smoke actually absorbed by the cutaneous tissue must be considered. From this point of

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view applying smoke to a pouch or to the cervix would tend to lead t o longer retention than applying the smoke to the lip or ear. It is apparent from this discussion that oral carcinogenesis in the experimental animal represents a far more difficult setting than that of skin carcinogenesis. The combination of various factors should be considered by investigators interested in this area of experimental carcinogenesis (see Sections IV,E,4 and 5). 4. Cervix

Cervical epithelium has also been used as a test site even though the sensitivity of this tissue, particularly in noncastrated animals, does not appear to be as high as that of mouse skin (Wynder et al., 1 9 6 3 ~ ) . Also, if the nicotine content of the smoke condensate is high, the material proves rather toxic because of faster resorption of nicotine through the cervical epithelium in comparison to the skin. Bogacz and Koprowska (1961) applied 0.15 mg. of cigarette smoke condensate 5 times a week for 92 weeks to two groups of mice. Among 11 C3H mice that survived 20 weeks, 3 mice had carcinoma in situ, 2 had cancer in situ with microinvasion, and 2 others had early invasive cancer. Among 19 ZBC strain mice surviving 20 weeks, 5 were found to have carcinoma in situ and 1 mouse early invasive carcinoma (Fig. 9 j . Chu et al. (1962) applied 100 paintings of a 50% “tar”-acetone solu-

FIG. 9. Carcinoma of the mouse cervix induced with cigarette smoke condensate (Bogacz and Koprowska, 1961).

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tion for 12 months to 30 Syrian hamsters. No invasive cancers were found, although 12 had evidence of malignancy on the basis of cytology. Fifteen of the animals had dysplastic and 3 had anaplastic epithelial changes, compared to none in the control group (20 hamsters, application of acetone alone). Whether the differences in these two studies of cervical application are due to different dosages or species differences cannot yet be ascertained. Perhaps the cervices of the hamsters might have responded similarly as the mouse cervix if the frequency of application had been the same as in the Bogaca and Koprowska study. 5. Lung Experiments using tobacco smoke aerosols will be reviewed subsequently. I n this section we deal with attempts to produce tumors of the respiratory tract and lung tissue with tobacco smoke condensate. Relatively few such studies have been done, largely because of obvious experimental difficulties. Della Porta e t al. (1958) used as test system the instillation of tobacco smoke condensate suspended in 1%gelatin colloid intratracheally into golden hamsters. I n this fashion they applied 50 pg. of DMBA once a week for 45 weeks. I n 10 animals surviving 20 weeks, they observed one cancer of the trachea and one of the larynx. Applying 200 mg. of tobacco “tar” twice a week for 32 weeks resulted in no tumors among 11 hamsters surviving 20 weeks. Animals receiving 100 pg. of DMBA for 17 weeks showed 4 cancers out of 7, while there were 3 cancers out of 9 animals receiving 100 pg. of DMBA and 500 mg. of tobacco “tar” once a week for 20 weeks. Relatively large amounts of DMBA are required to produce malignant changes of the respiratory epithelium under the conditions of the experiment. I n this setting i t may not seem surprising that a relatively weak carcinogen such as tobacco smoke condensate did not induce neoplastic changes. An interesting method involving thoracotomy and injection of smoke condensates directly into the lung was performed by Blacklock (1961) , using rats, guinea pigs, and rabbits. Realizing that cigarette smoke condensate is a relatively weak carcinogen, he set out to inject a dose as high as possible. Cigarette smoke condensate was mixed with eucerin. Each 0.2 ml. of mixture was claimed to contain the condensate from 40 cigarettes. The animals were permitted to live out their natural lives. It was attempted to inject the material into the hilar region. Among rats, 8 (11.1%) developed malignant tumors, 6 carcinomas (Fig. l o ) , and 2 sarcomas. Among the control animals that received eucerin alone (44 rats), 1 r a t developed a malignant tumor, and among 275 control ani-

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FIG.10. Carcinoma of lung in rat induced with injection of cigarette smoke condensate (Blacklock, 1961).

mals who received nothing, 4 (1.4%) developed spontaneous malignant tumors. Only among the animals receiving cigarette smoke condensate were tumors observed in rats less than 15 months old. Among guinea pigs, one of the rare times this animal has been used in experimental tobacco carcinogenesis, changes ranging from basal cell hyperplasia to squamous hyperplasia were observed, while among rabbits one carcinoma in situ was produced. These extensive experiments by Blacklock thus indicate that pulmonary cancer can be produced with tobacco smoke condensate if sufficient amounts can be applied. Rockey et al. (1958, 1962) have carried out extensive studies in which they applied cigarette smoke condensate to the medial wall of

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the left primary bronchus of 82 dogs. The great effort and high cost involved in this extensive research program is apparent. I n a five-year interval study, they reported that the dogs received a total of 11,748 “tar” applications, 0.1 cc. to 0.25 cc. three to five times weekly from 2 to 1203 days. Hyperplasia was found in 43%; hyperplasia with atypical features in 5% ; squamous metaplasia in 95%; squamous metaplasia with atypical features in 83%; precancerous changes in 24%; carcinoma in situ in 2.4%; and invasive carcinoma in 1.2% (Fig. 11). The latter occurred

FIG.11. Carcinoma of dog trachea induced with applications of cigarette smoke condensate (Rockey et al., 1962).

within such a short time period-after five “tar” applications in 11 days -that additional evidence must be awaited before i t can be stated that this technique will produce a significant number of invasive cancers. Knowing the length of latent period for cancer, we would predict upon the basis of present data that invasive cancers will develop if the study is continued for a sufficient length of time. The changes that are produced by Rockey et al. are indicated by them to be similar to those observed by Auerbach among heavy smokers (Auerbach et a&, 1961). Homographs of embryonic lung implanted subcutaneously or intramuscularly represent another approach for testing carcinogenic material. It should be recognized, however, that because of the relative resistance of this material to carcinogenic agents, as well as its apparent susceptibility to toxic influences of cigarette smoke condensate and its various

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fractions, it may not be an ideal test for tobacco smoke condensate when compared with single carcinogenic hydrocarbons. Lasnitzki (1958) observed the formation of new bronchioles and hyperplasia of the lining epithelium of individual bronchioles in tissue cultures of human fetal lung to which were added the neutral fraction of tobacco “tar” as well as a neutral fraction from which the hydrocarbons had been removed. Thus, the effects observed could not be due to B [ a ] P alone. I n a large-scale study by Hou and Willis (1963) various smoke condensates and tobacco fractions were incorporated into cholesterol pellets in a ratio of cholesterol to “tar” ranging from 1 :1 to 2: 1 ; as a control these authors used cholesterol-methylcholanthrene (MC) pellets consisting of three parts of the carcinogen and one part of the sterol. These materials were placed together with homographs of embryo lung subcutaneously and intramuscularly into rats. Although 4 out of 56 animals receiving M C developed highly differentiated squamous cell tumors, none of 644 rats implanted with various tobacco smoke fractions developed cancer. Without even considering the possible toxic effects of a t least some of the smoke condensate material, i t would appear obvious that the concentration of carcinogenic material, as is well known from chemical as well as biological studies, is not present in the same order of magnitude as the MC tested by these workers, which was in a higher concentration in the cholesterol pellets than cigarette smoke fractions. Furthermore, this experimental set-up does not consider the possibility that the extraction of MC from the cholesterol pellets and thus its contact with the homograph may be considerably longer for this single component than for the carcinogenic components present within a smoke condensate. For these reasons we must assume that the negative results of such studies (as these can be regarded), as a consequence of the difficulties of technique and dose involved, are no indication that tobacco carcinogens, which have been demonstrated in many other biological settings, could not produce tumors in this system if adequate doses could be directly applied. It may be assumed, for instance, that if a PAH concentrate obtained from cigarette smoke condensate were to be implanted with cholesterol in comparable concentrations, results similar to those obtained with M C could be expected. Results of studies investigating the effect of tobacco smoke condensate on cells in tissue cultures are also of interest in this respect. Nakanishi e t al. (1959) investigated the effect of cigarette smoke condensate on lung tissues obtained from a human fetus and young kittens. Human epithelial lung cells decreased in chromosome number to 76 from a modal value of 77 for the primary chromosomes not exposed to this condensate

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- a finding which the investigators attributed to the appearance of dicentric chromosomes in some of the passages. These investigators also found some irregularities of mitochondria of the fibroblast-like cells. Venema (1959) investigated the effect on allium cepa of cigarette smoke condensate solutions from which the higher aromatic polynuclear hydrocarbons had been removed and observed altered mitotic processes in which the prophases were decreased and the telophases increased. Venema considered that these abnormalities might be due to disturbances in the synthesis of ribonucleic acid (RNA). Bouchard and May (1960) observed increased mitotic abnormalities in mouse lung fragments that had been bathed 24 hours in the smoke condensate solution and subsequently grafted under the renal capsule of mice. Awa et al. (1961), using the same human cell strain as Nakanishi, observed more cell damage when the cells were exposed to paper smoke as compared to cigarette or tobacco smoke. All three systems, but especially the paper smoke, produced a decrease in the mitotic index and an increase in number of abnormal divisions. No conclusion can be drawn as to the effect of cigarette paper, since i t was burned in this study in a manner quite different from the way i t burns as part of a cigarette. Mizutani (1962), also using the Nakanishi cell strain, could not confirm the finding of Nakanishi et al. of a change in chromosome number when exposing the cells to cigarette smoke. He did, however, find significant cell damage which was far greater for unfiltered than for filtered cigarette smoke. The various studies, which have just been summarized, all indicate that cigarette smoke has a significant influence on cellular life when measured in terms of its effect on cells in tissue culture.

EXTRACTS B. TOBACCO Epidemiological studies have shown a relationship between chewing of tobacco and cancer of the oral cavity (Orr, 1933; Friedell and Rosenthal, 1941; G. E. Moore et al., 1953; Wynder et al., 1957c), but with betel nut chewing, seemingly only when tobacco is part of the quid (Khanolkar, 1959). For this reason studies testing the possible tumorigenic activity of different tobacco extracts are of particular interest. Such studies are, of course, of further interest in that they permit a comparison of possible tumorigenic agents that may be present in both tobacco extracts and tobacco smoke. An important variable that applies to tobacco extracts is the manner in which tobacco is extracted. I n most instances the method of extraction is quite dissimilar to that involved in tobacco chewing by man. Thus, the type of materials used in animal studies might well differ significantly

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in amount as well as in type to that involving the human tobacco chewers. Compared to studies with tobacco smoke condensate, studies with tobacco extracts are relatively limited, Application of tobacco extracts obtained by the use of different orgaiiic solvents to mouse skin suggest some, though relatively weak, carcinogenic activity for such extracts. Wynder and Wright (1957) applied a methanol extract to CAF, and Swiss mice. Among 40 CAF, mice receiving a 50% acetone-extract solution, 28% developed papillomas and no carcinomas, while among 40 Swiss mice, 8% developed papillomas and 376, cancer. Although this tumorigenic activity is lower than that obtained with cigarette smoke condensate, these extracts nevertheless exhibit definite tumorigenic activity. Khanolkar (1959) reported on large-scale studies with extracts from sun-cured Indian tobacco. The extracts obtained with successive extractions with petroleum ether, benzene, chloroform, and alcohol were applied to the skin of mice, often for more than 18 months, but no cancers were obtained. Ranadive e t al. (1963) applied alkaloid-free acetone extracts of an Indian type of tobacco cutaneously and intrascapularly to mice. The injected group received 0.1 cc. of a 2% solution once a month. The skin applications with extracts of unknown concentration were done twice a week and were followed by weekly painting with croton oil. I n another setting a single application of B [ a l p was followed by biweekly application of extracts. After the skin applications had been carried out for 60 to 95 weeks, 50% of the animals receiving tobacco extracts and croton oil had papillomas and 28.6% carcinomas, a significantly greater percentage of tumors than obtained with croton oil alone ( 3 mice out of 19). Extracts that were tested on mouse skin previously receiving B[a]P gave some tumors (9 mice out of 29) compared to none in the control group. While this study does not test the activity of tobacco extracts alone, i t does indicate tumorigenic activity when applied in conjunction with croton oil and also with B[a]P. The latter results are in line with similar findings for cigarette smoke condensate, whereas croton oil has not been found to increase the tumorigenic activity of cigarette smoke condensate alone (Gellhorn, 1958). This experiment should be repeated with extracts of Western types of tobacco. A water extract of betel-tobacco quid was indicated by Muir and Kirk (1960) to be tumorigenic to the ears of mice. After 2 years, they found two squamous cell carcinomas among 12 mice. Druckrey e t al. (1960) injected a 70% alcohol extract of cigarette tobacco subcutaneously into rats. The yield from 1 kg. of tobacco was

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about 200 g. of extract. A 33% solution, in alcoho1(70~)-glycerinas solvent, was injected weekly (45 mg.) for a total dose of 3.2 g. While the total tumor response is the same as reported by these authors for cigarette smoke condensate free of basic portion (22%), they claim the extract to be 10 times more active since the total dose given, 3.2 g., corresponds to only 16 cigarettes as against 160 cigarettes with smoke condensate. The “tars” and extracts tested by Druckrey et al. (1960) also were tested in our laboratory (Wynder and Hoffmann, 1963c) on mouse skin. I n view of the solubility problems involved in using a 3 to 2 solution of acetone/water-a factor certainly influencing the tumorigenic activity of a “tar”-the results of this study are difficult to evaluate. The various materials were applied in a 50% concentration to 50 Swiss (Millerton) mice by standard techniques. The basic-free portion of smoke condensate obtained from cigarettes made of United States blended tobacco produced 5 papillomas a t the end of 15 months. This compared with 4 papillomas in the group receiving an extract obtained with 100% alcohol. From a basic free portion of an extract of an Oriental cigarette, the papilloma yield was 5; with a 100% alcohol extract, 1 ; and with a 70% alcohol extract, also 1. Our standard cigarette smoke condensate in this acetone/water solution yielded 6 papillomas and 2 carcinomas among 50 mice. In this setting, the basic-free portion of smoke condensate (United States blend) had the same activity as the extract obtained with 100% and 70% alcohol, respectively, while that of the Oriental smoke condensate was insignificantly greater than that of the extracts. The relatively low tumor yield as evidenced by our standard ‘(tar” in this solvent setting makes a definite interpretation of these data difficult. Physical properties of a given material do affect sarcoma formation. The conclusion to be drawn from studies utilizing the subcutaneous route apply primarily to sarcoma formation and do not necessarily apply to the formation of carcinoma, a subject wh:ch we will discuss subsequently (Section VII) That certain tobacco extracts, however, do possess low tumorigenic activity is also clear on the basis of mouse skin studies.

.

C. TOBACCO SMOKE Throughout this discussion, it has been stressed that experimental settings should, as much as possible, simulate settings as applied to man. The direct inhalation of tobacco smoke, however, has not been effectively duplicated in the experimental animal, and it appears unlikely that this can be accomplished in the future. A method permitting direct applica-

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tion of cigarette smoke to mouse skin has recently been developed by Neurath and Kroger (1963). While this method is advantageous in that it prevents secondary reactions for particulate as well as gaseous constituents of the smoke, it may not yield appropriate tumor response due to lack of a vehicle. Even very strong skin carcinogens such as PAH rarely produce tumors by topical application without a vehicle. There is no epidemiological evidence that indirect inhalation of tobacco smoke contributes to the development of lung cancer. Most animal experimentation so far has involved this type of smoke exposure. I n this setting, we not only deal with an unknown quantity of smoke particles that will contact the bronchial epithelium, but because of the great volatility of some components, the disintegration of free radicals, and the interaction of other components, we deal with a different material compared to the one that would infringe on the bronchial epithelium upon direct inhalation in a closed system. A few comments on the physiology of inhalation, some aspects of which have recently been summarized by Brieger (1963) and by Hilding (1963), are relevant here. Indirect inhalation encounters a defense system that took nature undoubtedly millions of years to develop, a barrier which the human smoker intentionally bypasses. If particles of an insoluble dye, 2 to 7 p in diameter, are added to air, rats inhaling this “air” for 7 hours will, during and immediately after this period, have most of these particles on the skin. The second largest deposit will be in the gastrointestinal tract, and the smallest in the lung (Brieger and LaBelle, 1959). One day later the lung load is still slight. The substances that finally remain in the lung depend in part on the half-life of each particle as well as the size of the lung bronchus (LaBelle and Brieger, 1959). The size of the particles also affects their disposition in the lung. The point to be made is that clearly not all components inhaled indirectly from a smoke environment enter the lower respiratory system. Hilding (1963) presents a detailed summary of the defenses set up by the nose and its turbinates in particular, as well as by other areas where inhaled particles may settle, such as the nasopharynx, the base of the tongue, and the pyriform fossa. Morrow (1960) also presents a good review on how the nose and its turbinates serve as a protective barrier. The point to be stressed again is that studies on direct inhalation as carried out by man are very difficult, if not impossible, to duplicate in the experimental animal. Difficulties involved in experimental pulmonary carcinogenesis have recently been discussed by Shabad (1962) and by U. Klein (1962). A number of investigators exposed animals to tobacco smoke in

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attempting to produce lung tumors. Essenberg (1952, 1957) and Essenberg e t al. (1955, 1956) were among the first to produce an excessive number of pulmonary ademonas in mice exposed to tobacco smoke aerosol. Essenberg has pointed out the difficulty in obtaining an appropriate dose in indirect inhalation experiments. If the dose is too low the threshold for tumorigenic response may not be reached; and if it is too high it will be too toxic. Essenberg and his co-workers were able to increase the occurrence of adenomas and carcinomas of the lung in strains of mice where tumors occur spontaneously. They attribute this in part to the nicotine content of the smoke, but not to arsenic or cigarette paper. It is to be noted that this type of lung tumor induction appears to occur only in strains of mice that also develop these tumors spontaneously. Like the Essenberg group Muhlbock (1955) also found an increase in pulmonary adenomas among mice exposed to cigarette smoke. Scala and Vicari (1955) reported negative results even among mice exposed to tobacco smoke 2 hours a day for 120 days. Holland e t al. (1963) reported on a long-term study in which they had exposed rabbits to cigarette smoke. Although they found no neoplastic changes, there was a greater number of hyperplastic changes. The occasional pulmonary cancers reported among animals exposed to tobacco smoke (Komczymski, 1958; Guhrin, 1959) tend to be of the glandular or alveogenic type and are not comparable with true squamous cell bronchiogenic carcinoma, which represents the most frequent type of cancer occurring in man. It should be recognized that in most of these studies the amount of smoke aerosols actually deposited into the bronchial epithelium was probably too small to expect tumor development in these areas on the basis of the known carcinogenic potency of tobacco smoke condensate. P. R. Peacock (1955) attempted to have hens smoke cigarettes by direct inhalation for a relatively short time period without observing neoplastic changes. Of particular importance in this general area are the experiments by the Leuchtenbergers (1958, 1960a,b, 1963). Exposing mice to smoke aerosols (Fig. 12), they studied in detail early morphological and chemical changes in the bronchi. The findings ranged from no change to severe bronchitis, atypical basal hyperplasia, carcinoma in situ, an increase in deoxyribonucleic acid (DNA), an increase of nuclear volume, and in tyrosine and dry mucus. The pathological changes described by the Leuchtenbergers are similar to those reported by Auerbach et al. (1961) and by Chang (1957) in human smokers.

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FIQ.12. Smoking chamber as used by Leuchtenberger et al. (1958).

FIG.13. Inhalation chambers as used by Kuschner et al. (1957) a t New York University.

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29 1

Extensive inhalation studies dealing primarily with different components of air pollution, including PAH, have been carried out by Laskin, Kushner, and Nelson of the New York University group. Their elaborate inhalation chamber is shown in Fig. 13. A novel approach in this field is the spray technique as utilized by Dontenwill and Mohr (1962). These investigators sprayed tobacco smoke condensate in sesame oil three times a week into the oral cavity toward the trachea of hamsters. I n another experiment they placed hamsters into smoking chambers. After 12 months these workers found, among 37

FIG. 13a. Microphotograph of epidermoid carcinoma of the lung of a mouse exposed to cigarette smoke for 16 months (Otto, 1963).

animals, 7 with papillary, noninvasive tracheal tumors. It would be of interest to see the progression of those tumors if followed for a longer period of time. Among the hamsters placed in a smoking chamber for one year benign hyperplasia with focal papillary growth of the epithelium was found. The experiments of Dontenwill and Mohr again point to the emphasis that must be placed upon the manner in which tobacco smoke is applied to the respiratory tract. Otto (1963) has recently published a study in which he exposed mice to indirect “passive” inhalation for a t least 12 months. In one group of 30 mice that was exposed to an average of smoke from 12 cigarettes in a container of 400 1. volume for 60 minutes per day for about 24

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

months, he observed, in addition to 11 animals with pulmonary adenomas, 1 epithelial cancer (Fig. 13a). I n another group of 30 mice that were exposed to an average of 12 cigarettes per day for 90 minutes in the same manner, he observed 4 lung adenomas and another epithelial cancer of the lung. I n a group of 60 animals that was not exposed to smoke, he observed 3 lung adenomas. The finding of epithelial cancer of the lung by Otto represents the only incident in the literature where this type of cancer has been reported in the lung of animals exposed passively to cigarette smoke. These singular positive results in the production of cancer are perhaps related to the total duration and amount of cigarette smoke by the inhalation technique as well as the care with which the lungs were sectioned. I n line with our own views in this matter, Otto pointed to the fact that passive smoking exposes the mice to only a fraction of the dose compared to an active or direct inhalation of tobacco smoke. So far, true bronchiogenic cancer has in but rare instances been produced in laboratory animals through tobacco smoke exposure. The only way in which more positive results could be expected, considering the dose required, would be to blow tobacco smoke into the bronchus or trachea by tubes directly inserted into these areas. A study of this type, using dogs, is a t present being carried out by Auerbach and Cahan (1963). Through a tracheotomy these investigators are setting out to apply cigarette smoke to dogs sufficiently often and over a long enough period of time to expect the induction of malignant changes. The technical difficulties and the cost of such a study are apparent. That bronchial and tracheal epithelial tissues are susceptible to tobacco carcinogens is apparent not only from Otto’s study but also from those of Blacklock (1961) and Rockey et al. (1962), referred to in a previous section of this report. More studies such as those by Auerbach and Cahan using smoke aerosol should be encouraged. Only in this fashion can the volatile components of tobacco smoke be tested together with the particulate matter. I n view of the tumor promoting activity of some volatile components of smoke, such as the phenols, and the cilia-toxic nature of such volatile components as acrolein, it may turn out that the effect of smoke aerosols applied directly to bronchial epithelium will be greater than that of smoke condensate.

D. TOBACCO SMOKE CONDENSATE FRACTIONS While i t remained to the chemist to fractionate tobacco smoke products and identify chemical components, it is the task of the biologist to

EXPERIMENTAL TOBACCO CARCINOGENESIS

293

test these fractions and components for carcinogenic activity. These studies, as in fact the entire field of environmental carcinogenesis, require the close cooperation of scientists of various disciplines. The major purpose of these investigations is the identification of components or groups of components which might contribute to the established carcinogenic activity of tobacco products (see Section V) . It was apparent from the onset that the natural and treated tobacco leaves and their combustion products consist of hundreds of components, a t least some of which are structurally closely related, and that no single component can account for or even be held primarily responsible for the demonstrated carcinogenic activity of the whole material. The approach to such a task, therefore, lies in determining the relative activity of groups of components. The final evidence as to the relative importance of such components will be best indicated when their elimination or reduction results in a reduction of carcinogenic activity of the whole product. The available data in this regard are discussed in more or less general terms, since many of the details are presented in the tables that summarize these biological studies. The first large-scale studies on tobacco smoke condensate fractions were reported by Wynder and Wright in 1957, the biological results of which have been summarized in Fig. 14. This and subsequent studies by Wynder and Hoffmann (1959a) indicated that a high activity, though not the whole tumorigenic activity of tobacco smoke condensate, resides in one subfraction which is eluted with n-hexane during the column chromatography on silica gel of the neutral portion. I n this subfraction the majority of carcinogenic PAH resides. A further breakdown of this particular fraction (Fig. 15) demonstrates that the highest short-term activity in terms of hyperplasia and sebaceous gland destruction resides in the PAH-containing subfractivn. It is clear, however, that the activity of even this subgroup cannot be due solely to the PAH. Another group of components, also present in this fraction, possibly terpenes, might contribute to this activity. However, i t would appear that additional tumorigenic components are in this fraction. The role of PAH in tumorigenic activity of the fractions has been regarded by Wynder and Hoffmann (1959a) to be, though important, relatively small. It was estimated that B [ a ] P could not account for more than 2.4% of the tumorigenic activity of the whole condensate and not for more than 10% of that of the above-mentioned subfraction. Because of the known effect of even the most minute amounts of a tumor initiator (M. Klein, 1956; Hadler et al., 1959) when followed by application of a tumor promoter, the contribution of polynuclear aromatic hydro-

294

ERNEST L. WYNDER AND DIETRICH HOFFMANN

carbons in tobacco carcinogenesis cannot be given a numerical value. The studies by Wynder and Wright (1957) also indicated that the tumorigenic activity of the acidic plus the neutral fraction, as well as that of the nicotine-free basic portion plus the neutral fraction tended to be, though not to a statistically significant degree, greater than that of the neutral fraction alone when tested in concentrations present in tobacco smoke condensate. I n 50% concentration acidic and nicotinefree basic portions possessed minimal tumorigenic activity. These data suggest the presence of some type of tumorigenic components in these portions of tobacco smoke condensate.

7

c:

@

+kid* Partion

Parlion

-

P-

c+ P: +

I Satyr. NaHCO.-Wlion

*

f Naubal+ Phulolic

P

h+++

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n-naiana

2.0% C:

-

R-

I

n-Maianr

c. -. . P:

-

I

I

c+++

c-

n-Mrmna

R-

+++

I

I

I

CCI. /RONEM

Baruw

Wrta#aM

C:-

C:-

c-

7

MrWrokol

C:-

FIG. 14. Fractionation scheme of cigarette smoke condensate (Wynder and G. Wright, 1957; Wynder and Hoffmann, 1961b, 1963~).

Studies by Roe et al. (1959) and by Wynder and Hoffmann (1961b) have demonstrated tumor-promoting activity for the weak acidic fraction (“phenolic fraction”) applied to mice initiated with DMBA and with repeated applications of B [ a ] P in about the same concentration in which the fraction is present in tobacco smoke condensate. It is thought that this tumor-promoting activity may be due, at least in part, to phenol itself, as well as to substituted phenols, which Boutwell and Bosch (1959) and later also others have shown to possess (Wynder and Hoffmann, 1961b) tumor-promoting activity. Similar activity was also

295

EXPERIMENTAL TOBACCO CARCINOGENESIS

found for the acidic fraction. The concept of the weak acidic portion as tumor promoter was recently challenged (Bock and Moore, 1962). However, their data were based on a chemical separation of the whole “tar” and do not appear to permit such a conclusion. Clem0 and Miller (1960), using the PAH-containing subfraction of an air pollution sample as initiator, showed tumor-promoting activity for a fraction of the neutral portion. I n this regard it must be stressed that the terms neutral fraction, acidic, phenolic, or basic fraction of CCl,

Eluate from Silica Gel Chromatogram of the neutral portion

L

I I

B(a)p 250ppm 05% S T + H

1.2 Yo

,Dirtributlon cvclohexans

\

nitromet hone

FIG. 15. Benzolalpyrene and short-term activity (ST) of subfractions of fraction B of neutral portion (Wynder and Hoffmann, 1961a).

tobacco smoke do not necessarily mean the same fraction in terms of chemical characteristics in each experiment (Section V) . It also should be noted that chemical separations of fractions seem to lead to a loss of tumorigenic activity when these fractions are subsequently recombined again (Wynder and Hoffmann, 1 9 6 3 ~ )It . is not apparent why a similar loss of tumorigenic activity was not previously observed by Wynder and Wright (1957). Studies using the subcutaneous route in rats by Seelkopf et al. (1963) also demonst,rated a greater tumorigenic activity in terms of induced sarcoma for the neutral fractions containing most of the polycyclic

296

ERNEST L. WYNDER AND DIETRICH HOFFMANN

hydrocarbons, whereas relatively slight sarcogenic effect was observed among rats that had received the acidic and basic fractions. We can generalize from existing biological experiments with tobacco “tar” fractions that the major tumorigenic activity resides in the neutral fraction. This fraction also contains most of the initiating activity whereas the major tumor-promoting activity seems to reside in the acidic and weak acidic fractions. Some studies generally regarded to deal with whole smoke condensate do, in effect, apply to a fraction of the condensate. The detailed studies by Bock and Moore (1959, 1962) and Bock e t al. (1962) are to be regarded in this light. They used only the “heptane-soluble” components of the “tar” (“refined tar”) for their biological studies which they found to be of a higher tumorigenic activity than the whole smoke condensate. Thought must be given to possible antitumorigenic agents both in terms of “antiinitiators” as well as “tumor retarders.” The former fits into the general concept of competitive carcinogenesis between strong and weak PAH, as well demonstrated in studies by Steiner and Falk (1951) and recently by Kotin and Falk (1963b), using subcutaneous tissues as test tissue and our own studies (Wynder and Hoffmann, 1962b, 1963b,c) with epithelial tissue. Of particular interest is the inhibiting effect of benz[a]anthracene to B[a]P. The concept of antitumor promoters represents a n area in which very little has been done. Preliminary studies suggest that camphor may, to some extent, inhibit the tumor-promoting activity of phenol (Wynder and Lyons, 1961). It would not be surprising if inhibiting substances for phenols would also be present in combustion products. In terms of inhibition, one must also list substances that may interfere with the absorption of components. Studies by Hoffmann and Wynder (1963~) have shown that in pure form some of the long-chain hydrocarbons reduce the tumorigenic activity of B [ a l p , possibly by interfering with its absorption (see Section V ) . No single component has been identified in tobacco products which in the concentrations found, could account for the tumorigenic activity of these products, A number of carcinogenic PAH, a few carcinogenic heterocyclic hydrocarbons, and several tumor-promoters, as well as a number of sarcogenic components, have been identified in tobacco smoke condensate. Data on these components will be reviewed in Section V. While i t may be regarded as established that some of the possible constituents of tobacco smoke condensate, such as nickel tetracarbonyl and nitrosamines, are carcinogenic to laboratory animals, and that

EXPERIiVIENTAL TOBACCO CAHCINOGENESIS

297

arsenic is carcinogenic to man (see Section V ) , the tumorigenic activity of tobacco smoke condensate for laboratory animals cannot be due to these substances.

E. STUDIES OF SPECIAL FACTORS A number of studies have dealt with special factors, such as viral infection, nutritional deficiencies, radiation, trauma, and heat, that might affect the development of malignant tumors. Other experiments have attempted to study the relative initiating- and promoting-activities of tobacco products by combining their application with PAH and croton oil. Attempts also have been made to determine possible tumorigenic activity of tobacco products by short-term assays. 1. Short-Term Tests

Biological short-term tests for estimating tumorigenic activity of a given material have been established by different investigators-a subject extensively reviewed recently by U. Klein (1961). Some of these are: the sebaceous gland destruction test for tobacco smoke condensate by Suntzeff et al. (1955; 1957); GuBrin and Cuzin (1961); Wynder and Hoffniann (1961a) ; Kracht et al. (1961, 1963) ; and the triton test by Neukomm (1959). The sebaceous gland test is held by Kracht and Hiibner (1962), who have studied this subject recently in some detail, as relatively specific. I n contrast these investigators report negative experience with the triton test, which was originally reported by Neukomm (1959). Using the sebaceous gland short-term tests, Suntzeff et aZ. (1957) showed a relatively close parallel between the long-term results with tobacco smoke condensate and tobacco fractions. As reported by Wynder and Wright (1957), these short-term tests were positive for the fraction eluted from the neutral portion with carbon tetrachloride, which contained most of the aromatic hydrocarbons and which was found to have long-term tumorigenic activity. Kracht et al. (1961) utilized the sebaceous gland technique to investigate dose response and also observed the greatest sebaceous gland destructive activity in the “aromatic fractions.” Sebaceous gland tests may, therefore, serve as a guideline for the chemist, suggesting on which particular fraction to concentrate first, but they obviously cannot replace the long-term tests. Mellors e t al. (1957) studied the “fluorescence uptake” by the mouse skin of various cigarette smoke subfractions as used in the Wynder and Wright (1957) study. They found a correlation between “fluorescence uptake” and previously demonstrated tumorigenic activity.

298

ERNEST L. WYNDER AND DIETRICH HOFFMANN

2. Tumor Initiation and Promotion Tobacco smoke condensate has been definitely demonstrated to have promoting activity. Gellhorn (1958) has shown in a large-scale study that if mouse skin is treated twice with 200 pg. of B [ a ] P and then followed with 10 mg. tobacco smoke condensate given 5 to 6 times a week, a significantly greater tumor yield ( p < 0.01) is obtained compared to applying either of these components alone. Tumor-promoting activity of tobacco smoke was also demonstrated by using 300 pg. of DMBA as the single initiator followed by repeated applications (3 times weekly) of tobacco smoke condensate in as low a concentration as 10% (Fig. 16, Wynder and Hoffmann, 1962b). I N%smoke condensate ISC) II 30 (rg DMBA +lo% SC 111 30 pg DMBA +%%SC N 30pgDMBA

I

2

4

6

.

8

I

10

,

I 12

,

1

14

0

1 16

.

IN

18

Months

FIQ.16. Tumor-promoting activity of cigarette smoke condensate (Wynder and Hoffmann, 1962b).

Adding 1.25 pg. of B[a]P per application to 20% tobacco smoke condensate increased tumor yield over that of 20% condensate alone (Roe, 1962). The response represents according to Roe, a multiplication rather than a summation effect. When twice the amount of 17 different PAH’s as found in tobacco smoke condensate was added to the smoke condensate, a statistically significant increase in tumor yield was obtained with 50% (‘tar” as compared to that of 50% standard smoke condensate (Wynder and Hoffmann, 1 9 6 3 ~ ) . The painting of the oral cavity of mice with cigarette smoke con-

EXPERIMENTAL TOBACCO CARCINOGENESIS

299

densate increased the development of pulmonary adenomas in strain A mice which also received urethane intraperitoneally, an increase that is greater than could be explained on the basis of summation alone (DiPaolo and Sheehe, 1962). Experiments using croton oil applications following tobacco smoke applications did not result in an increased tumor yield. Gellhorn (1958) applied a 0.5% solution of croton oil 5 to 6 times a week to mice also receiving 10 mg. of tobacco “tar” 5 to 6 times a week. There was no significantly greater tumor yield observed than when each material was applied alone. The findings of Ranadive et al. (1963) of the synergistic effect of croton oil and nicotine-free extract of tobacco are contrary to the studies using croton oil and cigarette smoke condensate. Further work in this line with tobacco extracts appears indicated. The available biological data on initiators and promoters conform with the data from whole tobacco smoke condensates. Its relatively high tumor yield and relatively long “conversion time” from papilloma to carcinoma, as well as the observed tumor regressions, indicate that tobacco smoke is a more powerful promoting than tumor-initiating substance. On the other hand, it is apparent, in view of the fact that tobacco smoke condensate does produce carcinoma in a variety of animal tissues without any added components, that it must be regarded as a complete carcinogen although its promoting activity is to be regarded as greater than its initiating activity. 3. Virus

So far only one group of investigators has studied the possible coeffect of viruses and cigarette smoke on bronchial tissues, an area first introduced in terms of the interaction of virus infection and aerosols of ozonized gasoline by Wisely et al. (1961) and Kotin and Wiseley (1963). I n a recent study, C. Leuchtenberger et al. (1963) demonstrated that atypical proliferative changes in the bronchial mucosa of mice were lowest in frequency in mice that inhaled cigarette smoke only. It was more pronounced in those receiving influenza virus only, while a cumulative effect was observed in those that received both the influenza virus and cigarette smoke. In the latter group, squamous cell metaplasia and transgression of epithelium in the lung parenchyma were found more often in males than in females. Exposure to these substances was also associated with a moderate increase in DNA, RNA, and protein. Abnormal cellular RNA agglomerations also were noted in the animals exposed to virus alone. The authors suggest that because of the greater proliferative changes in male mice a study of the influence of sex hormones alone or in combination with virus and cigarette smoking may be in order.

300

ERNEST L. WYNDER AND DIETRICH HOFFMANN

4. Radiatio;n

Cowdry et al. (1961) observed that periodic P-radiation from strontium-90 to mouse skin in total doses of up to 20,800r produces additive effects, though not statistically significant in terms of skin cancer, when applied concurrently with cigarette smoke condensate. A synergistic effect between the two types of application was not obtained. Bock and Moore (1959) observed that intense irradiation of a small area of mouse skin increased the sensitivity of a distant area of the skin to cigarette smoke condensate, a sensitivity that was more pronounced among Swiss female than among C3H male mice. Salley (1963), applying ultraviolet (UV) radiation only to the ears of mice, could not demonstrate any statistically significant difference in neoplastic yield over that of UV and smoke condensate.

5 . Nutritional Deficiency The knowledge that nutritional deficiencies, particularly of the vitamin-B group, might relate to oral cavity cancer in man led Kreshover (1955), and Kreshover and Salley (1957, 1958) to a series of studies to determine the increased susceptibility that such deficient animals have to tobacco smoke condensates. They concluded that vitamin-B complexdeficient CAF, mice treated with smoke showed frequent cellular abnormality suggestive of precancerous changes or carcinoma in situ. Swiss strain mice were less responsive than were CAF, mice. Golden and albino hamsters showed no oral changes but the ears evidenced hyperkeratosis and hyperplasia. The authors do not give sufficient details about the nutritional part of this study to interpret them fully. However, on the basis of the long-term survival of their animals, i t would appear that the deficiencies were not very severe, since i t is known that on a complete B-complex-deficient diet, or for that matter, on a complete riboflavin-deficient diet, mice cannot survive more than 3 months. The basic concept, however, appears worthwhile pursuing in that i t is conceivable that nutritional deficiencies produce changes in the mucous membrane of the oral cavity of these animals, making them more susceptible to tobacco smoke as a carcinogen.

6. Trauma uzd Heat Exposing mouse skin to 58°C. for 3 minutes after application of Indian cigar “tar” on alternate days enhanced tumor response (Reddy e t al., 1960). After 4 months of Yar” and heat application, 6 of 14 female Swiss mice as well as 5 of 14 male Swiss mice showed malig-

EXPERIMENTAL TOBACCO CARCINOGENESIS

301

nant changes. Exposure to heat only produced a tendency to ulceration. Despite thc limited number of mice, this effect of heat to mouse skin, freshly painted with tobacco “tar” appears significant. Further studies in this general area would appear to be in order. To our knowledge, the only study dealing with trauma and tobacco “tar” was reported by Bock and Moore (1959), who sandpapered mouse skin prior to applying the “tar.” No qualitative differences in the biological response and no increased tumor yield were found. This study was done in part to evaluate the possible effect of shaving animals as a contributory factor to increased tumor response-a concept that may be ruled out by this study.

F. CILIA-TOXIC COMPONENTS The relationship of cilia-toxic agents in tobacco smoke and experimental tobacco carcinogenesis may appear only to be an indirect one. Yet, it cannot be emphasized too strongly that in the pathogenesis of bronchiogenic cancer, be it in man or in animal, without cilia stasis and concomitant mucus stagnation, subsequent metaplasia from ciliated to squamous epithelium and from there to epithelial cancer could not occur. A well-functioning ciliated epithelium, with adequate mucous flow, would tend to resist the absorption into underlying cells of tobacco smoke components and of particles that float on its surface. The destruction of this defense barrier, therefore, should be regarded as a first and obligatory step leading to neoplasia of bronchial epithelial tissue. Tobacco smoke is unique in that, in addition to tumor initiators and promoters, it also contains cilia-toxic agents in its vapor phase, as well as in its particulate matter. The cilia toxicity of tobacco smoke has been shown to apply to a variety of animal tissues as has tobacco smoke condensate in solution. Yroetz (1939) observed cilia stasis by blowing tobacco smoke on the upper respiratory mucosa of rabbits. Hilding (1956a-d, 1957, 1961), who has studied this problem in detail, has noted cilia stasis in the respiratory tract of freshly killed cows after introducing cigarette smoke. Mendenhall and Shreeve (1937, 1940) found cigarette smoke solutions to be toxic to cilia obtained from calves. Using cigarette smoke or smoke solutions, similar results were found by Falk et al. (1959, 1961, 1963), using frog esophagus and rat and rabbit trachea. Boche and Quilligan (1959) used cilia of chick lung and trachea tissue cultures; Dalhamn (1959), exposed trachea of living rats; Ballenger (1960), human respiratory cilia; Kensler and Battista (1963) dissected rabbit trachea; Wynder e t al. (1963a) and George (1963), gills of clams exposed to cigarette smoke and smoke solutions (Fig. 17); and Hornburger and Bernfeld (1963), adenosine triphosphate (ATP)-

302

ERNEST L. WYNDER AND DIETRICH HOFFMANN

qj ~1 ~

-

Water extracts of cigarette smoke condensate and its several fractions: T - - Standard condensate A- Acidic fraction P-- Phenolic fraction I -- Insoluble portion 6- Basic fraction N-- Neutral fraction

2M 100 0

I

A P I BNT

A P I E N 1

A P I B N

A P l B N

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n Phends and nicotine ETH-PH .-Ortho-ethyl phenol -CR------ Ortho-cresol Meta-cresol Parairesol 24 ,4-0'methyl phenol

______ *

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1.0%

0.5n

0.1n

Total stasis ltext step dl

n

Partial stasis (text stepc)

toss d metachronic wave in taterat ciiia(text step bl.

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FIa. 17. Tests of cigarette smoke components on the gills of the clam in tcrnis of possible cilia-toxic activity (Wynder et al., 1963a).

primed frog esophagus. Dalhamn (1963) has also demonstrated cilia stasis in rabbits with in viwo studies. Cilia stasis cannot be observed directly in man although this condition may be inferred from a number of clinical and post-mortem studies. A majority of heavy cigarette smokers reveal a history of persistent cough indicative of altered mucous flow in the respiratory tree (Phillips e t al., 1956; Hammond, 1961; Brinkman and Coates, 1962). Autopsy data have shown that with heavy smoking of cigarettes there is progressive destruction of the cilia in the trachea and bronchus. After 20-40 years of such smoking relatively few viable ciliated cells lining the hilar region remain (Chang, 1957; Knudtson, 1960; Auerbach e t al., 1960, 1961). The question now centers on what components in cigarette smoke are responsible for its cilia-toxic activity. Nicotine in the concentration

EXPERIMENTAL TOBACCO CARCINOGENESIS

303

present in cigarette smoke was not found to be cilia-toxic by Boche and Quilligan (1959). Rakieten et al. (1952) found that a 2% solution of nicotine stopped ciliary action of excised respiratory epithelium of rats, rabbits, and humans in 5 to 10 minutes, and that a 10% solution took 5 minutes. Falk e t al. (1959) attributed some of the whole smoke effect on ciliated mucus-producing epithelium to nicotine and other alkaloids. Nicotine, when given in amounts of 2.8 to 5.6 mg. exhibited a significant decelerating effect on mucous flow. The effect was similar when applied in aqueous solution by impingement or as an aerosol. These authors found no change in mucous flow after applying phenol in the concentration in which i t is found in cigarette smoke. Bernfeld et al. (1964) also found no effect of phenol vapor and phenol solution upon the rate of mucus transport in ATP-primed frog esophagi but morbid inhibitive effects were seen when phenol vapor was applied in the presence of cigarette smoke from a filter cigarette. Phenol did not potentiate the ciliatoxic effect of a nonfilter cigarette, probably because the smoke from this cigarette is already rich in phenols. Using the gills of clams as test organ, Wynder et al. (1963a) found strong activity for the acidic and phenolic fractions of cigarette smoke condensate as well as for phenols (Fig. 17). George (1963), using a similar test system but with mucus removed, confirmed these findings and also observed cilia-toxic effects for phenol vapor. Aliphatic acids in cigarette smoke (formic through butyric, also oxalic and benzoic) have displayed cilia toxicity in concentrations of 0.05% (Wynder et al., 1963a). Aldehydes have effects similar to that of phenol. Formaldehyde, acrolein, and croton aldehyde have the strongest toxicity on clam gills of the aldehydes tested (Fig. 17a) (Wynder et al., unpublished data). Among additives to smoke used, menthol was tested in 0.04% concentration on excised respiratory epithelium of humans, rabbits, and rats and produced no cilia-toxic effect (Rakieten et al., 1952). Guillerm et al. (1961) found cilia-toxic activity for acrolein and acetaldehyde both in liquid and gaseous states (Table I V ) . Recently Kensler and Battista (1963), using rabbit trachea and what is in essence a synthetic “mucus” (an isotonic solution of 2% egg albumin is continuously added to the mounted tissues), found particularly high activity for acrolein and formaldehyde, with less for phenol vapor (though these are still incomplete results). Using the clam gill test, Wynder and co-workers found acrolein to be a t least twice as cilia-toxic as phenol. Kensler and Battista’s experiments involve repetitive exposure of the tissue to toxic agents, in a fashion which tends to simulate human smoking habits. George (1963), using human ciliated tonsillar epithelium as a test system, found strong cilia-toxic activity for phenol and still stronger activity

304

I m 0 a 4 m

n a:

L

ERNEST L. WYNDER AND DIETRICH HOFFMAX"

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!

H-

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A c r Cr P H 0.05%

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(HWmwlRecovery o f n i e t a c h r o n i c w a v e

( t e x t step d l

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FIG.17a. Cilia toxicity of aldehydes and organic acids (Wynder et al., 1963a and Wynder et al., Cancer, in press).

g

x

306

ERNEST L. WYNDER AND DIETRICH HOFFMANN

for acrolein, thus qualitatively duplicating the findings obtained with clams. He found relatively low activity for acetaldehyde. I n regard to these studies it must be realized that such agents as nicotine, phenols, acids, and aldehydes have been tested in pure form. Their activity as part of tobacco smoke or smoke condensate almost certainly would be modified. A large portion of the nicotine in tobacco smoke, for example, is present in the neutralized form, and therefore tests TABLE IV LOWESTCONCENTRATIONS IN RINGERSOLUTION LEADINGTO O F RAT TR.4CHEAa Compound F’ormaldehyde Acetaldehyde Propionaldehyde Isobutyraldehyde Furf ural Aerolein Acetone Methyl ethyl ketone a

THE

STOPPAGE OF CILIA

Concentration (g./liter) 2x 3 x 3.5 x 4.5 x 7.5 x 9x

10-4 10-3 10-3 10-8 10-3 10-6 10-1

8 X

From Guillerm et al. (1961).

with pure nicotine may well be misleading as to its role as a cilia-toxic agent in cigarette smoke. Similar considerations are in order for other components that are being tested in pure form, either in the gaseous phase or in solution. The study by Guillerm et al. (1961) is important in this respect. After showing that cigarette smoke had cilia-static activity, a property also shown by several aldehydes and ketones, they demonstrated a synergistic effect of gaseous acrolein and acetaldehyde found in the condensate of a single cigarette. Recently, agents that may counteract the cilia-toxic effect of tobacco smoke have received attention. With epithelial tissue pretreated with acetylcholine, eserine, or arecoline prior to applying cigarette smoke, Falk et aZ. (1963) were able to obliterate its secondary inhibitory effect on mucous flow while leaving the primary acceleratory effect unchanged. Studies in our laboratory have suggested that camphor can partially counteract the cilia-toxic activity of 0.5% phenol. Falk et al. have attributed only a minimal response of the ciliated epithelium to the gas phase of tobacco smoke. They feel that filtration of smoke could be regarded as effective only if all particulate matter is removed and only the gas phase remains. Our own data, in agreement with those of George (1963) and Dalhamn (1963), indicate a decrease in cilia toxicity of smoke obtained from cigarettes with effective filter tips compared to those of nonfilter cigarettes.

EXPERIMENTAL TOBACCO CARCINOGENESIS

307

Quantitative differences between investigations in terms of specific cilia-toxicity may be due a t least in part to variations in the test systems used. Special studies appear in order with respect to the nature of the protective influence of mucus, some aspects of which have been recently well reviewed in a symposium on mucous secretions (New York Academy of Sciences, 1963). The solubility of various components in mucus, the type and amount of mucus, and the ability of tissue to respond with additional mucous secretion may also significantly influence the ability of agents to affect the underlying ciliated epithelium. The respective roles played by the bronchial glands and the goblet cells, as recently summarized by Hayek (1962) in producing mucus under normal and adverse conditions, are of great interest and require more study. Before the cilia static components can act, they must diffuse through the protective mucous coat, a covering whose composition is only partly known and is, in the smoker, heavily interlaced with nonphagocytized particles being transported out of the bronchi. All studies reported to date have shown that cigarette smoke affects the metachronic activity of cilia, a motion that is necessary to propel the viscid mucoid mass. During inhalation, in the absence of effectively beating cilia, mucous flow slows down and perhaps stops. At that time components in cigarette smoke may act upon the underlying cells as can the entrapped particles. The demonstration that cigarette smoke is cilia-toxic in all test systems is convincing, but further studies are necessary before some of the details may be accepted as proved. These include investigations of absorption rates of carcinogens by mucus-secreting epithelial tissue, as well as studies of the possible effects of cilia-toxic components on goblet cells, tracheal, and bronchial glands. The entire area from the basement membrane to the overlying mucous coat necessarily must be involved. In interpreting present biological studies, we must consider that we deal largely with the effect of gaseous components. As is, however, apparent from studies with water extracts, particularly of the weak and strong acidic fraction, nonvolatile components can also be cilia-toxic. An experiment that would most efficiently test the volatile as well as the particulate components would of necessity involve a system such as exists in the bronchial tree and is most closely approximated by Dalhamn (1959, 1963) in his studies. One area that deserves more attention than it has received in the past is the influence of tobacco smoke components on mucous integrity. It would appear that change in mucous flow cannot only result from diminished cilia activity, but also from an increase in viscosity of the mucus itself. Experiments, particularly with acidic components of tobacco smoke, indicate this likelihood. For tests of cilia-toxic components, Dalhamn’s system is perhaps the most elegant. Thus, the respiratory epithelium of animals may be used

308

ERNEST L. WYNDER AND DIETRICH HOFFMANN

in vivo to study many of the effects of cigarette smoke. We must, of course, consider that the human smoker exposes himself repeatedly over a long period of time to a complex of these components. The evaluation of short-term experimental results must be supplemented, therefore, by studies of the cumulative (long-term) effects on cilia of whole tobacco smoke, or that from which certain components have been selectively reduced or eliminated. V. Certain Constituents of Tobacco Products

A. SMOKECONDENSATE YIELDS The chemical composition of a tobacco product, as well as the physical variables, affects the amount of smoke produced. Physical variables include the amount of tobacco, volume of air participating in the combustion, moisture content of the tobacco, and relative humidity of the air and combustion temperatures. These variables are, of course, interrelated with such properties as geometrical shape of a tobacco product, tobacco cut, tobacco weight, pressure drop (draw resistance), volume, shape, and duration of puff, frequency of puff, and puff intermission. Most study groups attempt to simulate general human smoking habits, thereby maintaining certain standard conditions of duration, volume, and frequency of puffs. One should remember, however, that these chosen parameters by no means represent the standard for the individual smoker. On the other hand, the experimental conditions need never be set up to cover such extreme situations as taking a 2-ml. puff or smoking with continuous suction. 1. Cigarette Smoke Condensates

The yield of mainstream smoke condensate of cigarettes ranges from 1 to 6% of the weight of the original tobacco products, and ranges between 20 and 35 mg. per nonfilter cigarette. It is beyond the scope of this review to discuss in detail the multitude of data existing on cigarette smoke condensate obtained in various laboratories (Trifu and Dumitrescu, 1958; Wartman et al., 1959; Wynder and Hoffmann, 1960; Ogg et al., 1962). The short review of standard smoking procedures currently used in various countries (see Section 111) indicates some of the difficulties confronting the reviewer of such data. I n line with the definition of smoke condensate, as well as describing the dispersed and the condensable phases of cigarette smoke, it is pertinent to discuss which factors may influence the yield of condensate within a given standard method. The method of Wartman et al. (1959) is also used in our laboratory, where moisture conditioning and standard

EXPERIMENTAL TOBACCO CARCINOGENESIS

309

weighing procedures are observed. On the basis of equal tobacco weight per cigarette, comparisons of condensates obtained from cigarettes made exclusively of Virginia, Turkish, low-nicotine Burley, or Maryland tobaccos show that tobacco selection has a decisive influence on the yields of the condensate. Virginia tobacco cigarettes gave 33.4 mg. per unit; Turkish tobacco 31.5 mg.; cigarettes made of low-nicotine Burley yielded 25.6 mg.; and those of Maryland gave 21.2 mg. (Wynder and Hoffmann, 1963a). There has been some decrease of condensate content in popular brand cigarettes during recent years (Wynder and Hoffmann, 1960), attributable partially to changes in tobacco blend, and to the use of reconstituted tobacco; porous paper may play an additional role. Newsome and Keith (1957) published a detailed study on the effect of physical variables on the weight of smoke, including tobacco moisture and relative humidity. When standardizing for atmospheric and tobacco moisture, the amount of smoke collected a t a relative humidity of 90% is insignificantly lower (by about 2 mg. per cigarette) than the amount collected a t 20% relative humidity. Tobacco moisture exerts a much larger effect on smoke weight, according to Newsome and Keith. The weight of smoke decreases with increasing cigarette moisture content, but the authors do not state how much of the resulting mainstream smoke is actually dry condensate. Golaz e t al. (1959) studied relative humidity of the air and tobacco moisture in regard to condensate yield by electrostatic and by cold trap precipitation. They found the absolute “tar” quantity to be little influenced by relative air humidity and by tobacco moisture. They also reported that the higher the relative humidity and tobacco moisture, the lower the yield of nicotine in the “tar.” This is in agreement with findings by Neurath and Horstmann (1963), who compared yields of smoke condensate and smoke constituents on the basis of amount of tobacco contributing to the mainstream smoke. They found that the amount of dry condensate in the mainstream smoke remained a t a rather constant level for different moisture conditions. These authors also observed the diminishing effects of higher moisture contents on nicotine yields. Other factors influencing the nicotine content of cigarette smoke were studied by Kobashi e t al. (1959). Newsome and Keith (1957) worked under different standard smoking conditions from Neurath and Horstmann. The latter noted a strong influence of tobacco moisture on number of puffs, as is also described by Borowski and Seehofer (1962). Since Newsome and Keith took 12 puffs of 44 ml. volume with a frequency of 1 per 30 seconds, they have necessarily less tobacco burned in highly moisturized cigarettes than dry

310

ERNEST L. WYNDER AND DIETRICH HOFFMANN

cigarettes. The effect of moisture on individual smoke constituents will be discussed in regard to specific smoke components. Newsome and Keith find a linear relationship of puff volume and number of puffs to smoke weight. Wynder and Hoffmann (1960) reported higher yields of condensate a t higher puff frequency. Taking a 35 ml. puff of 2 seconds duration once per minute yielded 35.1 mg.; puffing twice a minute gave 53.0 mg.; and 3 puffs yielded 65.1 mg. condensate. These values were not quite in line with data expected from the amount of tobacco consumed. At higher level of puffs, the collection system apparently is not efficient in precipitating all particulate matter (see also Waltz et al., 1961). Increasing the puff volume and concurrently increasing the puff velocity also elevates condensate yield per cigarette in a linear relationship (Hoffmann and Wynder, 1 9 6 3 ~ ) . The first half of a cigarette yields less condensate on the basis of equal amounts of dry tobacco consumed than the second half of the same cigarette smoked under the same experimental conditions. This would be expected, owing to condensate deposit from the first half of burned tobacco (Wynder and Hoffmann, 1960). The ratio of condensate obtained from the first compared to the second 31-mm. section of a cigarette was 1:1.4. This finding merely reflects the pattern of smoke generated per section of cigarette as observed also by Newsome and Keith (1957). Recently, Ayres et aE. (1963) proposed an “equation” for the calculation of “tar” values obtainable under various smoking conditions. The application of such an equation appears to be limited, since “tar” is not a physicochemical entity, but an arbitrarily defined material. The conclusion that the smoke yield of a cigarette is dependent on the efficiency of the combustion process but independent of the distance between burning zone and cigarette end (Keith and Newsome, 1957) has been challenged by Meyer-Abich (1963) on the basis of theoretical considerations.

2. Smoke Condensate of Cigars An automatic smoking machine specially built for cigars (Schepartz, 1959) was designed to produce condensate from a large number of cigars rather than smoke for quantitative analysis from individual cigars and has, therefore, not been used to determine condensate yield. Other studies with cigar smoke condensate were designed to determine the tumor response on mouse skin in comparison to cigarette and pipe smoke condensate and describe the manner of smoking, but not the amount of “tar” obtained (Croninger et al., 1958). The lack of data on smoke condensate yield of cigars is not surprising since i t appears to be rather difficult to determine standards in

EXPERIMENTAL TOBACCO CARCINOGENESIS

311

smoking technique that simulate the habits of cigar smokers, and also because of the large variety of cigars. Kuratsune’s work (1956) indicated that Japanese cigars yield about 300 mg. condensate when smoked with a puff volume of 20 ml. a t rather frequent puffing. The smoke was collected in benzene-filled traps. The need for an analytically sound method has been met by Fishel (1960). The “anhydrous total particulate matter” increases with growing volume and higher frequency of puff. The determination of moisture is emphasized as a necessity in the estimate of total smoke solids since Fishel demonstrated that lower puff frequencies result in lower outputs of moisture in the mainstream smoke. This finding may be reflected in the chemical composition of the resulting smoke condensate. The puff volume seems to exert a smaller, though notable, effect on moisture variation in the smoke. Data for anhydrous smoke solids of cigars were reported graphically in this study; for comparison with data from our laboratory, we present values obtained a t 28-second puff intermission and 35-ml. puff volume. Under these conditions, Fishel’s “perfecto-shape cigar” yielded about 25 mg. dry condensate per gram of tobacco smoked. Hoffmann et al. (1963) obtained 13 mg. and 11 mg. dry condensate for 1 g. tobacco weight of a popular United States and Havana cigar, respectively. With smoking parameters set a t 35-ml. puff of 2 seconds duration twice a minute the individual United States cigar weighing 7.8 0.3 g. gave 101.4 mg. mainstream smoke condensate, the Havana averaging about 8.6 I+ 0.5 g. yielded 94.6 mg. condensate. Discrepancies between values reported by Fishel and those by Hoffmann et al. could be due to differences in cigar types smoked, as well as to differences in smoke solid collection systems (Cambridge filter vs. electrostatic precipitation). The amount of smoke condensate obtained on the basis of 100 g. cigar smoked, 1.3 g. for the United States and 1.1 g. for the Havana cigar, was compared with that of cigarettes smoked under the same conditions (2 puffs per minute). One hundred grams of cigarettes yielded 6.2 g. of condensate (1 puff per minute 4.1 g.). This comparison of condensate yield should not be overlooked in attempts to correlate data of other chemical constituents of different smoke products (Hoffmann et al., 1963).

3. Pipe Smoke Condensate Pipes smoked with pipe tobacco a t a rate of 2 puffs of 35 ml. (2 second duration) per minute yielded 67.5 mg. “tar” from 1.5 g. tobacco in the pipe; per 100 g. tobacco a yield of 4.5 g. “tar” (Hoffmann e t al., 1963). Under equal conditions when the pipes were fillled with 1.5 g. cigarette tobacco a t a moisture content close to that of the pipe tobacco

312

ERNEST L. WYNDER AND DIETRICH HOFFMANN

(13.1%) there was a yield of 43.5 mg. “tar.” One hundred grams of cigarette tobacco smoked in a pipe thus yielded 2.9 g. condensate, an amount considerably below the “tar” weight obtained from smoking this tobacco in the form of cigarettes (6.2 g.), owing to the more “complete combustion” achieved in the pipe bowl. 4. Oriental Pipe Smoke Condensate

Rakower and Fatal (1962) were among the first to set up a study on the “tar” content of smoke from the Narghile, the Oriental water pipe. The tobacco, generally used in Yemen, is tombac, a kind of makhorka. Rakower and Fatal compared its smoke with that of a popular brand of blended tobacco. The tombac smoked in the Boori (the pipe bowl) yielded only 161 mg. “tar” per 10 g. tobacco, a t a puff volume of 200 ml. with puff pauses of 60 seconds. The tobacco blend under these conditions yielded 262 mg. “tar.” Most remarkable, however, is the filter efficiency of the water in the Shishi, the water-filled main volume of the Narghile. When the Narghile is used with water, as normally practiced, “tar” yield from 10 g. of tombac is 84 mg. and from 10 g. of tobacco blend, 142 nig. I n other words, the percentage of ‘%ar” absorbed by the water is 82% in the case of tombac and 91% for the blended tobacco. That the water in the Shishi is a highly efficient filter medium for smoke condensate has been confirmed by Hoffmann et al. (1963). One hundred grams of Syrian tobacco smoked in a Middle East Arkileh without water yielded 1.7 g. of “tar,” whereas 650 ml. water in the Shishi retained 0.96 g. of this quantity. Thus, only 0.74 g. condensate is obtained when the Arkileh is smoked with parameters of 2 puffs per minute of 35 ml. and 2 seconds duration. Glowing charcoal was used to ignite the tobacco (2.2 g.) in the Boori. The authors reported experimental deviations to be higher than +5% owing to difficulties in securing continuous burning of the tobacco.

B. POLYNUCLEAR AROMATIC HYDROCARBONS (PAH) The demonstration of tumorigenic and cilia-toxic activity of various tobaccos and tobacco smoke products has presented a challenge to thc chemist to identify the causative agents. I n this attempt, the ‘?,ar” was fractionated and the end fractions were tested for their activities on mouse skin (Wynder and Wright, 1957; Wynder and Hoffmann, 1961a,b). Figure 14 summarizes the results of the tests expressed in relative carcinogenic and promoting activities compared t~ whole condensate. The most carcinogenic single fraction is fraction B, which contains all known

EXPERIMENTAL TOBACCO CARCINOGENESIS

313

carcinogenic PAH (Wynder and Hoffmann, 1959a), as well as terpenes, solanesol esters, and phthalates (Wynder and Hoffmann, 19634. 1. Isolation and Identification of PAH

Chemical-analytical studies with PAH originated with the discovery by Cook, Hewett and Hieger (1933) of benzo[a]pyrene (B[a]P) as the major carcinogen in coal tar. This milestone in chemical carcinogenesis was linked with the development of a new assay. The British investigators discovered that the carcinogenic fractions and subfractions from coal tar had benz [a] anthracene-like maxima in their fluorescence emission spectra which were found to stem from B[a]P. Since that time, fluorescence assays of carcinogenic materials containing PAH have been standard procedure in experimental carcinogenesis. During recent years much progress has been made with instrumentation and fluorescence spectrometry of PAH (Van Duuren, 1960, 1963; Sawicki et al., 1960). Other analytical methods and techniques, however, have also become widely used. The most important ones for PAH are column, paper, thinlayer, and vapor liquid chromatography, as well as ultraviolet spectrophotometry, countercurrent distribution, and the isotope dilution technique. Less than seven years ago, it could only be said that a “smoke factor” was isolated from tobacco “tar” which has an ultraviolet absorption spectrum of a “benzpyrene-like substance” (Fieser, 1957). Such a statement wouId today only reflect the incompetence of the investigator. Therefore, in evaluating analytical studies of PAH in tobacco smoke, the date of the experiment needs to be considered. As with coal tar, the first, conclusive chemical studies on PAH in tobacco smoke were carried out in England. A group of investigators, headed by Lindsey, was the first to succeed in the identification of PAH in cigarette smoke (Cooper and Lindsey, 1953; Commins et al., 1954; Cooper et al., 1954; Cooper and Lindsey, 1955). Smoke condensate was extracted with cyclohexane and after removal of acidic and basic components, the neutral portion, in cyclohexane, was examined by chromatography on alumina, followed by ultraviolet absorption spectrophotometry. Repeated column chromatography led to eluates which were typical of several hydrocarbons (acenaphthylene, anthracene, pyrene, and benzo[ghi]perylene), and B[a]P. The presence of B [ a ] P in the chromatography end fractions was confirmed also by fluorescence spectra. I n 1955, the presence of B [ a ] P in cigarette “tar” was reported by Seelkopf (1955). The enrichment of B [a] P was accomplished by vacuum distillation of the neutral portion, followed by repeated chromatography

314

ERNEST L. WYNDER AND DIETRICH HOFFMANN

on alumina. The end fraction gave in ultraviolet light three absorption maxima (405,387, and 365 mp) , comparable to those of authentic B [ a l p . With some modifications and improvements, Lindsey’s method was applied by various groups in the following years and led in most studies to tobacco smoke fractions highly enriched in PAH (Bonnet and Neukomm, 1956; Latarjet et al., 1956; Cardon et al., 1956; Lyons and Johnston, 1957; Wynder and Wright, 1957; Bentley and Burgan, 1958; Dikun and Chushin, 1959). However, others using very similar separation techniques were highly critical of their studies and did not claim to have identified B [ a ] P from smoke condensate (Kosak et al., 1956; Kuratsune, 1956; Rocchietta, 1956). Some of the claims made by investigators as having identified carcinogenic hydrocarbons in tobacco smoke cannot withstand objective criticism (J. W. Cook, 1961). The application of paper chromatography techniques for the isolation of PAH from column chromatography end fractions of smoke condensate represents a practical method for the isolation of individual compounds Van Duuren, 1958a,b; Ahlmann, 1958; Pietesch, 1958; Hoffmann and Wynder, 1960a,b; Kotin and Falk, 1960; Reid and Hellier, 1961; Grimmer, 1961; Barkemeyer, 1962; Neurath and Horstmann, 1963). The research laboratories of the French Tobacco Monopoly (S.E.I.T.A., 1961) accomplished the isolation of B [ a ] P from a column chromatographic end fraction by chromatography on powdered acetylated cellulose. Thin layer chromatography (TLC) on cellulose acetate does not, in our experience, result in the same resolution of PAH as chromatography on acetylated paper, since the length of the solvent run is only 10 cm. compared to 40 cm. of paper (Hoffmann and Wynder, 1 9 6 3 ~ )Even . repeated development of TLC plates with acetyl cellulose does not result in separations such as are achieved by paper chromatography. Recently, Lijinsky and Mason (1963) employed temperature-controlled gas chromatography with an ionization detector system. This analytical technique, however, allows the clear separation of PAH only from a high concentrate. It currently does not represent any real advantage compared to paper chromatography. I n the latter case, fractions which contain B [CLIP in a concentration down to 0.01% give an effective separation; the concentration for the gas chromatography technique has to be a t least 1% to obtain good resolution. Such a high concentration from tobacco “tar” demands not only relatively large amounts of starting material, but also an unusually time-consuming effort. Another disadvantage of the gas chromatography technique lies in its inability to separate certain pentacyclic hydrocarbons, for instance, B [ a ]P from benzo [el pyrene. It appears, however, that the method enables the investigators, for the first time, to separate clearly certain alkyl derivatives of 2-, 3-, and

EXPERIMENTAL TOBACCO CARCINOGENESIS

315

4-ring aromatic hydrocarbons (Johnstone and Quan, 1963). Some of these might be or are known to be carcinogenic to the experimental animal (e.g., alkyl derivatives of pyrene, fluoranthene, and chrysene) . Recently, Carugno and Waltz (1963) subjected the benzo[a]pyrene fraction of cigarette smoke condensate, obtained by column chromatography on silica gel of the neutral portion and by subsequent column chromatography on powdered acetylated cellulose (S.E.I.T.A., 1961) to vapor liquid chromatography. The application of these techniques was only meant to serve as a means of proper identification of B[a]P. As shown in this study for the pyrene fraction, column chromatography alone does not deliver a concentrate sufficiently enriched in PAH to make the application of the gas chromatography technique worthwhile. Recently, Demisch and Wright (1963) developed an analytical method for carcinogenic PAH by countercurrent distribution. It appears that the hexane and aqueous monoethanol-ammonium deoxycholate system has its limitations for such complex mixtures as found in the neutral portion of smoke condensate or its PAH-containing column fractions. Similar shortcomings are found with the solvent system nitromethane-cyclohexane (Hoffmann and Wynder, 1960a).

2. Qualitative Analysis of PAH An objective criticism on the analysis for carcinogenic PAH in cigarette smoke was offered by J. W. Cook (1961), with whose views we concur. A t least two, and even better, three analytical criteria are essential for the positive identification of traces of carcinogens in combustion material (Hoffmann and Wynder, 1960b, 1962a). Benzo [ a] pyrene was identified by UV-absorption and fluorescence spectra in 1955 by Cooper and Lindsey, Van Duuren (1958a), and by Wynder and Hoffmann (1959a). The last two publications presented the R f values and the ultraviolet and fluorescence spectra of isolated B[a]P and compared them with those of authentic material. Among carcinogenic hydrocarbons only B[a]P has been isolated from tobacco smoke in crystalline form, and its identity was established by melting and mixed melting point (Wynder and Hoffmann, 1959a). Dibenz [ a,h]anthracene, benzo [ j ]fluoranthene, chrysene, and benzo[elpyrene are four other carcinogens which Van Duuren (1958a,b) and Hoffmann and Wynder (1960b) identified in cigarette smoke with the same criteria as for B[a]P. Gilbert and Lindsey (195613) and Hoffmann and Wynder (1960b) identified benz [ a ]anthracene also, but Van Duuren was unable to detect this compound. He pointed to the similarity of its ultraviolet absorption spectrum to that of fluoranthene, also present in cigarette smoke. Column

TABLE V CARCINOGENIC HYDROCARBONS ISOLATED FROM CIGARETTE S M O ~ ~ Hydrocarbon

&nzo [ ca ] pyrene

Dibenz [ a,h ] anthracene

Structure

Relative carcinogenic activity b

+++

Micrograms isolated from smoke of 100 cigarettes

2.5

(3.920.3)'

d

+f+

0.4

m ta

ii U

Benzo [ b ] fluoranthene

++

0.3

Benzo[ jlfluoranthene

++

0.6

0

2

Dibenzo[a,Z Ipyrene

ft

Traces

Benz 1n ] anthracene

+

0.3

Chrysene

-t

6.0

Benzo [ e ] pyrene

+

0.3

3

0.4

Indeno [ 1,2,3-cd ] pyrene

023

aFrom Wynder and Hoffman (1963 a), 'Relative carcinogenic activity onmouse skin: +++high; ++ moderate; +weak (ace. to own experiments). 'Absolute amount, determined by CI4 - isotope dilution.

318

ERNEST L. WYNDER AND DIETRICH HOFFMANN

chromatography and subsequent paper chromatography, however, allow adequate separation of these two hydrocarbons. Pietzsch (1959) reported the presence of 7,lZ-dimethylbenz [a ]anthracene (DMBA) in cigarette smoke, but the formation of a dialkylated benz [ a ]anthracene during pyrolysis appears questionable (J. W. Cook. 1961). The presence of benzo [c] phenanthrene (Van Duuren, 1958b) and benzo [ b ]fluoranthene (Hoffmann and Wynder, 1960b) in cigarette smoke is not questioned by J. W. Cook (19611, nor is there doubt of the presence of beneo [ k ]fluoranthene, benzo [ ghi]fluoranthene and benzo[ghi]perylene in cigarette smoke (Van Duuren, 1958a,b; Hoffmann and Wynder, 1960a,b). However, these last three hydrocarbons are of questionable carcinogenic activity (Dannenberg, 1959; Wynder and Hoffmann, 1959b, 1963c) by the criteria of the Food Protection Committee (1959). The latest identified carcinogenic PAH in cigarette smoke is indeno[ 1,2,3-cd]pyrene (Hoffmann and Wynder, 1960b; Wynder and Hoffmann, 1963b). Recently Johnstone and Quan (1963) isolated a considerable quantity of naphthalene (0.17 p g . per cigarette) and six alkylated naphthalenes (together 3.52 pg.) ; unfortunately, their experimental data were limited. Table V summarizes the results of the qualitative analysis for PAH in cigarette smoke and their relative carcinogenic activities as found by Wynder and Hoffmann (1959b, 1 9 6 1 ~ )A . full account of all claims for the presence of PAH in cigarette smoke has been published by the Tobacco Research Council (Bentley and Berry, 1959, 1960; Berry, 1963).

3. Quantitative Analysis of PAH Methods: The critical reviewer of the various analytical methods will agree that absolute values for PAH in tobacco products can be obtained only with the aid of the isotope dilution technique. Results of other methods should be considered only as semiquantitative. Our experience with various methods when used for tobacco or other combustion material indicate that the loss of PAH cannot be predicted. For cigarette “tar” it was generally about 25-35%, and occasionally only 20% ; but in condensates with low concentrations of PAH from experimental cigarettes losses were up to 50%. In such studies the authors regard their values for PAH from tobacco products only as “minimal values” (Van Duuren, 195813) or “isolated quantities” (Onishi et al., 1958; Wynder and Hoffmann, 1963a). For the isotope dilution method the radioactive marker is added

EXPERIMENTAL TOBACCO CARCINOGENESIS

319

to the condensate in a concentration appropriate for the specific hydrocarbon as determined by a preliminary analysis. After isolation, the product must give an ultraviolet absorption spectrum identical with the authentic material down to 250 mp. The radioactivity of the isolated hydrocarbon in relation to that of the added labeled compound allows by simple calculation the determination of the absolute amount of the material originally present in the “tar.” So far, this method has only beeq applied to C14--labeledB [ a ] P (Wynder and Hoffmann, 1959a; Kotin and Falk, 1960; Reid and Hellier, 1961; Scherbak e t al., 1963). The experimental deviation was found to be, in extreme cases, not more than ?8% (Hoffmann and Wynder, 1960b). Recent studies showed that randomly tritiated hydrocarbons (Wilzbach, 1957; Dorfman and Wilzbach, 1959), which were highly purified (Giovanella et al., 1962), can also be used in the isotope dilution method using a liquid scintillation spectrometer (Hoffmann and Wynder, 19634. 4. Polycyclic Aromatic Hydrocarbons in Tobacco PAH in tobacco itself may derive from polluted air or from tobacco processing (curing, aging, and others). In a most extensive study by the Research Laboratories of the Tobacco Monopoly of Japan (Onishi et al., 1958), the investigators removed the alcoholic compounds from the neutral portion of the steam distillate of Japanese Burley leaf by precipitation, and chromatographed the rest repeatedly. They determined from 1 kg. of leaf 5.0 mg. phenanthrene, 4.21 mg. anthracene, 1.8 mg. pyrene, and 1.38 mg. fluoranthene in crystalline form. The authors consider the possibility that these 3- and 4-ring aromatic compounds “are framework compounds having similar structure to some important plant components.” Since these hydrocarbons were isolated in such unusually high concentrations as parts per million (p.p.m.), a history of the analyzed tobacco should have been given, especially since structures such as fluoranthene and pyrene appear to be rather unusual plant constituents. Campbell and Lindsey (1956, 1957b) isolated traces of acenaphthylene, phenanthrene, anthracene, pyrene, fluoranthene, benz [a ]anthracene and B [ a ] P from fresh and processed leaves. The B [a] P values ranged from three to six parts per billion (p.p.b.). In order to test whether these traces of hydrocarbons are derived from polluted air, the investigators analyzed cherry laurel leaves from three different locations in England and found traces of PAH in about the same concentrations. Bentley and Burgan (1958) and Wynder and Hoffmann (1961a) determined B[a]P in tobacco, and found up to 12 p.p.b. and 20 p.p.b., respectively. Studies by Lyons (1955) and Bentley and Burgan (1960a) have

320

ERNEST L. WYNDER AND DIETRICH HOFFMANN

BENZO[a]PYRENE Origin of cigarettes Bulgaria 63anada (presumably) Denmark (presumably) France

(a) Rhodopi 0 . S (b) Arda 0.7 3.35" 0.2 1.2 (a)" 1.0 (b) 0.8 (c) 0.9 2.7-5.4 1.6c 1.2 2.4~ 0.33

Italy

United Kingdom

United States

TABLE V I MAINSTREAM SMOKE

B[u]P isolated (pg./lOO cigarettes)

Germany

Switzerland (presumably) U.S.S.R.

I N THE

2.2

Belomorkanal 1.1 Aurora 1.6 Makhorka 2.6 Belomorkanal 7.5a 1.0 1.P 0.3-1. SC 2.9 f 0.3" 3.7 Rhodesia 0 . 5 4 . 6c (a) 10.0 (b) 12.25

(a) (b)

(c) (d)

1J.S. 1958

U.S.1960

(c) 8.0 0.4-1 .Oc 0.5 0.7 3.5a 3.5 f 0.5" 3.5 f 0.5a 2.7 f 0.3= 1.8 f 0 . 3 ~ 3 . 4 k 0.5a 3.8 k 0.30 3.9 k 0.3"

CIGARETTES

Reported by

Year of report

Alexandrov et al.

1961

Scherbak et al.

1963

Ahlmann

1958

Latarjet el al. Cuzin

1956 1960

Seelkopf Druckrey et al. (acc. to Grimmer) Pyriki Wynder and Hoffmann Scasselati-Sforzolini and Saldi Bonnet and Neukomm

1955 1960 1960 1961a 1961 1957

Dikun and Chushin

1959

Hoffmann and Wynder Cooper et ul. Lyons Bentley and Burgan Wynder and Hoffmann Ayres et al. Bentley and Burgan Cardon et at.

1961a 1954 1956 1958 l961a 1963 1958 1956

Bentley and Burgan Van Duuren Orris el al. Kotin and Falk Wynder and Hoffmann

I958 1958a 1958 1960 1960

Wynder and Hoffmann Wynder and Hoffmann

l961a 1963s

Determined by isotope dilution technique. Letters in parentheses refer to unidentified tobacco. c Estimated from data in publication. a

b

OF

EXPERIMENTAL TOBACCO CARCINOGENESIS

321

shown that these traces of PAH already in tobacco do not contribute an appreciable amount to the aromatic hydrocarbons in tobacco smoke. 5 . PAH in Cigarette Smoke Various factors can influence the yield of PAH in cigarette smoke. Rather than list all studies in this field, we shall make only a few comparisons to demonstrate how much caution should be exercised before quantitative values can be accepted. During the last decade publications appeared from all over the world concerning the concentration of carcinogenic hydrocarbons in cigarette smoke (Table VI). The cigarette paper contributes about 5% to the weight of the cigarette. Since no specific precursors for PAH are known to be present in standard cigarette paper, one is surprised at the emphasis given to this aspect (Cooper et al., 1955; Cardon et al., 1956). Bentley and Burgan (1960b) smoked cigarettes made completely from cigarette paper under standard conditions and found in the smoke of 100 g. paper only 2 pg. B[a]P. The authors present an “equation” in which the contribution of cigarette paper (4%) to the B [ a ] P in the mainstream smoke of 500 cigarettes is expressed: Combustion of paper (0.4~g.1

+ contamination +

+

(0.4~g.1

+

synthesis + mainstream during smoking smoke ~ z ~ g . --t 1 (5pg.)

While these figures may indicate the general situation, as it occurs during the smoking of a cigarette, such an equation needs further evaluation. Latarjet et al. (1956) pyrolyzed 300 g. of cigarette paper manufactured in the United States and two papers made in France and found in the ‘Lsmoke” 200, 250, and 70 pg. of B[a]P, respectively. One should be extremely cautious in adapting these figures for actual cigarette smoking conditions, since Wright and Wynder (1956) using cigarettes made entirely from cigarette paper, did not find any B [ a ] P in the smoke when the conditions employed were close to actual smoking conditions. Few studies deal with the quantities of polynuclear hydrocarbons in the sidestream of cigarette smoke. For 100 United States cigarettes, the values found by Kotin and Falk (1960) were 39 pg. pyrene and 13.5 pg. benzo [ a ]pyrene ; Wynder and Hoffmann (1961a) observed 30 pg. and 15 pg., respectively, and also 25 pg. chrysene. Bentley and Burgan (1960b) reported about 3.0 pg. B [ a ] P for the sidestream smoke of 100 British cigarettes. 6. PAH in Pipe and Cigar Smoke As expected, the smoke of tobacco products other than cigarettes also contains PAH. Campbell and Lindsey (1957a) compared their results

322

ERNEST L. WYNDER AND DIETRICH HOFFMANN

for four hydrocarbons in the smoke of cigarettes, cigars, and pipes (Table VII). Cigar smoke also was analyzed by Cardon et al. (1956). Though the results of the latter study are not in full agreement with those of Campbell and Lindsey (1957a), there are some similarities. COMPARISON

OF

TABLE VII MAINSTREAM SMOKE OF CIGARETTES, CIGARS,

Hydrocarbon Acenaphthylene Anthracene Pyrene Benso[a]pyrene

AND PIPESarb

Cigarettes

Cigars

Pipes"

5.0 10.9 12.5 0.9

1.6 11.9 17.6 3.4

29.1 110.0 75.5 8.5

Campbell and Lindsey (1957a). Micrograms per 100 g. of tobacco consumed. 0 Light pipe tobacco. 5

b

The significantly higher B[a]P value for the smoke of pipe tobacco, especially when compared with cigarette tobacco also smoked in a pipe, suggests that the additives for pipe tobacco, particularly the sugars, may be specific precursors for B[a]P upon pyrolysis (Hoffmann e t al., 1963) (Table VIII), TABLE VIII AMOUNTSOF CONDENSATE AND BENZO[a]PYRENE FROM 100 GRAMSOF TOBACCO PRODUCTS"

Tobacco productb

Amount of particulate materials (g.)

85 mm. plain U.S. cigarette (a) 85 mm. plain U.S. cigarette (b) U.S. cigar A (b) Havana cigar B (b) Standard pipe tobacco in pipe (b) Cigarette tobacco in pipe (b) Water pipec (b) Water piped (b)

4.1 6.2 1.3 1.1 4.5 2.9 0.74 1.7

BbIP (rg.)

4.7 7.8 5.1 4.0 27.0 10.5

1.7 4.1

From Hoffmann et al. (1963). Isotope dilution method: Smoking conditions: puff volume 35 ml,; (a) 1 puff per minute, (b) 2 puffs per minute; puff duration 2 seconds. c Water pipe smoked with water filling. d Water pipe smoked without water filling. b

EXPERIMENTAL TOBACCO CARCINOGENESIS

323

7. Precursors for PAH in Tobacco Smoke Does tobacco contain specific compounds that can be considered precursors for polynuclear aromatic hydrocarbons in tobacco smoke? This question is not only of academic interest, but may suggest an approach for the reduction of B [ a ]P in the smoke. This possibility has intrigued chemists of both academic and industrial institutions, and has led to valuable contributions to our present understanding of tobacco pyrolysis, as well as to combustion chemistry in general. The most extensive studies on the “mode of formation of carcinogenic hydrocarbons” during pyrolysis have been carried out by Badger and his co-workers (Badger, 1962a,b; Badger e t aE., 1962a,b). B [ a ]P formation was utilized in a working hypothesis which is summarized in Fig. 18.

IVI 1

(vn1

FIG. 18. Hypothesis of B[alP formation during pyrolysis (Badger, 1962b)

For pyrolysis the components were exposed for a short time to temperature around 700°C. in an inert gas phase. At this temperature carbonhydrogen bonds are readily broken to give free radicals, but aromatic ring systems have been shown to be relatively stable; only the carbonhydrogen bonds are broken to any significant extent (Badger, 1962b).

324

ERNEST L. WYNDER AND DIETRICH HOFFMANN

Badger et al. were able to prove a high probability for their hypothesis of B[a]P formation, as well as to indicate some probable modes of formation of other carcinogens, including benzo [ b ]fluoranthene, benzo[ j ]fluoranthene, benzo [ c ] phenanthrene, benz [ a ]anthracene, chrysene and indeno [ 1,2,3-cd] pyrene. Pyrolysis experiments with CI4-labeled precursors and the application of newest analytical methods contributed much to the established experimental data. Additional studies on the pyrolysis of PAH were published by Lang and Buffleb (1958, 1961) and Lang and Zander (1961). The first group of tobacco constituents studied by pyrolysis were the “tobacco paraffins,” which are n- and iso-hydrocarbons from C,? to CS5 (Lam, 1955a; Wynder et al., 1959). Both investigations point to paraffins as possible precursors of polycyclic hydrocarbons, a result which is in line with Badger’s studies. Wynder et al. (1958) extracted tobacco with n-hexane; the portion removed as extract (5.4%), contained paraffinic and polyenic hydrocarbons, glycerides and other esters, solanesol, phytosterols, and aliphatic esters. The extracted material was pyrolyzed a t various temperatures. The relative “tar” yields were as follows: a t 88O”C., 28%; 800”C., 28%; 720”C., 32%; 640°C., 35%; and 560”C., 50%. I n an additional experiment the extract was pyrolyzed a t 880°C. in the presence of air, giving a “tar” yield of 30%. Analytical studies of the 880” and 720°C. pyrolyzates revealed the presence of a broad spectrum of PAH; several of them are known carcinogens. Carcinomas developed on mouse skin after application of 1 and 5% pyrolyzate solutions in acetone. The 880°C. pyrolyzate in 1% concentration gave 97% papillomas and 77% carcinomas (in a group of 30 mice); the 800OC. pyrolyzate gave 97 and 60% and the 720OC. pyrolyzate 60% carcinoma response with the 5% concentration and no carcinoma response with the 1% concentration. With the 640°C. 5% pyrolyzate solution, the carcinoma response was 17% and with this pyrolyzate in 1% concentration as well as with both concentrations of the 560°C. pyrolyzate no carcinomas were obtained. The 880°C. pyrolyzate obtained in the presence of air had a carcinoma response of 67% in both concentrations tested (Fig. 19). Tests on rabbit ears gave comparable tumor response to solutions of the various pyrolyzates. These experiments not only demonstrate that the formation of PAH occurs especially from tobacco extracts rich in paraffinic hydrocarbons, but also clearly show the dependence of PAH formation on the conibustion temperature. Rayburn et al. (1958) challenged the concept, that tobacco paraffins are precursors for PAH in the smoke. Their experimental findings are partially based on “total polycyclic hydrocarbons of similar ultraviolet

EXPERIMENTAL TOBACCO CARCINOGENESIS

325

spectra” and not analytical data. The report did not mention counting techniques for C14-labeled paraffins, nor their quenching effects. These, as well as other factors, appear to weaken considerably the challenge of a concept based on extensive experimental data. Other experiments, concur to some extent with the ‘(tobacco paraffin” concept. Gilbert and Lindsey (1957) individually pyrolyzed a t about 650°C. ten of the major constituents of tobacco leaf. All constituents, eight of them without aromatic groups, gave traces of PAH upon pyrolysis. This is accepted as further evidence that traces of PAH are formed on “incomplete combustion” a t higher temperatures of organic matter. 80-

to60 K

-

w

8 I-

z W

40-

K

30-

0

w n

20

-

10

-

0

I[, 880.

,0

880. IN AIR

8000

I

/

/

720. /640° 560’

/

3 4

5 6

7

8 9 10 I I 12

A recent study by Grossman et al. (1963a) on the breakdown during pyrolysis of solanesol (known to be present only in tobacco) gives experimental proof for the formation of cyclic dipentenes, and thereby demonstrates solanesol to be a specific precursor for the cyelohexene ring. The authors found further that a t temperatures higher than 550°C. aromatization occurs. The first aromatized products will be, of course, alkyl and p-dialkylbenzenes, which, according to Badger (1962a), are necessary for the PAH pyrosynthesis. I n a recent meeting Grossman et al. (196313) reported the isolation of alkylated naphthalenes; but not of higher PAH from the pyrolyzate of solanesol. Wynder et al. (1959) also pyrolyzed a phytosterol fraction of tobacco

326

ERNEST L. WYNDER AND DIETRICH HOFFMANN

a t 850” and 720°C. The B [ a ] P values in the pyrolysis product were about 1% and O.l%, respectively. The phytosterol pyrolyzate applied to mouse skin as a 0.15% solution produced carcinomas in all animals. This study supports the view that phytosterols are precursors for PAH. It appears from all these studies that several components of tobacco may serve, to various degrees, of course, as precursors of PAH.

C. TERPENES, PHTHALATES, AND CERTAIN ESTERS 1. Terpenes The relatively high tumorigenic activity of fraction B to mouse skin (Figs. 14 and 15) cannot solely be explained by the presence of PAH (Wynder and Hoffmann, 1959a). Seelkopf et al. (1963) employed a different separation system for tobacco “tar” and applied the fractions subcutaneously to rats. Also, in this setting the fractions containing PAH (mainly distillation residue) exhibited tumorigenic activity unexplainable by the presence of known carcinogenic hydrocarbons alone. One group of compounds present in fraction B are terpenes. Recent studies employing countercurrent distribution and temperature-programed gas chromatography revealed the presence of a t least ten isoprenoid polyenes comprising about 25% of fraction B (Hoffmann and Wynder, 1 9 6 3 ~ ) . Identified as major compounds were dipentene and neophytadienes, as well as small amounts of a-pinene, myrcene, trans,trans- and cis,transallo-ocimene, squalene, and solanesene. With the exception of myrcene, a-pinene, and the allo-ocimenes, the other terpenes had already been identified in tobacco smoke (Van Duuren and Schmitt, 1958; Rodgman, 1959; Rodgman et al., 1961a; Johnstone et al., 1962). Few studies only have been reported on the biological effect of terpenes and these mainly on the eczematogenic action of A-3-carene containing turpentine. The active agent appears to be autoxidized A-%carene, perhaps a hydroperoxide (Hellerstroem et al., 1955). On mouse skin only A-3-carene (Wynder and Hoffmann, 1963c), d-limonene, myrcene and the two isomeric allo-ocimenes were tested. Even though myrcenc and the allo-ocimenes produced marked hyperplasia, only trans,transallo-ocimene showed slight promoting activity when tested as promoter in 2.5 and 1.0% concentration to skin initiated with 300 pg. DMBA. It cannot be excluded that the weak promoting activity derives from an autoxidation product, despite the fact that the test agent was freshly distilled and kept under nitrogen. Using the sebaceous gland short-term test, we found that a mixture of neophytadienes, isolated from cigarette

EXPERIMENTAL TOBACCO CARCINOGENESIS

327

smoke, did not show either sebaceous gland destruction or hyperplastic reaction. These results, as well as the fact that fraction B freed from terpenes did not significantly reduce the short-term activity compared to the complete fraction B, indicate that terpenes may not contribute significantly to the tumorigenic activity of tobacco smoke (Wynder and Hoffmann, 1963b). 2. Phthalates We have isolated from fraction B di- (2-ethylhexyl) phthalate (di-isooctylphthalate) (Wynder and Hoffmann, 1963b). The compound was identified and isolated by gas chromatography, and showed an infrared spectrum identical with that of a synthetic specimen. It appears that additional phthalates are also present in the fraction. Stedman and Dymicky (1959) identified n-propyl-, n-butyl-, and one unknown phthalate in flue-cured tobacco leaves. Recently, the isolation of ‘(isooctyl phthalate” from tobacco trichomes was reported (Chakraborty and Weybrew, 1963). Mallette and von Haam (1952) tested five phthalates for their skinirritating effects on white rabbits and human subjects. Dicapryl-, dioctyland butyl benzylphthalate showed in undiluted form a moderately irritating effect on the skin of rabbits and man. Our studies showed that from six tested phthalates in 5 and 10% acetone solution only diallyl phthalate exhibits some hyperplastic reaction on mouse skin. Di-n-butyl and di-n-octyl phthalate did not show tumor-promoting activity after 5 months when tested on mouse skin initiated with 300 pg. DMBA. All phthalates tested were not found to be complete carcinogens (Shubik and Hartwell, 1957). 3. Certain Esters

The infrared spectrum of fraction B shows a significant absorption band a t about 5 . 7 5 , ~indicating ~~ the presence of esters. Rechromatography of fraction B on silica gel yields, first, fractions containing paraffinic hydrocarbons, followed by fractions containing esters, terpenes, and PAH. From these fractions one can precipitate, with acetone a t low temperatures, white solids (15% and more of fraction B) from which, upon saponification, we have definitely identified solanesol, palmityl, and stearyl alcohol, as well as myristic, palmitic, stearic, oleic, and linolenic acid by gas chromatography. One can thus conclude that fraction B contains esters of Iong-chain fatty acids, in which the alcohol moieties a t least partially consist of long-chain alcohols and solanesol. The pres-

328

ERNEST L. WYNDER AND DIETRICH HOFFMANN

ence of phytosterol esters in the same fraction is indicated. Wynder and Wright (1957) tentatively identified in this fraction, the ester of hentriacontanyl alcohol and hentriacontanic acid. These data on fraction B are in agreement with studies of Rodgman and Cook (1959) and Rodgman et al. (1959, 1962), who found, in cigarette smoke, solanesyl esters of a t least six saturated and unsaturated long-chain acids; esters from dodecyl alcohol (G2)to l-heptacosanol (C2,) inclusive; and 17 acids from myristic (C14) to octacosanoic (CZ8)acids; oleic and linolenic acids, and several unidentified acids. Palmitic, myristic, and stearic were the major components of the series of acids. Recently, Rodgman and Cook (1963) reported the isolation of a considerable number of unsaturated alcohols (terpenols) from Turkish tobacco smoke. The esters of long-chain acids thus far tested are not complete carcinogens and have not yet been tested for tumor promotion (Hartwell, 1951; Shubik and Hartwell, 1957). Studies by P. Holsti (1958) indicate tumor-promoting activity for oleic and lauric acid in concentrations of 2076, a relatively high concentration, when applied to mouse skin initiated with a single application of 0.3% DMBA solution. In a recent report, P. Holsti (1961) presents some results on the hyperplastic effect of long-chain alcohols beginning with C, alcohol. One might expect, according to this finding, that some of the esters found in fraction B and in cigarette smoke may be tumor-promoting to mouse skin. Further experiments in this area are indicated. The relatively large amounts of esters of solanesol required for tumor-promoting tests and the unusually great effort to isolate large amounts of the alcohol from tobacco (synthesis does not represent a practical way; Ruegg e t al., 1960) make it unlikely, a t this time, that such tests will be performed. Solanesol itself gave negative results when tested on mouse skin for sebaceous gland destruction and hyperplasia in 2.5 and 5.0% concentration (Wynder and Hoffmann, 1962b).

D. PARAFFINIC HYDROCARBONS (ALKANES) 1. Waxes of Tobacco Leaf The coating of leaves with “waxes” is an almost universal phenomenon throughout the plant kingdom. The major wax constituents are alkanes (odd numbers C,,-C,,, even numbers C,,C3,, branches C,,-&), alcohols and acids (usually as esters), aldehydes (as polymers), and ketones (Eglinton et al., 1962a,b). Plants seem to manufacture their own specific wax composition (Purdy and Truter, 1961). After their biosynthesis, it

EXPERIMENTAL TOBACCO CARCINOGENESIS

329

appears that the waxes are not further affected by the plant metabolism as indicated by the lack of seasonal variation in wax composition (Chibnall e t al., 1934). Values scattered throughout the scientific literature indicate percentile differences in alkane content of tobacco varieties (alkanes are the major group of constituents in tobacco waxes). Carugno (1962) reported a detailed analysis by gas chromatography of the wax fraction of a tobacco blend used in Italian cigarettes. However, he did not calculate the percentile concentration of individual hydrocarbons in the tobacco blend. The first gas chromatographic separation of tobacco paraffins had been reported earlier by Carruthers and Johnstone (1959). They found that green tobacco leaf and black fermented tobacco of Argentinian cigarettes contain C,, to C,, paraffin hydrocarbons, A mass spectroscopic analysis allowed the differentiation between normal and branched hydrocarbons. About 50% of the wax fraction was made up of a- and iso-C31H64.Another successful method for the distinction between normal and branched long-chain hydrocarbons was found with X-ray diffractograms (Barbezat-Debreuil, 1958a,b; Cuzin et al., 1958; Kosak and Swinehart, 1960). The isolation of crystalline alkane fractions was reported by several groups for different tobacco types and grades (Gladding and Wright, 1959; Stedman and Rusaniwskyij, 1959b, 1960; and others), The content of crystalline alkanes was between 0.32 and 0.36% for Burley tobacco, 0.24 and 0.28% for flue-cured, 0.34 and 0.43% for Maryland, 0.36 and 0.37% for Turkish, and 0.30 and 0.32% for cigar types. The values for Burley tobacco grades were 0.30-0.34 for “Flyings,” 0.31-0.34 for “Cutters,” and 0.25-0.33 for “Leaf.” Recently, the Eastern Regional Research Laboratory reported the isolation of high-molecular-weight, cyclic hydrocarbons from flue-cured leaves. Their chemical nature, with molecular weights between 278 and 830, is not yet known (Stedman e t al., 1960a,b). Recently Mold e t al. (1963) identified from the extracts of different tobaccos various normal is0 (2-methyl) and anteiso (3-methyl) paraffin hydrocarbons. 2. Analysis of Tobacco Smoke Alkanes During the last few years various groups have analyzed alkanes in tobacco smoke condensate (Barbezat-Debreuil, 1958a,b; Cuzin e t aE., 1958; Carruthers and Johnstone, 1959; Kosak and Swinehart, 1960; Carugno, 1962). Most groups used isothermic gas chromatography. A recent method used by Spears e t al. (1963) presented a most comprehensive analysis of the alkane content in the smoke of Burley, flue-cured, Virginia, Turkish, and Maryland tobaccos, and a bIended cigarette. The authors used, for the separation of individual hydrocarbons from the

330

ERNEST L. WYNDER AND DIETRICH HOFFMANN

alkane concentrate, a vapor fractometer, equipped with flame ionization detector and temperature programmer. The starting temperature was 80°C. and the program rate was 14°C. per minute until 325°C. was reached. Normal and isoalkanes have been separated by molecular sieves (O'Connor and Norris, 1960). Identified were all n-alkanes from C,, to C,, and up to 13 branched isomers. The quantitative analysis was made with the isotope dilution technique using C14-labeled n-C32H66and tritiated n-CIZHz6.The alkanes in the smoke of cigarettes made exclusively from Burley, flue-cured, Turkish, and Maryland tobaccos as the sum of the individual hydrocarbons, amounted to 3.8, 2.8, 4.2, and 4.3%. The value for the smoke of a United States blend cigarette was 3.1%. For the isolation of a crystalline fraction of alkanes, the hydrocarbons were precipitated from methanol and the precipitate was chromatographed three times on alumina. Two further recrystallizations gave a fraction practically free of olefines, representing about 3.0% of the whole condensate. The analysis, according to Spears et al. (1963) showed n- and isoalkanes starting with c17 but going up to Ca6. The major individual hydrocarbons were n-C3,Hs4 (24.2%), n-C32H66 (14.4%), and d&Hs8 (9.2%). From C,, to C,, both normal and isoalkanes were found. 3. Reported Alkanes in Tobacco Carn'nogenesis The first pertinent study with paraffins was reported by Horton et al. (1957). They observed a relative tumor accelerating activity to the skin of C3H mice of 1.0, 1.9,2.3, and 1.7 when n-octane, n-decane, n-dodecane, or n-hexadecane was used as a solvent for B [a] P (1.0, no acceleration). The authors concluded from their results with n-paraffins and aromatic hydrocarbons with alkyl substituents that the molecular length must at least correspond to n-nonane in order to accelerate the activity of carcinogenic hydrocarbons. The alkanes tested proved to be noncarcinogenic to mouse skin, although they are primary irritants. As shown in a recent study by Spears et al. (1963) the alkane fractions of the smoke of four different tobaccos and one tobacco blend contain only a very small portion of paraffins with chain length below that of n-tetracosane (m.p. 51.1"C.) and practically none below n-hexadecane. The cZ7 to C,, hydrocarbons represent more than 80% of the paraffinic compounds with n-C3,Hs4 as the major single hydrocarbon. Using the same method, we also found traces of hydrocarbons up to CaG. Therefore, the effect on mouse skin of two representatives of the tobacco smoke paraffins (n-C31H64and n-C35H72)was simultaneously tested with B[a]P (0.005%) (Wynder and Hoffmann, 1962b). In 0.1 and 0.5%

EXPERIMENTAL TOBACCO CARCINOGENESIS

331

concentrations both hydrocarbons had a significant “inhibiting” effect on the carcinogenicity of B [ a ]P. When the application of paraffinic hydrocarbons was alternated with that of B[a]P their effect was significantly diminished. The effect of alkanes may not be inhibitory to tumorigenicity, but rather a consequence of having influenced resorption. The time-tumor response curves of these experiments clearly express a delay of tumor appearances, which we believe to be due to the retarding effect of the n-alkanes. The paraffin fraction of tobacco smoke condensate has been tested for its tumorigenic activity on mouse skin by Wynder and Wright (1957). Of 10 mice, only 2 developed papilloma; this occurrence may be attributable to other constituents, as it was shown that neophytadienes, as well as some PAH, are present in this fraction. How significant is the role of alkanes in the induction of skin tumors in mice by cigarette smoke condensate? For such an experiment, the content of about 3% of alkanes (C&&,) in “tar” was increased to 4% by adding crystalline paraffins from cigarette smoke condensate (for preparation see Section V,D,2). While the standard condensate, 50% acetone-hexane suspension, gave, after 19 months (15 months of application), 40% tumors (24% malignant) the condensate plus alkanes (51% acetone-hexane suspension) gave 24% tumors (18% malignant). Since each group was started with only 50 mice, a tumor response difference of 16% is not statistically significant ( p > 0.05) ; however, a reduction is indicated.

E. HETEROCYCLIC NITROGEN COMPOUNDS 1. T h e Basic Portion of Tobacco Smoke Condensate

The content of basic compounds in tobacco smoke condensate depends to a great extent on the concentration of alkaloids in the tobacco leaf itself. I n general, Burley and Maryland tobaccos contain more nicotine than Turkish and Virginia tobaccos, and in agreement with expectations, Wynder et al. (1957a) demonstrated in the Condensate of cigarettes from straight tobaccos exclusively basic portions of 20 and 15% for Burley and Maryland, and 10 and 11% for Turkish and Virginia, respectively. From these portions 48, 37, 24, and 40%, respectively, were nicotine and nornicotine. This finding may explain the different tumorigenic activities of condensates from the smoke of cigarettes made exclusively of each of the four predominant tobaccos. The condensate of the smoke of a United States tobacco blend cigarette in the above study contained a 16% basic portion of which about 40% were nicotine and nornicotine.

332

ERNEST L. WYNDER AND DIETRICH HOFFMANN

The high nicotine content does not permit the direct testing of the basic portion, but only that of the nicotine-free basic portion amounting to about 6,2% (Wynder and Wright, 1957). A 50% acetone solution of this fraction induced 2 carcinomas among 30 CAF, female mice and 6 papillomas among 30 Swiss female mice. When the skin of 30 Swiss female mice was initiated with a single dose of 300 pg. DMBA followed by three weekly applications of a 10% solution of the nicotine-free basic portion, 6 mice developed tumors, 5 of which were malignant; in the control group, only 1 mouse out of 30 developed a carcinoma (Wynder . summary, the nicotine-free basic portion of and Hoffmann, 1 9 6 3 ~ ) In smoke condensate is relatively weakly tumorigenic and has low tumorpromoting activity. Seelkopf e t al. (1963) fractionated a nicotine-free basic portion from cigarette smoke condensate into readily acid-soluble (5.294 and less acid-soluble subfractions (1.2%).When repeatedly injected into rats only the former fraction yielded a significant number of tumors (8 rats of 40).

2. Dibenzacridines Van Duuren e t al. (1960) distilled the basic portion of 250 g. of cigarette “tar” a t 100°C. and 0.5 mm. pressure. The residue (10 g.) was chromatographed on alumina and the benzene eluate repeatedly chromatographed on Whatman No. 1 paper and acetylated paper. Bands corresponding to that of the reference compounds dibenz [ a,h] acridine ( D B [ a $ ] AC) and dibenz [ a,j] acridine ( D B [ a,j] AC) were rechromatographed until the band delivered ultraviolet absorption and fluorescence spectra comparable with those of the reference compounds. By this means, 0.27 pg. D B [a,j]AC and 0.01 pg. D B [ a $ ] AC were isolated from the smoke of 100 cigarettes. With some modifications, this method was also applied by Candeli e t al. (1963) and enabled us to isolate from 100 cigarettes 1.0 pg. DB[u,j]AC. The presence of DB[a,h]AC in cigarette smoke could not be confirmed. DB[a,j]AC has been found to be carcinogenic to mouse skin and subcutaneous tissue of mice (Hartwell, 1951 ; Lacassagne et al., 1956). For a comparison with the activity of other carcinogens to mouse skin D B [a,j]AC was synthesized according to Blout and Corley (1947). DB[u,j]AC applied in 0.5 and 0.1% acetone solution thrice weekly to the backs of 20 Swiss female mice induced tumors after 12 to 14 months in 16 and 15 mice respectively (Wynder and Hoffmann, 1963c). In both groups 60% of the mice developed carcinoma. This relatively high tumor response in mouse skin (compared to Lacassagne e t al., 1956) might be partially explained by the high purity of the compounds, due mainly to the absence of the “Morgan’s base” a dihydro-DB [ a , j ] acridine.

EXPERIMENTAL TOBACCO CARCINOGENESIS

333

Only by repeated column chromatography on alumina can a D B [ a,j]AC be isolated which is free of the dihydroproduct (absence of N-H band in infrared spectrum). When mouse skin was initiated with a single application of 300 pg. DMBA and followed up thrice weekly with a 0.5% solution of DB[a,j]AC the same tumor response was observed as with the heterocyclic alone, except that the tumors appeared 2 to 3 months earlier (Wynder and Hoffmann, 1 9 6 3 ~ )This . result indicates there is no significant tumorpromoting activity of D B [ a,j]AC, despite its strong hyperplastic effect on mouse skin.

3. Dibenzocarbazoles From the benzene eluate of the neutral portion of cigarette smoke condensate, which was not found to be active on mouse skin (Wynder and Wright, 1957), Van Duuren et al. (1960) isolated, by repeated paper chromatography, a compound identified as 7H-dibenzo [a,g]carbazole by comparison of R f values, fluorescence, excitation, and emission spectra with an authentic specimen. Van Duuren could not identify 7Hdibenzo [a,g]carbazole from cigarette smoke (Van Duuren et al., 1960). The former dibenzocarbazole is known to produce epitheloma in mice and sarcoma in mice and rats dibenzocarbazole (Hartwell, 1951; Shubik and Hartwell, 1957) (Table IX). 4. Pyrolysis of Tobacco Alkaloids

I n addition to alkaloids, tobacco smoke contains several heterocyclic nitrogen compounds (Johnstone and Plimmer, 1959; Rodgman and Cook, 196213). One reason for several pyrolysis experiments with nicotine was the study of its degradation products (see Balasubrahmanyam and Quin, 1962). Jarboe and Rosene (1961) pyrolyzed nicotine a t 600-900°C. in an inert atmosphere and detected by gas chromatography several heterocyclic and aromatic hydrocarbons in the end product. Some of the major compounds were isolated in crystalline form: naphthalene, and pyridine and quinoline as picrates. The pyrolysis of nornicotine and myosmine led to ring systems such as quinoline and isoquinoline (Balasubrahmanyam and Quin, 1962), suggesting that tobacco alkaloids can, upon combustion, be precursors for some polynuclear aromatic and nitrogen heterocyclic hydrocarbons. This concept was confirmed for some N-heterocyclics. Van Duuren et al. (1960) found that a t 750°C. in an inert gas stream, pyridine, as well as nicotine, forms a highly fluorescent pyrolyzate containing the carcinogenic D B [a,j] AC and DB[a,h]AC; as in cigarette “tar,” the former was found in much larger quantities than the latter.

334

ERNEST L. WTNDER AND DIETRICH HOFFMANN

TABLE IX N-HETEROCYCLIC HYDROCARBONS IN CIGARETTE SMOKE Formula

Compound

Micrograms isolated from the smoke of 100 cigarettes

Dibenz [ n , h ] acridine

0. ola Not identifiedb

Dibenz [ a , j ] acridine

0.27;

Dibenzo [c,g]carbazole

0.07'

1.00

H 'Van Duurenef a l . ( l 9 6 0 ) . bCandeli el n 1 . ( 1 9 6 3 ) .

F. PHENOLIC COMPOUNDS AND CARBOXYLIC ACIDS 1. Phenolic Compounds

a. Polyphenols and Phenols in Tobacco. Early studies on phenolic constituents of tobacco derive from Shinuk and his group (1953). Although their main interest was academic, in 1927 they suggested the possibility that phenols might influence the quality of tobacco. With the studies of Koenig and D6rr (1933) investigations were started for the

EXPERIMENTAL TOBACCO CARCINOGENESIS

335

evaluation of polyphenol content and taste of tobacco smoke. During the following decades these studies led to a considerable accumulation of knowledge concerning the polyphenols in tobacco, a field recently reviewed by Stedman (1957), Johnstone and Plimmer (1959), and Herrmann (1961). Last year H. E. Wright, Jr. (1962) summarized present knowledge on the phenolics in tobacco including an annotation of all known tobacco phenols and polyphenols. The major phenolic constituents of tobacco are from the coumarin group, scopoletin ( I ) and scopolin (11) ; from the tannins, chlorogenic acid (111) (Yang e t al., 1958a) and some isomers, and from the flavonoids, glucosides and aglycones belonging to the flavonol type. These are mainly rutin (IV) (Weaving, 1960) and isoquercitin (V). A compound of minor importance is kaempferol (VI) (Fig. 20). Recently, another coumarin derivative, the esculetin (VII) glucoside RR'O '

'

o

m

o

1. Scopoletin: R' = OCH,, R" = H 11. Scopolin: R' = OCH,, R" =D-Glucose VII. Esculetin: R'=H, R"=H

I

OH

III. Chlorogenic acid OH

0

HO

Iv. Rutin:.

R' = OH,

V. Isoquercitin:R' = OH, VI. Kaempferol: R' = H,

'

R" = Rutinose (L-rhamnosido-D-glucose ) R" =D-glucose

R" = H

20. Polyphenols in tobacco.

336

ERNEST L. WYNDER AND DIETRICH HOFFMANN

cichoriin, was isolated from tobacco (Runeckles, 1962), and from aged flue-cured tobacco two high-molecular-weight benzopyran derivatives, a-tocopherol (vitamin E) and solanachromene (Rowland, 1958). Reid (1956) carried out comprehensive studies on polyphenols in tobacco. H e not only employed chemical-analytical techniques, but also studied enzymatic reactions of a tobacco extract fraction rich in polyphenol oxidase (s) with polyphenols. The results of polyphenol analyses of four typical English cigarettes are shown in Table X (Reid, 1959). TABLE X

ANALY SESa

O F CIGARETTES FOR POLYPHENOL CONTENTbvC

Brand

Components Y and Z

Total chlorogenic acids

Rutin

Total polyp henols

1 2 3 4

0.9 1.0 0.5 1.0

3.5 2.6 2.1 2.8

0.7 1.0 0.5 1.o

5.1 4.6 3.1 4.8

a In these analyses, components Y and Z were calculated with reference to chlorogenic acid as a standard. b From Reid (1959). 0 Results as wt. % on ash-free moisture-free basis.

These results should be applied only to this sample, since i t is known that chlorogenic acid is present in different concentrations, not only in various parts of the tobacco plant, but also in various parts of the leaf (Zucker and Ahrens, 1958). The concentration of polyphenols is strongly reduced when the short wavelength of the sunlight spectrum is excluded during the growing of tobacco (Frey-Wyssling and Baebler, 1957). During flue-curing the polyphenol content is only slightly changed. A considerable reduction occurs, however, during air-curing. This can be explained, at least partially, by the strong reduction in polyphenol oxidase(s) (Zelitch and Zucker, 1958). It appears that the oxidation of chlorogenic acid, mainly during the air-curing, yields brown pigments, which, in the case of air-cured Burley tobacco, contain an iron-protein-chlorogenic acid and rutin complex (H. E. Wright, Jr. e t al., 1960). The increase of the redox potential of the leaf during aircuring appears t o be a n important factor in the browning of tobacco (Birnstiel, 1960). The essential oil of Virginia tobacco leaves contains traces of simple phenols, such as phenol (6.2 p.p.m.) , m-cresol, guaiacol, eugenol, salicylaldehyde, and others. During aging of tobacco their concentration is reduced to about one tenth and less: m-cresol was not detected a t all

EXPERIMENTAL TOBACCO CARCINOGENESIS

337

(Naghski et al., 1944; Onishi and Yamamoto, 1956, 1957a,b). The origin of traces of phenols in tobacco is not yet understood. Melilotic acid(1) and caffeic acid(I1) are found in flue-cured tobacco (Penn and Weybrew, 1958; Geissmann and Hinreiner, 1952). CH= CH-COOH

CH= CH- COOH

I

I

HO

-Q

b. Polyphenols and Phenols in Tobacco Smoke. Of the polyphenols present in tobacco only scopoletin has been detected in cigarette smoke (Yang et al., 1958b; Reid, 1959). One gram of cigarettes yielded 14.0 to 27.4 pg. scopoletin, for cigarettes with filter tips the values ranged between 12.7 and 17.9 pg. On the assumption that all scopoletin derives from free phenol in tobacco and not from glycosides, it was calculated that 14.3-25.2% appears in the mainstream (Yang e t al., 195813). The first successful isolation of phenols from tobacco smoke was reported a s early as 1871 and 1876 (Vohl and Eulenberg, 1871; Ludwig, 1876). Since that time publications have dealt sporadically with smoke phenols. During the last decade several analytical techniques have been applied for phenol determinations. The first steps include mainly steam distillation and extractions. The enriched phenol-containing fraction was diazotized with p-nitroaniline (Rayburn e t al., 1953) or converted to the methyl ethers (Commins and Lindsey, 1956) and then paper chromatographed. The phenol ethers were determined quantitatively by ultraviolet absorption spectra of the phenol ether bands after extraction from the paper chromatogram. I n another method phenol, m-cresol, and guaiacol were identified as dinitrobenzoates. Salicylaldehyde, 2$-dimethylphenol, m-ethylphenol, and p-cresol were identified as phenyl azobenzene-sulfuric acid dye derivatives (Izawa et al., 1959a). For the quantitative determination of the methyl ethers of phenols, Carruthers and Johnstone (1960) employed gas chromatography. The application of the gas chromatography technique in the last step of the separation for free phenols represents the most advanced analytical method (Hoffmann and Wynder, 1961; Crouse et al., 1963; Spears, 1963a). For quantitative assessment in two of these methods, an internal

ERNEST L. WYNDER AND DIETRICH HOFFMANN

338

standard was added a t the beginning of the analysis; in the third method, the internal standard was added just before the final step of gas chromatography. Spears (1963a) reconfirmed his results with an internal standard by adding CI4-labeled phenol and using the isotope dilution technique. Table XI compares the results of the analyses for the major phenols from the smoke of 85-mm. American nonfilter cigarettes. Since all three groups used the same smoking technique, the observed agreement was to be expected. TABLE XI PHENOLS IN CIGARETTE SMOKE" Phenols per cigarette

Ab

Bc

Phenol (pg.) o-Cresol (pg.) m p-Cresol (pg.) 2,4 2,5-T>imethylphenol (pg.) m p-Ethyl phenol (pg.)

96.2-100.0 22.0-26.3 50.0-50.8 20.0-20.2 20.6-23.0

71.0-161.0 14.1-22.0 41.0-82.0 7.0-19.6 12.0-28.0

+ + + a b

C d

76.0-ICS.0 17.5-24.8 42.6-60.5 14.4-20.5 Not determined

85-mm. American nonfilter cigarettes. Result from 2 cigarettes (Hoffmann and Wynder, 1961, 1963a). Extreme values from 10 cigarettes (Crouse et al., 1963). Extreme values from 3 cigarettes (Spears, 1963a).

Investigators A further identified traces of salicylaldehyde, 3,4-dimethyl phenol and 2,3,5-trimethyl phenol. B found per cigarette, 19.746.0 pg. 2,6-dimethyl phenol, 9.2-14.0 pg. o-ethyl phenol and guaiacol, 0.4-6.7 pg. 2,3- and 3,5-dimethyl phenol, and up to 7.6 pg., 2,4,5 and 2,3,5-trimethyl phenol. C found 9.9-14.0 pg. guaiacol; and the presence of 2,6-dimethyl phenol, 2,4,6-trimethyl phenol, o- and p-methoxyphenol were reported. It is remarkable that in smoke derived from different tobacco products without major additives, there is a constant ratio of the major phenols (Spears, 1963a; Hoffmann e t al., 1963). Recently, Burdick e t al. (1963a) reported comparable results from blended cigarettes. Other phenols claimed to be present in Cigarette smoke include catechol, resorcinol, hydroquinone, m- and p-hydroxyacetophenone and a- and p-naphthols (Johnstone and Plimmer, 1959). For routine estimations of phenols in cigarette smoke, Lorentzen and Neurath (1963) suggested a method based on the color reaction of all non-p-substituted phenols with 4aminoantipyrine. Only two analyses have thus far been reported for phenols from the smoke of tobacco products other than cigarettes. One of these for cigar smoke appears not to fulfill the requirements for a quantitative analysis, since neither internal standard nor isotope dilution technique were em-

339

EXPERIMENTAL TOBACCO CARCINOGENESIS

ployed and since the gas chromatogram presented indicates strong background deriving from nonphenolic compounds (Osman et al., 1963). We regard the reported values as too low. The results of a quantitative phenol analysis of the smoke of cigarettes, cigars, pipes, and a water pipe are shown in Table XII. TABLE XI1 PHENOLS IN THE MAINSTREAM SMOKE OF TOBACCO PRODUCTS~~~ Tobacco product“ ~

1. 2. 3. 4. 5. 6. 7.

Phenol ~

85-mm. plain U.S. cigarette (a) 85-mm. plain U.S. cigarette (b) U.S. cigar A (b) Havana cigar B (b) Standard pipe tobacco in pipe (b) Cigarette tobacco in pipe (b) Water-pipe tobacco (b)

~~

11.7 25.4

10.7 7.4 68.7 21.2 1.8

:Lo”;

o-CresoI ~

2.6 4.9 2.1 1 9 8.7 4.1 Traces

~~~

5.8 11.6 6.2 4.2 25.3 9.8 0.8

+ p-Ethyl

vz

phenol ~

2.7 4.6 1.8 1.6 5.0 3.8 Traces

From Hoffmann et al. (1963). Milligrams per 100 g. of tobacco consumed. c Smoking conditions: (a) 1 puff per minute, duration 2 seconds, puff volume 35 ml.; (b) 2 puffs per minute, duration 2 seconds, puff volume 35 ml. Experimental deviations for experiments 1 to 4, f5%. For experiments 5 to 7: since the smoking of the pipe is not reproducible t o the same degree, the experimental deviations are considerably higher and not calculated. Q

b

The precursors in tobacco for the phenols in the smoke can only be conjectured. According to Wenusch (1939),i t appears that quinic acid (hexahydro-1,3,4,5-tetrahydroxybenzoicacid) , which is the alcoholic component of the ester chlorogenic acid, and the polyphenol itself are precursors to phenol and catechol. Lignin is a precursor for cresols and guaiacol, among other components. Since additives (mainly sugars) often constitute one third of pipe tobacco, they may be the source of the higher amounts of phenols in pipe smoke (Hoffmann et al., 1963). Recently, several pyrolysis products of rutin, quercitin, and chlorogenic acid were reported (Zane and Wender, 1963). Several nonvolatile dihydroxybenzene derivatives were identified in the pyrolyzate by paper chromatography, but the method applied was not designed for identification of phenols. Another study aiming a t this specific purpose is needed. c. The Tumorigenicity of Phenols. The weakly acidic (phenolic) fraction of cigarette smoke condensate possesses tumor-promoting activity (Section IV,D) . This portion was fractionated by vacuum distillation, as shown in Fig. 21. Fractions I1 and I11 in 5% acetone solution and the residue as a 33% solution in acetone were painted for 7 months thrice

340

ERNEST L. WYNDER AND DIETRICH HOFFMANN

weekly, together with 0.005% B [ a ] P (fraction I1 and control B [ a ] P , 20 mice each; fraction I1 and residue, 30 mice each). After another 4 months’ observation (a total of 11 months) the responses were as follows: fraction 11, 17% tumors (3% malignant), residue 40% tumors (10% malignant), and the control, 10% tumors (5% malignant) (Wynder and Hoffmann, 1 9 6 3 ~ )Since . only the distillation residue exhibited, in 33% acetone solution, a significant tumor-promoting activity on mouse skin

Neutral porllon 41.2%

I I

0.23% 1Wl.YJrnrn

Phenolic portlon

Basic portion 8.1%

*% ’

Acidic portion 2.2%

9.3% I I

I

II

111 0.67%

l,%iy 2109~5rnrn

InSOlUble portion

14.0% Residue

6”5*

FIG.21. Fractionation of cigarette smoke condensate (Wynder and Hoffmann, 1961~).

( p < 0.05) i t appears that for the other fractions only higher concentrations would have produced significant tumor-promoting activity, as shown for the whole weakly acidic portion (Wynder and Hoffmann, 1961b). The distillation residue contains, as the only known tumorpromoters, oleic and lauric acid. The other saturated and unsaturated C,,, C,,, and C,, acids, as well as the traces of polyphenols, are inactive, or not yet tested. Of the polyphenols present in tobacco only rutin has been tested (Hartwell, 1951; Shubik and Hartwell, 1957). It has not shown any tumorigenic activity in rats, guinea pigs, or rabbits (Wilson et al., 1947). However, chlorogenic acid belongs to the group of the hydrolyzable caffetannins, as do all other tannins in tobacco thus far identified. In mice and rats subcutaneous injections of condensed nonhydrolyzable tannins have given rise to sarcomas a t the site of injection, as well as hepatomas, whereas hydrolyzable tannins gave rise only to hepatomas (Korpassy, 1959, 1960; Kirby, 1960). No tests have been carried out with tannins from tobacco. Such tests are indicated and should include tannins from untreated and aged tobacco including the brown pigment. The toxicity of phenols was studied by Von Oettingen (1949) ; the cilia inhibiting activity by several groups, as shown in Section IV,F, and the tumor-promoting activity by Boutwell and Bosch (1959). Accord-

EXPERIMENTAL TOBACCO CARCINOGENESIS

341

ing to the latter, the relative tumor-promoting activity of tobacco smoke (active): phenol, 0-,m-, and p-cresol, 2,4-, phenols is as follows: 2,5-, 3,4-, and 3,5-dimethyl phenol; (low active): o-ethyl phenol; (questionably active) : 2,6-dimethyl phenol and resorcinol; - (not active) : 2,3,5-trimethyl phenol, catechol, hydroquinone, salicylaldehyde, and p-methoxyphenol ; (not tested) : 2,4,5- and 2,4,6-trimethyl phenol, m- and p-ethylphenol, guaiacol, m-methoxyphenol, m- and p-hydroxyacetophenone. Definite tumor-promoting activity for a variety of phenols may be regarded as established (Wynder and Hoffmann, 1961b). “Naphthol” is one of several agents reported to induce urinary bladder tumors (Temkin, 1963). I n opposition to this view is the fact that in England for the synthesis of certain dye-stuff intermediates the carcinogenic amine (P-naphthylamine) has to be by-passed, but still the syntheses start with ,&naphthol (Scott, 1962). Bonser et al. (1958) do not mention naphthols as agents for experimental induction of tumors of the bladder. All tests completed thus far with naphthols have not shown any tumorigenic activity in the experimental animal (Hartwell, 1951; Shubik and Hartwell, 1957). In view of the correlation of cigarette smoking to bladder cancer in man, further chemical studies as to possible bladder carcinogens in cigarette smoke should be carried out, particularly regarding o-hydroxyphenylamines (Bonser e t al., 1958) and their oxidation products, such as phenoxazines. These compounds have not yet been reported in tobacco smoke.

++

*

+

2. Carboxylic Acids

a. Organic Acids in Tobacco. A considerable number of organic acids appear in tobacco and tobacco smoke. If one were not to consider critically the analytical methods and techniques used, one might name as many as 100 different acids, but a critical review will still list about 40 to 50 (Bentley and Berry, 1959, 1960; Berry, 1963; Johnstone and Plimmer, 1959). Jarboe and Quinn (1960) reported on the major (malonic, oxalic, malic, citric) acids and minor (volatile, propionic, acetic, formic, maleic, succinic and glycolic) acids and their concentration as found in Maryland, Burley, cigar blend, Latakia, Turkish, and flue-cured tobaccos. The free, water-extractable acids as a whole were also determined by titration. Despite some reservations on the analytical methods, new facts became known as a result of this study. One would have anticipated appreciable “free acids” in flue-cured tobaccos, but not in air-cured tobaccos, such as Burley, Maryland, and perhaps also, cigar blends, Correlations between concentrations of acids and organoleptic differences, as well as biological activities of the smoke, still need to be

342

ERNEST L. WYNDER AND DIETRICH HOFFMANN

explored. The organic acids in tobacco contribute to the acidity of the smoke, and a better understanding of its relation to toxicities to ciliated epithelium is needed. Considerable difference exists between the total calculated acidities of tobaccos and the sum of known individual acids, only partially corrected by the recent finding of relatively high concentrations of glyoxylic and a-ketoglutaric acid in tobacco (Glock, 1957; Onishi and Yamasaki, 1956). A comparison of the “volatile organic acids” in essential oil of American and Japanese flue-cured tobaccos revealed that about 75% of the American and about 50% of the Japanese were made up of acetic acid (Onishi and Yamasaki, 1957a), the concentrations being 0.48 g. and 0.027 g. per kg. tobacco, respectively. Of further interest is the finding of 0.12 g. per kg. of formic acid in American, and only traces in Japanese tobacco. Total acids were, respectively, 0.62 g. and 0.055 g. per kg. tobacco, and during certain stages of the tobacco preparation, a considerable increase in the volatile acids occurs (Onishi and Yamasaki, 1957b). From the major di- and tribasic acids of tobacco, citric, malic, oxalic, malonic, and succinic acid, the first two appear to play an important role during the degradation of sugars (Frankenburg, 1950). After enrichment of the higher fatty acids of the hexane extract of tobacco and subsequent formation of the methyl esters, these can be determined by gas chromatography. Swain and Stedman (1962) found 6 major and 15 to 25 minor acidic components in tobacco. The major long-chain acids of seven different tobaccos are compared in Table XIII. b. Organic Acids in Tobacco Smoke. The first analysis of tobacco smoke acids was reported in 1871 for cigar smoke (Vohl and Eulenberg, 1871). For these and later investigations, classical methods were eniployed (Gabelya and Kipriyanova, 1929; Neuberg and Burkard, 1931). During the last decade new methods have come into use that yield more exact determinations for carboxylic acids in tobacco smoke. These are divided into volatile and nonvolatile acids, not merely from physicochemical considerations, but rather on the basis of methodology. After fractionation into condensate main fractions, the “tar acids” are subjected to steam distillation of the “volatile acids.” These acids are separated on specially prepared silica gel using a mixture of n-butanol and chloroform as elution agents (Buyske et al., 1957). The individual acids in each fraction are determined by paper chromatography and the quantitative analysis completed by titration. The results (Table XIV) indicate no major variations in the volatile acids of the smoke of three types of tobacco. The smoke of 100 cigarettes (70 mm. long) of Bright tobacco,

343

EXPERIMENTAL TOBACCO CARCINOGENESIS

CONTENT^

OF

TABLE XI11 CERTAINHIGHERFREEFATTY ACIDSIN AGEDOR FERMENTED TOBACCO~SC ~

~~

~

Type of Tobacco Fatty acid Myristic Palmitic Stearic Oleic Linoleic Linolenic

Bright 3 101 18 15 53 110

Fire- Cigar Burley Maryland Turkish cured filler 2 51 8 5 25 35

3 44 12 11 35 21

5 103 12 17 51 78

2 32 18 3 17 20

Cigar binder

3 41 8 5 8 2

4 59 18 7 20 21

Single determinations on an air-dried basis. From Swain and Stedman (1962). c Milligrams per 100 g. of tobacco. a

however, showed the highest total acid content (212 mg.) compared t o the smoke of Burley (176 mg.) and Turkish cigarettes (156 mg.). However, the average weight of the Bright tobacco cigarette was 1200 mg. compared to 900 mg. and 1100 mg. for the other two. Izawa and Kobashi (1958), using a similar technique, identified in addition p-methyl valeric acid and the ethyl esters of n-butyric, isoTABLE XIV STE.IM-~OLATILE ACIDSFOUND IN SMOKEFROM BRIGHT,BURLEY, AND TURKISH CIGARETTES~ Milligrams per 100 cigarettes Carboxylic acid Nonylic-capric and above Caprylic Heptylic Caproic Valeric-isovaleric Benzoic BUtyri c-isobutyric Propionic Acetic Formic Unknownb

Bright

Burley

Turkish

1.58 1.44 0.79 5.34 5.50 2.68 7.39 23.46 103.20 42.40 18.70

2.75 1.10 0.57 7.31 7.04 3.29 3.87 11.84 84.60 35.55 12.00

1.75 0.79 1.10 14.84 13.97 4.39 5.46 11.17 65.40 27.90 9.00

212.48

b

-

--

169.92

155.77

From Buyske et al. (1957). Molecular weight of 100 used to calculate milligrams of acid present.

344

ERNEST L. WYNDER AND DIETRICH HOFFMANN

valeric, and n-caproic and/or ,&methyl valeric acid. Recently, Burdick et al. (1963b) determined the volatile acids as free acids by gas chromatography. Highest volatile acid content was found for cigarette smoke deriving from flue-cured tobacco. The analysis of certain volatile acids in cigar smoke was reported by Schmeltz and Schlotzhauer (1962). Compared to cigarette smoke, including that from air-cured tobaccos, the reported values appear unusually low, on the basis of tobacco consumed. Since no confidence limits are given for the analysis a reconfirmation of these results is desirable. To date few publications have dealt with the content of nonvolatile acids in tobacco smoke. The methyl esters of acids between C,, and C,,, specifically, stearic, palmitic, myristic, and lauric, have been identified by gas chromatography (Van Duuren and Kosak, 1958). I n other publications indications are given for the presence of a t least 25 different acids. Saturated and unsaturated acids from C, to C,, (cerotic acid) were found; but only limited experimental details are given for the gas chromatographic separation of the methyl esters (Clemo, 1958, 1960). The results of a preliminary analysis of nonvolatile acids are reported by Quin and Hobbs (1958) for Bright tobacco cigarettes. The qualitative determination of 12 phenolic acids in the smoke of cigarettes was accomplished by paper chromatographic technique (Yang and Wender, 1962). The presence of several free amino acids in cigarette smoke has been demonstrated in Kobashi’s laboratory (Izawa and Taki, 1959) and qualitative and quantitative differences were reported for sun-cured and bulk-sweated tobaccos (Izawa et al., 1959b). Alanine was the most predominant free amino acid (10.5-268.2 pg. per cigarette), followed by proline (5.5-25.1 pg.), and glycine (4.7-22.5 pg.). The other acids were below 20 pg. per cigarette. It needs to be pointed out, however, that the cigarettes were constant-volume, continuously smoked, and the analytical techniques employed (chromatography and colorimetry) are not strictly quantitative. It is evident that new analytical techniques for the identification and quantitative determination of organic acids in tobacco and tobacco smoke, are necessary. c. Turnorigenic and Ciliastatic Activity. I n experimental tobacco carcinogenesis one might assume that the pH of the smoke condensate would affect the final tumor response on the test animal. Only one study thus far has been completed on the role of acidity in tumorigenesis (Wynder and Hoffmann, 1 9 6 3 ~ ) .The whole acidic fraction (11.5%) of a condensate was doubled by adding the acidic fraction from the same cigarettes. This increased the pH from about 6.8 to

EXPERIMENTAL TOBACCO CARCINOGENESIS

345

about 4.8. This “tar” plus control “tar” were each tested on 50 Swiss female mice under equal experimental conditions. At the end of the experiment (15 months’ application, 3 months’ observation) the tumor response was insignificantly increased from 34% papillomas and 22% carcinomas in the control group to 40 and 28% tumors, respectively, in the study group. Such a study should be repeated. Like the “phenolic portion” (weakly acidic portion) the acidic portion (2.2% of whole condensate) showed a significant tumor-promoting effect when applied in 10% concentration with O,OU5% B[a]P (Wynder and Hoffmann, 1961b). The control group gave 70% tumors, including 68% carcinoma on 40 mice tested and in the experimental group 97% papillomas and 93% carcinoma on 30 mice tested ( p < 0.01). I n addition, tumors appeared earlier when promoted by the acidic portion. The effects of individual acids on the tumor-promoting activity of the fraction have not been studied thus far. As discussed previously in this chapter, certain long-chain fatty acids are known to be tumor promoters to mouse skin (P. Holsti, 1959; Saffiotti and Shubik, 1963), and they might account for the weak tumor-promoting activity of tobacco extracts (Ranadive et al., 1963). A review of the general toxicity of saturated aliphatic acids and their esters is presented by von Oettingen (1959). Some data are available on the pH dependence of cilia movement and for the cilia toxicity of organic acids (Rivera, 1962; Sleigh, 1962; Wynder et al., 1963a) (for a further discussion of acids as ciliastatic agents see Section IV,F) . Formic acid exhibited the highest cilia-toxic activity of all acids tested. I n general, increase of the alkyl portion and decrease of volatility of an aliphatic acid reduces the cilia toxicity.

G. ALDEHYDES AND KETONES 1. Carbonyl Content of Tobacco The basic studies on tobacco a t the Kaiser-Wilhelm Institute for Biochemistry under Carl Neuberg (1925-1936) led to the isolation from the leaf of formaldehyde, acetaldehyde, n-butyraldehyde, benzaldehyde, methylglyoxal, diethyl ketone, dipropyl ketone, and diacetyl (Neuberg and Kobel, 1926; Kobel and Neuberg, 1935). About two decades passed until comprehensive studies were initiated on the carbonyl content of tobacco a t the Research Laboratories of the Tobacco Monopoly of Japan (Onishi and Nagasawa, 1955, 1957a,b,c; Onishi et al., 1956). From a neutral subfraction of the steam distillate of Virginia tobacco leaves (essential oil) the aldehydes were precipitated as 2,4-dinitrophenylhydraaones ( D N P H ). The hydrazones were separated

346

ERNEST L. WYNDER AND DIETRICH HOFFMANN

by chromatography on siIicic acid-celite. The infrared spectra of these fractions served for the identification of the hydrazones (J. H. ROSS, 1953) of acetaldehyde, isobutyraldehyde, benzaldehyde, furfural, 5methylfurfural, 5-hydroxmethylfurfural, an unknown C6-aldehyde, acetone, and 2-pyrrolmethyl ketone. The same investigators found, however, that furfural compounds are mainly formed during the steam distillation from certain carbohydrates of the tobacco leaf. Compared to cured tobacco during redrying and aging, furfural, isobutyraldehyde, and a (&-aldehyde decrease, whereas, 5-hydroxymethyl, furfural, and 5-methylfurfural increase; acetone was found only after redrying and aging of tobacco. Significant differences were found in the constituents and contents of carbonyl compounds in Japanese Burley and flue-cured tobacco. During the last few years Weybrew and his group have become interested in the carbonyl constituents of the volatile oils from tobacco and their possible contribution to the odor of tobacco. From unaged fluecured tobacco they identified as constituents in the volatile oils: formaldehyde, furfural, acetaldehyde, propionaldehyde, isobutyraldehyde isovaleraldehyde, acetone, and 2-butanone. For the identification of the carbonyl compounds, paper and vapor phase chromatography were employed (Shaw e t al., 1960) and for the gas chromatographic identification the investigators modified a rapid procedure (Stephens and Tessler, 1960) developed by Ralls (1960). The DNPH’s of the volatile carbonyl compounds are heated together with an excess of a-ketoglutaric acid; there occurs an exchange of the 2,4-dinitrophenylhydrazonegroup, and the liberated aldehydes and ketones volatilize directly into the gas chromatograph. By this method seven volatile carbonyl compounds were determined from a great variety of tobaccos (Weybrew and Stephens, 1962). One of the suggestions made by the authors, as a result of this study, is that acetone and 2-butanone “may be positively related to overall quality, while isobutyraldehyde and isovaleraldehyde appear to show a negative association.” The low-temperature (38°C.) reduced-pressure (49.7 mm.) distillate of tobacco (“aroma constituents”) has, among many compounds. furfural (Jones and Weybrew, 1962). 2. Aldehydes and Ketones in Tobacco S m o k e Reviewing the earlier studies on carbonyl compounds of the smoke, one is surprised a t the accomplishments achieved with the crude methods which contributed importantly to our knowledge of the subject (A, Trillat, 1904; Bogen, 1929; Neuberg and Burkard, 1931; Molinari, 1936; Wenusch, 1939; and others). Further progress has come from the development of gas chromatographic techniques and improved automat,ic smoking machines. One of

347

EXPERIMENTAL TOBACCO CARCINOGENESIS

the first studies using such improved methods was reported by Touey (1955) who formed bis (dimedon) derivatives from the aldehydes of the total smoke and determined these as “apparent acetaldehyde.” The values varied from 0.8-0.9 mg. per unfiltered, king-sized cigarette. Various groups precipitated carbonyl compounds as D N P H to separate and identify these finally by paper chromatography. Their quantitative analysis was accomplished by the ultraviolet absorption of the extracts of the paper chromatography bands (Buyske e t al., 1956; Martin, 1958; Mold and McRae, 1957). Others collected several carbony1 compounds from the gas phase of cigarette smoke by cold traps and, after fractionation, determined them by infrared absorption (Osborne e t al., 1956; Philippe and Hobbs, 1956). Vapor phase chromatography was first applied by Irby and Harlow (1959). The gaseous phase of the smoke is condensed, fractionated, and chromatographed a t constant temperature on three different columns. The only disadvantage of this elaborate method is the difficulty in its application to routine analysis (Table XV). TABLE XV CERTAINCARBONYL COMPOUNDS IN CIGARETTE SMOKE.

Compound

All tobacco (mg./cig.)

Plain composite tip (mg./ck.)

Acetaldehyde Propionaldehyde Acetone Acrolein Isobutyraldehyde Methyl ethyl ketone

0.73 0.05 0.39 0.07 0.03 0.08

0.66 0.04 0.37 0.06 0.03 0.07

(1

Impregnated composite filter tip (w./ c k ) 0.50 0.02 0.20 0.03 0.01 0.03

From Irby and Harlow (1959).

Employing Rall’s method (1960), Schepartz and McDowell (1961) identified formaldehyde, acetaldehyde, propanal, n-butanal, acetone, and methyl ethyl ketone in cigar smoke. Several other simple gas chromatography methods have been tried in various laboratories (Williamson et al., 1962; Grob, 1962a; and others). The use of Golay columns represents a real advance (Grob, 196213). The smoke from the puff before the last 250 mg. tobacco of a cigarette is filtered through a glass fiber filter a t 60°C. and an aliquot of the sample is used for the gas chromatographic separation. The author calculated semiquantitative values for one standard cigarette (Table XVI) . The identification was based on retention values obtained with

348

ERNEST L. WYNDER AND DIETRICH HOFFMANN

a t least two, usually three, columns with different liquid phases, and the components were partially classified with group reagents. Recently, Spears ( 1963c) incorporated further improvements which allow the direct, quantitative determination of several gaseous components including that of aldehydes and ketones without using a glass fiber filter for the separation of particulate matter. TABLE XVI ALDEHYDES AND KETONESI N CIGARETTESMOKE^ Aldehydesb (pg.) Acrolein Propion Isovaleryl Croton Isobutyr n-Butyr Methylacrolein n-Valeryl Pival a

b

Ketones (pg.) 45 40 20 16 12 8 8 8

Acetone Methyl ethyl Butenone Methyl propyl Diethyl Methyl isopropyl

360 80 28 12 12 6

4

From Grob (1962b). Acetaldehyde not determined.

Norman et al. (1963) trap the smoke vapors and determine direct from the collected materials by electrometric, colorimetric, or chromatographic methods various gaseous compounds including aldehydes and ketones. A most sophisticated analytical method was recently developed by Varsel e t al. (1963) employing low-voltage mass spectrometry. The values presently obtained by this method appear quite high. It seems that even after certain adjustments, this method will remain a research tool rather than an aid in routine analysis. Lindsey e t al. (1963) applied thin layer chromatography for the detection and semiquantitative determination of carbonyl compounds as DNPH. Diacetyl in tobacco smoke (Schmallfuss, 1950) as well as other a-diketones are sometimes t.hought to play a role as flavor components (Martin, 1958). Certain long-chain ketones, especially dipalmityl ketone, were also detected in cigarette smoke (Schurch and Winterstein, 1935; Van Duuren and Kosak, 1958). The concentration of volatile carbonyl compounds was significantly higher in the smoke of dry cigarettes (6.5% moisture) than in the smoke of cigarettes with 12% moisture (Pailer e t al., 1962). The tobacco chemist now has available excellent analytical tools for the determination of various gaseous components in tobacco smoke,

EXPERIMENTAL TOBACCO CARCINOGENESIS

349

especially volatile carbonyl compounds. This enables the investigator to test the effects of various materials designed to remove selectively certain compounds from cigarette smoke (see Section VI).

3. Cilia-Toxicity and Tumorigenic Activity of Aldehydes and Ketones The toxicity to ciliar movement of certain aldehydes has been extensively studied and has been reviewed in Section IV. One liter of unfiltered cigarette smoke, that is, the volume of about 30 puffs, contains between 100 and 450 @g.acrolein and between 30 and 60 pg. formaldehyde, both strong cilia-toxic components. Their reduction in tobacco smoke would thus be highly desirable (see Section V I ) ; however, aldehydes and ketones of various structures have not been found to be tumorigenic to the experimental animal, with the exception of some ketosteroids (Hartwell, 1951; Shubik and Hartwell, 1957). One recent study, however, showed that basal-cell hyperplasia and stratification of the epithelium were above normal in the trachea and major bronchi of C3H mice exposed to 50 pg. formaldehyde per liter air; 200 pg. formaldehyde per liter gave atypical metaplastic changes in the trachea which, however, did not progress to invasive carcinoma (Horton et al., 1963). These changes may have been a consequence of the cilia-toxic effects of formaldehyde, a possibility which should be explored further. H. STEROIDS 1. Phytosterols in Tobacco and Tobacco Smoke

Johnstone and Plimmer (1959) reviewed some of the chemicalanalytical and structural aspects of the steroids identified in tobacco and tobacco smoke. The more important ones in tobacco are stigmasterol (I),p-sitosterol (11), and y-sitosterol (111). Ergosterol (V) is present as a minor component. From flue-cured tobacco a A5-sterolglycoside with 2h-methyl configuration, and probably, unsaturation in the side chain (Dymicky and Stedman, 1959a) and free campesterol (IV) (Dymicky and Stedman, 1959b) were isolated. Compounds I, 11, and I11 have also been isolated from tobacco smoke. It appears that 3P-phytosteroids are present in tobacco as free alcohols, as glucosides, and/or esters of fatty esters (Rodgman et al., 1961b; H. E. Wright, Jr. et al., 1962) (Fig. 22). The isolation from Indian tobacco of four unidentified sterol glycosides, one of them a ketosterol glycoside, was reported by Divekar et al. (1961), without elucidating its structure. In recent years several studies were concerned with determinations of sterols in tobacco products (Stedman et al., 1958; Stedman and Rusaniwskyij, 1959a; Rodgman

350

ERNEST L. WYNDER AND DIETRICH HOFFMANN

e t al., 1959; Rodgman et al., 1961b). Rodgman et al. (1961b) isolated from the smoke of cigarettes made exclusively from Turkish, flue-cured, and Burley tobaccos, 315,200, and 300 mg. phytosterols, respectively, per kilogram of tobacco smoked. Phytosterol esters were detected in the smoke of Turkish tobacco; the smoke condensates of 1 kg. of flue-cured and Burley tobacco contained 65 and 15 mg. esters, respectively. The Turkish cigarettes were 70 mm. long without filter; the flue-cured 70 nim. long with a filter tip about 15 mm. I n the smoke of 1 kg. of 68-nim.

I. Stigmasterol:

,CHI R = -‘2%- CH= CH- CH- CH I C2H, ‘CH,

HO 11. 8-Sitosterol:

/CHI R = -CH-C&-CHZ-CHCH 1 I CH, C,H, ‘CH,

,CH,

III. 7-Sitosterol:

R = -CH-C&-C&-CH-CH I

CH,

.

CzH,

\

CH,

FIG.22. Major phytosterols in tobacco.

cigarettes made from cased commercial blend of tobacco they found 225 mg. phytosterols and 26 mg. phytosterol esters. The concentration of phytosterols in the condensates, unfortunately, cannot be calculated from the data presented. Assuming, however, that from 1 kg. of tobacco smoked 20-30 g. condensate was collected, one might expect a concentration of approximately 1% free phytosterols and that of the esters significantly lower. The major sterol esters were found to be palmitate, stearate, oleate, linoleate; traces were laurate, myristate, and linolenate (Rodgman et al., 1959). I n a recent communication the isolation of a sterol glyco-

EXPEEIMENTAL TOBACCO CARCINOGENEHK

35 1

side from tobacco and cigarette smoke was reported. After hydrolysis the sterol mixture was identified as consisting of stigmasterol, sitosterol, and campesterol (Kallianos et al., 1963). One might also expect, in tobacco smoke, some compounds deriving from phytosterols with an intact steroid ring system, but. with altered side chains, or such oxidation products as 3-keto-steroids, among others. As long as there is no special interest, in these steroids, their tedious isolation and identification probably will not be undertaken. 2. Tumorigenicity of Steroids

The limited experimental data available to date do not suggest an important role of steroids in epithelial tumor development. The neutral subfraction of smoke condensate (21.4% of whole condensate) containing the bulk of phytosterols was inactive when applied to mouse skin in 10% solution (Wynder and G. Wright, 1957). According to Fieser (19571, the dehydrogenation product of cholesterol ( I ) , A5-cholestene-3-one(111, can be oxidized with air to Sphydroperoxy-A4-cholestene-&one (111).

This first isolated steroid hydroperoxide (111) proved to elicit relatively rapidly fibrosarcoma in mice upon sucutaneous injection (Fieser et at., 1955). Other oxidation products of cholesterol are also cited as tumorigenic, all of them oxygenated a t position six as the hydroperoxide (111). Fieser (1957) suggests from these data as well as from the photosensitizing activity of methylcholanthrene and B [a]P for the peroxida-

352

ERNEST L. WYNDER AND DIETRICH HOFFMANN

tion of cholesterol that such “observations, regarding cholesterol, have a possible, if not very probable, bearing on the problem of lung cancer and tobacco smoke.” Since the three major sterols in tobacco and tobacco smoke, ergosterol, p-sitosterol, and y-sitosterol, have a hydroxyl group and a nuclear double bond in the same relative arrangement as in cholesterol, they appear to be capable of yielding oxidation products comparable t o those from cholesterol. How far these oxidation products are stable and formed during smoking cannot be determined a t this time. Even though such an investigation would require special and sophisticated isolation techniques, it would be worthwhile.

I. EPOXIDES, PEROXY COMPOUNDS, AND LACTONES

1. Epoxides and Peroxides in Tobacco and Tobacco Smoke Tobacco contains olefines and terpenes, some of which are readily oxidized, possibly to form epoxides during combustion, which would appear in the smoke. However, substantiation of this hypothesis, is still lacking. 2. Lactones in Tobacco and Tobacco Smoke Three diterpene lactones have been isolated from Turkish tobacco and its smoke (Giles and Schumacher, 1961; L. C. Cook and Rodgman, 1962; Giles et al., 1963). Two of these, a-levantenolide (I) and plevantenolide (11), are derivatives of 4-but-2-enolactone (Fig, 23). L. C. Cook and Rodgman (1962) isolated 19 mg. of a-levantenolide (I) and 1.4 mg. of P-levantenolide (11) from 1200 to 1250 Turkish cigarettes. Dickens (1962) discussed the possibility of formation of 4-pent-2enolactone (V) and 4-pent-3-enolactone (VI) (angelica lactones) from levulinic acid (Fig. 24). Levulinic acid has been detected in tobacco smoke (Quin and Hobbs, 1958) and is known to be formed from carbohydrates. 3. Tumorigenic Activity of Epoxides, Peroxides, and Lactones Van Duuren et al. (1963) discussed the chemical and biological reactivities of certain epoxides, hydroperoxides, peroxides, and lactones. Several of these are certainly highly reactive, for example, with the --SH groups of cysteine (Searle, 1961a,b) and some react with nucleic acids, thereby functioning as mutagenic agents (Kotin and Falk, 1963a). Of general interest in this regard are the studies by Dickens (1962),

EXPERIMENTAL TOBACCO CARCINOGENESIS

(I)

353

(n1

FIG.23. Two diterpene lactones, a-levantenolide (I) and /3-levantenolide (II), isolated from Turkish tobacco and its smoke (Giles and Schumacher, 1961).

I. 6-propiolactone

n. 4-butanolactone ( y-butyrolactone ) III. 4 -hex-4 -enolactone IV. 4-but-2-enolactone

v.

4-pent-2-enolactone (a-angelica lactone )

VI. 4-pent-3-enolactone (0-angelica lactone) FIQ.24. Carcinogenic lactones.

354

ERNEST L. WYNDER AND DIETRICH HOFFMANN

Dickens and Jones (1963), Kotin and Falk (1963a), and Van Duuren et al. (1963). Several investigators have considered peroxides and epoxides as possible neoplastic agents in the human environment, especially as it concerns air pollution (Hueper et al., 1962; Kotin and Falk, 196313). Haddow (1958) discussed the hypothesis of epoxide formation during metabolism of carcinogenic hydrocarbons. Some epoxides and diepoxides formed during oxidation of olefins are known carcinogens to mouse skin or sarcoma-producing agents in rats, when applied in relatively high concentrations (Kotin and Falk, 1963a ; Van Duuren et al., 1963). Some lipoperoxides gave rise to a few sarcomas in mice (Kotin and Falk, 1963a). I n feeding experiments with mice and rats only one gastric sarcoma (rat) and one early carcinoma (pregastric, mouse) were observed (Seelkopf and Salfelder, 1962). 7,12DimethyI-7,12-epoxy-benz[ a ]anthracene is known as a carcinogenic endocyclic epoxide (Badger et al., 1940). Of the peroxides tested only methyl ethyl ketone peroxide has given rise to a few sarcomas (Kotin and Falk, 1963a; Van Duuren et al., 1963). Known carcinogenic hydroperoxides are 6P-hydroperoxy-A4cholestene-3-one and l-hydroperoxy-l-vinylcyclohexene-3(Van Duuren et al., 1963). Some hydroperoxides, however, have induced a significant number of malignant lymphomas in mice upon subcutaneous injection (Kotin and Falk, 1963a). Kotin’s group succeeded in obtaining alveologenic adenocarcinomas in strain A and C57 black mice upon exposure to oaonized gasoline (Kotin et al., 1958) and squamous carcinomas in the lung of C57 black mice after successive infections with three mouse-adapted strains of influenza virus and continuous exposure to an aerosol of ozonized gasoline (Kotin and Wisely, 1963). All carcinogenic lactones except /3-propiolactone (I) (Searle, 1961b) are 7-butyrolactones (11). Except for 4-hex-4-enolactone (111) these are derivatives of 4-but-2-enolactones (IV) (Van Duuren e t al., 1963). The only known carcinogenic lactam is penicillin-G (Dickens and Jones, 1963). Since some derivatives of 4-but-2-enolactone have been shown to produce sarcomas in rats (Dickens and Jones, 1961) and since, as mentioned, two derivatives of this lactone have been found in tobacco and tobacco smoke, biological testing of these tobacco constituents (levantenolides) appears indicated. Dickens’ conclusion (1962) that lactones in tobacco smoke “might well constitute a new factor in the causation of lung cancer,” appears premature, since none of the angelica lactone9 * (Y- and p-Angelica lactones were recently found to be singularly noncarcinogenic in groups of 30 mice, each receiving 3 topical applications per week of 100 mg. of the compounds throughout their life span (Van Duuren et nl., 1964).

EXPERIMENTAL TOBACCO CARCINOGENESIS

355

(V,VI) suggested as being formed during smoking, have as yet been tested biologically (Fig. 24). Presently available evidence, both on the basis of presence in tobacco products, as well as established carcinogenic activity, indicates that the above-discussed components do not play a role in experimental tobacco carcinogenesis.

J. NITROSAMINES 1. A Group of “Procarcinogens” During an investigation of factors that might be responsible for liver damage in industrial workers, Magee and Barnes (1956) discovered that the feeding of dimethylnitrosamine (DMNA) to rats led to the development of hepatic tumors. This observation initiated extensive biological, biochemical, and chemical studies on a broad spectrum of nitrosamines (Weisburger and Weisburger, 1963). Based on metabolic studies with C14- and N15-labeled DMNA, the following intermediates were postulated:

alkylation of proteins, nucleic acids *Diazomethane is a resonance hybrid. Its structure can be presented by

+ - -

+

CH,=N=N c-) CH-NrN 1961; Huisgen, 1963).

and not by the cyclic form (L. F. Fieser and M. Fieser,

Another aspect in line with this postulate is the greatly accelerated incidence of spontaneous lung adenomata in A/2G mice, caused by diazomethane (Schoental, 1960). Druckrey et al. (1961a) extended this concept to nitrosamines in general. They proposed that only such dialkylnitrosamines can be carcinogenic which, after enzymatic oxidation of one alkyl group, can form diazoalkanes as intermediates. Their findings with various tests of nitrosamines on rats favor such a concept. It appears possible that the formed diazoalkanes alkylate essential cell constituents, such as nucleic acids, and thereby alter the genetic mechanisms and cellular information transfer processes (Weisburger and Weisburger, 1963). Based on these considerations, the active nitrosamines

356

ERNEST L. WYNDER AND DIETRICH HOFFMANN

are included in the group of “procarcinogens,” agents which presumably require metabolic activation. Reports on the induction of tumors in various sites and animals contribute further to the importance of this group. Tumors were induced with nitrosamines in the liver, kidney, lung, stomach, esophagus, and bladder of rats (Argus and Hoch-Ligeti, 1961; Druckrey et al., 1961a,b,c, 1962, 1963; Druckrey and Preussmann, 1962a), and carcinoma in the lung of hamsters (Dontenwill and Mohr, 1961; Herrold and Dunham, 1963). 2. Nitrosamines in Tobacco and Tobacco Smoke In a recent publication Druckrey and Preussmann (196213) considered the formation of nitrosamines in tobacco smoke as a most likely possibility, based on the presence of several secondary amines, such as diethylamine, piperidine, nornicotine, and anabasine (Johnstone and Plimmer, 1959), as well as nitrogen oxides in tobacco smoke (Haagen-Smit e t al., 1959; Bokhoven and Niessen, 1961). From both groups of compounds nitrosamines can be formed in vitro. Boyland et al. (1963) regard the formation of nitrosamines in cigarette smoke as a possibility, especially that of nitrosoanabasine and nitrosonornicotine ; this led them to develope a microtest for nitrosamines in general. So far, however, nitrosamines have not been identified in tobacco smoke. Tobacco itself seems to contain nitrosamines of still unknown nature (Druckrey and Preussmann, 1963). Another possibility of the formation of nitrosamines is the reaction of NO and/or NO, with cellular amines (Druckrey and Preussmann, 196213). This has recently been challenged by Henschler and Ross (1963). They exposed female mice twice weekly for 48 hours to 46 1j.p.m. as well as smaller amounts of NO,. After 6 to 15 months they observed 6 adenomas of the lung and 2 lymphosarcomas in a group of 51 mice. No squamous cell metaplasia or carcinomas were found. According to their calculations and comparisons with the diethylnitrosamine activity the authors regard it as relatively unlikely that nitrosamines can be formed in the tissues of the respiratory system after inhalation of nitrogen oxide gases. Recently, Herrold and Dunham (1963) reported a high tumor yield in the respiratory tree and ethmoturbinals of Syrian hamsters that received diethylnitrosamine independently applied intratracheally or intragastrically. These results encouraged the authors to propose that pathways other than the respiratory tract might carry the carcinogen to the human lung. While this concept is difficult to disprove, either experimentally or epidemiologically, it would seem to us that the direct effect of tumori-

EXPERIMENTAL TOBACCO CARCINOGENEMS

357

genic agents in tobacco products on adsorptive epithelial surfaces continues to be the most logical and proven route.

K. VOLATILECOMPONENTS IN TOBACCO SMOKE 1. Gaseous Hydrocarbons I n most studies of tobacco smoke, the investigator faces the difficulty of defining the group of “volatile compounds.’’ He will soon concludc that every grouping will be, to some extent, arbitrary and will not represent the true physicochemical conditions as they prevail in tobacco smoke. For studies of experimental tobacco carcinogenesis, we regard, as volatile components of tobacco smoke, all those which are lost in the collection and preparation of the condensate for application to the experimental animal. An analysis of this condensate shows that practically all those ‘(neutral components” are lost that appear during the gas chromatographic separation on Golay columns before the toluene peak is reached. For studies concerned with selective filtration of tobacco smoke constituents, the same arbitrary system of “volatile neutral components” was chosen and found to be helpful. I n other studies of tobacco smoke, one might find other arbitrary systems more suitable. One of the most detailed analyses for certain components in the gas phase of cigarette smoke was reported by Philippe and Hobbs (1956). Their results are based on the gaseous phase of a 70-mm. nonfilter cigarette smoked twice per minute with a duration of 2 seconds and a puff volume of 34 ml.; butt length was about 20 mm. The analytical method was based on an infrared compensation technique (Table XVII). Recently, Grob (1962b) determined cyclohexane by gas chromatography in the gas phase of cigarette smoke, (3 pg. per cigarette, about x lo-“ ml. per puff) and pentadiene (20 pg. per cigarette about ml. per puff). Johnstone and Plimmer (1959) further listed 0.6 x isobutane, cis- and trans-2-butenes and methylacetylene in tobacco smoke. No gas chromatographic analysis for hydrocarbons from cigar or pipe smoke has, as yet, been reported. A discussion of the effect of volatile hydrocarbons in experimental tobacco carcinogenesis must be speculative, due to lack of data. The most toxic hydrocarbon present in tobacco smoke is toluene. Cigarette smoke contains between 200 and 300 pg. toluene per liter, which is below the toxic level of 750 pg. per liter (Am. Conf. Govt. Ind. Hygienists, 1963). 2. Alcohols and Esters Among alcohols known to be present in tobacco products solanesol and cholesterols have already been discussed. Others, such as the

TABLE XVII SjUMMARY O F

ANALYSES BY

INFRARED

METHODS OF GASPHASE

Volume, cc./puff at NP T

OF

CIGARETTE

Mole % of total gas phase _ _ _ _ _ _ I -

Component COZ CO CHI Ethane Propane Butane Propane butane Ethylene Propylene Acetylene Isoprene Butadiene Acetaldehyde Acetone Methyl ethyl ketone Methanol HCN Diacetyl

+

cos

Methyl chloride Isobut,ylenec Benzene Toluene Fiiran 2-Methylfuran Condensed phase analyzed8 Condensed phase not analyzed' Total condensables Total noncondensables Total volume (NPT)

Blend 50% Bright-50% cased Burley 3.0 1 .4

Turkish 2.5 0.95

22.0 X 4.1 X10-2 1.6 X 0.2 x 10-4

-

2.3 0.68 0.38 3.1 0.17 9.7 1.4 0.18 0.85

3.8 0.09 0.07 3.2 0.07 0.15 0.04 0.11 -0.11

X lo-' X lo-' X X los2 X10-2 X X x 10-2 X X X X X X10L2 X lo-' X X X lo-"

16.0 X 3.2 X

-

-

2.5 X 1 .8 X 0.74 X 0.33 XIO-' 4.8 X 0.18 X 3.9 X 1.1 X10-2 0.08 x 10-2 2.2 X 0.26 X 0.09 X

0.23

x

1.6 X 0.26 X 10+ 0.11 X 0.15 X 0.08 X l O V 0.02 X

Blend 50% Bright40 % cased Burley

Turkish

8.7 7.3 4.2 2.8 0.64 0.47 12.0 X 9.3 X 10-2 4.7 x 10-2 0.5 X 7.2 x 6.8 X 5.4 X 2.0 x 2.2 x 1.1 X 10-2 1.0 X 10-2 9 . 2 X 10-8 14.1 X 0 . 5 X 10-2 0.5 x 28.3 X 11.3 X 4.1 x 3.1 X 0 . 5 x 10-2 0 . 2 x 10-2 2.5 X lo-% 6.5 x 10-2 11.2 X 10-2 0.8 x 0.3 X 0.3 X 0.2 x 0.7 X 9.3 X 4.7 x 0 . 2 X 10-2 0 . 8 X 10-2 0 . 4 X 10-2 0.3 x 0.1 X 10-2 0.5 x 0.3 x 0.2 X 4 . 3 X 0.07 X

3.3

2.7

9.7

8.0

0.3 3.6

0.4 3.1

0.9 10.6

9.2

30.6 34.1

31.2 34.3

89.4

90.8

1.2

From Philippe and Hobbs (1956). Average of mass spectral analyses of infrared inactive constituents (mole yo):Air -57% (k3%); nitrogen (excess) -24% (*I%); hydrogen -1.6%; argon -0.3%. Isobutylene was previously determined for cigarettes made from Bright and Burley tobaccos. The mole % concentrations were, respectively, 0.6 X and 0.7 X This compound was not determined for cigarettes made from caced Burley and from 50% Bright-50% Burley tobaccos. d Estimated directly from spectrum. Sum of volumes in cc./puff at NPT and of mole % of condensables quantitatively wcounted for. Essentially all CO and CH, were obtained in noncondensable fraction. f Based on mass spectral analysis, this material consists of approximately 80 mole % water and 20 mole % of other constituents, including met,hanol and acetone. a

358

EXPERIMENTAL TOBACCO CARCINOGENESIS

359

tobacco humectants glycerol, diethylene, and triethylene glycol (Puschmann and Miller, 1961; Bill et al., 1959) borneol, benzyl alcohol, and P-phenyl ethyl alcohol (Johnstone and Plimmer, 1959) are of no importance in line with the present discussion and their concentrations in tobacco smoke products are below the toxic level (Larson et al., 1961). However, Hueper (1963b) listed in a recent publication diethylene glycol as a suspected bladder carcinogen for smokers of cigarettes treated with this humectant. Traces of furfuryl alcohol have been isolated from tobacco leaves (Onishi et al., 1956). Guvernator (1963) compared furfury1 alcohol content in the smoke of Bright with that of Burley tobaccos, and found 14.6 pg. and 5.7 pg. per cigarette, respectively. Ethyl alcohol was found in traces (2 pg.) in cigarette smoke (Grob, 1962a,b). Methanol with reported values between 0.35 and 0.95 mg. per liter smoke, is above the nontoxic dose of 0.260 mg. per liter (Philippe and Hobbs, 1956; Grob, 1962a,b). Several volatile esters have been found in tobacco smoke (Iaawa et al., 1957; Iaawa and Kobashi, 1958). In the unfiltered smoke of one cigarette were detected: methyl formate (30 pg.) methyl acetate 10 and 8 pg., and ethyl acetate, 2 pg. (Irby and Harlow, 1959; Grob, 1962a,b). These values were far below the toxic levels of 255, 360, and 1440 pg. per liter, respectively (Von Oettingen, 1949). Suggestive evidence was also given for the presence of about 3 pg. methyl acrylate in the smoke of one cigarette (Grob, 1962b). 3. Organic Cyanides, Gaseous Heterocyclic Compounds, and Nitrites

Recently, several organic cyanides have been detected in cigarette smoke (Grob, 196213). Semiquantitative values given for the smoke of a cigarette are acetonitrile, 140 pg. ; propionitrile, 30 pg. ; isobutyronitrile, 8 pg.; acrylonitrile, 10 pg.; and methacrylonitrile, 3 pg. The concentration of acetonitrile is far above the permitted nontoxic level of 70 pg. per liter of air. Lately, the suggestion was made that the acetonitrile concentration in body fluids might be a good indicator for the actual degree of exposure t o tobacco smoke (McKee et al., 1962; J. K. Campbell et al., 1963). We concur with this concept. More knowledge should be obtained about such a correlation, including the metabolism of CH,CN. The analytical methods also have to meet standard requirements. Methyl chloride was found in cigarette smoke by Osborne e t al. (1956) and Philippe and Hobbs (1956) a t the concentrations of between 1.6 x and 9.3 X per puff, which is up to 30 times higher than the toxic level, of 0.210 mg. per liter of air (Am. Conf. Govt. I n k Hygienists, 1963). Various heterocyclic gaseous components were detected in cigarette

360

ERNEST L. WYNDER AND DIETRICH HOFFMANN

smoke, furan (18-30 pg. per cigarette), 2-methylfuran (20 pg.), dimethylfuran (16 pg.), tetrahydrofuran (8 pg.), thiophene (2 pg.), and tetrahydropyrane (2 pg.) (Philippe and Hobbs, 1956; Irby and Harlow, 1959; Grob, 1962a,b). A confirmation of the presence of some of these trace components would be desirable even though little seems to be known of their toxicity (Larson et al., 1961). Methyl nitrite in the smoke of cigarettes (Burley, 468 pg. per cigarette; Bright and Turkish, 16 pg. each) as well as methyl thionitrite and carbon disulfide were semiquantitatively determined by infrared spectrophotometry and the two latter components, by mass spectra also (Philippe and Hackney, 1959; Philippe and Moore, 1961). 4.Inorganic Components

a. Acids. The presence and potential toxicity of hydrogen cyanide (HCN) in tobacco smoke has been known since 1858 (Vogel). Recent values obtained by an infrared compensation technique vary between about 35 and 115 pug. per 34 cc. puff, which is far above the lowest toxic level (Osborne et al., 1956; Philippe and Hobbs, 1956). Present knowledge of H C N content of tobacco smoke and its effect on the living organism have been thoroughly reviewed by Larson et al. (1961). Kensler (1960) regards the hydrogen cyanide content of tobacco smoke as little, if any, hazard to the normal smoker. I n these considerations, however, the relative high toxicity of H C N to ciliated epithelium was not included (Rivera, 1962; Sleigh, 1962). Hydrogen sulfide and hydrogen thiocyanide are reported as constituents of tobacco smoke (Johnstone and Plimmer, 1959), but no quantitative data are available. b. Nitrogen Oxides. NO NO, in cigarette smoke is between 145 and 165 p.p.m.; in cigar smoke, between 167 and 1250 p.p.m.; and in pipe smoke, between 126 and 1154 p.p.m. (Haagen-Smit et al., 1959). Thc data are based on a semiquantitative colorimetric microdetermination. Upon deep inhalation, the nitrogen oxide content is reduced below the detectable level in the exhaled “smoke.” The smoke of “fast” puffed cigarettes contains a significantly higher level of nitrogen oxides compared with the smoke of LLslowly” puffed cigarettes. A more recent study reported for 85-mm. cigarettes, 80 to 120 p.p.m. NO, and 170 to 210 p.p.m. NO NO, (Bokhoven and Niessen, 1961). Actual inhalation studies showed 87 to 96% retention. The authors calculate that a smoker of 20 cigarettes absorbs about 3.0 mg. of nitrogen oxides (calculated as NO,). The maximal acceptable concentration of NO, is 5 p.p.m. according to the Am. Conf. Govt. Ind. Hygienists (1963). Nitrous oxide (N20) has not yet been detected in tobacco smoke.

+

+

EXPERIMENTAL TOBACCO CAHCINOGENESIS

361

c. Carbon Monoxide and Carbon Dioxide. Carbon dioxide has been reported to represent about 7 to 11% of the gas phase of cigarette smoke. The analysis of the same gas phase with an infrared compensation absorption procedure revealed values from about 2.9 to 5.170 CO (Osbornc et al., 1956); the values obtained with a gas chromatographic method were between 3.3 and 5.57% (Mumpower et al., 1962). The highest CO values were found for the last puffs of a cigarette, in the smoke of puffs drawn with high volumes and in the smoke of a cigarette with nonporous paper (Al-coated). While the CO content of tobacco smoke does not appear to play N role in experimental tobacco carcinogenesis, its relative level may serve as a useful indicator of “completeness” of combustion. L. ELECTRIC CHARGES, RADICALS, AND RADIOISOTOPES IN TOBACCO SMOKE 1. Electric Charges in Tobacco Smoke

a. Concentrations and Distributions. The first study concerned with the electric charge distribution in tobacco smoke was based on the Millikan method (Holmes e t al., 1959). The authors found tobacco smoke to be a lightly charged aerosol (3.2-12.2 x 10l2 electrons per 1 g. smoke). The aerosol as a whole is considered as electrically neutral. The conclusion drawn, based on their “aging experiments,” was that most of the charge of the tobacco smoke particles seems to be acquired through collision with atmospheric ions. This finding, however, cannot be regarded as substantiated. Another group found free charges (about 1OI2 electrons per cigarette) in cigarette smoke (Westermark and Lindroth, 1961 ; Westermark, 1961). The smoke from the tip of a cigarette contains both small ions (mobility greater than 0.4 cm.2 per second) and large ions (mainly formed by coalescence of smoke particles and small ions). Kingdon (1961) suggests that the ions are presumably generated by the thermal ionization of the potassium in the tobacco. While small ions are found around the glowing tip of a cigarette (concentration dependent upon the electric potential of the smoker), practically no small ions are observed in the smoke stream, especially after passing through a filter tip. The elimination of the small ions in the smoke appears to be caused by the Brownian motion and electrical forces while passing through the cigarette. Appleyard and Jaffe (1962) injected a 35-cc. puff of smoke into an insulated, hollow, conducting electrode, surrounded by grounded shield and connected with a high impedance electrometer. Their results are quite different from those of Holmes e t al. (1959). A 35-cc. puff of popular cigarettes, from five British cigarettes (Bright tobacco) gave mean values between +30 and +22 x 10-l‘ coulomb; from five American

362

ERNEST L. WYNDER AND DIETRICH HOFFMANN

cigarettes (tobacco blends) mean values between +13 and -13 X coulomb, and from two French cigarettes (Gauloise type) mean values of -22 and -24 x 10-l’ coulomb. I n their study, filtration did not significantly change the observed values and cigarette paper had no great effect either. By blending of the tobacco according to the charges, however, the smoke gave almost zero mean charge. All mean values are hased on 200 to 400 measurements. b. Possible Biological Eflects. Large ions are considered of little physiological importance. The condensation of the small ions with large ions is accompanied by the degrading of their electrostatic energy and partial loss of their charges by chemical reactions (Kingdon, 1960). As discussed before, tobacco smoke does not contain appreciable quantities of small ions, but some are found in sidestream smoke, which might be inhaled to some extent. Various groups demonstrated a decreasing of the cilia movement by positive air ions, and decrease in rate of mucous flow and contracture of the posterior tracheal wall. Negative ions, on the other hand, raise the rate of ciliary movement and accelerate mucous flow (Krueger and Smith, 1959; Rivera, 1962; Sleigh, 1962). It appears that negative ions act directly on cytochrome oxidase (Krueger and Smith, 1959). A correlation between the presence of ions in tobacco smoke and the induction of tumors must be considered to be based only on hypotheses (Westermark, 1961 ; Kingdon, 1961 ; Appleyard and Jaffe, 1962).

2. Organic Radicals in Tobacco Smoke

A suggested unifying concept for the mode of action of at least some carcinogenic agents is the intermediary formation and role of radicals. Certain carcinogens are activated metabolically to radicals which are reactive forms of these agents (Oppenheimer et al., 1953, 1955; Syrkin, 1960). Ingram (1961) considers two different theories of electron interaction during carcinogenesis. One is that free radicals, as such, take part in the mechanism of carcinogenesis. The other is that a high mobility of electrons in carcinogenic compounds plays an essential role. With the development of the electron resonance spectrometer (Ingram, 1959) it was possible to detect free radicals present in the human environment and to study the electron mobility of components which play a role in carcinogenesis. The first experimental evidence for the radical theory came from Oppenheimer et al. (1953, 1955). Irradiated plastic films containing free radicals gave higher tumor response when implanted in mice than nonirradiated materials. Free radicals in tobacco smoke were investigated by Lyons et al. (19581, Lyons and Spence (1960), and Ingram (1961). It was calculated

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363

that about 6 X 1015 free electrons are present in 1 g. of cigarette smoke condensate, when collected in traps cooled by liquid oxygen (the condensate contained large amounts of solid carbon dioxide and ice). Marsden and Collins (1963) used a similar method and found free radical concentrations in the smoke of four types of cigarettes between 1.6 and 3.4 X 1015 per gram of condensate. Warming yielded an aqueous phase and an organic or “tarry” phase. No radical concentration could be however, contained about 10“ detected in the aqueous phase. The free electrons per gram. These radicals appear quite stable and longlived. I n contrast to radicals in soot, the majority of the radicals in fresh trapped “tar” were found photosensitive and of low order of stability. Cigarette sidestream smoke was found to contain approxiiiiatcly 5 X lo’* free electrons per gram (Lyons and Spence, 1960). Upon separation of the cigarette “tar” by chromatography on alumina, n-hexane, benzene, and acetone eluates were obtained. No radicals could be detected in the n-hexane fraction, but about 35% in the benzene eluate and 50% in the acetone eluate. Based only on the results of these separations, Lyons and Spence (1960) found an indication “that some free electrons are trapped in individual aromatic structures containing as little as perhaps, four or five condensed nuclei.” Ingram (1961) elaborated more extensively on the occurrence of relatively stable radicals formed by electron trapping in condensed carbon rings. By pyrolyzing all types of organic matter, relatively high concentrations of long-lived radicals are formed. Maximal concentrations of radicals were produced by pyrolysis around 600°C. As the percentage of carbon in the pyrolysis product increases, the concentration of radicals also rises; the maxima was found around 90% carbon. These data suggested that the radicals are polycyclic hydrocarbons with trapped electrons. Ingram (1961) concluded his studies with these words: “All we can claim t o have shown in this work is that there are both active and stable free radicals present in cigarette smoke condensate.” Marsden and Collins (1963) concluded from their studies that the concentration of %table” radicals in cigarette smoke is directly in line with the concentration of a-activity in tobacco used, a suggestion which will be discussed subsequently. Ingram’s more promising conclusion concerning the high mobility of electrons in carcinogenic components is: “Carcinogenically active molecules, such as found in tobacco smoke condensate, contain both active and stabilized free radicals, as well as hydrocarbons, which may form complexes with protein molecules and in this process produce highly mobile electrons, It appears highly probable that either or both may play a very active part in the process of carcinogenesis. ”

364

ERNEST L. WYNDER AND DIETRICH HOFFMANN

Several facts seem to exclude an importance of radicals in tobacco smoke condensate carcinogenesis. Even though sidestream smoke condensate contains only about one tenth the amount of free radicals found in the mainstream smoke condensate, the tumor response of both sidestream and mainstream “tar” on mouse skin is not different statistically (Wynder and Hoffmann, 1 9 6 3 ~ ) .Furthermore, the sidestream smoke condensate contains four times the amount of PAH than does the mainstream smoke condensate. PAH, after all, are the best components known to trap free electrons and form radicals (Kotin and Falk, 1960; Wynder and Hoffmann, 1961a). I n addition, fractions with high polarity, such as acetone eluates, are completely inactive on mouse skin (Wynder and G. Wright, 1957), even though they contain relatively high radical concentrations (Lyons and Spence, 1960). The best known stable radical, apt-diphenyl-P-picrylhydrazylwas found to be inactive in 0.01 molar concentration to the subcutaneous tissue of mice (Boyland and Sargent, 1951). 3. Radioactivity in Tobacco and Tobacco Smoke

a. @-Activity of Tobacco Products. Tobacco products are reported to contain the radioactive elements or unstable isotopes of radium-228, radium-226, and potassium-40. Turner and Radley (1960) determined the presence of Razz&and RaZZ6in tobacco and tobacco smoke. Counting the a-radiation from cigarette ash resulted in constant values after 28 days, by which time the radon-222, caused by the loss during combustion, should have recovered its equilibrium with the radium-226 present in the ash. Based on their observation of constant values after 28 days, the authors concluded that the radioisotopes lead-210 and polonium-210 are not present in raw tobacco a t levels of activity of the same order as radium-226. One thousand and one hundred to 81 picrocwies (pc.) of a-activity were detected in 1OOg. of raw tobacco (material from ten different countries). All a-activity was found to remain in the cigarette ash. It was assumed that the amounts of radon-222 and its short-lived daughters radium A and C from tobacco lost during smoking were inhaled by the smoker. Based on this assumption, one may calculate that a smoker of 50 of the most “active” British cigarettes per day will inhale about 16 pc. radon222. Regarding 0.1 pc. radon per liter as the approximate average value for its concentration in the atmosphere and assuming that a man’s daily air exchange is 20,000 liters, the average daily intake of radon-222 would be about 2000 pc. I n view of this assumption, Turner and Radley calculated that the heavy smoker inhales less than 1% radon-222 from

EXPERIMENTAL TOBACCO CARCINOGENESIS

365

cigarette smoke as compared to the amount he inhales from the atmosphere. Recently these assumptions were strongly challenged by Marsden and Collins (1963). Based on the half-life time of 3.8 days for radon-222, one can calculate that man is not exposed daily to the radon content of 20,000 liters inhaled air, but maximal to that of 5 liters, which is about 0.5 pc. and not, as stated by Turner and Radley (1960), 2000 pc. One can apply the same calculation also to cigarette smoke. However, one should be aware that a higher percentage of radon from cigarette smoke is retained in the smoker’s lung, since radon might be absorbed by the particulate matter of cigarette smoke and the smoker tends to draw smoke deeply into his lungs. Marsden and Collins did not accept the data given by Turner and Radley as experimental proof for the absence of radiolead-210 and polonium-210, as well as other radioactive elements, another point with which one must agree. Recently Radford and Hunt (1964) found a polonium-210 content of 0.39 to 0.40 pc. in four brands of regular-sized U.S. nonfilter cigarettes; up to 25% of the Po-210 was found in the particulate matter of the mainstream smoke. Also, the counted a-radiation from tobacco is considered as too low, since Marsden and Collins stated that they determined in some samples, 16 pc. a-activity per gram of tobacco, a value 50 times higher than the maximum value in the earlier report. It has also been reported that often much higher values for a-activity were found in imported tobacco from New Zealand (experimental data are not given). The authors, furthermore, observed a correlation between a-activity of both stable and unstable radicals. It is hard to see why there ahould be any correlation between the a-particle activity in raw tobacco and the free radical concentration in “tar.” It is well known that pyrolysis within certain temperature ranges of most pure organic materials produces free radicals. The a-particle radiation will produce free radicals in the raw tobacco itself, and a simple calculation shows that for the amount of a-particles actually reported by Marsden and Collins, i t would take hundreds of years to reach the free radical concentration reported to exist in tobacco smoke, assuming that the radicals were stable. b. @-Activity of Tobacco Products. Two groups have studied the ,&radiation from tobacco (Ash, 1959, 1960; Runeckles, 1961). Certain inconsistencies appear in the studies by Ash. For this review article, we prefer to refer to Runeckles’ article only, since we can obtain from it the information needed for an understanding of the importance of P-radiation in experimental tohncco carcinogenesis.

366

ERNEST L. WYNDER AND DIETRICH HOFFMANN

The major and only ,&emitter in tobacco products is potassium-40 (K40). Runeckles found, per one American and one Canadian cigarette, 21.20 and 22.95 pc. ,&activity deriving from K’O. The activity in cigarette smoke was 0.159 and 0.094 pc., this represents a transfer of K4” from cigarette tobacco to smoke of 0.75 and 0.41%. The figure for the American cigarette (0.75%) is somewhat comparable with the Cogbill and Hobbs (1957) figure of K transfer of 0.5%. The concentration of K40 in cigarette smoke is about 2.4 x pc. per ml. to 5.0 X Mayneord (1960) found, as average value for the radioactivity of air, pc. per ml. However, while one might compare the physical data for the radioactivity of tobacco smoke and air, a comparison of these data in physiological studies is not permissible. For example, the major known radioactive element in air is radon-222, mainly an a-emitter with an E,,,. of 5.486 m.e.v., the major radioactive isotope in tobacco and tobacco smoke is K”, mainly a p-emitter with an E,,,. of 1.3 m.e.v., with about 10% of its activity appearing as 1.5 m.e.v. y-radiation. In addition, K40 can be quantitatively measured in the particulate matter of which up to 90% is retained by the smoker upon inhalation. These factors and others, such as resorption and elimination from the resorptive organ, remove any basis for a comparison of the radioactivity in tobacco smoke and air. Too many unwarranted generalizations are presented in the literature in connection with the radioactivity of tobacco and tobacco smoke. I n the biosphere a t large K40 is the major radioactive isotope. However, there are also other radioactive isotopes such as rubidium-87, carbon-14, tritium, and, perhaps, strontium-90. These radioactive isotopes have thus far not been studied in tobacco products. G. T h e Radioactivity of Tobacco and Its Importance in Carcinogenesis. It is apparent that dose-response relationships and threshold levels apply to the tumorigenic activity of radioactive materials (Glucksmann, 1958; Albert et al., 1961; and others). Certainly the different radiations, such as 0- and p-particles, electrons, neutrons, y - and X-irradiation have different effects on various organs. While tobacco carcinogenesis concerns mainly chemical carcinogenesis, nevertheless, one should be careful not to overloook other factors. I n chemical carcinogenesis irradiation can act as a “promoting” agent or as “initiator.” Mottram (1938) reported X-irradiation to increase the tumor response on mouse skin painted with B[a]P (irradiation as promoter), Shubik e t al. (1953) observed that ,&ray emission of ThZo4can be “tumor-initiating” to mouse skin, when application of croton oil follows. The effect of irradiation as “tumor initiator” in carcinogenesis might

EXPERIMENTAL TOBACCO CARCINOGENESIS

367

be more aptly described with the “hot spot” effect of irradiation as discussed by Alexander (1963). The known effects of the various irradiations on ciliated epithelium are covered in a review by Rivera (1962). Radium irradiation gave inconsistent results: X-ray treatment and exposure to light doses did not show any effect on cilia movement (Heine, 1936). I n experimental tobacco carcinogenesis two major studies have been reported on the combined effects of irradiation and tobacco ‘%ar.” Bock and Moore (1959) found a significant increase of tumor response on skin, when mice were irradiated once with 2600 r before receiving a 20% “tar concentrate” (see Section IV, Table 11).The usual high dose of X-irradiation apparently acted as a systemic initiating agent of carcinogenesis. Cowdry et al. (1961) reported a statistically insignificant increase in skin tumors when the backs of mice were concurrently irradiated with relatively high doses of P-radiation from strontium-90 in addition to being painted with 50% tobacco “tar” free of basic portion (see Section IV, Table 11). The percentage of tumors of the lung, leukemia, mammary gland carcinoma, and hepatoma was greater in the groups exposed to irradiation alone. I n a recent article Shabad (1962) discussed the difficulties the biologist encounters when he intends to induce carcinoma of the bronchi in the experimental animal. His own studies, as well as others, demonstrated clearly that a reproducible and high tumor response in the bronchi of animals can be produced only when relatively high doses of carcinogens are embedded for a long time. This general observation also applies to the induction of this type of tumor by radiation, being produced with certainty only when the radioactive elements are implanted in glass beads into the bronchus. On the basis of these results, one would be surprised to obtain tuiiiors in the bronchus of experimental animals by exposure to very low doses of K40, which is known to be very quickly eliminated from the resorptiw organ. I n summary, studies by Bock and Moore (1959), Cowdry et al. (1961) , and Shabad (1962) have demonstrated that the radioactive elements present in tobacco products are of minor, if any, importance in experimental tobacco carcinogenesis.

M. ARSENIC Arsenical compounds, given internally, are considered to contribute to cancer of the skin in man. Occupational exposure of arsenical compounds

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

provided corroborative evidence of the carcinogenic effect of trivalent arsenic on the skin (Neubauer, 1947). The association of arsenical exposure and cancer of the bronchus and lung stems from observations of effects of occupational exposures, particularly involving workers in smelting refineries and vineyards. Its significance, as interpreted by various investigators, has recently been summarized by Buchanan (1962) in a monograph on toxicity of arsenic compounds. Evidence of carcinogenicity has not been conclusive (Hueper, 1942a,b; Neubauer, 1947; Hueper and Payne, 1962). An observation t h a t deserves further study is that orally administered arsenic (13 mg./kg.) led to skin tumors in mice when cutaneous applications of croton oil followed (Van Esch and Van Genderen, 1960).4 When a correlation of cigarette smoking and lung cancer became evident, the investigation of a possible role of arsenic as a tobacco constituent became the subject of several publications, especially by Kennaway's group (Daff and Kennaway, 1950; Daff et al., 1951; Bailey et al., 1957). The presence of arsenic in tobacco was first demonstrated by Remington (1927), later by Satterlee (1956), Weber (1956), Holland et at. (1958b), and Guthrie e t al. (1959). Arsenic (As) as a plant constituent has been shown to originate from soil and insecticides (Guthrie e t al., 1959; Small and McCants, 1962a,b). The parallelism between use of arsenical insecticides and the As content of various tobaccos has been demonstrated in a number of studies (Satterlee and Blodgett, 1944; Satterlee, 1956; Weber, 1956; Holland et al., 1958b). The arsenic content of American cigarettes, according to Holland et al., rose from 7.5-30.0 p.p.m. in 1932/33 to 42.5-52.0 p.p.m. in 1957. From 1939 to 1957 Weber found between 7 and 51 p.p.m. arsenic in cigarette tobaccos in the United Kingdom, and reports a marked decrease since 1953, when alternative insecticides were available and when less American and Canadian leaf was used in the United Kingdom. Pavlu and Sula (1960), as well as Hjern (1961), applied silver diethyl dithiocarbamate as a color-complex-forming reagent for H,As for the determination of trivalent arsenic in tobacco products. (As3+is reduced to H,As.) This method has an experimental deviation of about 2 6 % and thus delivers results comparable to those obtained by the Gutzeit method used by Satterlee (1956), Weber (1956), and Holland et al. (1958b), but is less critical and much simpler.

'Contrary to these results stand the recent experiments of Baroni et al. (1963), who failed to show any carcinogenic, initiating, or promoting activity of arsenic to mouse skin.

EXPERIMENTAL TOBACCO CARCINOGENESIS

369

Swedish cigarette brands are slightly lower in arsenic than American ones. Hjern (1961) found an average for Swedish tobacco blends of 2.3 p.p.m., for Swedish oriental cigarettes 0.8 p.p.m., for Swedish fluecured 2.5 p.p.m., and 3.9 p.p.m. for American blend cigarettes. These values are far below those reported earlier by Satterlee (1956), Weber (1956), and Holland et al. (1958b). A lower use in the United States of arsenical insecticides may be reflected by these values; however, Cogbill and Hobbs (1957) found values closer to those reported by Hjern (1961), who found between 6 and 25 p.p.m. As in American cigarettes, using the molybdenum-blue photometric technique. A common finding by Satterlee (1956), Holland et al. (1958b), and Hjern (1961) is that the As content of oriental cigarettes is far lower than that of United States blend tobacco. Only one fourth to one fifth as much is found in Turkish tobacco. A question of major interest is the transfer of arsenic from cigarettes into the mainstream smoke. Cogbill and Hobbs (1957) investigated this matter in connection with a study of transfer of other elements, and found that the relative transfer amounted to about 4.4%. Holland et al. (1958b) studied the partitioning of arsenic trioxide in a cigarette when smoked: arsenic in a nonfilter American blended cigarette was 45 pg.; 14 pg. in the butt, 15 pg. in the ashes. The amount of volatilized arsenic, calculated by difference, was 16 pg. or about 36% of the total As. Similarly, Alexandrov (1960) found that in ten brands of Bulgarian cigarettes, between 11.4 and 37% of the arsenic was volatilized, between 12.3 and 28.6% remained in the butt, and up to 60% remained in the ashes. Bentley and Berry (1959) concluded from all the published work that 4 to 18% of the arsenic originally present in tobacco enters the mainstream smoke of a cigarette; for cigars and pipes the corresponding figures would be 6 to 12 and 19 to 26% respectively. In evaluating the direct exposure of a smoker to arsenic these figures must, of course, vary with individual smoking habits. Holland et al. (1958a) exposed rabbits in specially designed compartments to cigarette smoke containing Each cigarette was infiltrated with 100 pc. arsenic trioxide and the uptake per rabbit from three cigarettes corresponded to only about 0.01% of the As7* originally present. Although epidemiologically the role of arsenic, as a component of cigarette smoke in the development of lung cancer may be small (Daff et al., 1951) , the problem continues to interest epidemiologists (Buechley, 1963). Experimentally, however, it is certain that the tumorigenic response, a t least of cutaneous tissue in laboratory animals, cannot be attributed to arsenic.

370

ERNEST L. WYNDER AND DIETRICH HOFFMANN

N. METALLIC CONSTITUENTS I n view of the fact that certain metallic compounds, particularly in the vaporized state, appear to play a role in occupational cancer of the respiratory system (Hueper, 1942a; Wynder and Graham, 1951; Goldblatt, 1958a,b; Eckardt, 1959a,b; Rockstroh, 1959; Hueper, 1961 ; Brock, 1962) a brief review of pertinent studies of these compounds is indicated. Of the metallic compounds most extensively studied in occupational cancer, copper, cobalt, chromium, nickel, and beryllium and their oxides and salts appear to be of particular interest. Various metals and metallic compounds elicit neoplasms in guinea pigs, rats, and rabbits when inhaled or given by subcutaneous or intramuscular injection (Hueper, 1958; J. C. Heath, 1956; J. C. Heath et al., 1962; J. C. Heath and Daniel, 1962; Hueper and Payne, 1962; Gilman, 1962; Gilman and Ruckerbauer, 1962). Studies by Haddow (1959) and Haddow and Horning (1960) on iron-dextran and the role of metal chelation are also of interest in this regard. Tobacco, like other plant tissues, contains minerals and other inorganic constituents, deriving from soil or from fertilizers or agricultural sprays. Upon combustion, the metal compounds remain largely in the ashes, unless they become vaporized or transferred into the smoke stream entrained in microfragments of ash. Of main concern in respect to the question of carcinogenicity of metallic smoke constituents is the amount of such compounds appearing in the mainstream smoke. Cogbill and Hobbs (1957) found an average of 515 p.p.m. iron, 180 p.p.m. manganese, between 17 and 36 p.p.m. copper, 30 p.p.m. zinc in tobacco, and 80, 39, and 19 p.p.m. lead (for different tobacco samples). I n the mainstream smoke there appeared 1 p.p.m. iron, less than 0.6 p.p.m. manganese, between 1 and 4.9 p.p.m. copper, up to 2.5 p.p.m. zinc, and 4.3, 2.6, and 1.4 p.p.m. lead, respectively. The average of quantitative data for some metals in tobacco is given in Table XVIII. Voss and Nicol (1960) also found 1.2 p.p.m. cobalt, 0.87 p.p.m. molybdenum, 2.3 p.p.m. vanadium, and 3.4 p.p.m. tin. The unusual finding of titanium was related to soil contamination. Their relatively high copper value is possibly attributable to agricultural sprays. Sunderman and Sunderman (1961) suggest the formation of nickel carbonyl during smoking. They determined a mean content of 1.99 pg. nickel per cigarette for six brands analyzed. Twenty per cent appeared in the mainstream smoke, that is, 140 p.p.b. (0.14 pg.Jcigarette). Recently Pailer and Kuhn (1963) reported values of 100 p.p.b. of nickel in the mainstream smoke of a cigarette (=0.1 pg./cigarette). Even if only

EXPERIMENTAL TOBACCO CARCINOGENESIS

371

partially present as nickel carbonyl, this investigation holds special importance, since nickel carbonyl appears to be the most active form of the metal (Sunderman et al., 1959). Even though, nickel carbonyl may be formed, our knowledge of its decomposition under normal pressure a t 200°C. (Remy, 1956), makes its presence in smoke unlikely. Beryllium and its salts have been shown to produce osteosarcoma in various animals, especially rabbits (Tepper et al., 1961) and pulmonary neoplasms in rats (Vorwald, 1953; Vorwald and Reeves, 1959; Schepers TABLE XVIII OF TOBACCO METALCONSTITUENTS Values (p.p.m.) Metal

Cogbill and Hobbs (1957)

Voss and Nicol (1960)

Fe Mn Ti Zn Pb Ni Cu Cr

515 180 Not determined 30 19-80 1.9 17-36 1 3

765 135 I88 65 5 3 6 2 76.7 Not determined

et al., 1957; Schepers, 1961). The concentration of beryllium in tobacco is quite low, ranging in Bright, Burley, Turkish, and Maryland tobaccos from 0.015 to 0.075 p.p.m. A cased blend of cigarette tobacco contained 0.03 p.p.m. and upon combustion all of the beryllium was accounted for in the cigarette ashes and butts (Williams and Garmon, 1961). The chemical data show the concentrations of metallic elements in tobacco to be appreciable, but their occurrence in smoke is probably not significant, unless they are radioactive. However, since rather low amounts of nickel carbonyl can induce pulmonary tumors in the experimental animal, more detailed analytical and biological work on this subject may be in order.

0. SUMMARY A great many components of tobacco and tobacco smoke have been listed in this section which are considered to possess tumorigenic and cilia-toxic activity. Obviously, great differences exist in their relative activity in experimental tobacco carcinogenesis. Section VII will present our appraisal, from the biological and chemical point of view, of the various roles played by these components.

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

VI. Reduction of Tumorigenic Activity

Even though additional experimental work needs to be carried out to elucidate the roles of the various constituents involved in tobacco carcinogenesis, present evidence leaves no doubt that certain components are involved. A reduction of these specific substances as well as total smoke constituents is now a primary aim of investigative groups (Table XIX). TABLE XIX SOMESUGGESTED MEASURESOF REDUCING TUMORIGENIC ACTIVITYOF TOBACCO SMOKE A. Reduction of Total SmokCCondensate 1. Filtration 2. Tobacco selection 3. Tobacco extraction 4. Additives 5. Amount of (a) Tobacco (b) Homogenized leaf (tobacco sheets) (c) Stems 6. Tobacco cut 7. Porous paper

B. Reduction of Tumorigenic Agents 1. Modification of precursors (a) Process of curing (b) Extraction 2. Selective filtration 3. “Improved combustion” (a) Additives (b) Homogenized leaf (c) Optimum cutjpacking density ratio C. Reduction of Cilia-Toxic Agents 1. Selective filtration 2. Tobacco selection 3. Modification of combustion

A. REDUCTION OF TOTAL SMOKECONDENSATE It is apparent that a reduction of tumorigenic components can be most readily accomplished by reducing the total amount of smoke condensate or tobacco extract to which one is exposed. This has been clearly shown experimentally for smoke condensates by dose-response studies (Wynder e t al., 1957b; Wynder and Hoffmann, 1962a; Bock and Moore, 1962). These studies demonstrated that when 5 g. or less of whole smoke

373

EXPERIMENTAL TOBACCO CARCINOGENESIS 80

70 u

.E .- 60 L

0 n B

.3-

0 c C

z

30

L

u

n

20

2

3

4

5

6

7

8

9

10

Grams of cigarette smoke/mouse/yeor

FIG.25. Relationship of tumor yield and dose (Wynder et al., 1957b).

condensate is applied to mouse skin per year, no skin cancers will develop, and if 3 g. or less is applied, no papillomas are induced (Fig. 25). Recently Bock et aZ. (1962) demonstrated that the concentrates of smoke condensate from a given number of filter cigarettes will produce fewer tumors of the skin of mice than the “tar” concentrate from the same number of nonfilter cigarettes. I n this setting the group of animals receiving refined “tar” from cigarettes with filter tips obviously received a lesser amount of refined “tar.” These results are in accord with general experience in carcinogenesis studies. We msy recall in this context that human epidemiological data, both prospective and retrospective, also indicate an increase in the risk of cancer of the lung or the upper respiratory tract with increase in tobacco consumed. Effective filtration appears to be a practical step which has received wide acceptance. There exists, however, considerable variation in ‘‘tar” and nicotine content among various brands of filter cigarettes (Wynder and Hoffmann, 1960; Waltz and Hausermann, 1963a). As one might expect, considerable differences also exist in the “tar” yield of cigarette smoke as reported from different countries.

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ERNEST L. WYNDER AND DIETRICH HOFFMANN

A reduction of condensate from a given cigarette can be further accomplished by the use of tobaccos which are low in “tar” yield, by the amount of tobacco that is placed in a given cigarette, and also by the use of tobacco sheets (see Section 111) and tobacco stems, both of which are relatively low in yield of (‘tar” and nicotine. The following data demonstrate the influence of these factors. Cigarettes of 85-mm. length made of about the same weight and cut (30 cuts/inch) of tobacco but of different tobacco types yielded the following condensate in milligrams per cigarette (VCTynder and Hoffmann, 1963a): 33.4 for Virginia, 31.5 for Turkish, 25.6 for low-nicotine Burley, 21.2 for Maryland, and 28.8 for American blend. Newsome and Keith (1957) demonstrated for cigarettes with the same pressure drop, a linear correlation between amount of tobacco smoked and moist condensate collected. The use of homogenized tobacco and tobacco stems also can influence “tar” yield. An 85-mm. cigarette made exclusively of homogenized tobacco (tobacco sheets), though weighing 1.35 g. yielded only 16.1 mg. of “tar,” and one made of 100% tobacco stems weighing 1.78 g. yielded 13.0 mg. of “tar” (standard cigarette 28.8 mg.) (Hoffmann and Wynder, 19634. Few data are reported for condensate yield from pipe tobacco. For a pipe smoked under the same conditions, three different pipe tobaccos gave ‘ltar” values of 45.9, 48.6, and 51.3 mg. per gram of tobacco smoked (Miller and Leidl, 1962). I n another experiment 1 g. of popular United States pipe tobacco and 1 g. cigarette tobacco also smoked in a pipe under the same conditions were 45.0 and 29.0 mg., respectively (Hoffmann et aE., 1963). The higher “tar” value for pipe tobacco is probably due to its coarser cut as well as to additives, largely sugars. Some investigations have been directed toward the reduction of cigarette smoke condensate by the use of porous cigarette paper. Schur and Rickards (1957) (Fig. 26) and Lipp and Van Nooy (1962) (Table XX) have demonstrated that porous cigarette paper can contribute to the reduction of “tar” in cigarette smoke. The possible influence of porous paper on the tumorigenic activity of a smoke condensate obtained from cigarettes has also been tested by our group (Wynder and Hoffmann, 1 9 6 3 ~ )Although . there is some “tar” reduction when porous paper is used, neither the B [ a ]P nor the tumorigenic activity for mouse skin was changed when the “tars” were compared on a gram-to-gram basis. Sufficient L‘tars”were available for only 11 months of testing: a t that time, of 50 mice receiving standard “tar,” 12 had papillomas and 2 cancers, as compared to 16 mice with papillomas and no cancer among 50 mice that had received ((tar” from the same type of cigarettes except that it. was wrapped in porous paper. These differences are not significant.

37.5

EXPERIMENTAL TOBACCO C.4RCINOGENESIS

= c

I

3.8

d 3.6 E

Y

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2 3.0 C I n 4 2.81 2.61 c

M.L.P.

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MECHANICALLY

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I N H E R E N T L Y POROUS

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I 10 30 GREINER POROSiTY INDEX Decreasingly Porous ----t I 20

X

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1

I

FIQ.26. Effect of variations in cigarette wrapper on yield of particulates per puff (Schur and Rickards, 1957).

Newsome and Keith (1957) carried out extensive tests to correlate various physical parameters of a cigarette and smoking conditions with “tar” yields. A linear correlation was found between the amount of smoke produced, and the number of puffs, the per cent of a cigarette smoked, and the weight of cigarettes. Increase of smoking time for A POROSITY OF

Cigarette paper C D E F

F in yo of C

TABLE XX CIGARETTE PAPER AND

Porosity (%)c

Average no. of puffs

Nicotine (mg./cigarette)

Condensate (mg./cigarette)

12.2 11.7 11.3 10.7 88

1.06 0.93 0.90 0.78 74

20.6 1s.3 17.7 14.1

3.3 6.5 9.3 24.5 -

-

From Lipp and Van Nooy (1962). 85-mm. filter cigarettes. c Before igniting.

Q

“TAR” YIELUa’a

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376

ERNEST L. WYNDER AND DIETRICH HOFFMANN

given smoke volume, or increase of time interval between puffs for a given number of puffs, did not significantly influence the “tar” yield. However, i t should be recalled that if cigarettes are smoked down to the same butt length, an increase of number of puffs (shorter puff intervals) is correlated with significant increase in [‘tar” yield. This finding has also been well demonstrated by Segelken et al. ( 962) (Fig. 27).

1 1 1 1 1 1 1 1 1 1 1 , 1

2

3

4 5 6 7 Puff number

8

9

1011

FIG. 27. Condensate yield per consecutive puff from a nonfilter cigarette (Segelken et al., 1962).

Differences in the humidity of certain countries and the moisturc of tobacco smoked have been correlated with certain epidemiological findings (Sato et al., 1961). The authors suggest that lung cancer mortality is high in countries where overheating during wintertime conditions cigarettes to dryness and where the smoker is thus exposed to rather dry smoke. By presenting data of the Newsome and Keith study, it is not intended to take any position in this issue, but merely to show experimental results which are of importance in experimental tobacco carcinogenesis (Fig. 28). Finally, extraction of tobacco can also lead to a statistically significant reduction of “tar” yields (Wynder e t al., 1959; Cuzin et al.,

EXPERIMENTAL TOBACCO CARCINOGENESIS

\

-

D-MOUTURE 0 WWlOlTY

WEIGHT WOKE COLLECTED, MCMS./CK;.

FIG.28. Weight of smoke collected as a function of humidity and cigarette moisture (Newsome and Keith, 1957).

1963). When cigarette tobacco was extracted with boiling n-hexane, and made into cigarettes and smoked, i t yielded a smoke condensate of 24.7 mg., as compared with 31.0 mg. for cigarettes made of the unextracted cigarette tobacco (Wynder e t al., 1959). The reduction is most likely due to reduction in “tobacco waxes.” Additives, such as copper (1I)nitrate and nickel(II)acetate, when mixed in 4 or 570 concentration with cigarette tobacco will also significantly decrease “tar” yield. ‘$‘hese data make i t apparent that nonspecific reduction of smoke mmponents can thus be accomplished by a variety of means, some of practical and others only of academic significance. I

B. REDUCTION OF TUMORIGENIC AGENTS Chemical-analytical and biological studies with different tobacco types indicate that the curing process influences the chemical composit e of tobacco (see Section 111) as well as of certain precursors of tum&enic agents. Cigarettes of air-cured (Burley and Maryland} tobeoco not only deliver less condensate compared to flue- and mncuxed (Virginia and Turkish) tobacco, but their yield of certain tumori-

37s

ERNEST L. WTNDER AND DIETRICH HOFFMANN

genic components and the tumorigenic activities of their condensate to mouse skin are also significantly reduced (Fig. 29) (Wynder and Hoffmann, 1963a). For statistical purposes we have grouped together the data obtained with Virginia and Turkish cigarettes and the Burley and Maryland cigarettes. The papilloma yield was significantly different after 12 months ( p < 0.01) ; after 15 months, p is between 0.01 and 0.05. B [ a ]P and phenol have been chosen as first “chemical indicators” for the activity of smoke condensates. They indicate the concentration of carcinogenic PAH as a whole with B [ a ]P and tumor promotion of the phenol group with phenol itself (see Section V I I ) . Although extraction with boiling n-hexane yields a tobacco which upon combustion will yield less “tar,” it will not reduce the tumor yield, nor will it lower the PAH (Wynder et at., 1959). However, extraction with benzene-alcohol produced tobacco which gave a “tar” with a

C =Maryland D = Burley

V A L U S FW I GRAM S M G X CONDtNSAlL ~

11

I3

I5

I?

19

Months

FIG.29. Papilloma production on mouse skin with different cigarette smoke condensates (Wynder and Hoffmann, 1963a).

“statistically insignificant” lower tumor yield, compared to standard “tar” (Nicod, 1961; Uhlmann, 1963). Cuein et al. (1963) have tested the “tar” obtained from cigarettes made of dichlormethane-extracted tobacco by the sebaceous gland test and found no significant difference from tar” obtained from unextracted cigarettes. The values for anthracene, pyrene, and B [ a ] P of both “tars” were comparable. The value for B[a]P is in good agreement with that reported by Waltz and Hausermann

EXPERIMENTAL TOBACCO CARCINOGENESIS

379

(1963b). However, since the isotope dilution method was not applied, the reported PAH values in both studies do not indicate the absolute amounts present in the condensate, but only the amounts isolated even though the results were quite reproducible. When evaluating cigarette smoke condensate obtained from extracted tobaccos one should consider that the extraction may have changed the burning quality of the tobacco and thereby perhaps reverse the otherwise advantageous results of the extraction. Further work in this general area appears indicated. Smoking a tobacco product only two thirds or one half of its length is an obvious method for reducing “tar” and thus tumorigenic agents in the smoke of a given amount of tobacco. The first chemical analytical studies of this factor were reported by Lindsey (1959, 1962). Smoke condensate and B [ a ] P did not increase linearly with the amount of cigarette tobacco burned, but toward the end of the cigarette, “tar” and B [ a ] P increased sharply. Four times as much B [ a ]P was found in the condensate of a cigarette smoked down to 15 mm., compared to a butt length of 35 mm. Kotin and Falk (1960) reported 24% of the total B [ a ] P in the smoke of the first third of a cigarette, 29% in the smoke of the second third, and 47% in the smoke of the last third. Our own results are contrary in showing B [ a ] P concentration in the condensate of the first half of a cigarette of 1.85 p.p.ni. and the second half of 0.85 p.p.m. Considering the experimental deviations for “tar” of .t5% and B [ u ] P of 1-870 these results indicate the same amount of B [ a ] P in the first and second half (Wynder and Hoffmann, 1961a). Recently Ayres e t al. (1963) reported data which are in agreement with these latter findings, These data also find support in biological experiments which showed the condensate of the first half of an 85-mm. cigarette in 50% concentration to produce 40% papilloma and 16% carcinoma on mouse skin (Wynder e t al., 1958), a finding which is statistically not different with “tar” obtained from the second half of a cigarette. A comparison of phenol and condensate data for filter and nonfilter cigarettes is shown in Fig. 30. These values clearly show the efficiency of certain filter materials, especially in phenol removal. While selective filtration of tumor promoters such as phenols thus is possible (Wynder and Hoffmann, 1962b; Hoffmann and Wynder, 1963a; Spears, 1963b; Crouse e t al., 1963), selective filtration for B [ a ] P and other PAH, as well as arsenic(III)oxide, does not appear possible (Holland e t al., 1958b; Wynder and Hoffmann, 1960; Pyriki e t al., 1960). The reduction of phenols may not be large enough or may be “compensated” by other as yet unknown factors. This is apparent from biological studies in which the activities of condensates were compared on a gram-to-gram

380

ERNEST L. WYNDER AND DIETRICH HOFFMANN

basis by testing on mouse skin. A study by Wynder and Mann (1957 1 . recently confirmed by Wynder and Hoffmann (1963c), indicated a similar tumorigenic activity when tobacco smoke condensates from filter and nonfilter cigarettes were tested on a gram-to-gram basis under the same experimental conditions. However, the effects may be different for human inhalation, since the smoker fully benefits from the nonselective as well as the selective filtration. It appears most likely, according to our preliminary studies, that one might also succeed in selectively filtering out certain acids of cigarette smoke; this may already have been accomplished by some present filter cigarettes. However, only well-defined analytical studies can give the final answers. 15

I-

/nan

filter

0

mm of cigarette smoked

FIG.30. Smoke condensate (mg.) and phenol (pg.) as produced by different sections of filter and nonfilter cigarettes (Hoffmann and Wynder, 1963a).

Another approach to reduce specifically tumorigenic components in tobacco smoke lies in attempts to obtain more “complete combustion.” Muth (1955) was one of the first to consider the possibility that high combustion temperatures, in excess of 1000°C., might lead to more complete combustion and thus to a reduction of possible carcinogenic agents. Although the formation of tumorigenic components seems to take place largely when organic matters, particularly tobacco, is pyrolized at temperatures higher than 700°C. (Fig. 19; Wynder e t al., 1958), R

381

EXPERIMENTAL TOBACCO CARCINOGENESIS

lowering of the burning temperatures of tobacco appears a t this time not to be feasible or practical. It seems surprising that so far but few efforts have been directed toward obtaining more information about the degree of combustion of tobacco. Such information may be helpful in experimental tobacco carcinogenesis, a t least as far as it concerns PAH. The CO and CO, values per puff might serve as such indicators. Comparison of our B [ a ] P values for cigarette smoke of four different tobaccos (Wynder and Hoffmann, 1963a) and CO, and CO values for the smoke of cigarettes made exclusively of tobacco type Bright (2.5 ml. CO, and 1.0 ml. CO per puff), Turkish (2.5 ml. CO), and a one-to-one Bright-Burley blend (3.0 ml. CO,, 1.4 ml. CO) (Osborne e t al., 1956; Philippe and Hobbs, 1956) suggests that carbon oxides are a good choice as indicators of completeness of the tobacco combustion. Well-designed chemical-analytical studies in this field are desirable. Studies with tobacco cuts have been limited but may present a promising approach, as also suggested by Muth (1955). The assumption that fine-cut tobacco will burn more completely than coarse-cut. might be negated by the fact that the very fine-cut tobacco must be packed more tightly, thus adversely affecting the completeness of combustion. With an increase of the tobacco cut, a cigarette filled with the same weight of tobacco delivers significantly lower amounts of dry condensate and B [ a ] P (Table XXI). I n order to determine how far these results D R Y C O N D E N S A T E AND

TABLE XXI B[u]P FROM C I G A R E T T E S MADEFROM

D I F F E R E N T COTSarb

Tobacco type (cuts/inch)

Weight (g./cigarette)

Condensate (mg./cigarette)

BbIP (!4./100 cigarettes)

BbIP Gcg./1oog. tobacco)

8 20 30 50 60

1.17 1.12 1.235 1.185 1.20

29.1 27.3 25.4 24.4 23.0

3.7 3.4 3.3 2.3 2.1

3.2 3.0 2.7 2.0 1.7

From Hoffmann and Wynder (1963~). Cigarette length 85 mm.; all cigarettes same tobacco uncased. Smoking conditions: 1 puff per minute; puff duration 2 seconds; puff volume 35 ml.; butt length 23 mm. Experimental deviation: condensate +5%; B[a)P 8% (isotope dilution method). a

b

may affect the tumorigenicity of the various condensates, further chemical and biological studies have been set up in our laboratory. An experimental cigarette of very fine-cut tobacco, 64 cuts/inch, needed about 40% more tobacco than a standard United States cigarette (30 cuts/inch) . The higher tobacco content of many English cigarettes

382

ERNEST L. WTNDER AND DIETRICH HOFFMANN

is in part a result of the fine-cut 50/inch used for these cigarettes. It may be supposed that if the optimum level of cut and packing is obtained, the more efficient manner of combustion would result in a lower amount of PAH. Phenol data may parallel those of PAH. In the hope of affecting the combustion of cigarettes, a variety of additives has been studied. Bentley and Burgan (1960b) added 6 organic and 20 inorganic agents (7 of them ammonium salts) in 0.5 t o 5% concentrations to tobacco. They reported reduction for B [ a ]P from 57-67% for potassium nitrate as additive, 64434% for copper nitrate, 73% for sodium nitrate, 56% for ethylene glycol, and 62% for glycerol (3% zinc as additive). Recent findings from Scherbak et al. (1963) gave an insignificant increase of B [ a ]P in the smoke of cigarettes with tobacco with 3 and 6% glycol as additive. They found for 100 cigarettes 3.35 pg. B[a]P (standard), 3.48 pg. (3% glycerol), and 3.57 pg. (6% glycerol), using the quantitative isotope dilution method. Bentley and Burgan reported standard values from 0.9-1.4 pg. B [ a ] P per 100 g. tobacco (about 120 cigarettes). These figures represent only the amounts isolated but not those actually present in smoke condensate. Similarly, semiquantitative data are reported by Alvord and Cardon (1956), who found an 80% B [ a ] P reduction by a 2770 addition of ammonium sul f amate. The results of large-scale quantitative investigations of our group are summarized in Figs. 31 and 32. The additives were used in 4 to 5% concentration in tobacco (Wynder and Hoffmann, 1961a). The biological results are based on tests of the ‘ltars” on 50 mice each. From these

FIG.31. Benzo[alpyrene values of condensates from cignrrttes with :idditivrs (Wvnder and Hoffmann, 1961a).

EXPERIMENTAL TOBACCO CARCINOGENESIS

383

experiments only the results with calcium carbonate, copper (II)nitrate, and later nickel (11)acetate apprared of special interest. These tests were therefore repeated. Though we failed to reproduce a reduction of the tumor response with the “CaCO, cigarette tar,” the other “tars” gave a significant tumor reduction. Compared to 28% tumors (14% malignant) 60

I-

FIG.32. Per cent papillomas in mice after 15 months’ application of smoke condensates from cigarettes with additives (Wynder and Hoffmann, 1961a).

for the standard “tar” the “copper nitrate tar” gave 3% tumors (1% malignant), representing a statistically significant reduction, especially since each group consisted of 100 mice ( p < 0,001). The result for the “nickel acetate tar” (50 mice) was 12% tumors (6% malignant) ( p < 0.05). I n Fig. 33 the tumor yield and the values for the “indicators” B [a]P and phenol are presented (Wynder and Hoffmann, 1963a). Another additive which has been tested is ammonium sulfamate. Alvord and Cardon (1956) found that a 4.25% addition to cigarettes reduced B [a]P 45-60% in the smoke. Bentley and Burgan (1960b) found B[a]P reduced from 84 to 50% when ammonium sulfamate was added

384

ERNEST L, WYNDER AND DIETRICH HOFFMANN

in 5% concentration to tobacco. However, when added in 4% to cigarette paper these authors found no reduction. These results are in agreement with Cuzin e t al. (1963). Lindsey and his group, however, found a 60% reduction of B [ a ] P in the smoke of a cigarette wrapped in paper containing 4.25% ammonium sulfamate (Lindsey e t al., 1959; Candeli et al., 1960). It appears that ammonium sulfamate reduces B[a]P

l,,r

Gram condensate

IIIIV

j . -

v

I II

Copper nitrate

0

2

4

6

LO

8

12

14

16

18

Montnr

FIG.33. Papilloma production on mouse skin with cigarette smoke condensates (Wynder and Hoffmann, 1963a).

in the smoke when added t o tobacco. Moreover, this effect may have also caused positive results from Lindsey’s group in that in between shipping the cigarettes from Cleveland, Ohio, to London, a significant amount of the ammonium salt of the paper may have sublimated or diffused into the cigarette tobacco. It appears justified to conclude that the addition of certain inorganic salts and oxides to tobacco reduces the tumorigenicity of cigarette smoke. These studies should encourage further experiments in this field, which might lead not only to a better understanding of the formation of certain components from tobacco during burning, but might also lead to a practical cigarette additive. Basic conditions for these studies are the use of quantitative analytical methods and extensive animal tests. We previously reported the effect of porosity of paper on “tar” yield. One recent result indicates that the porosity of paper can affect the B[a]P concentration in cigarette smoke. Ayres e t al. (1963) reported an g. of one standard increase for B [ a ] P in the smoke from 3.7 X

EXPERIMENTAL TOBACCO CARCINOGENESIS

385

cigarette with standard cigarette paper to 8.6 X g. for the smoke of the same cigarette tobacco, but wrapped in nonporous paper. Our own studies comparing “tars” obtained from cigarettes made from highporosity paper with those of standard cigarette paper showed no appreciable difference in B [a]P values analyzed on a gram-to-gram basis (Hoffmann and Wynder, 1 9 6 3 ~ )Some . further studies in this area appear indicated. Attempts have been made to test smoke products other than tobacco. Some years ago a “cigarette” in which tobacco was replaced by different vegetable fibers (mainly corn silk) was on the United States market. The B[a]P concentration in the “tar” was 2.3 p.p.m., compared to 1.15 p.p.m. for the condensate of a nonfilter cigarette. For academic reasons, we also tested “spinach cigarettes.” Here again we found a relatively high B [a] P value, 1.9 p.p.m. (Wynder and Hoffmann, 1961a). The relatively high values might be explained partially by the fact that these “cigarettes” did not have the same burning quality as a tobacco cigarette, which was suggested by the relatively high percentage of coal particles and insoluble matter. The condensate of the spinach cigarette was not only low in “basic portion,” which can be explained by the absence of a specific “spinach alkaloid,” but also in the “weak acidic portion” (phenolic portion) as compared to cigarette smoke condensate. A short-term test for sebaceous gland destruction revealed that both “smoke products” when tested in 50% suspension, had only weak activity. Long-term biological tests with these “vegetable cigarettes” need to be completed before we can give a final evaluation of their tumorigenic properties. I n the preparations of such products one must, of course, overcome the poor burning properties of the vegetable fibers. C . REDUCTION OF CILIA-TOXIC AGENTS

As shown by several investigators there exists for cilia-toxic agents in cigarette smoke a dose response just as with tumorigenic agents. Our studies indicate a lower cilia-toxic response for smoke of an “effective filter” compared to a nonfilter cigarette when tested on clam gills (Wynder et al., 1963a). Use of the frog esophagus (Falk et al., 1961) did not establish significant differences in cilia-toxic response from smoke of filter and nonfilter cigarettes. On the other hand, Dalhamn (1963), using the cat trachea test in vivo, showed a significant difference in cilia-toxic response when comparing the smoke from cigarettes with “effective filter” with the smoke of nonfilter cigarettes. This finding appears to be paralleled by human epidemiological data, which show an improvement in cough when nonfilter cigarette smokers change to “effective filter” ciga-

386

ERNEST L. WYNDER AND DIETRICH HOFFMANN

rettes (Wynder and Hoffmann, 1960; Hammond, 1961). The selective reduction of cilia-toxic components from cigarette smoke is an area of considerable importance. In extensive studies by Hoffmann and Wynder (1962a), which have been confirmed by George and Davies (1962) and Spears (1963b), i t has been shown that cellulose acetate fibers can selectively remove phenolic components; a removal further improved by the addition of plasticizers. Haag e t al. (1959), and more recently Kensler and Battista (1963), have demonstrated that the cilia-toxic agents acrolein, acetaldehyde, and formaldehyde, present in smoke, may be selectively reduced by filtering through activated charcoal. Kensler and Battista demonstrated the efficiency of charcoal filtration in terms of reducing these volatile components by the rabbit trachea test. In attempting to interpret the efficiency of different filters i t is important, as we have stressed previously, that we use a test system that can adequately test the volatile as well as the particulate cilia-static components present in cigarette smoke. Recent work in our laboratories (Wynder and Hoffmann, 1964) has shown that the toxic effect of smoke to clam gill cilia can be decreased even further when the charcoal is compressed, and that drawing smoke through water is most effective in removing cilia-toxic components. This observation is in line with a decrease of aldehydes in the smoke of the respective cigarettes: The smoke of an 85 mm. nonfilter cigarette contains, for example, 10oO pg. acetaldehyde and 70 pg. acrolein ; a comparable tobacco blend in a granulated-charcoal filtertipped cigarette yields 300 pg. and 25 pg. of the aldehydes. A filter tip with compressed charcoal reduces these values to 100 pg. and 17 pg.; drawing the smoke through an excess of water gives 90 pg. and 9 pg. The possible use of components that may strengthen ciliary activity are also of interest in this respect. At present the findings of Falk e t al. (1961) with the parasympathetic agents acetylcholine, eserine, and arecolin seem to be only of academic interest but should be explored further. The possibility should also be tested as to how far the cilia toxicity of certain components may be inhibited; an example for such an effect is the “neutralizing effect of camphor” to phenol. The field of inhibition of cilia-toxic agents thus represents another area that deserves further attention. As was stated in Section IV,F (dealing with cilia-toxic Components), the best animal test systems would appear to be such as were utilized in the studies in vivo by Dalhamn. The in vitro investigations should be primarily regarded as screening techniques. Final proof for the effectiveness of filters must, of course, lie in long-term follow-up studies in man, both in terms of epidemiological studies on the reduction of cough among smokers changing to filter cigarettes, and long-term

EXPERIMENTAL TOBACCO CARCINOGENESIS

387

pathological findings like those of Auerbach to investigate the bronchial epithelium of individuals having smoked such cigarettes. The apparent fact that damaged ciliated epithelium may regenerate is indicated by the study of Ross and Kraus (1961). These investigators treated tracheal epithelium with cigarette smoke condensate, thus leading to metaplasia of the epithelial cells. They observed a reversihility of this metaplasia if no additional smoke condensate was applied to this system. The steps outlined here suggest a number of practical, and at the moment, academic measures which, if vigorously pursued, might well result in tobacco products the smoke of which would have less tumorigenic activity than standard tobacco smoke. VII. Interpretation of Experimental Findings

A. “A COMPLETE CARCINOGEN” Tobacco smoke condensate, from cigarettes, cigars, and pipes, and to a lesser extent also extracts of unburned tobacco, have been established as complete cancerigenic substances for a variety of epithelial tissues as well as for connective tissues of several animal species. Although tobacco smoke condensate must be regarded as a complete carcinogen, since i t produces transplantable carcinomas, we regard its promoting activity to be greater than its initiating activity. This view is based on experiments showing ( a ) promoting activity of tobacco smoke condensate to mouse skin initiated with DMBA or B[a]P; ( b ) on the relatively long time required for papillomas to become carcinomas ; ( c ) on the regression of papillomas if, up to a certain time period, “tar” application is stopped; and (d) on the chemical findings that smoke condensate contains relatively more known tumor promoters than tumor initiators. Chemical studies have attempted to establish which components of tobacco and tobacco smoke are carcinogenic, and which components are only promoters. As is apparent from Section V there are literally dozens of components that when given in a high concentration will produce tumors, often only sarcomas, in experimental animals. Do these components contribute to the formation of epithelial cancer in the concentration in which they are present in tobacco products? We have summarized and critically reviewed in Table X X I I available data in this regard. It is apparent that quantitative values are not always available and that much work remains to be done. On the other hand, the wealth of data that are a t hand have impressively demonstrated, during the last decade, that tobacco products contain many components

EVALUATION OF

THE

TABLE XXIIO RELATIVE ROLEOF TOBACCO SMOKECONSTITUENTS IN EXPERIMENTAL TOBACCO CARCINOGENESIS i.

1. Section

v

B

2. Name of component or group of components PAH

Benzo[a]pyrene Dibenz[u,h]anthracene Dibenzo[a,Z]pyrene Benzo[b]fluoranthene BenzoGlfluoranthene Benzo[e]pyrene Benz[a]anthracene Chrysene Indeno[1,2,3-cd]pyrene Eenzo[c]phenanthrene Methylbenzo[u]pyrene Methylchrysene Dibenzo[a,zlpyrene Dibenzo[a,h]pyrene Total PAH

c,1

TERPENES

trans, trans-Alloocimenc Terpenes of unknowri structure

3. Certainty of presence

+ + + + + + + + + + + + + ? + + +

4. Amount per 100 cigarettes

3.9 pg. 0 . 4 pg. Trace 0.3 pg. 0 . 6 pg. 0 . 3 pg. 0 . 3 pg. 1.5-2.0 pg. 0.1-0.15pg. Trace 0 . 1 px. 1.5-2.0 pg. 0.02 pg. ND NI>

N1) ?

5. Concentration in condensate

(%I

6. Relative imporNature tance in experiof mental tobacco agent carcinogenesis

++ +? + +?

1.15 x 1.0 x
lo-** c

C

TI

+++

N I>

P

P

+

10-5 10-5 10-5 10-5 10-5 10-5

10-4

c c c c c c, I c

C C

C 10-4

c

C C

TI TI TI TI TI TI TI TI TI TI TI TI TI ?

8. Remarks

?

+ +

May act also as inhibitor

? ? ? ? ?

P(?) ?

Promoter with terpenelike structure probably present

PHTHALATES

Di-(2ethylhexyl)phthalate Phthalates

+ +

P(?) ? P(lj :’

ND

ND

CERTAIN ESTERS AND ALCOHOLS

High-molecular esters Esters of long-chain acids with long-chain alcohols Oleic alcohol Long-chain unsaturated alcohols PARAFFINIC HYDROCARBONS

Hentriacontane (12rC31H64) Pentatriacontane (n-CtsH,z) N - and isohydrocarbons from CmHaz-C3aH68

+ + +

ND

ND

P

P?

:’

ND ND

ND ND

P P(?)

P? P?

? ?

0.04 3.5*

R R R

R R

+ ?

ND

C

TI? ? TI? } TI? ?

+ + +

19.4 mg.* -1.0 mg. 85.2 mg.*

+ + +

0.01 pg. 1.0 pg. 0.07 pg.

0.8*

R

Evidence not conclusive

++

HETEROCYCLIC HYDROCARBONS

Dibenz[u,h]acridine Dibenz[u,j]acridine Dibenzo[c,g]carbazole PHENOLS

Phenol 0-Cresol m . p-Cresol . 2,PDimethyl phenol 2,5-Dimethyl phenol Total phenols o- Aminophenols

+

+ + + ++

+I ?

10.0 2.5 5.0 2.0

mg.* mg.* mg.* mg.*

25-30 mg.* ND

For definitions of symbo!s, see key at end of table, page 392.

3 . 0 x 10-5 ND

0.35* 0.09* 0.18* 0.07* -1 .O*

ND

c

C

P P P P P P BC

+

p p p p ?

Substances not tested for tumor-initiating activity

+1

+I + I

+I ?

Phenols also contribute to cilia-toxic activit.y of cigarette smoke

TABLE XXII (Continued)

1. Section

v

F,2

2. Name of component or group of components CARBOXYLIC ACIDS

Lauric acid Oleic acid Linoleic acid Linolenic acid Total long-chain acids G-C2*) Formic acid Acetic acid

G

ALDEHYDES A N D KETONES

Formaldehyde Acrolein

H

+ + + + + + + +

+

4.

Amount per 100 cigarettes

5. Concentration in condensate

(7%)

P

?I

P?

?

w

(0

0

8. Remarks

ND ND ND ND ND

ND ND ND ND ND

P P P? P? P

p

++

30-50 mg. 60-1000 mg.

ND ND

IR IR

IIR R

++ +

3.0-12.0mg. 7.0 mg.*

ND ND

IR IR

IR +?) I R +?

Both aldehydes contribute to the cilia-toxic activity of cigarette smoke

ND

ND

C

?

No conclusiveevidence at

Quantitative analysis and biological tests not done

I

Both acids inhibit cilia movement

STEROIDS

Hydroxyperoxides of Sterols

I

3. Certainty of presence

7. 6. Relative imporNature tance in experiof mental tobacco agent carcinogenesis

?

?

hand

z

EPOXIDES, PEROXY

2:

COMPOUNDS, AND LACTONES

Epoxides and peroxy compounds of unknown nature a-levantenolide 8-Levantenolide

?

+ +

ND -20.0 mg. 2.0 mg.

ND -0.8

0.08

C?

C?

C?

- ? ?

?? )

Isolated from smoke of Turkish tobacco; perhaps weak carcinogens, not tested

ND

Unknown angelica ketones

ND

C?

-

-

Suggested aa carcinogens without any evidence. This group is not likely to contribute to tumorigenic activity of tobacco "tar" in the concentration it is contained therein

J

NITROSAMINES

C C(?) C(?) C(?)

Diethylnitrosamine Nitrosonornicotine Nitrosoanabasine Unknown nitrosamines

K

GASEOUS COMPONENTS

Nitrogen oxide

Hydrogen cyanide

L

+

? ? ? ?

") ?

m e formation of nitrosamines and, if so, their stability in smoke appear doubtfui

M

$z F5Z

2p e

0

r Q

-150-200

?

rg.

+

3.5-115 mg.

+

ND

-

+

Suggested to act in the respiratory system with s e c o n d a r y amines to nitrosamines. Available results make the occurrence unlikely Strong cilia toxic agent

O"

ji z M

8

RADICALS AND RADIOACTIVE COMPONENTS

Radicals

6.0 X 10l6 radicals/g. condensate

?

?

-

Suggested aa possible carcinogen, in disagreement with the experiments

% C-L

TABLE XXII (Continued) 1.

1.

Section V

2. Name of component or group of components

RaZZ6; Rnm and other a-ray emitters KU and other &ray emitters M N

ARSENIC

3. Certainty of presence

+ +

+

4.

Amount per 100 cigarettes

5. Concentration in condensate

(%)

6. Relative imporNature tance in experiof mental tobacco agent carcinogenesis

M

SJ

8. Remarks

Large spectrum of metal aa!ts and oxides

?

+

NU

C

Up to 17.5 pc

ND

C

Up t.o 1.5 mg.

Up t o 0.08

?J

C

TI(?) ?

C

-

C(?)

-

Suggested as carcinogens; however, for experimental studies unimportant

See Section V,N

See Section v,N

Presence in tobacco smoke most unlikely

-

+,

+ +,

3

$

2

_

KEY:Values in columns 4 and 5 are isolated amounts; quantitative values are marked*. Column 3: presence rtssured; ?,presence questionable. Columns 4 and 5: ND, not done; -, not found. Columns 6 and 7: C, carcinogen; TI, tumor initiator; P, tumor promoter; I, tumor inhibitor; R, resorption delaying agent; BC, bladder carcinogen; IR, irritant, leading t o reversible cell changes; sign with ?, activity suggested, not proved. Column 7: f, contributing to activity; important contributor to tumorigenic activity of smoke decisive contributor t o tumorigenir activity of smoke cmdensate. rondensate;

+ ++,

c3

r A

Up t o 3 2 p c

METALLIC CONSTITUENTS

Nickel tetracarbonyl

2rni

x x

EXPERIMENTAL TOBACCO CAHCINOUENESIS

393

that are tumorigenic to some types of animal tissues, even though relatively few of these components are carcinogenic to epithelial tissue. It is furthermore true that none of the agents is carcinogenic in the concentrations in which they are present in tobacco products. Probably, the carcinogenic effects of tobacco products are the result of the combined effectsof different components. A decision on which components are most important can come only after fractionation and testing of the relative tumorigenic activities of these various components. Although no single component in tobacco smoke can by itself or jointly with other components account for the established tumorigenic activity of tobacco products, one cannot deny that these products are tumorigenic. Chemical identification of single carcinogenic components is not necessary to establish biological activity of the mixture. At that, chemical data have gone far to explain the tumorigenic activity of different tobacco smoke products in terms of various agents. It appeared a t the outset that the relatively weak carcinogenic activity of tobacco smoke condensate could be attributed to interactions of the different polynuclear aromatic hydrocarbons, to effects of various components on absorption, and to effects of various tumor promoters and inhibitors. It is therefore not surprising that no single component or group of components could be held solely responsible for the established tumorigenic activity of tobacco smoke condensate. Our present view is that tobacco smoke condensate contains tumor initiators, in the form of a variety of polynuclear and heterocyclic aromatic hydrocarbons, If they were present in large concentrations, they would, of course, be complete carcinogens, In addition, tobacco smoke condensate contains tumor promoters of which the phenols are one major group. Doubtlessly other tumor initiators and promoters in tobacco smoke condensate remain to be identified. Substances such as the paraffinic hydrocarbons may interfere with the absorption of tobacco smoke condensate, certain polynuclear hydrocarbons such as benz [ a ]anthracene may interact competitively with benzo [ a ]pyrene, and possibly phenolic Components may interact with phenol itself. To completely unravel this complex situation seems too time-consuming if not indeed futile. One should rather attempt to establish chemical indicators on the basis of which the tumorigenic activity of a given ‘(tar” could be predicted. I n this manner we regard benzo [ a ]pyrene as an “indicator” of initiating polynuclear aromatic hydrocarbons, and phenol as an “indicator” of tumor-promoting phenolic components. They are not to be regarded, and this needs to be emphasized, as being by themselves responsible for the tumorigenic activity of tobacco smoke condensate. Kotin and Falk (1960) believe that “cigarette smoke serves car-

394

ERNEST L. WYNDER AND DIETRICH HOFFMANN

cinogenesis by its adverse epithelial effect,” with a concomitant impairment of ciliary action and slowing of mucous flow, and that thus carcinogenic particulate matter is more readily retained. We agree that tobacco smoke as a cilia-toxic agent plays a role but, as both laboratory and epidemiological data indicate, that tobacco smoke, as well as tobacco itself, possesses carcinogenic properties. Even though in special circumstances viruses may contribute to the development of pulmonary lesions, there is a t present no experimental evidence that the production of cancer with tobacco products so far accomplished is due to an infectious virus. It is now certain that tobacco smoke contains known tumorigenic agents. Changes in chromosome number occur when human lung cells in tissue culture are exposed to tobacco smoke condensate. Injury to mitochondria has been noted. Interference with cellular respiration associated with such damage may well play a role in the tumorigenic activity of tobacco products. Some day all of these facets may fall into place. Whatever the final cause of cancer may be-biological, chemical, or physical-the fundamental fact remains that a variety of tobacco products are carcinogenic to several experimental animals. It must be our task to reduce this activity. This can be accomplished even though we may not fully comprehend a t this time the nature of neoplastic disease. The truth of this concept is borne out by some of the oldest lessons of preventive medicine.

B. INHALATION STUDIES Epidermoid cancer of the lung has so far not been produced in the experimental animal by inhalation of tobacco smoke. Since it is difficult, if not impossible, to obtain direct (active) inhalation through the mouth in experimental animals, and experiments must therefore be limited to indirect (passive) inhalation, it is unlikely that sufficient unaltered smoke condensate can be brought into contact with the bronchial epithelium to induce neoplastic changes. Tobacco smoke condensate is, after all, a relatively weak carcinogen as shown by laboratory, as well as epidemiological studies. Since, however, tobacco smoke condensate has been shown to be carcinogenic to mouse skin, rabbit skin, mouse cervix, subcutaneous tissue of rats, and, if directly applied, also to dog trachea and hilum of rats, it would appear likely that it would also prove carcinogenic to bronchial epithelium if sufficient amounts could be deposited. C. STATISTICAL CONSIDERATIONS In comparing results of different studies, a plea is made for proper statistical evaluation of the data, in addition to proper standardization

EXPERIMENTAL TOBACCO CARCINOGENESIS

395

of experimental variables. While statistical methodology has played an important role in clinical research, its application to experimental data in tobacco carcinogenesis has been surprisingly limited. Some of the differences reported among various investigators when subjected to appropriate statistical analysis may be attributable to chance alone. A factor limiting the usefulness of statistical procedures when the results of various investigators are being compared is that the conditions of experimentation vary from one laboratory to another. Such studies are likely to involve differences in more than one variable. Even within the same laboratory it is difficult to maintain standard experimental conditions, especially when one is working with such complex mixtures as tobacco smoke condensates. To the extent that experimental details have been reasonably well standardized, it becomes possible to compare results obtained with different smoke condensates and to make meaningful statistical evaluations. It is important also to consider the number of animals used, in appraising the statistical significance of the results obtained. Investigators interested in experimental tobacco carcinogenesis would do well to bear in mind the factors that are involved in establishing statistical significance when planning experiments or analyzing experimental findings.

D. FUTURE S~TDIES Because of experimental difficulties, the tumorigenic activity of tobacco smoke has so far not been studied properly. Since the volatile components of tobacco smoke contain a t least some known tumor promoters, as well as cilia-toxic components, we may predict that the tumorigenic activity, especially in respect to ciliated epithelium, would be greater for the total smoke than for the smoke condensate alone. Experiments with whole tobacco smoke should be encouraged. Because of the prominent role that ciliastasis and subsequent mucus stagnation appear to play in the pathogenesis of bronchiogenic carcinoma, further investigations in this area are indicated. The short-term experiments involving tobacco smoke and ciliary action are an area where practical experiments can well be designed. Long-term studies with tobacco smoke, in attempting to produce neoplastic lesions of the bronchus through indirect inhalation, appear to us not to be a fruitful area of research for reasons discussed. Experiments designed to induce squamous bronchiogenic cancer by direct smoke inhalation appear futile, owing to the practical difficulties, but these may be avoided by applying smoke directly by tracheotomy. Further work on testing tobacco smoke as a possible bladder carcinogen is desirable. A correlation of cigarette smoking to bladder cancer has

396

ERNEST L. WYNDER AND DIETRICH HOFFMANN

been made on the basis of several epidemiological studies, both retrospective and prospective. We wonder whether the oral route may be less effective in absorbing tobacco smoke than the respiratory tract. The possibility that the mouse may not metabolize a procarcinogen as effectively as man, a concept well-learned from studies with p-naphthylamine, must also be taken into consideration. Similarly, studies dealing with tobacco smoke condensate as a carcinogen to the oral cavity should be encouraged, Human experience suggests that nutritional deficiencies tend to increase the susceptibility of the oral mucosa to tobacco carcinogens. Additional studies combining vitamin deficiencies and tobacco “tar” application should be carried out. Studies on determining tumorigenic agents in tobacco extracts are to be encouraged. Such studies may add to our knowledge of such agents in tobacco smoke itself as well as giving information on precursors of tumorigenic components of tobacco smoke. Practical means of reducing tumorigenic and cilia-toxic components have been reviewed in a previous section. These areas deserve the greatest attention of investigators concerned with the field of experimental tobacco carcinogenesis. We are optimistic that considerable advances can be made in addition to those already accomplished.

E. RELATIONTO HUMAN DATA Biological experiments have indicated that certain modifications of tobacco products, the manner of combustion, and the type of filtration used can affect the tumorigenic activity of the resulting smoke condensate. These experiments provide strong impetus for further experimental work on tobacco carcinogenesis. A pertinent question that needs to be asked in this respect is: What is the relationship between these experimental studies and the established correlation of tobacco smoking and cancer in man? A thoughtful answer to this inquiry involves all of cancer research, be i t chemotherapy, immunology, virology, or any of its other areas. It involves, in fact, research in relation to other disease entities, Not all of the substances proved tumorigenic to the experimental animal need necessarily be so in man. This would appear particularly to be true if different types of tissues were involved. Thus, connective tissue tumors in the experimental animal would be less likely to be related to epithelial tissue tumors in man than tumors of animal epithelial tissue. Parenteral injection, a method recommended by Boyland (1958) and Hueper (1963a), has been regarded by the Food Protection Committee (1959) as “not to withstand a critical approval insofar as this recommendation applies to repeated subcutaneous injections.” The committee suggested that if the subcutaneous route is to

EXPERILMENTAL TOBACCO CARCINOGENESIS

397

be taken, i t should consist of a single injection of not more than 100 mg. of the test substance in powdered form. Studies by Nothdurft (19561, Oppenheimer et al. (1959), Alexander et al. (1960), and Truhaut (1963) also support this point of view. Graffi and Bielka (1959) have also stressed the importance of tissue specificity of carcinogens. We do not want to delve into this important issue concerning experimental carcinogenesis in this review except to stress that we have limited our own studies to epithelial tissue, for the reasons just discussed, because this is the type of tissue that is primarily involved in the correlation of tobacco use and human cancer. The association of cigarette smoking to lung cancer has been regarded as causative by a number of public health authorities that have reviewed the evidence, as emphasized most recently by the Royal College of Physicians of London (1962) as well as by the United States Surgeon Generals Report on Smoking and Health (U.S. Health Service Publ. N o . 1103, 1964). Epidemiological data have also demonstrated correlations of tobacco smoking to cancer of the mouth, larynx, and bladder. A correlation of tobacco chewing to cancer of the oral cavity has also been shown by several epidemiological studies. The epidemiological and laboratory studies may be thus regarded as mutually supportive. On this basis, if a given modification of a tobacco product would lead to a reduction of its tumorigenic activity to laboratory animals, we would recommend that such modifications, insofar as they are practical, and do not have any other toxic side effects, be accepted. The final proof, whether man would similarly benefit, would demand a long-term follow-up study of man himself. It is along these lines that further efforts in the field of tobacco carcinogenesis need to be directed. VIII. Postscript

Students of preventive medicine and public health authorities who are convinced that tobacco habits contribute to the development of certain types of human neoplasms may well propose that public education on this issue should be extended so as to discourage smoking. It appears, however, that efforts toward “modified” smoking products might prove to be a more promising immediate measure that could benefit the greatest number of people. Such an approach does not negate efforts in the field of education, which might parallel expanded efforts in the field of modification of tobacco products. Wherever practical measures can be realized, they should be put into practice. Sope progress, such as in the areas of effective, as well as selective, filtration, has already been made. Since complete cessation of the human smoking habit appears not to

TABLE I1 A SELECTIVE SUMMARY OF EXPERIMENTS. PART1: EPIDERMAL CANCER Smoking and collection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco product used (B) A

Shotadze (1953)

B Tobacco from Lagodekh dist., Georgia

A Passey et al. (1954) B Cig.

A

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. (C)

Stoneware container A with burning tobacco connected via glass tube B to glass f l a k connected C to water suction pump. Dense portion of “tar” deposited in glass tube; liquid emulsion flowed into flask B Not given; inferred to be <350”C.

( a ) 80 white mice (b) 20 white mice Not given 11 mo.

D

E F

A 5 dfl. mouse strains Automatic smoking I 50 mice machine, smoking cig. I1 51 mice a t approx. rate of man’s habit (no details given) B Not given B Up to750”C. C Lifespan

D

A

~

A Gwynn (1954) B Cis.

Type of material applied (D). Solvent (E), Application prodecure (F)

E F ~

Reaults

Tobacco “tar” applied None used (a) Painting of lower lip with glass rod 3 X weekly for 11 mo. (b) Painting of tobacco “tar” on skin of backs for 4 mo.

(a) No neoplastic changes (b) No neoplastic changes

I Whole “tar” I1 Neutral fraction (18 g. from

No malignant tumor observed in I or 11‘ One small papilloma in group I. but regressed 3 wk. later

10,OOO cis. ) Not given Twice weekly painting of the back (not shaved) ~

~

~

~

A Extn. of butts of A Mice (no. not given) D 1 Whole ext. No tumors by any of these methods naturally smoked cig. B Not given 2 Whole ext. and croton oil C Test 1: 246 days 3 Whole ext. and DMBA by methanol (40% of dry butt) in Soxhlet for Tests 2 and 3: 161 days E Methanol 1 hr. F Weekly appln. 5% methanol soln. to B Not given backs of mice I Ext. from butts of Virginian cigarettes I1 Ext. from butts of Rhodesian cigarettes 1 Weekly appin. 5% soln. in MeOH 2 Weekly appln. of soln. (Mon.) and croton oil (Thurs.) 3 Weekly appln. of soln. after init. treatment with DMBA

~

~~

A Kakhiani (1955) A Pipelike app. B 1 Trapesund of B 500-600”C. Lagodekh dist., Georgia 2 Samsun of Ahhaziya dist.. Georgia 3 Trapesund of Adshar dist., Georgia A Wynder B Cig.

et al.

(1955)

A Wynder et aZ. (1953) B 884 f30”C.

A C

a 0

A

App. specifically deskned for study B Not given

W

A Sugiura (1956) B Cig.

A

B

Wynder et d. (1953) 880°C.

D

1 yr.

E F

1 Brown liquid 2 Thick brown “tar” 3 Both, 1 : l None used Rubbing of “tar” on skin 290 times

No neoplastic changes in any group

D Whole cond. E Acetone (50% s o h ) F 3 X weekly painting with 40 mg. “tar” of shaved back (I and 11). 3 X weekly painting with “tar” and once weekly with 5% croton oil (111 and IV)

I 22 papillomas, 12 carcinomas I1 10 papillomas, 2 carcinomas 111 4 papillomas, 1 carcinoma IV 4 Papillomas, 1 carcinoma

1 201 Swiss mice 2 Hamsters; no. and strain not given B 10-12 wk. C 80days

D

No tumors 1 (a) Histologically, epidermis on smoked side of ear shows epithelial byperphis (b) Vitamin %complex deficiency and 38 smoke appln.: Ulceration with epithelial hyperplasia (c) Riboflavin deficiency and 40 smoke appln.: Marked degree of keratinisation, epidermal hyperplasia. acanthosis (d) Pyridoxine deficiency and 36 smoke appln.: Hyperplastic epithelium, moderate degree keratinization 2 Vitamin B-complex-defioient hamsters show no cutaneous tissue changes after receiving twice as many smoke appln. as did mice

A 60 8,Rookland Swiss albino mice B 35 days C 2 yr.

D Whole cond. E Acetone

A

I 86 Swiss mice

I1 89 C57BL mice B C

A Kreshover (1955) B Cig.

Mongrel white mice

B 2-3 mo.

111 13 Swiss mice IV 24 C57BL mice 8-12 wk. More than 2 yr.

A

Whole tob. smoke

E None F Appln. whole tob. emoke t o posterosuperior region (I%.ear) for 5 sec. (comprising smoke flow of 17.5 cc./ see.) for 5 exposures

F

16 papillomas, 12 carcinomas

Approx. 80 mg. “tar” applied with brush 3 X weekly to sbaved backs of mice (Continued1

TABLE I1 (Continued) Smoking and collection technique (A). Reported combustion temp. (B)

Reference (A) and Tobacco product used (B)

A R

Hamer and Woodhouse (1956) Cig.

rp-

8A

Wynder et al. (1956) B Cig.

A Gwynii and Salaman (1956)

A

All-glass app., 20 cig. A 140 outbred albino smoked concurrently: strain mice, 3 rabbits every 25 sec. 1 puff. (Dutch breed’ (13-2 sec. duration). B Not given Smoke led through C 18 mo. acetone/dry-ice cooled, and acetone-filled traps a t room temp. 30-35 g. “tar”/1000 cia. R Peak temp. during puff. 7FO”C. a t center, 640°C a t outer rim of cia.

A Wynder rt al.

Cis. smoked on 2 automatic machines under conditions closely resembling those of “normal smoking.” “Tar”colln. Pyst. not described B Not given

A

Wynder and G. Wright A

B Cig.

(1953):

A

Cia.

n

Type of material applied (D), Solvent (E), Application procedure (F) D

E F

Wyndar et ul (1!)5ti) 884 f 30°C

A

Acetone Twice weekly dropping of 0.3 ml. of ZOYo soh. on unshaved backs (mice); rabbits received 0.5 ml.

No. with No. animals papillomas . -

“Tar” (mice) ‘q-ap(rabbits) * ‘ T ~ and ~ ” croton oil (mice) B[a]P for 1 wk., then “tar” (mice) B[alP for 1 wk. (mice)

50 38 30

0 2

30

4

30

0

1

* 5 sites per rabbit. D

E F

A 40-5 strain mice B Not given C Not given

D E F

A

D

New Zealand white rabbits (I-I\‘ and VI-IX. 3 rabbits; V, 2 rabbits) B Not given

Results

(1) Whole tar ( 2 ) 0.3Y0 w/v B[ulP (3) 0.3% w/v croton oil in acetone

-

I 40 Swiss mice but machine smokes 100 I1 50 CAFi mice cig. at a time; uses a B 12 wk. %see. puff every 18 sec. C 2 yr B 884 f30”C.

B

(1957)

Strain and animal used (A), Age of animal at onset of expt. (B), Duration of expt. (C)

Whole cond. Acetone 3 X weekly, about 65 mg./painting of back

I 45% papillomas, 35% carcinomas I1 46% papillomas, 12% carcinomas

Whole “tar” Acetone Painting of back 1-2 X weekly; 0.06 ml. “tar” dild. 3-5 X with acetone “Tar“ alone “Tar” after 1 appln. 0.2 ml. 0.15% DMBA “Tar” with intervening appln. 0.3 ml. 0.17Y0 croton oil in acetone

No tumors appeared in mice of ether group treated with “tar” alone A number of tumors appeared in mice t r e a t d with “tar” and croton oil; not greater than that compared to incidence from crotori oil alone In group treated with “tar” and DMBA, 2 cancers appeared on 2 mice in 25th wk.

I Whole coiid. I1 Methylene chloride, insol. portion I11 Acidic neutral portion I V Nicotine-free basic neutral portion

+

+

I 50 papillomas after 9 mo. I1 No tumor I11 -50papillomas after 12 mo. I V 50 papillomas after 8 mo. V 1 papilloma after 0 mo.

C

Until 50 papillomas were observed in 3 rabbits or up t o 15 mo.

V VI VII VIII

Nicotine-free basic portion Acidic portion Neutral portion Hexanc fraction of neutral portion IX Carbon tetrachloride frz-tio.. of neutral portion E Acetone: I-VII, 50Tosoln.; VIII and IX, 10% soh. F 5 X weekly painting of nape of neck

V I 5 papillomas after 15 mo.

VII 50 papillomas after 11 mo. V I I I 50 papillomas after 13 mo. IX 26 papillomas after 5 mo.

~~

A

Grahtriu rt al. (1957b) A B

B Cig.

Wynder zt al. (lY53) 835°C.

A

54 albino New Zaaland

B rabbits C

9-12 wk. To72mo.

D

E F

Whole Cond. I 41 papdomas, 5 carcinomas Acetone I1 4 1 papillomas. 2 carcinomas 5 X weekly painting both ears I11 No tumors I 41 animals: eig. “tar” (50% soh.) I1 10 animals: cig. “tar” once weekly, croton oil (5Y0) in mineral oii 111 3 animals: acetone once weekly croton oil

+

+

I&

A Wynder and Mann A Wynder el al. (1956) (1957) B 884 3 ~ 3 0 ° C . B Regular- and kingsized filter cig. compared t o regular- and king-sized nonfiltcr cig. ; various U.S. brands URI: unfiltered reg. UKz: unfiltered king UK3: unfiltered king FRI: filtered reg. FRP: same as FRL FR2: filtered regular FKa: filtered king FKI: filtered king

A

CAFi (Jackson) and Swiss (Millerton) mice; 20 to 50 animals in each group B 6-8 wk. C Appln. for time of survival of animals

D Whole cond. E Acetone F Painting of 50% soln. to shaved backs of mice 3 X weekly

Mice with skin tumors Cig.

No.

URL

40 Swiss

URi UP;% UKz UK3 FRI FRi FRl* FRI~ FRe FR2 FKa Fl‘& FK4 FKI

50CAFi 30 Swiss 30CAFi 50 Swiss 20 Swiss 25 CAFi 40 Swiss 40 CAFi 20 Swiss 25CAFi 30 Swiss 30 CAFi 40 Swiss 40 CAFl

Papilloma Cawinom:r

(% )

(% )

45 46 40 53 28 30 60 40 37 60 48 33 53 43

35

.50

-

12

8 27 14 25 20 30 8 25 4 20 13 28 5 (Continued)

TABLE I1 (Continued)

Reference (A) and Tobacco product used (B)

Smoking and collection technique (A), Reported combustion temp. (B)

A Wynder et a2. (1957b) A Wynder et a2. (1956) B Cig. B 884 f30’C.

A Wynder et al. (1957s) A Wynder et al. (1956) B 884 rt3O”C. B Burley, Maryland, Turkish, and Virginia tob. cig.

Strain and animal used (A). Age of animal at onset of expt. (B), Duration of expt. (C)

Type of material applied (D), Solvent (El, Application procedure (F)

A

530 Swiss (Millerton) D E mice B Not given F C Groups 1-6, 11, and 12: lifespan Group 7, 3 mo. Group 8, 6 mo. Group 9, 9 mo. Group 10, 12 mo.

A

CAFi (Jackson) and Swiss (Millerton) mice (4 groups of each strain with 40 animals each) B 6-8 wks. C Appln. throughout Pfeapan

Whole cond. Acetone Painting of back (1) 5 X weekly, “tar”-acetone soh. 1 : l (50%) (2) 3 X weekly, “tar”-acetone soln. 1 :1 (3) 2 X weekly, “tar”-acetone soh. 1 : 1 (4) 1 X weekly, “tar”-acetone Soh. 1:1 (5) 3 X weekly for alternating 2 wk. periods (6) 3 X weekly for alternating 4 wk. periods (7-11) 3 X weekly (12) 3 X weekly “tar”-acetone 1:2 (33%)

D Basic-free portions of “tar” E Acetone F

Painting 50% “tar”-acetone soln. 3 X weekly to shaved backs

Results

% Mice with Group

No. Mice

Papilloma Carcinoma 12 38 10 6 5 15 0 6 34 58 45 42

8 16 3 0 0 0 0 0 12 30 35 12

% Mice with Tar (hasic-free)

NO.

Burley Burley Maryland Maryland Turkish Turkish Virginia Virginia

40 Swiss

mice

40 CAFl 40 Swim 40 CAFl 40 Swiss 40 CAFl 40 Swiss 40 CAFl

Papilloma Carcinoma 43 55

70 33 65 53 43 55

8 28 18 10 18 10

10 35

A Engelbreth-Holm and A Absorption of smoke by A Ahlmann (1957) cotton wool, 2 puffs/ B Blended tob. (Danish) min., 1 sec. duration B (YO!. not described). C Cond. extd. with acetone a t 20°C. and 56-57°C. B Not described

Mice St/Eh Strain D Acetone-sol. material of smoke cond. (59 0 and 55 3) E Acetone Not given F 2 X weekly painting of 50% soln. 13 mo. for most, up t o on backs 23 months for remaining

A

Gubrin and Cuzin (1957) B 6 types of cig.

A Mice (392 3,392 0 ) A Automatic smoking divided into 7 groups machine, 30-1111. puff, of 112 each; Inst. 1.5 sec. duration a t Pasteur strain 30-540.intervals. Smoke B 3mo. trapped in glass beadC Over 1 yr. filled column, then in boiling acetone trap. Solid particles are captured by condn. of acetone-laden smoke particulate phase at -25°C. B Not given

D Whole cond. E Acetone F Painting about 0.14.25 ml acetone soln. t o neck of mice 2 X weekly

A Passey (1957) B I English cig. I1 American cig.

A See Passey et al. (1954) A B Upto750T.

D Whole “tar” (I and 11) E Acetone F 3 X weekly painting of 50% soln. t o shaved back

A Wynder and Wright (1957) B King-sized cig. and cig. tob. for pipe

A A Automatic machine smoking 150 cig. a t one time. 35 ml. average puff; butt lgth. 25 f 5 mm., puff duration 2 sec. B 884 + 30°C. (cig.) 750”-950”C. (cig. tob. for pipe)

b b

W 0

Swiss mice (from U.S.A.) I 50 mice I1 50 mice B Not given C About 2yr.

I 30 CAFI, 30 Swiss mice I1 40 CAFI, 40 Swiss mice 111 30 CAFI, 30 Swiss mice IV 25 CAFI. 25 Swiss mice V 30 CAFI. 30 Swiss mice VI 30 CAFI, 30 Swiss mice VII 30 CAFI. 30

D

Papillpornas in 13 of 38 animals after 6-7 mo. of treatment. I n 6 of 13, papillomas became carcinomas after 16-23 mo. of treatment. An episootic of ectromelia killed more than 50% of animals during first 12 mo. of expt. Group I. Caporal: 47 survivors. 1 epithelioma (20t mo.) 2 papillomas (14 mo.) 1 sarcoma (15+ mo.) Group 11. S.O. France: 20 survivors. 2 papillomas (121 and 24 mo.) Group 111. Algier: 34 survivors. 1 papilloma (154 mo.) 1 epithelioma (151 mo.) Group IV. Bone: 37 survivors. 1 papilloma (131 mo.) 2 epitheliomas (18 mo.) Group V. Orient: 42 survivors. 1 papilloma (15 mo.) 1 epithelioma (16f mo.) Group VI. Camel: 40 survivors. 3 papillomas (17, IS+, 23 mo.) Group VII. Acetone: no tumors I 3 papillomas, 2 epitheliomas

I1 6 papillomas, 3 epitheliomas, 1 sarcoma

I Whole cond. C AFI mice I1 Nicotine-free cond. 111 Nicotine-free recombined PapilCarciIV Methylene chloride insol. loma noma V Methylene chloride insol., Group (%) (%) H20 sol. VI Acid neutral portion I 53 27 VII Nicotine-free basic neutral I1 73 25 portion In 76 30 VIII Acidic portion IV v IX Nicotine-free basic portion X Neutral portion VI 47 13 XI Hexane eluate of neutral portion VII 50 17

+

+

Swiss mice Papilloma

Carcinoma

(%)

(70)

53 43 33 8

10 20 13 4

-

-

50 43

23 37 (Continued)

TABLE I1 (Continued!

Reference (A) and Tobacco product used (B) Wynder and Wright (1957) continued:

Smoking and collection technique (A), Reported combustion temp. (B)

Strain and animal used (A), Age of animal at onset of expt. (B), Duretion of expt. ( C )

Type of material applied (D). Solvent (El, Application procedure (F)

XI1 CClr eluate of neutral portion Swiss mice XI11 Benzene eluate of neutral VIII 30 CAFI, 30 portion Swiss mice XIV Ethylacetate eluate of neutral 1X 30 CAFI, 30 portion Swiss mice XV Methanol eluate of neutral X 30 CAP,, 30 portion Swiss mice XVI Pyridine eluate of neutral XI 10 CAFI mice portion XI1 10 CABl mice XVII Neutral portion of cig. smoke X l I I 10 CAP1 mice cond. XIV 10 CAFI mice XVIII Neutral podion of pipe cond. XV 10 CAFI mice XIX Cig. tob.; methanol ext. XVI 10 CAFl mice XVII 30 CAFI mice, E Acetone 30 Swiss mice F 3 x weekly painting of mice (shaved back) XVIII 30 CAFl mice. 5 x weekly painting of rabbits 30 Swiss mice (shaved back) XlX 40 CAFI mice, 40 Swiss mice 111, IV, VI, VII, VIII. IX. x, XI, XII: 3 albino New Zealand rabbits each B Mice: 6 wk. Rabbits: 2-3 mo. C Mice: lifespan Rabbits: until 50 papillomas appeared but not longer than 15 mo.

Results CAP, mice Papilloma

Carcinoma

Swiis mice Papil- Carciloma noma

Group

(5%)

(%)

(%I

(%)

VIII IX X XI XI1 XI11 XIV XV XVI XVII XVIII XIX

37 0 30

7 7 3

20 20

0 0 33

20

-

100

63

80

-

-

-

-

30 60

20

63 63

28

0

8

3

33 50 3

Group

No. papillomas (rabbits)

I11

50 (9mo.) 0 (15 mo.) 50 (1'2 mo.) 50 (8 mo. I 5 (15 mo.) 1 (9 mo.) 50 (11 mo.) 50 (13 mo ) 26 (5 mo.)

IV VI VII VIII IX X XI XI1

A Graham et al. (1957a) A Wyndcr et a!. (1956) B 884 5 3 0 ° C . B Cig.

A

B

Kreshover and Salley (1957) Cig.

A B

Not given Not given

A 74 CAFI mice I 34 mice not further painted I1 33 mice painted for another 12 mo. B 8-10 wk. C I 12 mo. I1 24 mo. A

B

C

A Gellhorn (1958) R Cig.

(a) CAFl and Swiss mice 75 animals (b) Golden and albino hamsters (a) 8 wk. (b) Not given (a) 76 wk. (b) 48 wk.

A All-glass smoking A Smiss mice (B[a]P and machine with alc./dry“tar” expt.); Paria RIII mice (all other ice traps; 11 puffs of 75 ml./min. expt.) B 500-700”F. (255370°C.) B 6 wk. C 35-1CQwk.

D

E F

D

E F

D

E F

Whole cond. Acetone 3 X weekly painting 50% s o h . to shaved back

(1) Whole tob. smoke (2) BIaIP in acetone None used (1) Daily appln. whole tob. smoke t o lips and ears (2) 3 X/wk. painting of palatal mucosa and ears (3) Daily appln. whole tob. smoke t o cheek pouch (4) 3 X/wk. painting with B[u]P t o cheek pouch

I 12 papillomas. 8 carcinomas

I1 18 papillomss. 14 carcinomas

(a) (1) Vitamin B-deficient CAFl mice treatei with smoke showad csllular abnormalitis suggestive of precancerous changes or carcinoma in situ. Swiss strain less affected. (b) (1) No oral changes; ears: hyperkeratosis and hyperplasia (h) (2) 24 wk. treatmsnt: no oral tissue changes but ears showed ulceration, thickening, and occasional papillomatous formation (b) (3) Infection and edema after 8 mo. (b) (4) Epidermoid carcinoma after 4 mo.

Whole cond. Benzene or acetone 5-6 X/wk. appln. of ‘tar” and croton oil to intencapular reaion; 200 pg. B[u]P as 1%soln. applied for 2 succesdve days at start of expt. BblP B[u]P “Tar“ “Tar”

No. mice 529 581 595 croton oil 200

+ “tar”

+

No. mice with papilloma

No. mice with carcinoma

20 61

5 35

3 10

2 0 (Continued)

TABLE I1 (Continued) Smoking and collection technique (A). Reported combustion temp. (B)

Reference (A) and Tobacco product used (B) A Moore and Miller (1958) B Cia.

A Orris et d. (1958) B (a) Popular U.S. cig. blend (NYU) (b) Popular US. cig. blend (SKI)

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. (C)

Manifold smoking A I 80 golden hamsters machine, 3 puffs/min.. I1 13 golden hamsters duration 2 sec., vol. B Av. 3 mo. 30 cc., butt lgth. 10 mm. C Varying per group Colln. syst. not described: 35 cig. gave about 1 g. “tar” B Av. 700°C.

A

A

B

Type of material applied (D), Solvent (E), Application proeedurr, (F)

D Whole “tar” None I Cotton and gauze wads (1 cm. diam. impregnated with 440 mg. “tar”) secured with single suture into extremity of rt. pouch. Wads often extruded and had t o be replaced about 8 X for animal living 2 yr. 11 Hamster pouches painted 3 X weekly with “tar” for up to 30 mo.

E F

(a) Manifold smoking A Swiss Millerton Q mice A Whole cond. (a) NYU machine, 1 puff/ in groups of 50 animals B 8 wk. (b) SKI min., 2 sec. duration, 35-40 ml. vol.; C 24 mo. B Acetone cold traps C 3 X weekly painting of shaved backs of mice with 50’% soln. (b) Manifold smoking machine 3 puffs/ min., vol. 35 ml. (a) Not given (b) 884 f30”C.

A Croninger et ~ l (1958) . A Cigarettes (Wynder A B Cigar, pipe, cig. and et al. 1953). Cigars smoked with 50-ml. puff all-tob. cis. “tar.” Cig. 3 X/min., 4 sec. dura(Wynder et al., 1953); tion (smoking time av. B 26 min.) C All-tob. cig. smoked under previously described conditions (Wynder et al., 1953); they burned 161 min./cig.

Originally only CAFi mice used; all expt. repeated using Swiss Millerton Q mice 6 wk. At least 19 mo.

Results

Type of treatment Plain cotton wad B[a]P cotton wad “Tar” cotton wad Control, untreated left pouches “Tar”-painted pouches

Pouches showing Duration histological (mo.) change 2-25 5-23 1-30

4 (17%) 9 (75%) 32 (600/0)

22-27

1 (llo/o)

11-16

6 (46%)

(a) 12% papillomas, 8% carcinomas (b) 18% papillomas, 18% carcinomas

Mice with

D

I Cig. smoke cond. (fresh)) I1 Cis. smoke cond. (old acetone soln.) I11 All-tobacco cig. smoke cond. GroJp I V Cigar smoke cond. (nicotine-free) V Pipe smoke cond. (nicotine-free) I VI Acetone E Acetone I1 F Soln. applied as whole “tar” in 2 diln. I11 IV (1:l and 1:2), 3 X weekly to shaved backs of mice; 1: 1 soln. applied with V glass rod to 1.5 cm.2 area, (60 mg./ VI

No.

86 92 54 78 89 23

Papilloma

Carcinoma

swiss 47 53 35 33 30

37 36 26 19 16

0

0

painting) 1 :2 soln. applied to 2 X 2 cm. area. 25 mg./painting

Pipe: 30-ml. puff every 10 sec.; duration 1 sec. "Tar" colln.: cis. and cigar smoke were collected in solvent; pipe smoke in a seriea of cold traps without solvent Old "tar" stored in acetone B Cigars: 910°C. C i . : 884 &30°C. Pipes: 776 f30"C. All-tob. cia.: 650-1025°C. A Wynder et al. (1958) B Hot hexane ext. cig.

@b

rl 0

Hot hexane ext. 2200 A 1 30 Swiss mice/group 2 3 New Zealsnd rabking-sized cig. pyrolyeed bit /group in nitrogen and for comparison pyrolyeed in air B Mice: 6-8 wk. Rabbits: 2-3 mo. B In nitrogen: (a) 880; C 12-13 month8 (b) 800; (c) 720; (d) 640; (e) 580°C. In air: (f) 880°C.

A

D

E

F

1 Pyrolyeates (a-f) applied in concn. of 5 and 1% to the backs of mice; pyrolyeate (a)also applied in 0.1 and O.Ol%o concn. 2 Pyrolyeates (a-e) applied in 5% concn. to rabbit ears Acetone 1 3 X weekly skin painting soln. to shaved backs of mice 2 5 X u-eekly appln. soln. to inner surface of rabbit ears

I I1 111 IV V VI

88 87 55 90 86 24

CAFi 52 48

43 44 29 10 13

53 50

43 0

0

Mice with tumors 5% concn.

Pyrolyzate (a)

(b) (C)

(d) (e) (f)

Papilloma (%)

1 Yo concn.

Carcinoma

Papilloma

Carcinoma

(%)

(%)

(%)

80 77 93 67 33 3 3 0 0 0 83 67 0.1% concn.

97 77 97 43 0 7 0 0 0 0 97 67 0.01% concn. 0

0

Effect of 5% concn. on rabbit ears

No. rabbits Total no. with papilloma Pyrolyzate papilloma (after 12 mo.) (a ) 3 41 3 29 (after 9 mo.) (b) (C ) 1 5 (d) 2 2 (el 1 2

TABLE I1 (Continued) Smoking and collection technique (A). Repcrted combustion temp. (B)

Reference (A) and Tobacco product used (B) Wynder et at. (1958) B U.S. cig.: King-sized filtered (KSF) King-sized unfiltered (KSUF) Reg.-sized unfiltered (RSUF) A

A

* 0 m

Strain and animal used (A). Age of animal a t onset of expt. (B), Curation of expt. (C)

A For smoking machine B and colln. of cond. see C Wynder et a?. (1956) (1) High-vol. puff (60 ml.); total 15 puffs/ cig. t o smoke 70% of cig. (2)Low-vol. puff (11 ml.) total 28 puffs/ cig. to smoke 70y0 of cig. (3) (a) KSF smoked to halfway mark (b) KSF smoked to butt (23 mm.) (4) (a) KSUF, halfway (b) KSUF, butt ( 5 ) (a) RSUF. halfway (b) RSUF, but,t

30 Swiss mice/group 6-8wk. Lifespan

Type of material applied (D), Solvent (E), Application procedure (F)

D Smoke cond. E F

Soln. made u p so that material is compared on g.-g. basis: concn.: 50% Acetone Skin painting shaved backs of mice; frequency of appln. and total dose not given (Author’s note: 3 X weekly)

Bock and Moore (1959)

13 Cig.

Manifold smoking ma- A chine. One 35-ml. puff/ min., duration 2 see., butt Igth. 23 mm., about 20 mg. cond./cig. 1% Not given

A

Effect cig. smoke cond. obtained by (1) High-vol. puff (2)Low-vol. puff 28 Papilloma 24 Papilloma (%) (%) Carcinoma 14 Carcinoma 8

(%)

(%) Papilloma

Carcinoma

(%)

(%)

36 38 40 30 30 35

16 18 16 16

(3)(a) (b)

(4)(a) (b) (5)(a) !b)

~

A

Results

Swiss 0 mice for groups D I-XI. C3H a* mice for groups XII-XI11 E Group A F I 30 mice shaved on alt. Mon. prior to painting with soln. A I1 30 controls shaved on alt. Mon. prior t o painting with acetme 111 30 mice shaved

Soln. A: Whole cond. Soln. B: Conc. whole cond. Acetone Groups I, 11, 111, V, VI, VII. and VIII painted 10 X a wk. Group I V : 5 X a wk. Groups IX-XI11 painted 5 X a wk. with 0.25 ml. of soln. B Groups X-XI11 irradiated HVI, 0.1 mm. Al (1M) Kv. unfiltered) and a dose rate of 780 r/min. a t distance of 20 cm. Single dose of 2500 r delivered when animals were 55 days old

16 26

Mice with skin cancer (carcinomas or papillomas)

~ ~ _ _ _ _ _ _ _ _ _ Group

I

111 IV V VII

No.

%

No.

%

1 1 2 7 5

3 3

6 5

7 23 17

8 10 9

20 17 27 33 30

IY V

VI VII VIII

PX

l a l Fri. ea. mo., but not painted until following Mon. with soln. A 30 mice shaved when nec.; painted with soln. A 30 mice Fubbed with sandpaper 3 X weekly prior to painting with soh. A 10controls treated like V but painted with acetone 30 mice never shaved; painted with soln. A 10 control mice never shaved; painted with acetone Group B 50 noriirradiated Swiss 0 mice painted with soln.

B X 65 irradiated Swiss 0 mice painted with soln. B XI 39 irradiated Swiss 0 mice painted with acetone XI1 45 irradiated C3H 67 painted with sohi. B XI11 19 irradiated C3H 8 ;painted m7aith acetone €3 Not given C Lifespan

Mice with skin tumors after 64 wk. Group

No.

u/O.

IX

13 44

27

X XI XI1 XtII

0 12 0

68

29

-

TABLE I1 (Continued)

Reference (A) and Tobacco product used (B)

Smoking and collection technique (A). Reported combustion temp. (B) ~~~

. A Wynder et al. (1959) A Wynder et ~ l (1956) B Treated and untreated B 884 f 30°C. U.S. cig. tob. compared t o hot hexaneextd. and cold hexaneextd. tob. King-siae (KS) cig. and reg.size (RS) cis.

Strain and animal used (A), Age of animal at onset of expt. (B). Duration of expt. (C)

Type of material applied (D), Solvent (E), Application procedure (F)

Results

~

A 6 groups Swim 2' mice, D (total: 370) B 6-8 wk E C Appln. of material throughout lifespan F

2 c

Grorip 1-111: methylene Chloride-sol. portion of cond. Group IV-VI: whole cond. Acetone Painting of 50% soln. 3 X weekly t o shaved backs I Cond. from KS cis. made from untreated tob. I1 Cond. from KS cig. extd. with cold-hexane before smoking I11 Cond. from KS cig. extd. with hot hexane before smoking IV Cond. from RS cig. made from unextd. cased tob. V Cond. from RS cig. made from unextd. uncased tob. VI Cond. from RS cie. made from cased tob. extd. with hot hexane

% ' mice with ~

Group I I1 I11 IV

5

No.

Papilloma

Carcinoma

0 50

28 20 14 30 14 40

14 10

50

100

v 5 0

VI

70

10 22

0

27

I

Wynder and Hoffmann A See Wynder et al. (1956)A Swiss (Millerton) '2 (1959a) 85 mrn. Am. nonfiltered mice B Cig. tob. (U.S. blend) cis. smoked in autoB 6-8wk. matic smoking machine; C Throughout lifespan 1 puff /min. duration 2 sec., vol. 35 ml., butt lgth. 35 mm./cig. about 30 mg. particulate ma& ter colld. in cold traps B See W.ynder e l al. (195fi)

A

D

E F

Whole cond. compared to CCb-eluate of neutral fraction and compared t o pyrolyzate of hot bexane-extd. tob. Acetone 3 X weekly painting of (a) 50% acetone soln. smoke cond. (b) 10% acetone soln. CCkeluate of neutral portion ( c ) 0.01% acetone soln. of pyrolyzate of hot bexane-extd. tob. to backs of mice

% mice with No. (a ) (b) (c)

100 12 20

Papilloma Carcinoma

30 100 85

22 100 70

A Koerbler et al. (1959) B Pipe tab. (untreated)

A B

Roe et a2. (1959) Cig.

e

Pipe smoked by man B Not given A

20 albino mice, strain not given; 248 control mice (untreated) B Not given C >13 ma.

A

D

Residue formed in pipe smoked by man E Human saliva F Daily appln. behind ears (dosage not given); 10 with saliva and smoke cond., 10 with smoke cond.

Automatic smoking ma- A 0 and 8 mice of “101” D inbred strain chine, 4 puffs/min. 15I 30 8 and 32 0 ml. vol. puff duration 2 mice aec.. butt Igth. 20 mm. I1 30 8 and 33 0 Colld. in cold traps B Not given mice 111 3 and 0 mice (a) 30; (b) 30; (c) 50 E F B Not given C I and 11. 40 wk.; 111, 37 wk. A

c

I Whole smoke cond. I1 Neutral portion 111 Smoke “phenol fraction” (a) DMBA smoke “phenol fraction“ (b) Smoke “phenol fraction” (c) DMBA Acetone 3 X weekly painting of shaved back I 2 5 3 3 % (each appln. 25-40 ms.) I1 2 5 3 3 % (2540mg.) 111 12.525% (6-12 mg.) (a) 0.2 ml. 0.15% DMBA in ace12.525% smoke tone “phenol fraction” (b) 12.525% smoke “phenol fraction” (0) 0.2 ml. 0.15% DMBA in acetone

+

No skin cancers, but subsequent to licking by animal of painted areas, 1 mouse with scirrhous carcinoma of lower jaw after 10 ma., 1 mouse with planocallular carcinoma of lower jaw with metastask to lung after 134 mo. Controls neg. I 2 mice each, with 1 papilloma, no carcinoma I1 1 mouse with 1 papilloma 111 (a) 15 mice with papilloma, 1 with carcinoma (b) no mice with papilloma or carcinoma (c) 4 mice with papilloma, no carcinoma

+

A B

Reddyetal. (1960) Indian cigars

Cigars continuously smoked; smoke led through acetone B Not given

A

D Whole cigar “tar” (a) E None (h) Foundation Lab. strain F Mice and rats painted on shaved back (I. 11. 111). 31 albino (e) every 2nd day rats m i t a r strain) (a) Tob. tar B Mice about 12 wk. (b) Tob. tar exposure t o heat (d) (58°C.) for 3 min. C Mice: 4 ma. (e) (c) Heat exposure only Rats: 7 ma. (d) Acetone (e) Control

A

48 Swiss mice. 3 groups of 14 eaeh Rockefeller

+

No neoplastic change 6 0 and 5 8 mice with malignant changes Tendency t o ulceration with healing signs No abnormal changes No abnormal changes

(Continued)

TABLE I1 (Continued)

Reference (A) and Tobacco product used (B) Gritsiute and Mironova (19EO) B (a) Russian cig. (Belomorkanal) (b) Russian cigars (Aurora) (c) Shag whiff tab.

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. (C)

Smoking and collection technique (A), Reported combus t ion temp. (B)

Type of material applied (D), Solvent (El, Application procedure (F)

Cig. and cigars smoked A lP3 ”high-cancer” mice D by automatic machine (line A ) 227 “low-cancer” mice using cold traps for (line D ) colln. of cond. Toh. 12 rabbits “tar” extd. (method not described) B Mice: 2 ma. Rabbits: not given E Pyrolyzate of tab. C Mice painted through- F prepd. by destructive out 10 ma. distn. after Roffo Rabbits treated for 6 B Not given mo.

A

A

A B

Muir and Kirk (19t0) A Singapore betel/tob. quid.

(a) Smoke cond. Belomorkanal cig. (h) Smoke cond. Aurora cigan ( c ) Pyrolyzate tob. (d) Extd. from smoke conden. (a) (e) Extd. from smoke conden. (h) (f) Ext. from shag whiff tab. “tar” No solvent; undild. “tar” or ext. used Mice: 3 X weekly appln. of soln. with glass r o d to posterior third of back. Total dose: 1.4-2.6 g. “tar” Rabbits: treated daily or alt. days by painting of inner surface of both ears. Total 120 paintings (4.8-7.2 g. “tar”)

6 ts

B

Water ext. of quid: to 5.5 g. betel/toh. mimt. (ground with mortar and pestle) 2 ml. Hx0 added; resultant dark red mass squeezed by fingers. Fluid so obtained (28°C.).used to paint ears of mice

Swiss wnite mice I 12 I1 41 B Not given C I 22-24 ma. I1 2 yr., (still nnder ohs. )

A

u E F

Water ext. No addtl. soh. used Painting of the water-ext. on ears of mice

Results (a) 2 mice (of 94) of line A with tumors a t site of treatment: 1 mouse (of 84) with tumor a t site of treatment (h) Line A: no tumors at site of treatment (73 mice) Line D: no tumors a t site of treatment (75 mice) ( c ) Line A: no tumor a t site of treatment (16 mice) Line D: 1 tumor a t site of treatment (68 mice) (d) Line A: no tumors a t site of treatment (10 mice) Line D : no tumors at site of treatment (40 mice) ( e ) Line A: no tumors at site of treatment (13 mice) Line D: 1 tumor at site of treatment (37 mice) ( f ) Line A: no tumors a t site of treatment (16 mice) Line D: 1 tumor a t site of trestment (68 mice) Of 12 rabbits treated with pyrolyzate 5 developed multiple papillomas on the ears; these -~ papillomas persisted only in 2 rabbits

I 2 squamous cell carcinomas and 2 squamous papillomas I1 1 papilloma. 30 of 41 mice still under obs.

A

R

Procedure simulates av. A 153 C57BL mice. 68 0, D nat. smoking (no de89 ~3 tails). &step absorbing B 3-6 wk. C Lifespan syst.; methanol as absorbing solvent B Not given

Clemo and Miller (1960) Cig.

A

Roe (1960)

A

5 w

A 13

Cie.

Automatic: smoking ma- A Stock albino mice chine, 4 puffs/min.. 15- B Not given ml. vol. 2 sec. duration, C Not given butt Igth. 20 mm.; cond. colld. in cold traps B Not given

(a) Fraction C derived from r i t j smoke (air pollutants) applied as 1% soln. in benzene (b) Croton oil 0.5% soln. in acetone ( c ) Neutral fraction of cig. smoke condens. 10% soln. in benzene E Benzene resp. acetone F Group ( 1 ) : 21 mice painted 3 X weekly for 2 wk. only with fraction C. Group (2):21 mice painted with fraction C. After 3 wk. interval neutral fraction smoke cond. 3 X weekly till death. Group (3): 24 mice painted with fraction C. After 3-wk. interval, painted with croton oil 2 X weekly till death. Group ( 4 ) :22 mice painted 3 X weekly for 2 wk. only with neutral fraction. Group ( 6 ) :21 mice painted as group (4). After 3-wk. interval. painted with fraction C 3 X weekly till death. Group (6): 25 mice painted as group (4) after 3-wk. interval, painted with croton oil 2 X weekly till death. Group (7): 23 mice painted with croton oil 2 X weekly till death

D

E F

Whole cond. and neutral portion Acetone Init.: 225 pg. DMBA; later (a) whole cond.. 3 X waekly (40 mr.) (b) neutral portion, 3 X weekly

Mdignant skin tumor7

(a) Skin tumors developed after 22 wk. (b) Greater no. skin tumors than expected by addition of carcinogenic effects of DMB.4 and neutral fraction

(Continued)

TABLE I1 (Continued)

Reference (A) and Tobacco product used (B) A w Cowdry et al. (1961) B Cis.

Smoking and collection technique (A), Reported combustion temp. (B)

Strain and animal used (A). Age of animal at onset of expt. (B), Duration of expt. (C)

A See Wynder et al. (1956) A 397 0 Swiss mice B Not given B 2mo. C Painted for 12 mo.. obsd. foi lifespan

Type of material applied (D), Solvent (E), Application procedure (F)

Results

D Cond. free of basic portion, appl. as 50% soln. E Acetone F Painting of shaved backs of mice. 200 roentgen (a) “Tar” 3 X weekly equivalent physical (r.e.p.), 2 X weekly (b) “Tar” 3 X weekly 1000 r.e.p., 1 X monthly kc) “Tar” 3 X weekly (d) 200 r.e.p.. 2 X weekly (e) 1000 r.e.p., 1 X monthly (f) Acetone 3 X weekly

+

No. of mice

G~~~~

Mice killed or dead after 6 mo. (no.)

Mice with skin cancer

1%)

-

+

76 74

(a) (b) ( c) (d ) (e)

51 76 73 47

(f)

75

61 56 43 12

73 49 73 62 44

0 7

“Tar” and @-radiation gave additive not synergistic effect in the production of epidermal carcinomas Per cent of tumors of lung, leukemia, mammary gland carcinoma, and hepatoma was greater in groups in which percentage of malignant tumors of skin was low Wynder and Hoffmann A Wynder and Hoffmann A Swiss (Millerton) 0 (1961a) (1959a) cig. smoked by mice, 50,’group B Cig.. exptl. cis. machine: B 6wk. I Drawing 3 puffs,’ C 15 mo. min. I1 Drawing 1 puff/min. B 884°C. f30

A

D E F

Whole cond. Acetone 3 X weekly painting of shaved back with 50% soln. cond. of cig. I (a) Aluminum oxide trihydrate (b) Cobalt(II1)oxide (0) Boric acid (d) Aluminum silicate (4%) (e) Calcium carbonate (f) Magnesium oxide (9) Copper(1I)nitrate (h) Whole cond. (control) I1 (a) Calcium carbonate (b) Copper (11)nitrate ( c ) Whole cond. (control)

yo Alice with Additive

+:

I

papillomas

(a) (b)

58 44 42 23 37

(C )

(d 1 (e)

28 28

(f)

(g) (h )

~

-~

44

% Mice with

% Mice with

papillomas

cancers

(a) 18 (b) 6

2

44

14

~~

I1

(P)

2

_

_

D A Wynder and Hoffmann A Automatic smoking ma- A Swiss (Millerton) 0 mice, 30/group; (1961b) chine; 1 puff/min.. duration 2 mu., vol. 35 B 6wk. B 85 mm. Am. non3 mo. filtered cig. ml., butt lgth. 23 mm.; C 12 mo. appln., obsn. about 30 mg. particuE late matter/& colF lected in cold traps B 884 f30"C.

+

75 fig. DMBA Phenol: 5 and 10% soln. Cis. smoke cond. 50% soln.; phenolic fraction: 5, 10, and 25% soln.; B[u]P: 0.005% soln.; acidic fraction: 10% soln. Acetone (1) 75 fig. DMBA appld. once (2) Phenol 5% appld. 3 X weekly (3) Phenol 10% appld. 2 X weekly (4) Phenol 10% appld. 3 X weekly (5) Cig. smoke condens. 50%, 2 X weekly (6) 75 Fg. DMBA once only followed by cig. smoke cond. 50% 2 X weekly (7) Cig. smoke cond. 50% 3 X weekly (8)75 ~ g DMBA . once only followed by cig. smoke cond. 50% 3 X weekly (9) Phenolic fraction 10% 3 X weekly (10) 75 fig. DMBA once only followed by phenolic fraction, 10% 3 X weekly (11) Phenolic fraction 2570 3 X weekly (12) 75 fig. DMBA once only followed by phenolic fraction 25% 3 X weekly (13) B[u]P 0.005% 3 X weekly (14) B[alP 0.005% 3 X weekly phenolic fraction 5% 2 X weekly (15) B[ulP 0.005% 3 X weekly phenolic fraction 10% 2 X weekly (16) B[alP 0.005% 3 X weekly 4- acidic fraction 10% 2 X weekly

+ +

% Mice with Group (1I (2 ) (3) (4 )

(5) (6) (7 ) (8) (9)

(10) (11) (12) (13) (14) (15) (16)

Papillomas

Carcinomas

10

-

-

7 3 10 43 44

63

30 7 53 70 93 97 93

7

3

3 27 22 37

-

3 68 77 93

80

TABLE I1 (Continued)

Reference ( A ) and Tobacco product used ( B ) A B

Gudrin (1961) Cig.

Smoking and col!ection technique (A ), Repoited combustion temp. ( B ) A See Gu6rin and Cuiiii (1957) B Not given

Strain and animal used (A), Age of aiiimal at onset of expt. (B), Duration of expt. (C) A 4 groups of 60 mice Swiss strain, (40 0 , 4067) B About 3 mo. C Not given

Type of material applied (D), Solvent 03). Application procedure (F)

D

E F

Whole cond. Condensate and B[a]P Condensate and croton oil Acetone B[a]P: 0.25 ml. of 1% soln. (1 ) Applied to mouse back with pipette once weekly for 1 mo. (10 mg. total dose) (2)B [ ~ ] P “tar” from 2 cig. painted once weekly for 4 a k . , 2 X weekly for next 4 wk., 3 X weekly thereafter (3) “Tar” from 2 Lip. painted once weekly for 4 wk., 2 X weekly for next 4 wk.. 3 X weekly thereafter (4) “Tar” from 2 cis. painted once croton oil weekly for 4 wk. 0.25 mi. soln. on back 2 X weekly for 4 wk., 3 X weekly thereafter

+

Results

Group (1 1

No. survivors 63 69 79

(2) (3) (4)

78*

% Mice with skin tumors 22 57.9 20.2 3.8

* High

mortality during first 3 ma. Only 50% survivors a t 6 ma.: nine lived longer than 1 yr.

+

A Svoboda (1961) B Cig.

A Automatic smoking ma- A B chine, 1 puff/min.. 2 C 8ec. duration, 22 ml. vol.. butt Igtb. 22 Irm. Smoke led through cold traps (-30°C.) B Not given

Swiss 0 mice 10-12 wk. 12 ma. painting, 3 mo. ObEervation after

D E

F

Cond. free of basic and water-sol. compd. Benzene ( a ) 100 mice painted with 50% tob. “tar” s o h 3 X weekly 40 mg. per applic. (b) 100 mice painted with tob. “tar” soln. 4-mg. pellet of 25% stilbestrol in cholesterol implanted S.C.

+

% ’ Mice with Group

Paoillomas

Carcinomas

(a 1 (b

16

9 0

~~

>

4

Wynder and Hoffmann A See Wynder and Hoffmann (1961a) (1962a) B 884 f 30°C. B Cig. A

200 Swiss (Jlillerton) 0 D mice. E B About 6 wk. F C 15 mo., 3 mo. further observation.

A

Whole smoke cond. Acetone Painting of shaved back 3 X weekly I 33% soh. I1 25Y0 soh. 111 10% soln. 1V 5% s o h

07, ,” Mire ----

Group

Papillomas

Homburger et ul.

R

(1963) (1) Cig. made from

A B

No details given Peak temp. (1) 867°C. (2) 889°C.

cigar tob. (2) Cig. made from

(3)880°C.

500 3 and 500 0 CAFl D Whole cond. E Acetone mice B 8-12 wk. F Whole cond., 50% soin., backs painted C More than 2 yr. 2 X weekly. Total dose durinn 120 wk., 8 g.

____ 34

20

11 I11

18 6 0

8

A +

r;

Bock et al. (1962)

B 4 brands 70 mm. US.

A B

cig.. groups (a-d) 2 brands filter-tipped 80 mm. U.S. cig., groups (e-g)

Roe Cig.

(1962)

-

croup (1) (2)

(3) Manifold-type smoking A ICR Swiss 0 mice; 6 D “Refined tar”: heptanesol. material groups with 30 each: machine 35-ml. puff, from heptane/acetone: water partition duration 2 scc., fre2 control groups with of cond. 66 mice each E Acetone quency 1 min. Colln. B 38-45days cond. in glass syst. F Painting 0.25 ml. “refined tar” noln. cooled by alc.-dry ice. C 1 yr.: surviving animals 10 X weekly to shaved backs of sacrificed at that time Colln. of 1st puff mice. Dose/mouse during first 2 wk.: avoided “tar” equiv. 8.3 smoked cig./day, then B Not given “tar” equiv. of 6.6 smoked c i d d a y throughout study

A

A Cig. smoked in autoA Albino mice, 5 groups matic smoking machine, each with 20 0 and 20 no details given cp B Not given B Not given C 84wk.

(a) 40 mg. smoke cond. with 0.025 m g . B[u]P (b) 40 mg. smoke cond. with 0.06 mg. B[u]P ( c ) 40 mg. smoke cond. with 0.25 mg. B[u]P (d) 40 mg. smoke cond. with 1.25 mg. WalP (e) 1.25 mg. B[a]P soln. E Acetone F Painting of shaved back 3 X weekly

0

0

yo of Mice with skin tumors

A

pipe tob. (3) Commercial cig. A l l cis. 70 mm.

Carcinomas

I IV

A

with

Group

_ I _

8

0

64 62 58

55 57 56

% Mice with skin cancer -

(a) (b)

25

5 33

(C)

(d) (e) (f)

23

(9)

-

7 3

D

Group

NO. survivors

Yo Mice with skin tumors

TABLE I1 (Continued) ~~

Smoking and collection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco product used (B)

Strain and animal used (A). Age of animal a t onset of expt. (B), Duration of expt. (C)

C. Moore and A See C. Moore and Miller A 20 hamsters (10 golden Christopherson (1962) (1958) and 10 albino) B Cig. B Av.700’C. B 34mo. C Up t o 7 2 6 days

A

Nicod (1961) A Not given B Cig. tob.: B Not given J Treated Oriental 0 Nontreated Oriental A Treated Am. N Nontreated Am. A

A White 0 mice E strain B 141 days C 750days

Type of material applied (D), Solvent (E), Application procedure (F) D Whole cond. E None F Painting of shaved skin of back ant. t o tail; 40 mg. cond./appln. D E F

“Tar” A1c.-benzene mixt. 1:2 2 X weekly painting of shaved back of neck

Results No neoplastic changes

NO. G r n n =---naintings --l_r

J 0 A N

b

Do

NO. animals

206 206 198 186

21 17 34

39

yo Skin cancers 19.0 41.1 11.7 23.0 ~

A Neukomm (1962) B Am. cig.

A

A Xensler (1962) B Cig. (a-f), cigars (g)

A Mechanical smoking

B

Notgiven Not given

machine. N o detaila given B Notreported

A 39 white mice E strain B 34mo. C Not given

D E F

A 4 groups of CAFI hybrid mice (>lo0 mice/ group) 3 groups of Swiss mice (>lo0mice/

D Whole smoke cond. E Acetone

group)

B Not given C Lifetime

Whole “tar” Benzene 2 paintings weekly on neck over 2 om.* surface (30 mg. “tar”/appln.

23% cancers after latency period of 55 wk.

Group

76 Papilloma

% Carcinoma

CAFI mice 41 46 53 Sa iss mice 34 40 42

14 17 25

33 28 41

TABLE I1 A

Smoking and collection technique (A). Reported combustion temp. (B)

Reference (A) and Tobacco product used (B)

A Gritsiute and Mironova (1960) B (a) Russian cig. (Belomorkanal) (b) Russian cigars (Aurora) (c) Shag whiff tob.

SELECTIVE SUMM.4RY O F EXPERIMENTS. P A R T

-4 Cig. and cigars smoked by automatic machine using cold traps for cond. colln. Tob. "tar" extd. (method not described) Pyrolyzate tob. prepd. by destructive distn. after RoEo B Not given

Strain and animal used (A), Age of animal at onset of expt. (B), Duration of expt. (C)

A

2:

SUBCUTANEOUS S K I N CANCER

Type of material applied (D). Solvent (E). Application procedure (F) (a) Smoke cond. Belomorkanal cig. (b) Smoke cond. Aurora cigars (c) Pyrolyzate shag whiff tob. (d) Ext. cond. (a) (e) Ext. oond. (b) ( f ) Ext. tob. pyrolymte E No solvent: undil. "tar" or ext. used F Injection 40 mg. undild. "tar" once every 7-10 days beneath skin rt. flank (same spot each time); total 50 injections = 2 g. "tar"

(a) 25 rats: no tumors ( b ) 26 rats: 1 sarcoma (c) 25 rats: no tumor a t site of trestment (d) 28 rats: no tumor a t site of treatment (e) 35 rats: no tum3r at gite of treatment (f) 12 rats: 2 tumws a t site of treatment

D

(a)

White rats (of nonD deacript breed) B Not given (wt. 100-120 B. )

C

1 yr.

Results

~

A Druckrey et al. (1960) A B (a) Commercial cig. (b) Special cig. W

Automatic smoking ma- A chine with electrhstatic pptn. (2 g. cond. from 100 cig.) Colln. cond. B for biol. expt. in cold C traps

Rats, BD-inbred strain (a) 75 (b) 74 Not given 60 wk.

E F

Denicotinized cond. 70% alc., 30% tricaprylin 50 mg. once weekly injected S.C. (3.2g. total dose)

~~

20% malignant tumors 6 )18% malignant tumors

~~

Seelkopf et al. (1963) A Automatic smoking ma- A Rats, B D T strain B Cig.: blend of Virginia chine: 603 g. "tar" from B 2 mo. (av. wt. 137 9.) and Orient tob.. 8900 cig. C >2yr. commercial B Not given A

* Parenthetical value represents total

dose/rat.

D

Distn. residue of neutral (a) portion (b) Distillate neutral portion (b.p. 70-150°C.,3 mm. Hg) (c) Distillate of neutral portion (b.p. 15&18o"C., 3 mm. Hg) (d) Acidic portion (e) Nicotine-free basic portion (f) Nicotine-free weak basic portion E (a-c) Olive oil (d-f) Propylene glycol F* (a) 31 67 (920 mg.); 33 0 (730mg.) (b) 25 67 (990mg.); 16 0 (795mg.) (c) 25 67 (510mg.); 16 0 (435mg.) (d) 17 67 (920mg.); 24 0 (750mg.) (e) 20 67 (350mg.); 20 0 (272mg.) (f) 17 d (230mg.); 15 0 (197mg.) Divided doses given t o each rat S.C.

Sarcomas [some fibromss e~pecially in ( c ) ]

(%)

(a) 46 (b) 15 (0) 19 (d) 10 (e) 2 (f) None No tumors among controls injected with olive oil and propylene glycol

(Continued)

TABLE I1 A SELECTIVE SUMMARY OF EXPERIMENTS. Part 3 : ORALCAVITY(BLADDER) Smoking and cdlection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco product used (B)

Strain and animal used (A), Age of animal a t on-et of expt. (B), Duration of expt. ( C )

Total "tar" 10% of treated animals with papillary carNone cinoma of bladder; 87.5% of mice develDally swabbing of lips and oral cavity oped benign papillomatosis of urinary bladder, 6 converted to malignant stage. of mice (about 0.03 g./appln.) No changes in oral cavity

(a) CAFiandSwiss mice (75 animals) (b) Golden and albino hamsters B (a) 8 wk. (b) Not given C (a) 76 wk. (b) 48 wk.

(1) Whole tob. smoke

A Tob. "tar" prepd. in glass app. resembling pipe. Details not reported. "tar" colld. by coolers and glass filters D Not given

A

A

A Not given B Not given

A

B

Kreshover and Salley (1957) Cia.

C. Moore and Miller (1958) B Cig. A

Mariiiold smoking ma- A '36 BALD/c mice chine, 3 puf€s/min., puff B Not given duration 2 see., puff vol. C Lifespan 30 cc.. butt lgth. 10 mm. B Av. 700°C.

A

Results

60 mice, albino: D mixed, known strain. 40 E control mice of same F age, sex, strain B Adult C 140 (consecutive) days' swabbing; 12 mo. after outset of expt. all mice killed

A Holsti and Ermala (1955) I3 H p e tob.

l3

Type of material applied (D), Solvent (E), Application procedure (F)

D E

F

_.

( 2 ) B[a]P in acetone None (l),acetone ( 2 ) (1) Daily appln. of whole tob. smoke to lips and ears (2) 3 X weekly painting of palatal mucosa and ears (3) Daily appln. of whole toh. smoke to cheek pouch (4) 3 X weekly painting with B[alP soh. of cheek pouch

L) Whole "tar"

E F

None Painting 3 X weekly of shaved back; each appln. about 40 mg.

___~_..

-

(a, 1 ) Vitamin B-deficient CAFl mice showed cellular abnormalities suggestive of precancerous changes or carcinoma in situ; Swiss strain leas affected (b, 1 ) No oral changes; ears: hyperkeratosis and hyperplasia (b, 2) 24 wk. treatment: no oral changes; ears showed ulceration, thickening, and occasional papillomatous formation (b, 3) Infection and edema after 8 mo. (b. 4 ) Epidermoid carcinoma after 4 mo. Many animals developed papillomas after 2 mo.; these disappeared in 6 wk.; 2 squamous-cell carcinoma of the skin: 1 after 14 mo.. the other after 18 mo.

A

DiPaolo and Moore (1959)

B

Am. cig.

A

Manifold smoking mchine B Not given

A

B C

Mice, Swiss ICR/Ha strain, 25 8.25 O / group; controls: 25 8, 25 0

D E

F

lmo. 13 mo.

Whole cond. Acetone Painting of lips and oral areas (a) 5 X weekly 20-30 mg. (20% “tar” soln.) (b) 5 X weekly 60-90 mg. obtained via evaporation of acetone from (a) (c) Acetone (control)

NeODlasmS

Skin Lung= Bladder Oral-cavity

3‘ 17

26 10

-

-

I*

-

2

-

2 carcinomas, 1 papilloma. Carcinomas. 0 Lung adenomas. d Anaplastic sarcoma.

b

A Mody and Ranadive

&B

c.’

(1959)

Tob. (vaadkan) type grown in southern India

See Khanolkar (1957) A Albino mice “A” (a) Extn. dried tob. leaf (Strong) strain and with petr. ether, Swiss strain benzene, chloroform 241 (8 0) 207 (8f 0 ) and alc. (b) Extn. with benzene; B 2-3 mo. deliberation of ext. C 12-18 mo. from alkaloids ( c ) Water ext. of tob. made alkaloid-free B Not given

A

+

D E F

(a) Whole ext. (incl. alkaloids) (b) Whole ext. (without alkaloids) (c) Water ext. (without alkaloids) Benzene and chloroform (I) Appln. of ext. to shaved back of 12-15 mice from each strain (11) Painting of buccal mucosa

(I) No. mice with skin changesa Group (a) (b) (c)

Intlammation 2 2

(6) (6)

Not tested

Hyperplasia 11 18

(12) (13)

Not tested

(11) No. mice with oral changes.

Group (a) (b) (c)

Hyperkeratinieation 1 2

(1) (4)

Not test.ed

Hyperplasia 5 1

(4)

(2)

Not tested

4 First col.: “A” strain; in paren: Swiss mice.

(Continued)

TABLE I1 (Continued) Smoking and collection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco product used (B) A

B

Peacock and Brawley (1959) (a) Snuff (b) Chewing tob.

A B

None None

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. (C) A

(a) 50 hamsters (b) 50 hamsters

B Not given C Lifespan

Type of material applied (D). Solvent (El, Application procedure (F) D

E F

Results

(a) Snuff paste (a) No neoplasm of any type in snuff or (b) Chewing tob. plug control pouch (b) No neoplasm of any type in tob. or None In approx. of animals, right control pouch pouch implanted with control substance (nonspecific irritant, sand or other bland substance). Implantation into left oral pouch of (a) 10 cc. thick snuff paste (b) 2 X 1 X 1 cm. plug chewing tob.

+

-

3

A Peacocket at. (1960) B (a) Snuff (b) Chewingtob.

A B -

A

A Akamatsu (1960a) B Cig.

A Smoke-blowing app. B Not given

A 60 # Syrian golden hamsters B 3mo. C 70 wk.

(a) 60 golden hamsters D (b) 64 golden hamsters E F Implantation [see Peacock and Brawley (1959)l

D

Whole cig. smoke

E -

~

No neoplasms (a) and (b)

Macroscopically, no marked changes. Microscopically, after 5 wk. epithelial hyperplasia; after 10 wk. epithelial cornification. After 70 wk.. no tumor formation

F

Smoke from 1 cig. blown 3 X daily into left hamster pouch

D sters (b) Syrian golden ham- E sters F (27 # ) B 3mo. C 70wk.

Acetone tob. ext. prepd. by soaking cig. in acetone for 1 wk. (a) None (b) Acetone Daily painting of acetone tob. ext. t o mucous membrane of hamster cheek pouch.

No marked changes of mucous membrane of pouch: epithelial cornification evident. No tumor formation

A 20 Albino hamsters (random-bred) B 3-61110. C Up to 638 days

Wbole cond. None Painting 3 X weekly on exteriorized hamster oral pouch (40 mg. cond. painting)

Hyperkeratosis and slight hyperplasia

~~

A Akamatsu (1960h) B Cig. (Peach brand)

A B

-

C. M o o n a n d A See Moore and Miller (1958) Christopherson (1962) B Cig. B Av. 700°C.

A

A

(a) Syrian golden ham-

D E F

TABLE I1 A SELECTIVE SUMMARY OF EXPERIMENTS. PART4. CERVIX Smoking and collection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco product used (B)

Strain and animal used (A). Age of animal at onset of expt. (B), Duration of expt. (C)

A Koprowska and Bogacz (1959) B Cig.

A

Manifold smoking machine 2-sec. puff once/ min.. 35-40 ml. vol.; smoke led through sy& tem of cool traps B Not given

A C3H 0 virgin mice B 4-6 wk. C Variable

A Bogace and Koprowska (1961) B Cia.

A

See Wynder and Hoffmann (1961a) B See WynderandHoffmann (19618)

A

I C3H mice (16) I1 ZBC mice (20) B 6-8wk. C 92 wk.

Type of material applied (D), Solvent (E), Application procedure (F)

Results

D E F

Whole “tar” (I and 11);B[u]P (111) I 9 mice carcinoma i n situ, 6 mice carNone for ”tar.” acetone for B[a]P cinoma in situ with microinvasions. 4 mice with invasive carcinoma 5 X weekly appln. about 0.15 mg. I1 2 mice carcinoma in situ, 2 mice car“tar” to cervix I Without thread (20 mice) cinoma in situ with microinvasion. I1 Thread inserted into endocervi5 mice invasive carcinoma III(a) Lesions rep. of developmental stages cal canal (10 mice) of induced cervical carcinoma 111 B[a]P as 1% acetone soln. III(b) All had invasive carcinoma appld. 2 X weekly (50 mice) IV No demonstrable lesions III(a) 20 of 50 mice treated until sacrificed: 2 mice sacrificed every other week from 3rd-21st wk. of B[u]P appln. 11103) 30 of 50 treated for 19 wk. and then observed for variable lgth. of time to 11 wk. IV No treatment appld. to 5 control mice; sacrificed after 46-58 wk. obsv.

D

Whole cond. None Appln. “tar” on surface of cervix with wire loop: about 0.15 mg. 5 X weekly

E F

%

%

C3H ZBC mice mice (a) Invasive squamous cell carcinoma (b) Early invasive sqnamons cell carcinoma ( c ) Carcinoma in situ with microinvasion (d) Carcinoma in situ (e) Basal cell hyperplasia and/or dysplasia

-

-

12

7

12

-

32 13

25 15

(Continued)

TABLE I1 (Continued)

Reference (A) and Tobacco product used (B) A Chu et al. (1962) Cis.

6B I+

Smoking and collection technique (A), Reported combustion temp. (B) A Nodetails B Not given

Strain and animal used (A), Age of animal at onset of expt. (B), Duration of expt. (C) A

Syrian 0 hamsters (N.C.l. strain)

B 4-6wk. C

About 12 mo.

Type of material applied (D), Solvent (E), Application procedure (F)

D

E

F

Results

(1) Tob. “tar” ( 2 ) Acetone

Acetone 100 paintings of cervix with wood applicator (1) 30 animals recd. tob. “tar” 50% soln. ( 2 ) 20 animals recd. acetone (3) 6 animals recd. painting with wood applicator (trauma)

No. cytologically malignant (1 )

12

(3)

-

(2)

0

15 dysplastic and 3 anaplastic epithelial changes; no invasive lesions. With control animals (only acetone) no significant abnormal epithelial changes

A ~

~

TABLE I1 EXPERIMENTS, P.4RT 5. LUNG (INHALATION)

~~

Smoking and collection technique (A). Reported combustion temp. (B)

Reference (A) and Tobacco product used (B) A B

SELECTIVE SUMMARY O F

Essenberg (1952) Cis.

A

Smoking machine, continuous suction B Not given

Strain and animal used (A). Age of animal at onset of expt. (B), Duration of expt. (C) A 72 strain A mice B Not given C 14 mo.

Type of material applied (D), Solvent (E), Application prodecure (F) D

F

Results

Smoke Not given

No. mice

Lung tumors

%

Exptl. group 23 21 91.3 Control group 32 19 59.4 No epidermoid type of cancer; papillary adenocarcinoma was most common type of tumor %A B

Miihlbock (1955) Cig.

A

B

A B

Essenberg et al. (1956) A I Low-nicotine cig. B I1 Same as I plus 0.55 mg./cig. arsenic trioxide

20-ml. puffs blown into A 020strain 0 mice, DBA D strain # mice F 2250-m1. glass cages I 17 0 and 12 d’ mice housing 4 mice each for 2 hr. daily; 10 puffs (exptl.) I1 19 0 and 13 d’ mice obtd. from each cig. (control) Not given B Not given C 2 yr.

Whole smoke I Exposed to cig. smoke in sealed cages I1 Control

D F

Smoke Exposure of 1/3-1/2 of cig./hr. for first 2 days: then full dwage (12 cig./day ) for 6 days/wk. for 1 yr.

Smoking machine smok- A A/Jax mice 36 study, 36 controls ing 12 cig./day, l/hr. Not given B Not given C 1 yr.

All lung tumors (I and 11) carcinomas, probably derived from alveolar epithelium. No other tumors found I 29 mice, 23(79y0) lung tumors I1 32 mice, 10(310/,) lung tumors

I Exptl. Control I1 Exptl. Control

No. animals

yo Tumors

28 30

64 40

No. animals

yo tumors

24 28

62 39 (Continued)

TABLE I1 (Continued) Smoking and collection technique (A), Reported combust.ion temp. (B)

Reference (A) and Tobacco product used (B)

A

Essenberg (1957)

B Cig.

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. (C)

Smoking machine so A constructed that smoke B from cig. and atm. air C mingled freely; both entered animal chamber well-mixed and cool B Not given A

Type of material applied (D), Solvent (E), Application procedure (F)

A/Jax albino mice Not given Not given

D

I 50 mice of author’s 5-yr.inbred strain (#+ 9) I1 20 mice of author’s

D F

F

Results

Smoke Inhalation I Smoke of high nicotine content (2.07%) I1 Smoke of low nicotine content (0.78%)

I 32% lung tumors I1 24% lung tumors

Smoke I Exposure to concd. smoke I1 Exposure t o dild. smoke. Expoaure time t o smoke increased step by step t o 200 min./day

In groups I and 11, inflammatory changes were seen in the bronchioles, usually with atelectatic foci of lung tissue. No difference found between I and 11. 1 mouse developed carcinoma of bronchial origin. Control group (70 mice): no tumors. I n 2000 mice of same breed not 1 case of carcinoma of lung

~~

A

R

Komczymski (1958) Cig. (Polish “sport”)

A

B

Cig. smoke drawn into glass cage with aid of filter pump Not given

A

6-yr. inbred strain

+@

53

(al+ 0 ) B Not given C 703 days A Leuchtenberger et al. (1958) B Cig.

A

B A R

Guerin (1959) Cig.

Smoking machine, modi- A 275 0 albino CFI mice fication of Essenberg B 3-9 mo. (1952) C Variable: 11-201 days Not given

Smoking machine for A 80 rats, Institut du Cancer strain (IC) 60 1 4 cig.; smoke going t o 2 plastic containers; 1 rats, Wistar strain puff of 2-see. duration, B 2-6 mo. C > 2 yr. puff vol. 30 ml./min. B Not given

A

D Smoke

F

Inhalation cip. smoke from 6-8 cia. daily

Histopathological findings of 23 mice: 15, basal cell hyperplasia: 14, atypical basal cell hyperplasia; 9, dysplasia (2 of which are questionable); 2, squamous cell metaplasia

D C i . smoke F Exposure of rats to smoke, approx. 45 min. daily

PulSurviOral maNo. vow cavity nary rats (5t ma.) tumors tumors

-

Exptl.

IC

60

W

40

53 15

3 2

4 0

3 IC rat: papillomatosis of buccal osteodentoma of the maxilla; mucosa 1 0 rat: buccal papilloma; another: epithelioma infiltrating musculature. Wistar rats: keratinizing epithelioma invading the jaw bone. Another rat; undifferentiated epithelioma invading both salivary glands extensive cervical lymph and jaw bone node metastases 1

+

+

Leuchtenberger et al. (1960a) B Cis. A

A

A Smoking machine as B Leuchtenberger et ~ 2 . (1958) except present C machine has 30% larger smoking chamber

CFI 0 mice Not given 1 m0.-2 yr.

D

Cig. smoke Inhalation of smoke daily 5-6 cis, No.

No.

151 150 36 36 34 51 63

25-1526

Lgth. exposure

Cessation

Severe bronchitis, peribronchitis; atypical epithelial proliferation ~

30

(8)

B

7

(a) (b) (c)

(d) (e) (f) (g)

A

B

Leuchtenberger et al. (195Ob) Cig.

Leuchtenberger et al. (1958) B -

A

A CFI 0 mice B 3-28mo. C Time exposure Cig. smoke (days)

-

1W200 250-500

60&1600 1W00 100-400

1-23 O(contro1s) 1-3 4-8 9-23 3-8 ZO(t300 3-6

D Cig. smoke F

Daily exposure (except for weekends and holidays) to smoke of +-8 cig. hourly intervals

(d) (e)

7 8 4

(f)

17

(9)

Incidence of grossly visible adenomatous lung tumors in study and control group essentially the same

-

No. mice

(a) None (control) (b) 17-99 ( c ) 1W199 (d) 200-800

Mice with pulmonary adenomatous tumors -

examined

No.

%

(C)

81 39 35

46 16 13

(d)

51

34

56 41 37 66

(a)

(b)

(Continued)

TABLE I1 (Continued) ~

~~~

Smoking and collection technique (A). Reported combustion temp. (B)

Reference (A) and Tobacco product used (B)

Strain and animal used (A), Age of animal a t onset of expt. (B). Duration of expt. (C)

Type of material applied (D), Solvent (E), Application procedure (F)

A Automatic smoking ma- A 12 d golden hamsters chine. 1 puff/min., 2 sec. B About 42 days duration, 35-ml. vol.. C 12 ma. butt lgth. 23 mm. B Not given

D

B

Dontenwill and Mohr (1962) Cig.

A B

Holland et al. (1963) Cig.

A

D F

A

R

Smoking machine deA 40 rabbits (strain not given): study group: 31 signed by Holland. 30controls 60 ml. puff, 2 8ec. duration, 1 puff/min. B Not given Not given C 2-5: yr.

F

Cig. smoke Blowing of smoke of 10 cig. daily through cage contg. 10 hamsters

Results Most hamsters developed benign metaplasia with focal papillary growths of epithelium, especially within bronchi

“Normal” cig. smoke 30 of original 40 “smokers” survived 2 years: After 1st yr., rabbits allowed t o inhale 31 of controls 2 puffs smoke/min. instead of 1 puff Cytological findings in tracheobroncbial mucosa No. No. “smokers” controls

7

Normal Focal hyperplasia General hyperplasia General hyperplasia, atypical cells Squamous metaplasia ~

Leuchtenberger et al. (1963) I3 Cis. A

10 9

21 6 3

3 1

1 0

~

A B

Leuchtenberger et al. (1960a) Not given

A CFI mice; 308 0 , 200

d

B C

Not given 1-14 mo. for rig. smoke

D

(a) Cis. smoke (b) PR8 influenza virus (c) P R 8 influenza virus cig. smoke (a) Mice exposed daily t o smoke from f-3 cis. for 30-420 days (b) Influenza virus: mice reed. intranasal innoculation under ether 1-3 X intervals of 1-16 wk. (c) Both: virus given 4-8 days before inhalation of smoke began

% ’ Atypical epithelial proliferation in bronchi

+

F

Negative

Group

100 98 88 96 74 100 49 42 a

Mild

_____ Ma S b Ma Sa

Major bronchus

* Small bronchus.

Marked

92 - 8 94 2 6 74 12 2 68 4 20 _ _ 66 13 2 13 11 62 - 14 _ 27 23 5 25 9 20 29 10 29 29

yo Squamous cell metaplasia in bronchi Group Controls

0

d (a)

(b) (c)

0 d 0

Negative

Mild

100 98 97 96

-

Marked -

2

-

3 4 5

d

95

5

11 -

0

68

3

54

23 17

9 29

84

~~~

Otto (1963) Nonfilter cig.. (German commercial type)

D Smoking of cig. in glass A Inbred strain (Pathol. F Anat. Inst. Erlangen) distributor. Suction apalbino mice plied by means of subatm. pressure in inhala- B young mice tion chamber (600-650 C Duration expt. I Lifespan (12-24 mm. Hg). Smoke from mo.) 12 cig. led into chamber I1 Most mice died by opening of valves during first 2 mo. "in usual rhythm" of Longest survival: smoking during 60- or 12 mo. SO-min. period 111 BIulP and smoke: Chamber vol.. 400 liters B Not reported 30 mice, 12 cig./ 90 min./day for 19i mo. IV Smoke: 12 mice, 12 cig./90 min./ day for 9 mo. V Smoke: 27 mice, 12 cig./60 min./ day for 8 mo. VI Smoke: 20 mice, 12 cis./ 60 min./ day for 4 mo.

A

Smoke (arbitrary) I 60 mice not treated (control of spontan. tumor rate) I1 0.5 ml. satd. soh. B[ulP in paraffin/mouse intraperitoneally 111-XI see left column, C.

Increased epithelial proliferation in aged mice in control group; less pronounced, than that in experimental groups I 3 with lung adenoma I1 1 lung adenoma (11 mo.) 111 6 lung adenomas (partially occurring as multiples) IV 1 lung adenoma (9 mo.) V No tumors; expt. terminated because of bronchopneumonia V I No tumors; expt. terminated unexpectedly VII No tumors; premature termination of expt. due to death of animals as in V I VIII 5 mice with lung adenoma most prevalent in age and after long exposure IX No tumors (bronchopneumonia) X 4 lung adenomas, 1 epithelial carcinoma (keratinizing of lung) XI 11 lung adenomas, 1 epithelial carcinoma of lung

.~_

(Continued)

TABLE I1 (Continued)

Reference (A) and Tobacco product used (B) Otto (1963), continued.'

Smoking and collection technique (A), Reported combustion temp. 03)

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. ( C )

VII Smoke: 30 mice, 12 cig./60 min./ day, incr. to 12 cig./90 min./day; total 2? ma. V I I I Smoke: 40 mice, 12 cig./90 min./ day; total 24 mo. IX Smoke: 30 mice, 12 cig./SO min./ day; total 17 ma. Intervals (up to 2 mo.) without treatment X Smoke: 30 mice, 12 cig./90 min./ day; total 2.5 ma. XI Smoke: 30 mice, 12 ek./60 m i x / day for most of appln.; total 24 mo.; initially higher, during last ma. longer intervals without treatment

Type of material applied (D), Solvent (El, Application of procedure (F)

Results

A

~~

TABLE I1 SUMMARY O F EXPERIMENTS. PART6. LUNG(OTHER

Smoking and collection technique (A). Reported combustion temp. (B)

Reference (A) and Tobacco product used (B) A P. R. Peacock (1955) B Cig.

SELECTIVE

A

Hand glass pump: 20ml. syringe. Y-shaped Pyrex glass tube with nonreturn valves. Cig. inserted into limb of app, can be smoked by means of syringe used as pump; 15ml. puff vol. 33 Not given

Strain and animal used (A). Age of animal at onset of expt. (B), Duration of expt. (C) A 4 white Leghorn hens B Not given C 3 mo.

THAN INHALATION)

Type of material applied (D), Solvent (E), Application procedure (F)

Results

D F

Whole smoke Expelling smoke through surgically created stoma from axilla t o thoracic air sac; every 2nd day 10 injections. 15 ml. smoke each (half a cig.)

No neoplastic changes

(1) Smoking machine: 1 A White rats, Chester puff, 2 sec. duration Beatty strain every 45 sec., puff (a) 10 vol. 25 ml. Cond. (b) 8 colld. in acetone, B 3 4 mo. (40 me./&.) C Not given ( 2 ) Smoke obtained by man’s smoking through cartridgetype filter, cond. extd. from filter B Not given

D E F

Denicotiniaed cond. Olive coil Injection denicotinired cond. (a) 0.1 ml. olive oil contg. “tar” from 4 cig. (1) (b) 0.1 ml. olive oil contg. “tar” (2) 0.01 mg. killed from 4 cig. tubercle bacilli

(a) (1) No tumors (b) ( 2 ) 1 sarcoma, 1 carcino!r:a

A

D Whole “Tar” E No solvent used I Bronchial painting with “tar” F

~

A Blacklock (1957) B Popular brand British cig.

A Rockey et al. (1958) B Cig.

A

Manifold smoking m a chine: 2 sec. puff once/ min. vol. 3-0 ml., butt I&. 25 mm. B Not given

I 7 dogs I1 6 dogs B Not given C I 178-320days I1 6 5 2 2 3 days

A

+

(0.05 cc.) for first 3-6 appln.; then 3-5 X weekly 0.1 cc. I1 Control group

.

Long-term painting with “tar” resulted in extensive squamous metaplasia in 7 dogs (2 of the treated dogs died accidentally at days 178 and 303) Controls neg. (Continued)

TABLE I1 (Continued) Smoking and collection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco product used (B) A B

A B

DellaPorta et aZ. (1958) Popular U.S. cis. blend

Rigdon (19EO) Cig.

Manifold smoking machine. Cold traps; 1 puff/min., 2 sec. d u r n tion, 3 5 4 0 ml. vol. B Not given

A

A B

Mechanical smoking of cig. Not given

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. ( C ) A Syrian golden hamsters B Not given C 12-55 wk.

Type of material applied (D), Solvent (E). Application procedure (F) D

E F

(a) DMBA (b) Whole oond. 1% aq. gelatin soh. Tob. “tar” instillation into tracheobronchial tree (1) 21 8:DMBA 50 pg. weekly for 45 wk. ( 2 ) 21 0 : tob. “tar” 200 pg. 2 X weekly for 32 wk. (3) 10 8:DMBA 50 pg. weekly for 12 wk. (4) 10 0 DMBA: 50 M. weekly for 12 wk., then tob. “tar” 200 fig. 2 X weekly for 30 wk. (5) 9 8,11 0 : DMBA 100 pg. weekly for 17 wk. (6) 10 8,10 0 : DMBA 100 pg. and “tob. tar” 500 pg. weekly for 20 wk.

A White Pekin ducks D Whole cond. I 26 recd. tob. cond. E Not given in liq. petr. F Dil. cond. (concn. not given) in 0.5 ml. liq. petr. once daily except I1 2.5 rccd. liq. petr. 111 99 controls unholidays) intratracheally: 7 ducks recd. 1 injection, 5 were treated B Not given killed 5-15 min. later 7 ducks recd. 1 injection, 1 was C Variable:
Results (1) 2 carcinoma (1 trachea, 1 larynx) (2) No histol. demonstrative lesions (3) No histol. demonstrative lesions

No histol. demonstrative lesions (5) 4 carcinomas (2 traehea, 1 larynx, 1 esophagus trachea) (6) 2 carcinomas (1 stem bronchus. 1 larynx) (4)

+

No neoplastic ohangrs

A Blacklock (1961) B Cig.

A

B

B Cig.

D Whole condensate Chester Beatty Inst. E Eucerin breed F Inoculation into left lung after (2) (a) 72 white rats, thoracotomy (0.2 ml. Eucerin-cond. Chester Beatty milt. representing about 40 cig.)a Inst., pure strain Reviewers remark: Amount of “tar” (b) 88 guinea pigs, from cig., calcd. to be 2.5-5.0 mg., appears t o be rather low. hybrid strain (0) Rabbits, hybrid strain (no. not given) B Rats: 8-12 wk. Guinea pigs: 3 4 mo. Rabbits: Fr6 mo. C (1) Rats killed at intervals of 1 wk. t o over 2 yr. after inoculation (2) (a) Rats: lifespan (h) Guinea pigs: 1-52 wk. ( c ) Rabbits: not given ( 1 ) 176 white rats,

Automatic smoking ma- A (1) 6 r a t s (2) 6 rats chine, 4 puffs per min.; (3) 9 rats puff vol. 15 Id.;puff (4) 11 rats duration 2 secs.: butt (5) 5 rats length 20 mm. Conden(6) 6 rats ate collected in traps (Chester Beatty breed) immersed in dry ice/ B 8-12 wk. acetone. C Up to730days B Not given

A Blacklock and Burgan A (1962)

Automatic smoking ma- A chine. 1 puff/45 sec. 2 sec. duration; 25-ml. vol., butt lgth: 15 mm. Not given

D

E F

(1) Phenolic; (2) acidic; (3) neutral fraction; (4) neutral fraction minus hydrocarbons; (5) hydrocarbon-containing fraction; (6) “wax” fraction Eucerin Inoculation of Eucerin suspensions in left lung (1) 100 mg. phenolic fraction in 0.2 ml. Eucerin (2) 8 mg. acidic fraction in 0.1 ml. Eucerin (3) 2 rats, 80 mg. neutral fraction in 0.1 ml. Eucerin; 7 rats, 40 mg. in 0.1 ml. Eucerin (4) 60 mg. in 0.1 ml. Eucerin (5) 90 mg. hydrocarbon fraction in 0.2 ml. Eucerin (6) 7 mg. “waxes” in 0.1 ml. Eucerin

(1) Rats showed proliferation of basal cells of bronchial epithelium, squamous metaplasia followed by hyperplasia, carcinoma in situ, and finally squamous epitbelioma (2) (a) Rats: 6 lung carcinomas, 2 lung sarcomas (b) Guinea pigs observed to have proliferation of basal cells of bronchial epithelium and metaplasia t o a hyperplastic squamous type (0) Rabbits: 1 with carcinoma i n situ

(1) 1 vascular sarcoma (2-6) No tumors

(Continued)

TABLE I1 (Continued) Smoking and collection technique (A), Reported combustion temp. (B)

Reference (A) and Tobacco pmduct used (B) A

B

Dontenwill and Mohr (1962) Cis.

A

B A Rockey et a!. (1962) R Cig.

Strain and animal used (A), Age of animal a t onset of expt. (B), Duration of expt. ( C )

Automatic smoking ma- A chine, 1 puff/min. 2 sec. B duration, 35-ml. vol.. C butt lgth. 23 mm., cold trap colht. Not given

A Not given B Not. given

31 c? golden hamEters About 42 days 12 mo.

Type of material applied (D), Solvent (E), Application procedure (F)

D Whole cond. E F

Sesame oil 3 X weekly spraying of cond. in sesame oil into trachea 70 mg. cond./animal

A No details given B Not given

(1) 10 Syrian hamsters (5 0 , 5 d ) ( 2 ) 10 Syrian hamstem (5 0 , 5 8 ) B 1-2 ma. C (1) 6-10 mo. (2) 10 mo.

A

Of 31 hamsters, 7 developed papillary noninvasive tracheal tumors and metaplastic epithelial changes in trachea and lung

-

A Mongrel dogs first submitted t o tracheal fenestration; 55 cj’ and 27 0 dogs B Not given C Inferred to be a t least 4 yr.

Cis. smoke cond. E None F (1 ) Dogs treated with cig. smoke cond. Expt. 1 49 dogs recd. 0.1-0.25 mi. cond. t o mucosa of mesial wall, left primary bronchus 3-5 X weekly; total no. treatments, 2-682 Expt. 2 20 dogs recd. 0.1-0.25 ml. cond. 3-5 X weekly only after biopsy site had healed; total no. treatments 2-673 Expt. 3 13 dogs recd. 0.1-0.25 ml. cond. 3-5 X weekly Discontinued treatments: 32-121 (2) Dogs subjected t o rubbing manipulations only

D

~~

A Herrold (1963) B Cig.

Results

Group 1 Expt. Expt. Expt. 1 2 3 47 Hyperplasia (%) Squamous metaplasia (%) 94 Precancerous changes (%) 18 Carcinoma in situ (%) 2 Invasive 2 carcinoma (yo)

40

31

100

92

25 0

23 0

0

0

~

Group 2: No neoplastic changes

~

D

E F

( I ) Nicotine-free cond. (2) Control Tween 60 (12.5% aq. soh.) (1) Intratracheal instillation about 0.05 ml. of 50% suspension/ animal once weekly (2) Tween 60 (12.5% aq. soln.) as control

(1) 6 hamsters survived 6 mo. treatment. Basal eel1 hyperplasia and prominent increase in mucus-secreting cells (2) No changes in tracheal epithelium

EXPERIMENTAL TOBACCO CARCINOGENESIS

435

be in the realm of reality, a t least in the foreseeable future, a reduction of the tumorigenic activity of tobacco products seems to be the only possible and practical alternative. To achieve this goal will require continued efforts of scientists of independent research groups, as well as the cooperation of the tobacco industry, public health authorities, and the public a t large.

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AUTHOR INDEX Numbers in italic indicate the pages on which the complete references are listed

A Ando, T., 78, 118 Andrewes, C. H., 2, 32, 36, 39 Abbe, R., 250, 4% Abdel-Kader, M., 224, 225, 226, 227, 228, Andrews, F. N., 229, 248 Anguera, G., 376, 378, 384, 43s 246 Appleyard, J. H., 361, 362, 485 Abel, P., 2, 39 Antoni, F., 110, 115 Abell, C. W., 319, 440 Archetti, I., 29, 36 Acevedo, A. R., 368, 369, 379, 442 hffmann, E., 4.35 Adamek, S., 347, 359, 360, 361, 381, 447 Argus, M. F., 356, 436 Adams, H. A,, 55, 87, 113, 114, 117 Ash, M., 365, 436 Afkham, J., 356, 439 Ahlmann, J., 261, 272, 314, 320, 403, 436, Arky, I., 110, 115 Armstrong, J. A,, 32, 39 439 Arnoult, J., 26, 37 Ahn, K. J., 282, 283, 292, 434, 448 Aronson, M. M., 158, Is9 Ahrens, J. F., 336, 453 Asao, T., 224, 225, 226, 227, 246 Aiaawa, M., 237, 248 Ashley, L. M., 195, 200, 201, 203, 246 Akamatsu, E., 279, 422, 435 Ashworth, C. T., 125, 184 hkiyama, V., 125, 187 Asplin, F. D., 216, 246 Albanese, M. P., 131, 166, 184 Atkinson, W. O., 254, 435 hlbert, R. E., 366, 4% Atwood, M. B., 193, 220, 246 Alexander, P., 367, 397, 435 Aub, J. C., 47, 89, 118 Alexandrov, X., 320, 369, 435 Alfert, M., 49, 69, 79, 80, 86, 112, 131, Auerbach, L., 128, 184 Auerbach, O., 283, 289, 292, 302, 435 132, 185 Alleroff, R., 210, 213, 214, 215, 218, 224, Austin, C. R., 130, 184 Austwick, P. K. C., 210, 212, 213, 246 225, 226, 228, 238, 239, 646, 248 Avery, 0. T., 80, 113 Allen, M. J., 237, 246 Allfrev, V. G., 43, 47, 51, 52, 57, 59, 60, Awa, A., 285, 435 65, 72, 80, 83, 84, 107, 116, 123, 214, Axelrad, A. A., 2, 15, 35, 58 Ayerst, G., 210, 212, 213, 246 115, 118, 119 Ayres, C. I., 255, 310, 320, 379, 384, 435 Allman, D. R., 347, 462 Almeida, J. D., 11, 15, 16, 19, 20, 29, 36, B 38, 99, 40 Bacon, C. W., 253, 254, 440 Almquist, H. J., 224, 246 Badger, G. M., 263, 323, 325, 354, 45'5 Altschuler, B., 366, 435 Alvord, E. T., 314, 320, 321, 322, 379, 383, BadrC, R., 303, 306, 441 Baebler, S., 336, 440 435, 437, 448 Baghirzade, M., 297, 444 Amano, M., 166, 187 Rahr, G. F., 152, 184 Ambaye, R. V., 349, 439 Bailey, E. J., 368, 435 Amenta, P. S., 129, 184 Bakay, B., 68, 92, 93, 115 Ainer, S. M., 43, 70, 113 Baker, J. R., 272, 273, 417, 442 hnderson, D. C., 73, 107, 108, 109, 113 Baker, N., 116 Anderson, N. G., 45, 120 Baker, W. K., 183, 184 Anderson, T. F., 4, 5, 17, 18, $6, 38 455

456

AUTHOR INDEX

Balasubrahmanyam, S. N., 333, 436 Baldwin, R. S., 229, 248 Ballenger, J. J., 301, 436 Baltimore, D., 112, 115 Bandel, D., 255, 449 Banfield, W. G., 15, 27, 36 Bang, F. B., 5, 36 Barbczat-Debreuil, S., 329, 436 Barkeley, W. H., 2, 20, 37 Barkemeyer, H., 314, 436 Barnes, J. M., 355, 445 Barnett, S. R., 45, 115 Baroni, C., 368, 436 Barson, G. J., 229, 248 Baserga, R., 86, 113 Bates, W. W., 308, 446 Battista, S. P., 301, 303, 386, 443 Bauer, H., 132, 186 Bavley, A,, 253, 255, 325, 436, 438, 441 Bayley, S . T., 89, 104, 118 Bearcroft, W. C. C., 165, 184 Beard, D., 17, 26, 36, 37, 38 Beard, J. W., 17, 26, 36, 37, 38 Beaudreau, G. S., 26, 36 Becker, C., 26, 36 Becker, H. J., 150, 156, 157, 158, 160, 161, 178, 184 Beermann, W., 132, 147, 152, 153, 154, 155, 156, 157, 158, 159, 164, 165, 167. 168, 175, 184, 186 Behrens, M., 44, 47, 59, 62, 113 Beinhart, E. G., 337, 446 Bell, J. H., 329, 330, 450 Bellin, A., 328, 449 Belousov, A. P., 63, 117 Bendoraitis, J. G., 329, 451 Bentley, H. R., 262, 314, 318, 319, 320, 321, 341, 369, 382, 383, 436 Bergel, F., 237, 246, 271, 398, 403, 447 Berman, C., 237, 240, 246 Bernal, J. D., 4, 36 Bernfeld, P., 301, 303, 436, 442 Bernhard, W., 14, 15, 20, 21, 22, 26, 27, 36, SY, 39, 48, 50, 113, 122, 125, 184 Bernstein, M. H., 62, 113 Berry, E. G. N., 318, 341, 369, 436 Berry, R. C., Jr., 336, 349, 453 Berwick, L., 5, 17, 18, 36 Besse, P., 194, 247 Bessman, M. J., 57, 113

Beuthner, H., 263, 265, 276, 277, 286, 287, 320, 419, 439 Bibao, J. A., 452 Bielka, H., 397, 441 Bijvoet, P., 91, 92, 113 Bilbao, J. A., 332, 333, 334, 462 Bilinsky, W. R., 265, 436, 451 Bill, M. E., 359, 436 Billen, D., 83, 84, 113 Bils, R. F., 9, 36 Bird, A. F., 165, 184 Birnstiel, M., 336, 436 Bischoff, L., 351, 440 Biserte, G., 100, 113 Bissinger, L. L., 285, 446 Black, M. M., 109, 113 Blacklock, J. W. S., 281, 282, 292, 431, 433, 435 Blackmore, R. H., 255, 308, 436, 446 Blake, V., 191, 246 Bloch, D. P., 80, 85, 86, 113 Blodgeth, G., 368, 449 Blohm, S. G., 326, 441 Blout, E. R., 436 Blount, W. P., 192, 246 Blum, G., 356, 439 Bocciarelli, D. S., 29, 36 Boche, R., 301, 303, 436 Bock, F. G., 261, 262, 272, 273, 295, 296, 300, 301, 367, 372, 373, 408, 417, 436 Bogace, J., 280, 423, 436, 444 Bogen, E., 259, 346, 436 Bokhoven, C., 356, 360, 436 Bollum, F. J., 58, 118 Rolognari, A,, 122, 124, 125, 129, 131, 166, 184

Bonar, R. A,, 17, 26, 36, 37 Bond, V. P., 165, 186 Bonner, J., 43, 82, 83, 84, 91, 104, 113, 116, 165, 184 Bonnet, J., 265, 314, 320, 436, 4.66 Bonser, G. M., 241, 246, 278, 341, 436 Booker, L., 289, 428, 442 Bopp-Hassen Kamp, G., 130, 184 Borgese, N. G., 2, 39 Borman, G. S., 2, 20, 37 Borowski, H., 266, 269, 309, 436 Bosch, D. K., 294, 340, 437 Bouchard, J., 285, $36, 437 Bouchard-Madrelle, C., 437

457

AUTHOR INDEX

Bouroncle, B. A., 126, 186 Boutwell, R. K., 294, 340, 437 Bowling, J. D., 253, 254, 440 Boykin, M. J., Jr., 303, 448 Boyland, E., 237, 846, 271, 356, 364, 396, 398, 403, 437, 447 Brachet, J., 122, 129, 184 Bradbury, E. M., 79, 113 Bradford, J. A., 437 Brain, P. W., 208, 846 Brawley, B. W., 279, 422, 447 Breedis, C., 5, 17, 18, 36 Brenner, S., 5, 29, 36, 38 Breuer, M. E., 154, 155, 156, 184, 188 Bridges, C. B., 157, 186 Brieger, H., 288, 437, 444 Briggs, R., 134, 146, 149, 179, 180, 181, 182, 185, 187, 183 Brindley, D. C., 15, 36 Brink, R. A., 173, 186 Brinkman, G. L., 302, 437 Brock, H. J., 370, 437 Brookshire, S. R., 255, 437 Brosch, A,, 250, 437 Bross, I. J., 277, 285, 463 Brown, C. H., 124, 185 Brown, E. V., 218, 219, 246 Bruckner, H., 266, 268, 437 Brummond, D. D., 46, 116 Brunelle, M. F., 356, 360, 441 Brunish, R., 72, 113 Brunton, D. C., 242, 247 Buchanan, W. D., 368, 437 Buchi, G., 224, 225, 226, 227, 228, 246 Buechley, R. W., 369, 437 Buffett, R. F., 15, 37 Buffleb, H., 324, 444 Bullock, M. W., 158, 186 Bunting, H., 19, 36, 39 Burdette, W. J., 158, 185 Burdick, D., 265, 338, 344, 437 Rurgan, J. G., 261, 262, 294, 314, 319, 320, 321, 382, 383, 411, 433, 436, 449 Burhan, A., 448 Burkard, J., 342, 346, 446 Burnet, F. M., 2, 36 Burnside, J. E., 193, 220, 246 Burow, F. H., 446 Burrows, I. E., 348, 446 Burton, W. W., 336, 349, 453

Busch, H., 43, 44, 45, 46, 48, 49, 50, 52, 55, 56, 58, 65, 70, 72, 73, 74, 77, 78, 82, 85, 86, 87, 90,94, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 116, 116, 117, 119, 122, 186 Butenandt, A,, 158, 185 Butler, G. C., 91, 92, 96, 100, 104, 117, 119 Butler, J. A. V., 43, 54, 73, 77, 78, 86, 89, 92, 94, 96, 97, 98, 100, 103, 105, 108, 109, 111, 114, 116, 116, 117, 118 Buu-Hoi, N. P., 332, 444 Buyske, D. A,, 342, 343, 347, 437 Byers, T. J., 69, l l 4 Byvoet, P., 50, 55, 58, 87, 113, 11.4, 122, 186

C Cabasso, V. J., 29, 37 Cahan, W., 292, 436 Cairn, J., 112, 114 Calkin, J. G., 255, 437 Callan, H. G., 150, 185 Callanan, M. J., 78, 114 Campbell, A., 172, 187 Campbell, J. G., 192, 2-48 Campbell, J. K., 359, 437, 446 Campbell, J. M., 319, 321, 322, 437 Campbell, P. N., 108, 114 Candeli, A., 334, 384, 437, 446 Cardon, J. Z., 382, 383, 436 Cardon, S. Z., 314, 320, 321, 322, 437, 448 Carleton, H. M., 129, 186 Carll, W. T., 193, 220, 246 Carlson, L., 158, 189 Carlton, W. W., 219, 247 Carnaghan, R. B. A., 193, 210, 213, 214, 215, 216, 218, 224, 225, 226, 228, 238, 239, 246 Carpenter, R. D., 255, 456 Carroll, W. R., 78, 114 Carruthers, W., 326, 329, 337, 437, 443 Carugno, N., 315, 329, 437 Caruer, M. J., 62, 114 Caspar, D. L. D., 3, 4, 5, 9, 11, 12, 19, 36, 38 Caspersson, O., 172, 186 Caspersson, T., 122, 127, 172, 174, 185 Ceretto, F., 192, 194, 244, 246

458

AUTHOR INDEX

Chakraborty, M. K., 327, 4% Chalvet, O., 444 Chamberlin, R. I., 371, 461 Chambers, R., 128, 143, 186 Chang, S., 224, 225, 226, 227, 228, 246 Chang, S. C., 289, 302, 43Y Chapell, C. K., 349, 350, 449 Chargaff, E., 52, 55, 62, 80, 82, 91, l l 4 Chase, M., 80, 116 Chauveau, J., 43, 45, 114 Chayen, J., 129, 187 Chen, T. T., 138, 185 Chevallier, M-R., 104, l l Y Chibnall, A. C., 328, @Y Chien, S. C., 99, 100, 111, 113 Chouinard, L. A,, 124, 129, 130, 132, 18Y Christian, L. C., 135, 188 Christofano, E., 290, 444 Christopherson, W. M., 279, 418, 422, 446 Chu, E. H. Y., 142, 186 Chu, E. W., 280, 424, 437 Chushin, S. G . , 314, 320, 439 Cinader, B., 15, 36 Clar, E., 266, 438 Clark, D. A,, 368, 379, 442 Clark, P. C., 273, 296, 373, 417, 436 Clark, P. J., 261, 462 Clayson, D. B., 241, 246, 341, 436, 438 Clemo, G. R., 295, 344, 413, 438 Clever, U., 158, 159, 176, 186 Coates, F. O., Jr., 302, 437 Cobb, J. P., 144, 186 Cogbill, E. C., 262, 308, 366, 369, 370, 371, 438, 446, 45g Cohen, J., 261, 294, 411, 449 Cohn, P., 77, 86, 89, 114 Cole, A., 50, 119 Collins, M. A., 363, 365, 445 Commins, B. T., 313, 337, 438 Condra, N., 462 Connell, G. E., 100, 104, 117 Consbruch, U., 258, 449 Cook, H. A., 92, 93, 96, l l Y Cook, J. W., 313, 314, 315, 318, 354, 455, 438

Cook, L. C., 326, 328, 333, 349, 350, 352, 438, 448, 449

Cooper, E. A., 266, 268, 438 Cooper, N. S., 273, 448 Cooper, R. L., 313, 315, 320, 321, 438

Copeland, D. H., 219, 243, 246, 248 Corlette, S. T., 152, 153, 154, 189 Corley, R. S., 4.36 Cotte, G., 124, 186 Cottone, M. A., 45, 115 Couch, J. F., 337, 446 Cowdry, E. V., 297, 300, 367, 414, 4SS, 451

Cramer, R., 15, 36 Crampton, C. F., 52, 54, 55, 77, 80, 82, 91, 93, 94, 95, 97, 103, 114 Crawford, E. M., 15, 36 Crawford, L. V., 9, 10, 15, 18, 20, S6, S8 Crawford, N., 229, 248 Crayton, F., 253, 438 Creedon, F., 371, 449 Crick, F. H. C., 4, 36 Crider, W. L., 258, @S Croninger, A. B., 251, 261, 266, 267, 269, 271, 272, 273, 274, 275, 297, 300, 310, 367, 399, 400, 401, 405, 406, 414, 438, 441, 461,453 Crosby, A. R., 135, 139, 185 Crosby Longwell, A., 139, 186 Crouse, R. H., 337, 338, 379, 438 Cruff, H. J., 91, 93, 97, 99, 104, 109, 114 Cudkowicz, G., 194, 197, 246 Cummins, J. T., 85, l l Y Cuain, J. L., 259, 267, 297, 314, 320, 321, 329, 376, 378, 384, 403, 416, 438. 461, 444, 451 D Daff, M. E., 368, 369, 438 Dalhamn, T., 301, 302, 306, 307, 385, 438 Dallam, R. D., 63, 64, 114, 119 Dalton, A. J., 14, 24, 25, S6, SY Daly, M. M., 72, 80, 92, 95, 97, 107. 212, 114

Dan, K., 64, 117 D’Angelo, E. G., 151, 185 Daniel, M. R., 370, 441 Danishetsky, I., 362, 397, 44Y Dannenberg, H., 318, @S Darrhun, V., 293, 441 Darkis, F. R., 253, 438 Darlington, C. D., 133, 137, 185 D’Arrigo, D., 144, 186 Das, N. K., 129, 131, 132, 167, 1R.5

AUTHOR INDEX

Daudel, R., 332, 444 Davidson, J. N., 51, 65, 72, 107, 117, 119 Davies, D. F., 438 Davies, H. G., 44, 119 Davies, H. J., 440 Davies, M. C., 29, 37 Davis, J. M. G., 124, 129, 131, 185 Davis, J. R., 43, 44, 45, 73, 77, 78, 90,98, 99, 100, 107, 108, 109, 110, 111, 113,

459

Doolin, P. F., 289, 290, 426, 427, 444 Dorfman, L. M., 319, 439 Dorr, W., 334, 444 Doty, P., 54, 120 Dounce, A. L., 44, 45, 46, 48, 54, 64, 78, 94, 115, 117 Dourmashkin, R. R., 15, 16, 26, 27, 37 Dowd, J. E., 273, 296, 373, 417, 436 Doxtader, E. K., 340, 452 114, 116 Druckrey, H., 258, 263, 265, 276, 277, 286, Davison, P. F., 54, 89, 92, 93, 96, 97, 103, 287, 320, 355, 356, 419, 439, 449 Dukes, C . E., 237, 397, 246, 435 114 Dawkins, A. W., 208, 246 Dulbecco, R., 2, 4, 12, 35, 36, 37, 39 Dawe, C. J., 2, 15, 36, 37 Dumitrescu, M., 308, 451 de Albertini, M., 50, 114 Dunham, L. J., 356, 442 Dearing, W. H., 128, 185 Dunn, C. G., 209, 214, 225, 248 Debov, S. S., 62, 64, 114, 120 Duran-Reynals, F., 1, 27, 37 De Harven, E., 22, 24, 37 Durkan, T. M., 371, &9 de la Burde, R., 253, 438 Duryee, W. R., 150, 185 Delahant, A., 371, 449 Dutton, A., 441 Della Porta, G., 281, 432, 438 Dymicky, M., 265, 327, 349, 439, 450 deLoze, C., 79, 116, 116 E Demisch, R. R., 315, 438 Eckardt, R. E., 370, 439 de Mende, S., 91, 115 Eddy, B. E., 2, 20, 37, 39 Denman, D. T.,330, 442 Edstrom, J-E., 166, 167, 168, 186, 186 Denoix, P. F., 277, 438, 450 Edwards, G. A., 15, 37 Derrick, J. C., 257, 258, 443 Egerton, A., 268, 439 Desjardins, R., 49, 115 Eglinton, G., 328, 439 de Souza, J. E., 319, 320, 382, 449 Eirich, F. R., 362, 447 Deszyck, E. J., 325, 441 Eisenbud, M., 258, 439 de The, G., 26, 38 Eldredge, N. T., 92, 93, 96, 117 Dhom, G., 277, 295, 332, 419, 450 DiBerardino, M. A., 133, 180, 181, 182, Elsdale, T. R., 122, 164, 181, 186 Enders, F. F., 2, 36 185 Dirkens, F., 224, 228, 229, 230, 231, 232, Engbring, V. K., 63, 115 Engel, R. W., 243, 246 246, 352, 354, 438, 439 Engelbreth-Holm, J., 261, 403, 439 Dikun, P. P., 314, 320, 439 Englert, M. E., 29, 37 Dingle, J. T., 370, 441 Enomoto, M., 193, 234, 235, 236, 243, 247 DiPaolo, J. A., 278, 299, 421, 438, 439 Epstein, M. A., 29, 37 Divekar, V. D., 349, 439 Ermala, P., 266, 268, 278, 420, 439, 442 Dmitrieva, N. P., 55, 130 Dmochowski, L., 14, 15, 22, 37, 125, 185 Ermolaeva, L. P., 47, 62, 115 Errea, M., 145, 146, 165, 186, 188 Dobrowsky, A,, 267, 439 Eschenbrenner, A. B., 218, 219, 246 Doll, E. R., 193, 220, 246 Essenberg, J. M., 289, 425, 426, 439, 440 Doll, R., 251, 368, 369, 438, 439 Estable, C., 122, 124, 125, 186 Dollar, A. M., 198, 200, 246 Eulenberg, H., 337, 342, 452 Donelly, A. J., 371, 451 Evans, J. H., 54, 81, 86, 87, 115, 116 Donelly, J. K., 323, 435 Everett, 6 . I,., 271, 398, 403, 417 Dontenwill, W., 291, 356, 428, 434, 439

460

AUTHOR INDEX

F

Fredericq, E., 54, 115 Falk, H. L., 296, 301, 303, 306, 314, 319, Freer, R. M., 45, 115 320, 321, 352, 354, 379, 385, 386, 393. Frenster, J. H., 51, 116 Frey-Wyssling, A., 336, 440 440, 44.3, 444 Friedell, H. L., 285, 440 Falk, R., 173, 186 Friend, C., 24, 87 Fankhauser, G., 134, 186 Fujita, H., 193, 236, 237, 247 Fankuchen, I., 4, 36 Fukuyama, T., 376, 449 Farnham, A. E., 23, 38 Fukuzumi, T., 265, 318, 319, 345, 359, Farr, W. K., 258, 440 447 Fatal, B., 312, 448 Fawcett, D. W., 21, 37 G Febvre, H. L., 15, 26, 27, 36, 37 Gabelya, Yu. O., 342, 440 Feinendegen, L. E., 165, 186 Gaffney, E., 289, 425, 440 Feldman, D., 303, 448 Gall, J. G., 150, 151, 186 Feldman, R. M., 277, 285, 463 Gard, S., 2, 36 Felix, 77, 78, 116 Gardiner, M. S., 122, 186 Fell, M. B., 128, 185 Garfinkel, L., 283, 289, 302, 436 Fenje, P., 29, 39 Garmon, R. G., 371, 452 Ferguson-Smith, M. A., 141, 186 Garner, J. W., 337, 338, 379, 438 Ficq, A., 154, 186 Garner, W. W., 253, 254, 256, 259, 440 Fieser, L. F., 313, 355, 440 Gates, R. R., 122, 186 Fieser, M., 351, 355, 440 Gaulden, M. E., 145, 165, 186 Fikitan, F., 257, 449 Gay, H., 169, 186 Finamore, F. J., 128, 166, 286 Gaylord, W. H., 21, 3Y Finnegan, J. K., 386, 441 Geissmann, T. A,, 337, 440 Firszt, D. C., 43, 70, 113 Fischberg, M., 132, 146, 164, 165, 181, 186 Gelber, D., 17, 39 Gellhorn, A., 286, 298, 299, 405, 440 Fischer, H., 77, 78, 115 George, W. T., 301, 303, 306, 386, 440 Fishel, J. B., 311, 440 Georgiev, G. P., 47, 55, 62, 116, 120, 166, Fisher, H. W., 47, 116 Fisher, M. A., 257, 444 Fisher, R. A., 440 Fitzhugh, 0. G., 204, 246 Flamant, R., 277, 450 Flint, F. A,, 126, 187 Flory, C. M., 266, 440 Fontana, F., 122, 186 Fordyce, W. B., 255, 310, 320, 379, 384, 435

Forgacs, J., 193, 220, 241, $46 Forth, J., 15, 37 Fowler, P. R., 299, 46.3 Fraenkel-Conrat, H., 80, 115 Frajola, W. J., 126, 186 Frank, P., 278, 411, 444 Frankel, 0. H., 126, 186 Frankenburg, W. G., 256, 342, 440 Franklin, R., 4, 37 Franklin, R. M., 112, 116, 144, 188

186

Gerhardt, P. R., 251, 445 Geschwind, I. I., 49, 69, 79, 112 Ghittino, P., 192, 194, 244, 246 Gibbons, A. P., 46, 116 Gibbs, A. J., 11, 39 Gibson, J. E., 362, 446 Gierer, A,, 80, 87, 116 Gilbert, J. A. S., 315, 321, 325, 488, 440 Giles, J. A., 352, 353, 440 Gillette, K. G., 229, 248 Gilman, J. P. W., 370, 440 Giovanella, B. C., 319, 440 Girardet, A., 309, 440 Gladding, R. N., 329, 440 Mock, E., 342, 440 Gloor, U., 328, 449 Gliicksmann, A., 366, 440 Gochenour> A. M., 2, 39

46 1

AUTHOR INDEX

Godman, G. C., 80, 113 Godward, M. B. E., 136, 186 Godwin, J., 220, 221, 222, 223, 224, 248 Golaz, P., 309, 440 Goldberg, M., 6, 7, 37 Goldblatt, M. W., 370, 440, 441 Goldfarb, A. R., 366, 460 Goldhaber, P., 278, 441 Goldstein, H., 251, 446 Goldstein, L., 69, 114, 165, 166, 186 Goldstein, M. N., 125, 144, 186, 187 Gonzales, A. G., 328, 438 Gonzales-Ramirez, J., 125, 127, 186 Goodman, D., 301, 302, 303, 305, 345, 385, 4c53 Gorrod, J. W., 356, 437 Gothoskar, S., 286, 299, 345, 448 Gottlieb, S., 331, 402, 463 Graffi, A., 125, 186, 397, 441 Graham, E. A., 251, 261, 266, 267, 269, 271, 272, 273, 274, 275, 310, 370, 399, 400, 401, 405, 406, 438, 441, 461, 463 Grampp, W., 166, 186 Granboulan, N., 21, 22, 36, 39, 48, 50, 113 Grand, L. C., 242, 247 Green, M., 35, 37 Greenburg, B. G., 279, 422, 447 Greene, C. R., 266, 268, 269, 441 Greene, T. W., 351, 440 Greengard, O., 108, 114 Greenstein, J. P., 78, 116 Greenwood, F. C., 73, 110, 117 Gregoire, J., 92, 97, 116 Greider, M. H., 126, 186 Grener, C., 271, 400, 401, 402, 405, 410, 414, 463 Grey, C. E., 15, 37 Griffith, F., 80, 116 Grimmer, G., 297, 314, 441, 444 Gritsiute, L. A,, 271, 273, 276, 412, 419, 441

Grob, K., 347, 348, 357, 359, 360, 441 Gross, A. L., 359, 437, 446 Gross, L., 2, 14, 24, 37 Grossman, J. D., 325, 349, 441, 460 Grove, J. F., 208, 246' Griinberger, I., 262, 348, 447 Grubbs, G. E., 2, 20, 37, 39 Gukrin, M., 22, 36, 278, 289, 297, 403, 416, 426, 441

Gugan, K., 268, 439 Guillard, M., 267, 438 Guillerm, R., 303, 306, 441 Gulick, A., 62, 64, 117 Guljamov, T. D., 109, 116 Guthrie, F. E., 368, 441 Guvernator, G. C., 111, 359, 441 Gwynn, R. H., 271, 398, 400, 441 Gurdon, J. E., 181, 186

H Haag, H. B., 359, 360, 386, 441, 444 Haagamen, C. D., 22, 39 Haagen-Smit, A. J., 356, 360, 441 Habermann, J., 259, 441 Hackney, E. J., 253, 360, 438, 447 Haddow, A., 191, 246, 354, 370, 441 Hadler, H. I., 293, 441 Haguenau, F., 14, 17, 25, 26, 37 Haley, D., 259, 443 Haley, M. I., 92, 93, 96, 117 Hall, C. E., 5, 9, 11, 36, 38, 170, 190 Halter, C. R., 242, 247 Halver, J. E., 192, 195, 196, 198, 199, 200, 201, 202, 203, 205, 206, 207, 246, 248 Ham, A. W., 2, 15, 35, 38 Hamdan, A. A., 218, 219, 246 Hamer, D., 92, 95, 116, 271, 273, 400 441 Hamilton, M. G., 52, 118 Hamilton, R. J., 328, 439 Hamm, H. R., 446 Hammarsten, E., 55, 116 Hammond, E. C., 283, 289, 302, 436, 441 Hanmer, H. R., 337, 437, 448 Handmaker, S. D., 141, 186 Hannover, R., 59, 118 Hanson, J. B., 85, 116, 118 Haque, A., 137, 186 Hara, J., 356, 360, 441 Hardcastle, J. E., 257, 361, 442 Hardy, H. L., 371, 4661 Harlan, W. R., 337, 437, 446, 448 Harlow, E. S., 262, 263, 266, 268, 269, 308, 347, 359, 360, 441, 443, 462 Harper, H. A., 95, 117 Harris, H., 47, 116, 130, 186 Harris, J. I., 4, 38 Harris, J. L., 170, 189 Hartwell, J. L., 327, 328, 332, 333, 340, 341, 349, 441, 460

462

AUTHOR INDEX

Hashimoto, C., 78, 113 Hart, R. G., 12, 38 Hartley, J. W., 14, 38 Hartman, F. W., 229, 247 Hauschka, T., 230, 247 Hausermann, M., 260, 310, 373, 378, 452 Haun, C., 452 Hauser, T. R., 313, 449 Hayek, H., 307, 441 Haynes, L. J., 224, 847 Heath, D. F., 441 Heath, J. C., 370, 441 Heidelberger, C., 319, 440 Heine, L. H., 367, 441 Heine, U., 26, 38, 125, 186 Heitz, E., 130, 132, 135, 141, 186 Hell, A,, 145, 146, 186, 188 Hellerstroem, S., 326, 441 Hellier, D. N., 314, 318, 448 Helmcke, H.-J., 125, 186 Heming, H. G., 208, 246 Hendry, J. A., 229, 248 Henschler, D., 356, 441, 449 Herring, A. S., 193, 246 Herrmann, K., 335, 442 Herrold, K. McD., 280, 356, 424, 434, 437, 442 Hershey, A. D., 80, 116 Hertl, M., 122, 186 Herzberg, K., 13, 38 Hew, H. Y. C., 85, 113 Hewett, C. L., 313, 354, 435, 438 Hichens, M., 99, 116 Hidvegi, E. J., 110, 115 Hieger, I., 313, 438 Higginson, J., 240, 247 Higman, H. C., 339, 447 Hilding, A. C., 288, 301, 442 Hill, A. B., 251, 439 Hilleman, M. T., 20, 39 Hindley, J., 91, 93, 97, 114 Hinreiner, E., 337, 440 Hirose, T., 282, 283, 292, 434, 448 Him, C. H. W., 105, 116 Hirst, E. L., 266, 438 Hirst, G. K., 2, 36 Hitchcock, R., 314, 320, 321, 322, 437 Hjern, L., 368, 369, 442 Hlinka, J., 297, 445

Hnilica, L. S.,43, 47, 55, 58, 74, 77. 83, 84, 85, 94, 99, 100, 101, 102, 103, 101, 105, 106, 111, 113, 114, 116 Ho, R., 68, 213 Hobbs, M. E., 257, 342, 343, 344, 347, 352, 357, 358, 359, 360, 361, 366, 369, 370, 371, 381, 437, 488, 447, 448, 452 Hoch-Ligeti, C., 356, 435 Hoffmann, D., 257, 258, 262, 264, 273, 275, 280, 287, 293, 294, 295, 296, 297, 298, 301, 302, 303, 305, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320, 321, 322, 326, 327, 328, 330, 332, 333, 334, 337, 338, 339, 340, 341, 344, 345, 364, 372, 373, 374, 378, 379, 380, 381, 382, 383, 384, 385, 386, 410, 414, 415, 417, 423, 437, 442, 463 Hogeboom, G. H., 45, 57, 58, 116, 118 Holbrook, D. J., Jr., 54, 81, 86, 87, 109, 115, 116, 118 Holland, R. H., 289, 368, 369, 379, 428, 449 Hollender, A. J., 297, 446 Hollman, K., 17, 38 Holmes, J. C., 257, 264, 361, 446 Holoubek, V., 86, 116 Holsti, L. R., 266, 268, 278, 328, 420, 442 Holsti, P., 345, 442 Homburger, F., 272, 274, 301, 303, 417, 436,442 Homer, R. F., 229, 248 Honig, G. R., 108, 109, 110, 118, 114, 116 Hopkins, J . W., 60, 116 Horn, E. C., 69, 80, 116 Horne, R. S., 3, 5, 39 Horne, R. W., 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, 15, 23, 28, 29, 36, 38, 39, 40 Homing, E. S., 237, 370, 846, 441 Horstmann, E., 130, 186 Horstmann, H., 267, 309, 314, 446 Horton, A. W., 330, 349, 442 Horowite, M., 289, 425, 440 Hoskins, G. C., 149, 186 Hotta, Y., 65, 117 Hou, L. T., 284, 442 Hou, W., 126, 188 Houlan, K. G., 347, 462 Howatson, A. F., 2, 6, 9, 10, 11, 15, 16. 18, 19, 20, 26, 28, 29, 35, 36, 38, 40

463

AUTHOR INDEX

Hudack, E. D., 46, 116 Hiibner, E., 297, 444 Huebner, R. J., 21, 38 Hueper, W. C., 194, 195, 197, 200, 24Y, 251, 354, 359, 368, 370, 396, 442 Huggins, C., 242, 24Y Hughes, I. W., 255, 310, 320, 379, 384, 435 Huisgen, R., 355, 443 Humphrey, R. R., 134, 146, 185, 186, 189 Hunt, V. R., 365, 448 Hunter, C. G., 204, 2'4Y Hurlbert, R. B., 46, 119 Huxley, A. H., 127, 189 Huxley, H . E., 5, 11, 38 FIyde, B. B., 50, 116 Hpmer, W. C., 47, 116

Jacob, J., 135, 177, 1W,189 Jaffe, A. A., 361, 362, 436 Jakowska, S., 194, 247 James, D. W. F., 89, 92, 114 Jarboe, C. H., 333, 341, 445 Jenkins, F. P., 192, 213, 216, 217, 218, 221, 231, Z4Y Jensen, C. O., 256, 259, 443 Johansen, D. A., 126, 1W Johns, E. W., 43, 78, 89, 94, 96, 98, 99, 100, 103, 104, 105, 111, 115, 116, 113 Johnson, C. L., 195, 200, 201, 203, 246, 247 Johnston, H., 314, 446 Johnstone, R. A. W., 252, 315, 318, 326, 329, 333, 335, 337, 338, 341, 349, 356, 357, 359, 360, 43Y, 443 Jones, L. A., 346, 443 Jones, H. E. H., 108, 114, 224, 228, 229, 230, 231, 232, 246, 354, 438, 439 Jordan, L. E., 9, 38 Joseph, C. A,, 332, 333, 334, 452 Journey, L. J., 125, 144, 187 Jukes, T. H., 204, 24Y Jull, J. W., 241, 246, 341, 436

I

K

Hoyle, L., 13, 38 Hsc, T. C., 50, 119, 126, 155, 174, 186, 187, 189 Hsiung, G. O., 21, 37 Huang, R. C., 43, 82, 83, 84, 91, 104, 113, 116 Hubert-Habart, M., 314, 320, 321, 438,

444

Kahler, H., 14, 17, 38, 258, 443 Ikeda, R. M., 325, 441 Kahn, J., 134, 18Y Ikegami, R., 193, 236, 237, 24Y Kaiser, H. E., 301, 302, 303, 305, 345, 385, Imanishi, M., 193, 236, 237, 247 453 Ingelstedt, S., 269, 443 Kajland, A., 438 Ingram, D. J. E., 362, 363, 443, 445 Kakhiani, Z. H., 271, 399, 443 Inone, T., 128, 187 Kallianos, A. G., 351, 443 Irby, R. M., 263, 347, 359, 360, 443 Kallner, G., 275, 448 Irvin, E. M., 109, 116, 118 Kaplan, N. M., 2, 36 Irvin, J. L., 54, 81, 86, 87, 100, 109, 115, Kaplan, W. D., 133, 138, 141, 142, 143, 11Y, 118 188 Ishigura, H., 26, 38 Karlson, P., 158, 185, 187 Ishii, S.-I., 78, 113 Karney, D. H., 144, 189 Ishiko, T., 193, 234, 235, 236, 243, 24Y Kass, S. J., 11, 17, 18, 40 Isler, O., 328, 449 Kato, K. I., 177, 189 Ivankovic, S., 356, 439 Katz, M., 198, 200, 204, 246 Iwai; K., 78, 115 Kaufmann, B. P., 132, 133, 153, 18Y Izawa, M., 266, 309, 337, 343, 344, 359, Kaushiva, B. S., 126, 187 443 Kawade, Y., 54, 116 Kawai, A., 193, 236, 237, 247 J Kawamata, J., 193, 236, 237, 246 Jacob, F., 4, 58, 42, 116, 171, 172, 173, Kay, E. R. M., 48, 1lY Keir, H. M., 43, 57, 58, 116 175, 176, 187

464

AUTHOR INDEX

Keith, C. H., 257, 258, 260, 309, 310, 348, 374, 375, 377, 443, 446 Kelley, A. R., 229, 247 Kendrew, J. C., 170, 187 Kennaway, E. L., 354, 368, 369, 436, 438, 443

Kennaway, N. M., 354, 435 Kensler, C. J., 243, 247, 272, 274, 301, 303, 360, 386, 418, 443 Kent, P. W., 99, 116 Kenyon, O., 259, 443 Khanolkar, V. R., 257, 264, 285, 286, 299, 345, 349, 421, 439, 443, 448 Khouvine, Y., 54, 91, 116, 116 Kilham, L., 33, 38 Kimber, R. W. L., 323, 435 Kimura, K., 166, 169 Kimura, M., 78, 113 Kincaid, J. F., 371, 461 King, T. J., 149, 179, 180, 181, 182, 185, 187

Kingdon, K. H., 361, 362, 443 Kinosita, R., 133, 138, 141, 142, 143, 243, 188

Kipriyanova, G. I., 342, 440 Kirby, K. S., 70, 116, 340, 443 Kirk, R., 286, 412, 446 Kiriyama, M., 124, 190 Kirkham, W. R., 63, 64, 119 Kirschner, L. B., 68, 93, f l 3 Kissling, R., 259, 443 Kit, S., 66, 116 Klamerth, O., 79, 116 Klein, M., 293, 443 Klein, U., 288, 297, 443, 444 Kleinfeld, R. G., 143, 187 Kleinschmidt, A., 13, 38 Klug, A., 3, 4, 5, 9, 11, 12, 20, 36, 38 Kluss, B. C., 58,118 Klyszejko, L., 54, 91, 116, 116 Knight, C . A., 4, 11, 17, 18, 38, 40 Knight, G. R., 86, 119 Knobloch, A., 104, 117 Knopp, A,, 130, 186 Knudtson, K. P., 302, 443 Kobashi, Y., 266, 309, 337, 343, 344, 359,

443 Kobayashi, J., 144, 187 Kobayashi, Y., 234, 235, 243, 247 Kobel, M., 345, 443, 446

Koch, H., 241, 246 Kodani, M., 141, I M Koenig, P., 334, 444 Koerbler, J., 278, 411, 444 Kofler, M., 328, 449 Kohen, E., 43, 70, 113 Kolb, J. J., 92, 113 Kolb, L., 281, 432, 438 Komczymski, L., 289, 426, 444 Konikova, A. S., 109, 116 Kopac, M. J., 49, 116, 122, 123, 125, 127, 128, 147, 148, 149, 150, 151, 171, 187, 188

Kopf, P., 372, 373, 402, 453 Koprowska, I., 280, 423, 436, 444 Kornberg, A., 57, 113 Korpassy, B., 340, 444 Kosak, A. I., 259, 261, 271, 282, 314, 320, 329, 344, 348, 406, 431, 444, 447, 448, 452

Kossel, A., 77, 80, 90, 95, 116 Kotin, P., 258, 296, 299, 301, 303, 306, 314, 319, 320, 321, 352, 354, 379, 385, 386, 393, 440, 442, 444, 452 Koulish, S., 143, 187 Koslowski, E. J., 289, 428, 442 Kracht, J., 297, 444 Kraus, G. E., 387, 449 Kraybill, H. F., 202, 203, 204, 215, 241, 242, g47, 848 Krekels, A., 77, 78, 115 Kreshover, S. J., 277, 279, 300, 399, 405, 420, 444, 449 Kritzman, M. G., 109, 116 Krivshenko, J., 139, 187 Kroeger, H., 149, 158, 161, 162, 163, 176, 178, 179, 187 Kroger, R., 288, 446 Krueger, A. P., 362, 444 Kuhle, E., 119 Kuhn, H., 262, 348, 370, 644, 447 Kummerow, F., 200, 248 Kupke, D. W., 92, 93, 96, 117 Kuratsume, M., 311, 314, 444 Kuschner, M., 282, 290, 431, 444, 448 Kutosumi, K., 125, 187 Kutecher, F., 77, 116

L LaBelle, C. W., 288, 487, 443

465

AUTHOR INDEX

Lacassagne, A., 332, 444 LaCour, L. F., 129, 137, 187 Lafontaine, J. G., 124, 129, 130, 131, 132, 187

Lam, J., 265, 266, 273, 324, 325, 376, 377, 378, 379, 380, 407, 408, 410, 444 453 Lamb, F. W. M., 266, 438 Lamb, W. G. P., 51, 116 Lammers, W., 238, 247 Lancaster, M. C., 192, 213, 216, 217, 218, 221, 231, 247 Lane, W. T., 21, 38 Lang, D., 13, 38 Lang, H., 107, 116 Lang, K. F., 57, 107, 116, 119, 324, 444 Langer, G., 257, 444 Langemann, A., 328, 449 Langseth, L., 354, 452 Lanz, H. C., 369, 379, 442 LaRoche, G., 200, 201, 203, 247 Larson, C. P., 192, 194, 195, 248 Larson, P. S.,359, 360, 386, 441 Lasgargues, E. Y., 22, 39 Laskin, S., 290, 444 Laskowski, M., 63, 115 Lasnitzki, I., 284, 444 Lassiter, C. W., 329, 330, 460 Latarjet, R., 314, 320, 321, 438, 444 Latimer, P. H., 328, 349, 350, 449 Laurence, D. J. R., 73, 78, 103, 108, 109,

Le van Thoi, 329, 438 Levin, E., 63, 117 Levin, M. L., 251, 445 Lewis, E. B., 183, 188 Lewis, G. E., 271, 398, 447 Lewis, J. S., 361, 446 Lezzi, M., 178, 188 Liau, M., 48, 114 Lieser, R. C., 260, 261, 446 Liyinsky, W., 314, 445 Lillick, L., 109, 113 Lima-de-Faria, A., 173, 188 Limozin, M., 92, 97, 115 Lin, M., 129, 138, 139, 188 Linde, J. A., 238, 247 Lindner, A., 86, 117 Lindsey, A. J., 313, 315, 319, 320, 321, 322, 325, 334, 337, 348, 379, 384, 437, 438, 440,

443, 445

Lindroth, H., 361, 462 Lipp, G., 374, 375, 445 Lipsey, A., 43, 70, 113 Lipshitz, R., 52, 55, 80, 82, 91, 114 Lisco, H., 366, 450 Litt, M., 48, 117 Littau, V . C., 43, 52, 60, 83, 84, 113, 118 Liu, T, T., 155, 186 Lloyd, B. J., 14, 17, 38, 258, 443 Lloyd, L., 150, 185 Lockwood, K., 277, 445 Logan, R., 51, 65, 117 114, 116 LoGrippo, G., 229, 247 Lavit-Lamy, D., 444 Loosmore, R. M., 214, 247 Law, L. W., 2. 37 Lax, R. E., 351, 443 Lorentzen, G., 338, 445 Leaver, J. L., 104, 114 Lopez, G., 351, 440 Lou, T. Y., 126, 187 Leavitt, A. M., 289, 44V Love, R., 129, 188 LeBlond, C. P., 166, 187 Lowe, D., 208, 246 Lee, K., 293, 441 Lucius, S., 57, 107, 116, 119 Lehman, I. R., 57, 113 Luck, J. M., 72, 92, 93, 96, 113, 117, 118 Leidl, E., 374, 445 Lucy, J . A,, 89, 92, 117 Lellouch, J., 277, 45V Ludwig, E., 337, 445 Lenormant, H., 79, 116 Luibel, F. J., 125, 184 Leslie, I., 80, 116, 117 Lupberger, A., 271, 400, 401, 402, 405, Lettrk, H., 46, 47, 117 410, 414, 453 Lettrk, R., 125, 127, 188 Leuchtenberger, C., 289, 290, 299, 426, LwoK, A,, 2, 4, 12, 36, 38 Lyon, M. F., 173, 188 427, 428, 444, 445 Leuchtenberger, R., 289, 290, 299, 426, Lyons, M. J., 296, 314, 319, 320, 362, 363, 364, 445, 453 427, 428, 444,446 Lyons, M. I,.,23, 24, 38 Levaditi, J., 194, 247

166

AUTHOR INDEX

M McAllister, H. C., 81, 86, 100, 116, 117 McCall, M. S., 368, 369, 379, 442 McCalla, T. M., 229, 247 McCammon, C. J., 354, 444 McCants, C. B., 368, 441, 450 McConnell, W. V., 445 McCarty, M., 80, 113 McCIintock, B., 123, 130, 131, 132, 134, 147, 164, 172, 173. 188 McCulloch, E. A., 2, 15, 35, 38 McDowell, P. E., 347, 449 MacGillivray, A. J., 73, 110, 117 McIlwain, H., 85, 117, 120 Mclndoe, W. M., 72, 107, 119 McKernan, W., 225, 248 McKee, H. C., 359, 445 Maclean, E. C., 11, 38 McLeish, J., 137, 188 MacLeod, C. M., 80, 115 McMaster-Kaye, R., 165, 188 McNally, P. N., 269, 445 MacPherson, I. A., 15, 40 McRae, M. T., 347, 445 Ma, S. C., 126, 188 Magadia, N. E., 280, 465 Magee, L. A,, 15, SY Magee, P. N., 355, 445 Maggio, R., 48, 117 Mahlandt, B. G., 193, 246 Mallette, F. S., 327, 446 Mann, J., 273, 380, 401, 465 Mantel, N., 277, 453 Mantieva, V. A., 63, l l Y Mantieva, U. L., 47, 62, 115 Mantieva, V. L., 166, 186 Marek, J., 262, 444 Marko, A. M., 91, 119 Markson, L. M., 214, 247 Marsden, E., 363, 365, 445 Martin, A. P., 46, 118 Martin, I., 347, 348, 445 Martin, J. B., 271, 398, 403, 447 Martin, R. H., 354, 455 Mason, G., 314, 445 Mateyko, G. M., 49, 116, 122, 125, 127, 128, 147, 148, 187, 188 Matsuura, H., 132, 138, 188 Mattern, C. F. T., 9, 10, 20, 58

Mattingly, A., 86, 117 Mauritzen, C. M., 77, 91, 93, 97, 99, 109, 114, i i r Mavioglu, H., 48, 55, 58, 74, 77, 85, 102, 105, 114 May, R . M., 284, 436 Mayer, D. T., 62, 63, 64, 117, 119 Mayer, E., 282, 429, 448 Mayneord, W. V., 366, 445 Mayor, H. 0..9, 20, 58 Mazia, D., 62, 64, 113, 117 Means, R. E., 329, 351, 443, 445 Mechelke, F., 154, 155, 188 Medina, D., 2, 89 Meissner, H. U., 255, 445 Meitus, R. J., 228, 248 Mellow, R. C., 297, 446 Melnick, J. L., 9, 18, 19, 20, 29, 38, 39 MendenhaI1, W. L., 301, 445 Metz, C. W., 135, 188 Meyer, G. F., 150, 188 Meyer-Abich, K. M., 310, 445 Micou, J., 165, 166, 186 Middleton, F. C., 271, 398, 403, 447 Mihama, K., 259, 451 Miller, A. J., 279, 406, 418, 420, 422, ,456 Miller, E. C., 218, 219, 246, 247 Miller, E. W., 295, 413, 438 Miller, H. E., 359, 448 Miller, J. A., 218, 219, 247 Miller, J. E., 261, 374, 445 Miller, M. W., 237, 247 Miller, R. L., 265, 337, 437 Mims, S. S., 326, 328, 4 9 Mironova, A. I., 271, 273, 276, 412, 419,

441 Mirsky, A. E., 43, 47, 50, 51, 52, 57, 60, 61, 62, 65, 72, 80, 83, 84, 92, 95, 97, 107, 112, 113, 114, 115, l l Y 118, 119 Mitchell, E. R., 78, 114 Mitchell, R. I., 257, 259, 361, 449, 446 Mitchley, B. C. V., 356, 397, 436, 437 Miyake, M., 193, 234, 235, 236, 243, 24Y Mixutani, M., 284, 285, 445, 446 Mody, J. K., 264, 421, 446 Mohr, U., 291, 356, 428, 434, 439 Mold, J. D., 329, 347, 351, 443, 445 Moldenhauer, W., 379, 448 Molinari, E., 346, 446

AUTHOR INDEX

Moloney, J. B., 24, 25, 37, 39 Monod, J., 42, 116, 171, 172, 173, 175, 176, 187 Montgomery, P. O’B., 144, 145, 149, 188,

467

Nakagawa, Y., 335, 337, 453 Nakanishi, Y. H., 284, 446 Navaschin, M., 134, 188 Naylor, J. M., 153, 154, 158, 189 189 Nazimoff, O., 194, 247 Montgomery, T. H., 122, 128, 188 Neelin, E. M., 89, 96, 100, 104, 117, 118 Monty, K. J., 45, 48, 115, 117 Neelin, J. M., 89, 100, 104, 117, 118 Moore, C . , 279, 418, 420, 422, 446 Negroni, G., 15, 16, 37 Moore, D. H., 17, 18, 22, 23, 24, 38, 39 Nelson, A. A,, 204, 246 Moore G. E., 261, 262, 272, 273, 278, 285, Nelson, N. L., 271, 290, 320, 352, 354, 295, 296, 300, 301, 367, 372, 373, 408, 406, 444, 447, 452 417, 421, 436, 499 Nesbitt, B., 214, 225, 226. 247 Moore, H., 360, 406, 447 Neubauer, O., 368, 446 Moore, S., 93, 94, 95, 97, 103, 104, 106, Neuberg, C., 342, 345, 346, 443, 4.66 114, 115, 119 Neukomm, S., 265, 297, 314, 320, 418, 436, Moree, S., 329, 438 446 Morrell, F. A., 348, 462 Neurath, G., 267, 288, 309, 314, 338, 445, Morris, D., 369, 442 446 Morris, M. D., 95, 117 Newberne, P. M., 219, 220, 243, 247 Morris, R. M., 264, 446 Newman, W., 366, 435 Morrow, P. E., 288, 446 Newsome, J. R., 260, 309, 310, 348, 374, Mortarotti, T . G., 340, 452 375, 377, 443, 446 Moseley, J. M., 446 Nicod, J. L., 378, 418, 446 Mottram, J. C., 366, 446 Nicol, H., 370, 371, 452 Mode, Y., 43, 45, 114 Nielson, R. S., 196, 247 Mouron, J. C., 265, 446 Niemejer, J. A., 91, 1lY Muhlbock, O., 289, 425, 446 Niessen, H. J., 356, 360, 436 Muller, F. H., 251, 446 Nigrelli, R. F., 194, 247 Muller, M., 355, 356, 439 Nilson, H. W., 202, 203, 241, 247 Muller, R., 265, 266, 379, 446, 448 Nishibori, A., 236, 247 Muel, B., 314, 320, 321, 438, 444 Niven, J. S. F., 32, 39 Muir, C . S., 286, 412, 446 Nixon, C.W., 303, 436 Muller, E., 57, 118 Nixon, H. L., 11, 39 Muller, L., 57, 119 Noguchi, Y., 193, 234, 235, 243, 247 Mumpower, R. C . 11, 266, 267, 268, 361, Noll, H., 87, 180 Nooij, E. H., 91, 1i7 445, 446, 451 Muramatsu, M., 43, 44, 48, 49, 86, 87, Norman, V., 348, 446 i14, 117 Norris, G. L. F., 208, 246 Murray, K., 96, 100, 104, 117, 118 Norris, M. S., 330, 446 Murray, M. R., 22, 39 Norstadt, F. A., 229, 247 Muth, F., 263, 265, 276, 277, 285, 287, Nothdurft, H., 397, 446 320, 380, 381, 419, 439, 446 Novack, R. M., 218, 219, 246 Novotny, J., 323, 436 N Nunez, B. A,, 127, 186 Nagasawa, M., 265, 318, 319, 345, 446, Nyari, E., 260, 310, 452 Nyhan, W. L., 43, 70, 108, 109, i13, f l 7 , 447 119 Naghski, J., 337, 446 Nagington, J., 13, 99 0 Nair, P. V., 108, 109, 113 Nakabayashi, N., 193, 236, 237, 247 Oberling, C., 22, 26, 96. 122, 188

468

AUTHOR INDEX

O’Connor, J. G., 330, 446 O’Donnell, E. H. J., 125, 136, 188 Oettle, A. G., 240, .947 ogg, C. L., 308, 446 Ohno, S., 133, 135, 138, 141, 142, 143, 188

Ohnuki, Y., 285, 4% Okamura, N., 90, 119 O’Keeffe, A. E., 260, 261, 446 O’Kelly, J., 193, 210, 214, 218, 225, 226, 247

Olah, L. V., 128, 129, 131, 136, 188 Onderdonk, J., 277, 453 Q’Neill, H. J., 337, 338, 379, 438 Onishi, I., 265, 318, 319, 337, 342, 345, 359, 446, 447 Oppenheimer, B. S., 362, 397, 447 Oppenheimer, E. T., 362, 397, 447 Orr, I. M., 285, 447 Orris, E., 352, 354, 458 Orris, L., 271, 320, 354, 406, 447, 45.9 Osawa, S., 60, 65, 112, 113, 117 Osborne, J. S., 347, 359, 360, 361, 381, 447 Oser, B. L., 204, 241, 848 Oser, M., 204, 241, 248 Osman, S., 339, 447 Ostler, D. C., 216, 248 Otto, H., 291, 429, 430, 447 Owen, L. H., 346, 437

P Paget, G. E., 213, 248 Pailer, M., 262, 348, 370, 447 Painter, R. B., 165, 186 Pallade, G., 48, 117 Palmade, C., 104, 117 Palmes, D., 352, 354, 452 Pame, T. B., 349, 439 Pardee, A. B., 172, 188 Parmele, H. B., 256, 443 Parsons, D. F., 23, 25, 26, 33, 36, 39 Passey, R. D., 271, 398, 403, 447 Pate, S.,45, 115 Pavan, C., 154, 155, 156, 184, 186, 188 Pavlu, J., 368, 447 Payne, W. W., 194, 195, 197, 200, 247, 354, 368, 370, 442 Peacock, E. E., Jr., 279, 422, 447 Peacock, P. R., 263, 289, 431, 447 Prclchv, R. D. G , 465,184

Peacocke, A. R., 78, 117 Pedersen, P. M., 324, 448 Peirce, W. E. H., 449 Pelling, C., 165, 188 Penn, P. T., 253, 337, 447 Pereira, H. G., 32, 39 Perevoschchikova, K. A., 57, 120 Perone, V. B., 228, 248 Perry, R. P., 145, 146, 165, 166, 186, 188, 189

Persaud, K., 334, 384, 437, 445 Perugnini, S., 78, 117, 118 Peterman, M. L., 77, l i d Peters, D., 13, 27, 39 Petersen, E. L., 308, 446 Pfendt, E., 144, 186 Pfyl, B., 259, 447 Philippe, R. J., 347, 357, 358, 359, 360, 381, 447 Phillips, A. M., 302, 447 Phillips, R. W., 302, 447 Phillips, D. M. P., 43, 55, 77, 78, 84, 89, 94, 96, 100, 103, 104, 105, 106, 111, 116, 118

Philp, J. McL., 192, 213, 216, 217, 218, 221, 231, 24Y Pianese, G., 122, 188 Pierce, K. R., 220, 221, 222, 223, 224, 248 Pietzsch, A., 266, 268, 314, 318, 447 Pinteric, L., 29, 89 Piper, S. H., 329, 437 Platt, D. B., 69, 114 Plimmer, J. R., 252, 333, 335, 338, 341, 349, 356, 357, 359, 360, 443 Plowright, W., 13, 39 Pogo, A. O., 52, 118 Pogo, B. G. T., 52, 118 Pogosova, A. V., 109, 116 Pollard, E. C., 22, 89 Pollister, A. W.. 52, 61, 117 Polydorova, M., 259, 447 Pollard, A., 329, 437 Pomerat, C. M., 144, 188, 284, 285, 435, 446

Pontag, J., 259, 448 Popper, H., 204, 248 Poort, C., 46, 118 Porter, R. R.. 77, 89, 118 Pott, P., 250, 448 Potter, V. R., 68, 118

469

AUTHOR INDEX

Poulson, D. F., 135, 188 Powell, H. M., 4, 39 Prescott, D. M., 58, 69, 86, 118 Prestidge, 172, 188 Preussmann, R., 355, 356, 439 Price, C. C., 70, 119 Price, W. C., 79, 113 Prickett, C. O., 219, 220, 243, 248 Proehl, E. C., 285, 446 Proetz, A,, 301, 447 Purdy, S. J., 328, 448 Puschmann, H., 359, 447 Pyriki, C., 266, 320, 379, 448

Q Quan, P. M., 315, 318, 326, 443 Quass, F. W., 239, 248 Quilligan, J., 301, 303, 456 Quin, L. D., 333, 344, 352, 436, 448 Quinn, A. D., 341, 443

R Radford, E. P., 365, 448 Radley, J. M., 364, 365, 451 Rains, A, J. H., 229, 248 Rakieten, M. L., 303, 448 Rakieten, N., 303, 448 Rakower, J., 275, 312, 448 Ralls, J. W., 346, 347, 448 Ranadive, K. J., 264, 286, 299, 345, 421, 446, 448 Rand, H. J., 314, 320, 321, 322, 437, 448 Randt, 8., 125, 186 Rapoport, E. A., 109, 116 Rao, P. R., 269, 300, 411, 448 Raphael, R. A,, 328, 439 Rasch, E., 104, 118 Rasmussen, P. S., 92, 93, 96, 117, 118 Rathkamp, G., 257, 275, 311, 312, 322, 338, 339, 374, 442 Rayhurn, C. H., 324, 337, 448 Reddy, B. D., 269, 300, 411, 448 Reddy, D. G., 269, 300, 411, 448 Reeves, A. L., 371, 452 Regamey, R., 309, 440 Reid, W. W., 314, 319, 336, 337, 448 Reich, E., 144, 188 Rekemeyer, M. L., 147, 164, 165, 189 Remington, R. E., 368, 448 Remy, H., 371, 448

Rendi, R., 60, 118 Resende, F., 137, 188 Resnik, F. E., 348, 359, 436, 462 Revere, A., 258, 440 Reuss, K., 13, 38 Reynaud, J., 92, 97, 116 Reynolds, R. C., 144, 188, 189 Rhoads, C . P., 242, 247, 273, 448 Rhoades, J. W., 359, 437, 445 Rice, R. L., 319, 320, 382, 449 Rich, A., 170, 190 Richards, J. C., 261, 374, 375, 460 Richardson, J., 204, 247 Richardson, L. R., 211, 220, 221, 222, 223, 224, 248 Richter, W., 255, 446 Ricken, W., 277, 295, 332, 419, 460 Rigdon, R. H., 432, 448 Ris, H., 50, 51, 62, 72, 80, 95, 114, 117, 124, 186 Ritchie, A. C., 366, 460 Ritossa, F., 158, 189 Rivenbark, W. L., 85, 118 Rivera, J. A,, 345, 360, 362, 367, 448 Rivers, T. M., 1, 39 Roberts, D. C., 229, 248 Robinson, A. M., 354, 456 Robinson, J., 204, 247 Rocchietta, S., 314, 448 Rockey, E. E., 282, 283, 292, 431, 434, 448 Rockstroh, H., 370, 448 Rodgman, A., 326, 328, 333, 349, 350, 352, 448, 449 Rodkiewicz, B., 125, 189 Roe, E. M. F., 271, 398, 403, 413, 447 Roe, F. J. C., 229, 648, 261, 294, 298, 356, 411, 417, 437, 449 Roffo, A. H., 259, 445 Roodvn, D. B., 44, 45, 118 Roof, B. S., 47, 89, 118 Rose, F. L., 229, 248 Rosenherg, S., 255, 449 Rosene, C. J., 333, 443 Rosenthal, L. M., 285, 440 Ross, C. A., 387, 449 Ross, J. H.. 346, 449 Ross, W., 356, 441, 449 Rotherham. J., 109, 118 Rothman. N.. 158. 189 >

,

470

AUTHOR INDEX

Rouiller, C., 43, 45, 114 Rowe, W. P., 14, 21, 38 Rowland, R. L., 336, 449 Rowlette, W., 301, 306, 440 Royer, R., 314, 320, 321, 438, 444 Rubin, H., 34, 39 Ruch, F., 289, 299, 428, 445 Rucker, R. R., 197, 248 Ruckerbauer, G., 370, 440 Rudkin, G. T., 152, 153, 154, 189 Ruegg, R., 328, 449 Runeckles, V. C., 336, 365, 449 Rupp, J. J., 351, 440 Rusaniwskyij, W., 265, 329, 349, 450, 451 Russell, G. E., 12, 38 Russell, W. C., 5, 9, 40 Ruth, J. M., 329, 445 Rylander, R., 438 Ryser, G., 328, 449

Satterlee, H. S., 368, 369, 449 Sauer, L. A., 46, 118 Sautihre, P., 100, 113 Savatinova, I., 320, 435 Sawada, T., 124, 190 Sawicki, E., 313, 354, 442, 449 Scala, O., 289, 449 Scanes, F. S., 78, 118 Scasselati-Sforzolini, G., 320, 449 Scheel, L. D., 228, 248 Scheparte, A. I., 261, 310, 347, 449 Schepers, G. W. H., 371, 449 Scherbak, M., 319, 320, 382, 449 Schildbach, A., 276, 4% Schleiden, M. J., 122, IS9 Schlotzhauer, W. S., 344, 449 Schmahl, D., 258, 263, 265, 276, 277, 286, 287, 320, 355, 356, 419, 439, 449 Schmallfuss, H., 348, 449 Schmeltz, I., 265, 338, 344, 437, 449 Schmeltz, J., 338, 447 Sabin, A. B., 28, 39 Schmitt, F. L., 271, 320, 326, 352, 354, Sachs, L., 2, 33 406, 447, 462 Saetren, H., 47, 57, 112, 129 Schmitt, O., 259, 447 Saffiotti, U., 345, 368, 436, 449 Schneider, W. C., 45, 57, 58, 59, 116, 118 Schoental, R., 213, 216, 217, 238, 2.48, 355, Sahai, P. N., 329, 437 Saito, M., 234, 235, 236, 243, 247 449 Schor, N., 166, 186 Sakaguchi, S., 266, 309, 337, 359, 443 Schramm, G., 80, 115 Sakai, F., 193, 234, 235, 236, 243, 247 Schroder, R., 376, 460 Sakai, V., 234, 235, 236, 247, 376, 449 Salaman, M. H., 229, 248, 261, 271, 294, Schultz, J., 122, 140, 141, 158, 172, 174, 186, 189 400, 411, 441, 449 Schiirch, O., 348, 450 Sakata, S., 257, 449 Schumacher, J. N., 352, 353, 440 Saldi, G., 320, 449 Schur, M. O., 261, 374, 375, 460 Salfelder, K., 354, 450 Salley, J. J., 277, 279, 300, 405, 420, 4$4, Schwartz, D., 277, 438, 450 Scolari, C., 194, 197, 246 449 Scott, T. S., 340, 450 Salmon, W. D., 219, 220, 243, 246 Samarina, 0. P., 46, 52, 109, 118, 120, Scrimshaw, N. S., 209, 214, 225, 248 Searle, C. E., 352, 354, 460 166, 186 Seehofer, F., 266, 269, 309, 376, 436, 44.9 Sanders, E., 125, 184, 266, 438 Seelkopf, C., 266, 277, 295, 313, 320, 332, Sanger, F., 105, 118 354, 419, 450 Sano, I., 257, 449 Sargeant, K., 193, 210, 214, 218, 225, 226, Segelken, D., 376, 450 Serra, J. A,, 124, 189 228, 246 Setterfield, G., 89, 104, 118 Sargent, S., 364, 437 Shabad, L. M., 288, 367, 450 Sarkisov, A. K. H., 193, 948 Shaffer, C. B., 204, 247 Sasaki, T., 243, 248 Shaffer. P., 289, 290, 426, 427, 428, 444, Satake, K., 93, 96, 227. 228 %to, T., 376, 44.9 445

s

AUTHOR INDEX

Sharp, D. G., 17, 39 Sharpe, S. H., 229, Z48 Shatkin, A. J., 144, 188 Shaw, E. W., 19, 39 Shaw, W. G. T., 346, 450 Sheehe, R. R., 299, 439 Shelburne, F. A,, 351, 443 Sheridan, A., 193, 214, 225, 226, 247 Shikata, T., 193, 234, 235, 236, 237, 243, 347

Shimkin, M.B., 215, 242, 248 Shmuk, A. A,, 334, 450 Shotadze, D. D., 271, 278, 398, 450 Shreeve, K., 301, 445 Shreeve, W. W., 165, 186 Shrewsbury, J. F. D., 229, 248 Shooter, K. V., 89, 92, 114, 118 Shope, R. E., 17, 18, 39 Shubik, P., 281, 327, 328, 333, 340, 341, 345, 349, 366, 432, 438, 449, 460 Sibatani, A,, 70, 118, 166, 189 Siebert, G., 43, 44, 48, 57, 58, 59, 60, 107, 116, 118, 119 Siebs, W., 125, 127, 188 Siekevitz, P., 48, 117 Signoret, J., 146, 189 Siller, W. G., 216, 248 Silverstone, H., 270, 451 Silvette, H., 359, 360, 444 Simova, P., 320, 435 Siminovitch, L., 2, 15, 35, 38 Simms, E. S., 57, 113 Sims, P., 271, 398, 403, 447 Simson, P., 43, 77, 78, 89, 94, 96, 100, 103, 104, 105, 106, 111, 114, 116 118 Simons, P. J., 26, 37 Sippel, J. E., 193, 220, 246 Sirlin, J. L., 86, 119, 122, 127, 125, 166, 177, 1S7, 189 Sleigh, M. A., 345, 360, 362, 460 Small, H. G., Jr., 368, 441, 450 Smetana, K., 43, 44, 45, 46, 48, 49, 50, 55, 58, 65, 87, 114, 115, 117 119, 122, 185

Smellie, R. M. S.,43, 57, 58, 72, 107, 116, 119

Smillie, L. B., 91, 92, 11.9 Smith, D. B., 92, 119 Smith, G. A. L., 348, 446 Smith, R. F., 362, 444

471

Smith, R. H., 225, 248 Smith, S., 164, 186 Smith, W. E., 273, 448 Smyth, C. N., 269, 460 Sniessko, S.F., 196, 248 Sommering, S.Th., 250, 450 Solaric, S., 300, 367, 414, 438 Soldati, M., 78, 117, 118 Somers, C. E., 50, 119, 174, 187, 189 Sommer, J. R., 26, 36, 38 Sorof, S., 68, 113 Sotelo, J. R., 122, 124, 126, 186 Spackman, D. H., 106, 119 Spahr, P. F., 170, 18.9 Spears, A. W., 329, 330, 337, 338, 348. 379, 450 Speer, F. D., 109, 113, 282, 283, 292, 434, 448 Spence, J. B., 362, 363, 364, 445 Spicer, S. S., 77, 119 Spinar, L. H., 258, 438 Spotswood, T. McL., 323, 436 Staberg, E. M., 262, 460 Staehelin, T., 87, 120 Stanley, T. W., 313, 449 Starbuck, W. C., 43, 45, 99, 108, 113, 119 Stebbins, M. R., 29, 37 Stedman, Edgar, 42, 61, 62, 77, 80, 81, 90. 91, 92, 93, 97, 99, 109, 114, 117, 119 Stedman, Ellen, 42, 61, 62, 80, 81, 90, 92, 119

Stedman, R. L., 253, 265, 327, 329, 335, 338, 339, 342, 343, 344, 349, 436, 437, 439. 447, 460, 541 Steele, W. J., 43, 46, 48, 49, 50, 52, 55, 56. 58, 65. 70, 72, 74, 77, 78, 85, 87, 90. 102, 105, 114,115, 119 Stein, W. H., 93, 94, 95, 97, 103, 104, 106. 114. 115, 119 Steinberg, A. G., 173, 189 Sternberg, S. S., 204, 246 Steiner, P. E., 296, 451 Stemmer, K. L., 349, 442 Stenius, C., 135, 142, 188 Stephano, C. S., 269, 451 Stephens, R. L., 254, 346, 460, 451, 452 Stern, H., 47, 57, 60, 112, 213, 11.9 Stevens, B. J., 129, 130. 132, 15.9 Stevens, R. K., 329, 351, 443. 445 Stewart, S. E., 2, 39

472

AUTHOR INDEX

Stich, H. F., 153, 189 Stich, M., 122, 154, 158, 189 Stiles, E. P., 81, 86, 116 St. Lawrence, P., 140, 141, 189 Stob, M., 229, Z48 Stoker, M. G. P., 2, 4, 12, 15, 36, 39, 40 Stolz, E., 46, 118 ’ Stone, R. S., 17, 18, 39 Stout, A. P., 283, 289, 302, 362, 397, 435, 447 Strauss, M. J., 19, 39 Striebich, M. J., 45, 116 Striegel, J. A,, 452 Strozier, V. N., 43, 70, 119 Sugai, M., 200, 248 Sugihara, R., 124, 190 Sugioka, M., 124, 190 Sugiura, K., 243, 247, 399, 451 Sula, J., 368, 447 Sullivan, P. J., 348, 445 Sullivan, R. D., 273, 448 Sunderman, F. W., 370, 371, 451 Sunderman, F. W., Jr., 370, 451 Suntzeff, V., 271, 297, 300, 367, 414, 438, 461 Sussman, R., 172, 187 Suauki, T., 376, 449 Svihla, G., 139, 185 Svoboda, D. J., 416, 451 Swain, A. P., 230, 247, 265, 329, 342, 343, 451 Swanson, H. R., 85, 115 Sweet, B. H., 20, 39 Swift, H., 50, 79, 119, 122, 128, 129, 132, 152, 154, 189 Swinehart, J. S., 261, 314, 329, 444 Swint, T. B., 46, 119 Syrkin, A. B., 362, 451

T Taber, D., 261, 314, 444 Tabor, E. C., 354, 448 Takahashi, T,, 46, 119, 166, 189 Takata, K., 65, 117 Takayanagi, J., 376, 449 Takeda, J., 237, 248 Taki, M., 344, 442 Taki, S., 251, 461 Tamura, T., 78, 113 Tanaka, K., 289, 299, 428, 445

Tanaka, T., 289, 299, 428, 445 Tandler, C. J., 131, 132, 189 Tannenbaum, A., 200, 240, 248, 270, 451 Tatsuno, T., 193, 234, 235, 236, 243, 247 Tatum, E. L., 144, 188 Taylor, C. W., 48, 55, 58, 74, 77, 85, 99, 100, 102, 104, 105, 106, 111, 113 114, 116

Taylor, G., 2, 21, 39 Taylor, J. H., 166, 173, 189 Tazawa, Y., 77, 119 Temin, H. M., 34, 39 Temkin, I. S., 341, 451 Tepper, L. B., 371, 461 Tertian, L., 259, 451 Tessman, I., 11, 3s Testa, S.,376, 378, 384, 438 Teszler, A. P., 346, 451 Thiers, H. D., 211, 248 Thomas, L. E., 62, 63, 64, 114, 117, 119 Thompson, J. L., 302, 447 Thompson, S. A., 282, 283, 292, 434, $48 Thyresson, N., 326, 441 Timonen, S.,60, 119 Tishkoff, G. H., 45, 115 Tobias, P. V., 133, 138, 141, 189 Toennies, G., 68, 92, 113, 230, 247 Tomita, H., 265, 318, 319, 345, 359, 447 Tooze, J., 44, 119 Torelli, U., 78, 117, 118 Toschi, G., 29, 36 Touey, G. P., 263, 266, 267, 268, 347, 361, 445, 446, 451 Tournier, P., 2, 4, 9, 12, 20, 21, 36, 38, 39 Tozer, B. T., 78, 118 Tregier, A., 272, 273, 417, 442 Tremer, H. M., 301, 303, 385, 386, 440 Trentin, J. J., 2, 21, 39 Trifu, I. S., 308, 451 Trillat, A,, 346, 451 Trillat, J. J., 259, 451 Trim, A. R., 12, 38 Trivedy, J., 299, 452 Tromans, W. J., 11, 39 Trosset, R. P., 330, 442 Truhaut, R., 397, 461 Trujillo, J. M., 138, 141, 142, 143, 188 Truter, E. V., 328, 448 Turner, R. C., 364, 365, 461 Turner. V., 278, 411, 444

473

AUTHOR INDEX

Tsuchiyama, H., 200, 248 Tsukioka, M., 193, 234, 235, 236, 243, 247 Tsunoda, H., 234, 235, 243, 247 Tsvetikov, A. N., 93, 96, 117 Tuite, J., 229, 848 Tye, R., 349, 448

W

Waknian, S. A., 236, 848 Walker, D. G., 144, 185 Wallace, H., 127, 129, 132, 139, 146, 164, 165, 186, 189 Wallenius, K., 269, 443 Waller, R. E., 313, 320, 438 U Walpole, A. L., 229, 248 Walter, J. P., 170, 189 Uhlmann, W., 378, 451 Waltz, P., 260, 310, 315, 373, 378, 437, Ui, N., 91, 92, 119 Urquhart, M. E., 368, 435 452 Wan, Y. C., 100, 117 Umana, R., 64, 94, 125 Wang, T-V., 51, 63, 64, 66, 119 Umezawa, H., 236, 248 Uraguchi, K., 193, 234, 235, 236, 243, 247 Ward, C. L., 80, 216 Ward, P. F. V., 99, 116 Uretz, R. B., 145, 189 Warner, B. R., 257, 452 Ursprung, H., 183, 189 Warner, J. R., 170, 190 Ushida, T., 236, 247 Wartman, W. B., Jr., 262, 308, 324, 448, 452 V Waterson, A. P., 5, 12, 13, 23, 29, 38, 59 Valentine, R. C., 3, 5, 32, 39 Watson, D. H., 3, 15, 29, 56, 39 Van Duuren, B. L., 271, 313, 314, 315, Watson, J. D., 4, 36, 39, 169, 190 318, 320, 326, 332, 333, 334, 344, 348, Watson, J. G., 237, 846 352, 354, 406, 447, 451, 452 Watson, M. R., 104, 180 Van Esch, G. J., 368, 436, 452 Weaving, A. S., 253, 335, 452 Van Genderen, M. H. C., 368, 452 Webb, M., 370, 441' Van Nooy, H., 374, 375, 446 Webb, B. D., 211, 248 Varsel, C., 348, 452 Weber, J. H., 368, 369, 452 Varteresz, V., 110, 115 Webster, G. W., 60, 120 Vasquez, C., 9, 20, 36, 3.9 Weil, C. S., 458 Vendrely, R,, 104, 117 Weiler, C., 142, 188 Venema, G., 284, 452 Weinberg, F. J., 268, 439 Vibert, R., 194, 247 Weinstein, D., 26, 36 Vicari, F., 289, 449 Weisburger, E. K., 355, 452 Vigier, P., 26, 36 Weisburger, J . H., 355, 452 Vignon, V., 303, 306, 441 Weiss, S., 83, 120 Vilcins, G., 359, 436' Wells, R. L., 257, 452 Vincent, W. S., 122, 127, 130, 189 Welshons, W. J., 172, 190 Vogel, A., 360, 468 Wender, S. H., 335, 337, 339, 344, 453 Vohl, H., 337, 342, @2 Wenusch, A,, 259, 339, 346, 452 Vogt, M., 2, 35, 37, 39 West, B., 371, 461 Vogt-Kohne, L., 158, 172, 185, 189 Westermark, T., 361, 362, 462 von Borstel, R. C., 147, 164, 165, 178, Wettstein, F. O., 87, 120 183, 189 Weybrew, J. A., 253, 254, 327, 337, 346, von Haam, E., 143, 287, 327, 446 43Y, 443, 4.47, 4 0 , 462 von Essen, E., 438 White, M. J. D., 137, 154, 190 Von Oettingen, W. F., 340, 359, 4 5 6 White-Stevens, R. H., 241, 246 von Planta, G., 328, 449 Whitman, G. J., 255, 437 Vonvald, A. J., 371, 452 Whitmore, G. F., 26, 38 Voss, R. C., 370, 371, 468

474

AUTHOR INDEX

Wick, E. L., 209, 214, 224, 225, 226, 227, 228, 246, 248 Wickham, J. E., Jr., 264, 446 Widmark, C., 326, 441 Wilbur, K. M., 45, 120 Wilder, P., Jr., 342, 343, 347, 437 Wildy, p.,2, 3, 4, 5, 6, 7, 9, 11, 12, 15, 23, 28, 29, 36, 38, 39, 40 Wilkes, S., 220, 221, 222, 223, 224, 248 Wilkins, M. H. F., 74, 78, 79, 82, 90, 91, 120

Wilkinson, G. R., 79, 113 Willhite, M., 397, 447 Williams, E. F., 329, 437 Williams, J. F., 371, 452 Williams, M. G., 19, 20, 29, 36, 40 Williams, R. C., 5, 11, 17, 18, 40, 80, 116 Williamson, D. H., 230, 246 Williamson, J. T., 261, 347, 462 Willis, R. A,, 284, 442 Wilson, E. B., 122, I90 Wilson, H. R., 78, 120 Wilson, R. H., 340, 368, 369, 379, 442, 462

Wilsbach, K. E., 319, 462 Winterstein, A,, 348,. 460 Wiseley, D. W., 299, 462 Wiseley, D. V., 354, 444 Witting, L. A., 200, 248 Witler, R. F., 45, 116 Wolfe, L. S., 85, 190 Wogan, G. N., 209, 214, 219, 224, 225, 226, 227, 228, 246 Wood, E. M., 192, 194, 195, 198, 199, 248 Wood, T. A., Jr., 280, 424, 437 Woodall, A. N., 192, 198, 199, 248 Woodard, J. W., 104, 118 Woodhouse, D. L., 271, 273, 400, 441 Woodman, R. J., 85, 117 Woodruff, H. B., 236, 248 Woods, D., 166, 189 Woods, R. D., 12, 39 Wright, G. F., 261, 265, 273, 286, 293, 294, 295, 297, 314, 315, 321, 324, 325, 328, 331, 332, 333, 351, 364, 376, 377, 379, 380, 400, 402, 403, 404, 407, 408, 410, 438, 453 Wright, H. E., 329, 440 Wright, H. E., Jr., 335, 336, 349, 463 Wright, J., 348, 446

Wynder, E. L., 251, 257, 258, 261, 264, 265, 266, 267, 269, 271, 272, 274, 275, 277, 280, 284, 286, 287, 294, 295, 296, 297, 298, 301, 302, 305, 308, 309, 310, 311, 312, 313, 315, 317, 318, 319, 320, 321, 322, 325, 326, 327, 328, 330, 331, 332, 334, 337, 338, 339, 340, 341, 344, 351, 364, 370, 372, 373, 374, 376, 378, 379, 380, 381, 382, 383, 384, 386, 399, 400, 401, 402, 403, 404, 406, 407, 408, 410, 414, 415, 417,

262, 273, 293, 303, 314, 324, 333, 345, 377, 385, 405, 423,

437, 438, 441, 442, 461, 463

Y Yale, Y., 2, 21, 39 Yamamoto, K., 337, 446 Yamana, K., 166, 189 Yamasaki, K., 265, 342, 446, 447 Yamasaki, M., 78, 113 Yanagisawa, K., 42, 120 Yang, C. H., 335, 337, 344, 453 Yasutake, W. T., 192, 198, 199, 245 Yazueumi, G., 124, 190 Yeates, J. S., 138, 190 Yerganian, G., 141, 190 Yermolayeva, L. P., 55, 120 Yoshida, T., 243, 848 Young, G. W., 328, 352, 449 Young, N. F., 243, 247 Young, R. D., 2, 20, 37

Z Zajdela, F., 332, 444 Zalokar, M., 126, 165, 190 Zander, M., 324, 444 Zane, A,, 339, 463 Zbarsky, I. B., 47, 55, 57, 62, 63, 64, 109, 115, 120, 166, 186 Zebrun, W., 289, 427, 428, 445 Zelitch, I., 336, 463 Zenkteler, M., 130, 190 Zhdanov, V. M., 2, 36 Ziegler, H., 372, 373, 402, 463 Zirkle, C., 138, 190 Zubay, G. L., 11, 38, 54, 74, 78, 79, 82, 90, 91, 104, 113, 120 Zucker, M., 336, @3 Zuniga, M., 127, 186

SUBJECT INDEX A 2-Acetylaminofluorene, as trout carcinogen, 205, 207 Acid phosphatase, in nucleus, 60 Actinomycins, effects on nucleoli, 144 tumor production by, 236-237 Adenosine deaminases, in nudeus, 60 Adenocirus, negative st,aining of, 5 Adenovirus type 12, classification of, 33 structure of, 21 Aflatoxin(s), amino acid effects on growth of, 209-

Arsenic, in tobacco, 367-369 as tobacco carcinogen, 392 Aspergillus Pavus Link ex Fries, toxin from, 193, 208 (See also Aflatoxin) Aspergillus niger, in moldy peanuts, 212 Aspergillus tamarii, in moldy peanuts, 212 Avian sarcoma-leukosis viruses, structure of, 2 6 2 7 Azure C, as nuclear stain, 44, 46

B

Ben2 [a1 anthracene, in cigarette smoke, 210 317 characterization of, 225-228 Benzo [ b l fluoranthene, in cigarette isolation of, 225-228 smoke, 316 metabolism of, 213-215 occurrence and development in plants, Benzo [jl fluoranthene, in cigarette smoke, 316 210-213 Benzo [a1 pyrene, production of, 208-210 in cigarette smoke, 320 role in African liver cancer, 240 in cigarette “tar,” 313, 316 species susceptibility to, 213-215 in cigarettes, additive effects on, 384 “turkey X disease” from, 193 as “indicator” of tobacco PAH, 393 Aflatoxin B, in tobacco cancerigenesis, 388 carcinogenic activity of, 231 in various tobacco products, 322 formula of, 227 Benzo [el pyrene, in cigarette smoke, properties of, 226-227 317 Aflatoxin G, Beryllium, in tobacco, 371 carcinogenic activity of, 231 Bladder cancer, tobacco and, 395-396 formula of, 227 Bovine papular stomatitis virus, strurproperties of, 226-227 ture of, 13 Aldehydes and ketones, cilia-t,oxicity and tumorigenic activity y-Butyrolactone, as carcinogen, 231 of, 349 C in tobacco cancerigenesis, 390 Aminoazotoluene, as trout carcinogen, Calcium carbonate, as cigarette additi\ e, 383 205, 206 Ammonium nitrate, as cigarette additive, Cancer, 383-384 liver, incidence of, 237-238 &-Angelica lactone, carcinogenic activity (See also Carcinogenicity, Tobacco cancerigenesis, Tumors) of, 231 Capsid, Anucleate genomes, fate of, 16s165 definition of, 4 Aramite, as carcinogen, 204, 241 475

476

SUBJECT INDEX

viral, classification scheme based on, 31 Capsomeres, definition of, 4 Carbarsone, as trout carcinogen, 205, 206 Carbon dioxide, in cigarette smoke, 361 Carbon monoxide, in cigarette smoke, 361 Carbon tetrachloride, aa trout carcinogen, 205 S-2-Carboxyethyl-~-cysteine, as carcinogen, 230, 231 Carboxylic acids, in tobacco cancerigenesis, 390 tumorigenic and ciliastatic activity of, 344 Carcinogenesis, from contaminated foods, 191-249 tobacco, see Tobacco cancerigenesis Carcinogenicity, structure related to, 233 Celery “pink rot,” fungal metabolites in, 228-229 Chicken pox virus, see Varicella virus “Chromosin,” 61, 62 Chromosomes, activation of, reversal and recapitulation of, 17&184 isolation of, 50-51 lampbrush, see Lampbrush chromosomes loci of, serial activation of, 175-177 nucleolar, see Nucleolar chromosomes polytene, 151-163 hormone effects on, 158-162, 163 puffing patterns in, 154-157 “Chromosomin,” from nuclear residues, 6, 62 Chrysene, in cigarette smoke, 317 “Chutta,” 269 Cigar (91, production of, 256 smoke, benzo [ a ] pyrene in, 321-322 condensate of, 310-311 Cigaretteb), additives in, benzo [ a ] pyrene values in, 382-383 arsenic in, 368-369 artificial, 385 corn silk, 385

filter, phenol and condenEate data on, 379-380 fine-cut, 381-382 modified, 397 paper, smoke condensate and, 374-376 polyphenols in, 336 production of, 254-256 smoke, aldehydes and ketones in, 348 benzo [a1 pyrene in, 320, 321 carbonyl compounds in, 347 carcinogenic hydrocarbons from, 316-317 condensates of, 308, 374-375 dibenzocarbazoles in, 333 N-heterocyclic hydrocarbons m, 334 temperature of, 266-268 “tar,” dibenzoacridines in, 332-333 “spinach,” 385 Cilia-toxic agents, reduction of in tobacco, 386 Citric acid cycle enzymes, of nuclei, 5960 Clavacin, see Patulin Copper nitrate, as cigarette additive, 382 Corn, moldy, in African diet, 239 Cottonseed meal, role in trout hepatomas, 192, 207-208 Cyanides, in tobacco smoke, 359-360 Cytonucleoproteins, 69-70 U

DDT, as trout carcinogen, 205, 206 Deoxyribonucleic acid nucleotidyl transferase, isolation of, 57-58 Deoxyribonucleoprotein, complex, acidic proteins of, 70-77 isolation of, 52-55 Dibenzacridines, in cigarette “tar,” 332333 Dibenz [a,hl anthracene, in cigarette smoke, 316 Dibenzocarbazoles, in cigarette smoke, 333 Dibenzo [a,U pyrene, in cigarette smoke, 317 Diethylstilbestrol effect on trout, 206 Dimethylaminoazobenzene,

477

SUBJECT INDEX

carcinogenesis, protection against, 243 as trout carcinogen, 205 Dimethylnitrosamine, as trout carcinogen, 205-207 Diphosphopyridine nucleotide synthetase, isolation of, 58-59

E Ecdysone, effect on polytene chromosomes, 158-162 Epoxides, in tobacco, 353 in tobacco cancerigenesis, 390 tumorigenic activity of, 353-355 Esterases, in nucleus, 60 Ethylene glycol, as cigarette additive, 382

F Fibroma virus, classification of, 30 Fish meal, role in trout hepatomas, 192, 207-208 toxicity of, 241 Foods, contaminated, carcinogenesis by, 191-249 Fungal metabolites, carcinogenesis by, 191-249

G Gene, loci, activation of, 171-175 Globulins, nuclear, 63-65 amino acids of, 64 terminal amino acids of, 67 Glucosed-phosphate, in nucleus, 60 p-Glucuronidase, in nucleus, 60 Glutamic dehydrogenase, nuclear, 59 Glycerol, as cigarette additive, 382 Glycolytic enzymes, nuclear, isolation of, 59 Gross leukemia virus, structure of, 2426

H Heterocyclic hydrocarbons, in tobacco cancerigenesis, 389 4-Hex-2-enolactone, as carcinogen, 230 4-Hex-4-enolactone, as carcinogen, 230, 231

Histones, nuclear, 77-111 amino acids of, 95, 97, 98 chemistry of, 88-90 DNA and, 78-79, 83-85 extraction of, 90-91 functions of, 79-85 genetic control and, 80-82 metabolism of, 107-109 nomenclature of, 77-78 origins of, 85-88 peptides of, 104-106 RNA synthesis and, 82-83 separation of, 91-100 of tumors, 98-99 Human adenovirus type 12, 2 Human wart virus, capsomeres of, 9 structure of, 19-20 8-Hydroxyquinoline, as carcinogen, 24

I Indeno [1,2,3-cdl pyrene, in cigarette smoke, 317 Influenza virus, classification of, 30-32 Isocitric dehydrogenase, nuclear, 59

K K virus, capsomeres of, 9 “Khaini,” 257 L Lactones, carcinogenic, 228, 232, 352 reaction with proteins, 230, 232 in tobacco, 353 in tobacco cancerigenesis, 390 tumorigenic activity of, 353-355 Lampbrush chromosomes, 1-151 Leukemia viruses (mouse), 2426 classification of, 30, 31 Liver, cancer, incidence of, 237-238 cells, nuclei isolation from, 45 tumor, nucleoprotein amino acids of, 71-77 Luck6 kidney tumor virus, classification of, 31 structure of, 21 Luteoskyrin, toxicity of, 235-236

478

SUBJECT INDEX

M Malic dehydrogenase, nuclear, 59 Mammary tumor virus, structure of, 21-24 Measles virus, fliamentous internal component of, 12-13 Mengo virus, effects on nuclear metaholism, 112 8-Methoxypsoralen, in celery “pink rot,” 228 Microsomes, amino acids of, 64 Mold, food infected by, carcinogenesis from, 191-248 Molluscan contagiosum virus, classification of, 32 Mo!onry leukemia virus, structure of, 24-26 Monkey poxvirus, classification of, 32 Mouse leukemia viruses, structure of, 24-26 Mycotoxicoses, health implications of, 237-240 nutritional considerations in, 220-225 pathology in, 215-220 carcinogenesis, 216-220 toxicity, 215-216 Mycotoxins, carcinogenesis and, 191ff. occurrence and development of, 208213 Myxoma virus, classification of, 32

N Naryhile, smoke condensate of, 312 Neoplastic cells, nuclear proteins of, 41120 Nickel acetate, as cigarette additive, 383 Nickel carbonyl, in tobacco smoke, 370-371 Nicotine, in cigarette smoke, 331-332 Nitrites, in cigarette smoke, 359-360 Nitrogen oxides, in cigarette aniokr. 360-361 4-Nitroquinoline-N-oxitie, effect on nucleoli, 144 Nitrosamines, in tobacco, 355-357 in tobacco rancerigenesis, 3!)1

Nuclear proteins, acidic, 61-63 antitumor agents effect on, 70-72 of deoxyribonucleoprotein complex, 70-77 fractionation of, 73 in tumors, 62-63 cyto-, 69-70 globulin type, 63-64 amino acids of, 64 histones of, 77-111 lipo-, 62-63 of neoplastic cells, 41-120 nuclei isolation and, 44-48 ribo-, 65-70 ribosomal, 66, 67 Nuclear sap, amino acids of, 64 proteins of, terminal amino acids of, 67 Nuclei, isolation of, 44-48 Nucleocapsid, definition of, 4 Nucleolar bodies, 123-132 extrusion of, 126-127 pre-, 129-132 structure of, 124-126 variation in different tissues, 127-129 Nucleolar chromosomes, 121-190 human, 140-143 non-nucleolar chromosomes and, 139140 nucleolar-chromosomal complrx of, 132-135 nucleolar organizers and, 135-139 Nucleoli, rhemical modification of, 143-144 experimental studies on, 143-150 genetic modification of, 146-147 isolation of, 48-50 transplantation of, 147-150 UV effects on, 144-146 Nurleolochromosomal apparatus, isolation of, 55-57 5‘-Nuc.leotidases, in nwlrns, 60 Nucleus, acidic proteins of, 61-63 rnzvmes of, isolation of, 57-61 globulins of, 63-64 isolation of componrnts o f , 48-57

479

SUBJECT INDEX

chromosomes, 50-51 deoxyribonucleoproteins, 52-55 nucleoli, 48-50 niicleolochromosomal apparatiis, .i.i57 ribonucleoprotein network, 50 ribosomes, 51-52 scheme for, 53 summary of, 56 lipoprot.ein of, 62-63

0 Orf papular stomatitis virus, structure of, 13 Osmotic shock, use on nuclear preparations, 46

P PAH, see Polynuclear aromatic hydrocarbons Papilloma virus, classification of, 30, 31 “Papova” viruses, 29 Paraffinic hydrocarbons, in tobacco, 328-329 in tobacco carcinogenesis, 389 Patulin, as carcinogen, 229, 231 Peanut butter, aflatoxin in, 238 Peanut meal, aflatoxin extraction from, 210 hepatic carcinoma from, 192 Penicillic acid, carcinogenic activity of, 230, 231 Penicillin G, carcinogenic activity of, 230, 231 l’enicillium commune, toxic metabolites of, 234 I’enicillium islaadicum Sopp., toxic metabolites of, 233-235 Penicillium mnrtensii. toxic isolate from, 213 Penicillium urticne Bainer, phytotoxic metabolite from, 229 Peroxy compounds, in tobacco, 353 in tobacco cancerigenesis, 390 tumorigenic activity of, 353-355 Phenols, as “indicator” of tobacco cancerigenesis, 393

in tobacco cancerigenesis, 389 tumorigenicity of, 339-341 “Philadelphia chromosome,” 142, 143 Phoma sp., from moldy peanuts, 212 Phosphatases, in nuc~hseti:,60 Phthalates, in tobacco, 327 in tobacco cancerigenesis, 389 Phytosterols, in tobacco, 349-351 “Pink rot,” of celery, fungal metabolites of, 228 Pipe, Orient’al, smoke condensate of, 312 smoke, benzo[alpyrene in, 321-322 condensate of, 311-312 temperatures, of 328 Poliovirus, capsid symmetry of, 11 Polynuelear hydrocarbons (PAH), in pipe and cigar smoke, 321-322 precursors in tobacco smoke, 323-326 qualitative analysis of, 316318 quantitative analysis of, 318-319 in tobacco, 319-321 in tobacco cancerigenesis, 381 in tobacco smoke, 312-326 Polyoma virus, capsomeres of, 9, 10 classification of, 31 discovery of, 2 structure of, 14-17 Potassium nitrate, as cigarette additive, 382 Poxviruses, 13 structure of, 27 /3-Propiolactone, as carcinogen, 229-231 Proteolytic enzymes, nuclear, 60 Psoralen compounds, toxicity of, 228

R Rabbit papilloma virus, structure of, 1719 Radicals, in tobacco cancerigenesis, 391 Ribonuclear protein network, of nuclei, isolation of, 50 Ribonucleic acid, effect on genetic information, 42 m-, production of, 165-169

480

SUBJECT INDEX

Ribonucleic acid nucleotidyl transferase, isolation of, 57-58 Ribonucleoproteins, nuclear, 65-70 Ribosomes, nuclear proteins of, 66 amino acids of, 64 isolation of, 51-52 site of formation of, 169-171 terminal amino acids of, 67 Rous sarcoma virus, 1 “RP2-L,” in tumors, 109-111

S Salivary gland nucleus, transplantation of, 163 Sch&?tosoma haemotobium, carcinogenic implications of, 242 Selerotinia sclerotiorum, celery “pink rot” from, 228 Shope fibroma virus, properties of, 27 Simian vacuolating virus SV 40, 2 capsomeres of, 9 classification of, 31 structure of, 20-21 Sodium nitrate, as cigarette additive, 382 Solanesol, in tobacco, 325-326, 328 Smoke, tobacco, see Tobacco smoke Smoking, machines, 259-262 public education against, 397, 435 variables in, 262 (See also Tobacco) Steroids, in tobacco, 353 in tobacco cancerigenesis, 390 tumorigenicity of, 351-353

T Tannic acid, as trout carcinogen, 205, 206 Terpenes, in tobacco cancerigenesis, 388 in tobacco smoke, 326-327 Thioacetamide, effect on nucleoli, 143 hepatomas from, 204 as trout carcinogen, 206 Thiourea, hepatomas from, 204 as trout carcinogen, 205, 206

Thymus, isolation of nuclei from, 47 Tobacco, aldehydes and ketones in, 345349, 390 alkaloids, pyrolysis of, 333-334 arsenic in, 367-369, 392 carbonyl content of, 345-364, 392 carboxylic acids in, 341-345, 390 characteristics of, 252269 chemical composition of, 354 chewing, production of, 257 extraction, effect on “tar” yield, 376377 extracts of, 264-265 epoxides in, 353, 390 lactones in, 353, 390 metallic constituents of, 370-371, 392 nitrosamines in, 355-357 organic acids in, 341-342, 390 “paraffins,” 324-325 phenolic compounds in, 334341, 389 phytosterols in, 349-351 smoke, see Tobacco smoke PAH in, 319-321 pipe, production of, 256-257 production of, 252-254 steroids in, 349-353 tumorigenic agents in, reduction of, 377-379 waxes in, 328-329 (See also Cigar, Cigarette, Pipe) Tobacco cancerigenesis (experimental), 249-453 alkanes in, 330-331 biological tests for, 269-308 of cervix, 280-281 of epidermis, 271-275 of lung, 281-285 nutritional deficiency and, 300 of oral cavity and bladder, 277-280 radiation effects on, 300 short-term, 297 of smoke condensate, 271-287 of smoke condensate fractions, 292297 of subcutaneous tissue, 276-277 of tobacco extracts, 285-287 of tobacco smoke, 287-292 trauma and heat effects on, 300-301

SUBJECT INDEX

tumor initiation and promotion in, 298-299 variables in, 270 virus effects on, 299 of bladder, 395-396 of cervix, 280-281, 423 of epidermis, 271-275, 398-418 subcutaneous, 419 future studies on, 395-396 history of, 250-251 human data on, 396 inhalation studies on, 394, 425-430 of lung, 281-285, 425-434 of oral cavity (bladder), 420-422 reduction of, 372-387 cilia-toxic agents in, 385-387 relative role of carcinogens in, 387 statistical considerations of, 394-395 Tobacco mosaic virus, helical symmetry of, 11-12 Tobacco smoke, alkanes in, 329-330 basic portion of, 331-332 cilia-toxic components in, 301 condensate, reduction of, 372 constituents of, 308-371 electric charges in, 361-362 heterocyclic nitrogen compounds in, 331-332 inhalation studies on, 394 inorganic components in, 360-361 nicotine in, 331 organic acids in, 342-344, 390 organic radicals in, 362-364, 391 particle sizes in, 257-258 phenolic compounds in, 337-341, 389 phytosterols in, 349-351 polynuclear aromatic hydrocarbons in, 312-326, 388 precursors, 323-326 preparation of, 262-264 radioactivity in, 364-367 retention on inhalation, 258-259 temperatures of, 266-269 terpenes, phthalates, and esters in, 3 2 6 328, 388-389 volatile components in, 357-361 (See also Smoking) Toluidine Blue, as nuclear stain, 46

481

“Tri.Pr.,” 61-62, 63 4,5’&Trimethylpsoralen, in celery “pink rot,” 228 Triphosphatases, in nucleus, 60 Trout, insecticide effects on, 204-205 h e r tumors in, 191-192 bacteria of viral invasion and, 197 chemical carcinogens and, 204-206 ,diet carcinogens and, 203-204 dietary factors and, 198-203 genetic predisposition and, 195-197 pathology of, 194-195 from processed rations, 194-208 projected research on, 207-208 radiation effects on, 206-207 Tumor(s), acidic nuclear proteins in, 62-63 histones of, 98-104 purification of, 100-101 isolation of nuclei from, 45-47 nuclear proteins of, electrophoretic studies on, 68-69 “RP2-L” in, 109-111 (See also Cancer, Walker tumor) Tumor viruses, development of, 14-27 structure of, 1-39 Turkey X disease, 191 from toxic peanuts, 193 Turnip yellows mosaic virus, capsomeres of, 11 Tyrosinase, in nucleus, 60

U Urethane, as trout carcinogen, 205, 206 V Vaccinia virus, structure of, 13, 29 Varicella virus, capsomeres of, 9 Virion(s), 3-4 complex, 13 definition of, 4 with helical symmetry, 11-13 with icosahedral symmetry, 5-11 symmetry properties of, 4-5 Virus(es), classification of, 2-3 scheme for, 28-32

482

SUBJECT INDEX

structural elements of, 3-4 tumor, see Tumor viruses

Vitamin E, role in liver cancer, 244

W Walker tumor, histones of, 99, 102-104

nuclear proteins of, 101 amino acids of, 71-77 terminal’amino acids of, 67 nucleoli isolation from, 48-49 RNA extraction from, 52, 53 “RP2-L” in, 109-111 Water pipe, see Narghile Waxes, of tobacco leaf, 328-329