New Developments in Chemical Carcinogenesis by Polycyclic Hydrocarbons and Related Heterocycles: a Review*

CANCER RESEARCH

VOLUME24 OCTOBER 1964 NUMBER 9 New Developments in Chemical Carcinogenesis by Polycyclic Hydrocarbons and Related Heterocycles: A Review*

N. P. Buu-HoÃ¯ (Centre National de la Recherche Scientifique, Paris, France)

When the last of the famous series of surveys on chemi five. Almost all the then known active hydrocarbons con cal carcinogenesis by Cook and Kennaway appeared in the tained a phenanthrene system, and this fact promoted, American Journal of Cancer in 1940 (18), it brought to an and justified, the theories according to which the meso- end the first chapter of research in that domain, which phenanthrenic double bond (called "K-zone") would play a had seen the discovery of the first families of chemical prominent role in the biochemical interactions between hy carcinogens and had brought to light the first relationships drocarbon and cell components which constitute the early between chemical structure and oncogenic activity. Since stages in the carcinogenic process. The introduction into then, a considerable acceleration of progress in our knowl the molecule of electron-releasing groups (especially methyl edge of chemical carcinogenesis has occurred, with the groups) enhances the 7r-electron density, and hence the discovery of many prolific new families of carcinogens as chemical reactivity, of the K-zone, and should conse well as a wider knowledge of those already known, the quently increase carcinogenicity. Contrariwise, the pres acquisition of the first clues as to the mode of biochemical ence of electron-accepting groups (aldehyde, ketone, nitrile, interaction between carcinogens and cells, and, finally, the carboxyalkyl, etc.), or the replacement of a nuclear first practical applications of the science of chemical car â€”CH= group by a nitrogen atom, depresses the reactivity cinogenesis to the field of cancer chemotherapy and chemo- of the K-zone, and thus a decrease in carcinogenicity prophylaxis. should be expected. A similar negative effect would also Because of the scope of the progress made and the pro ensue when the interaction between the K-zones and the gressive specialization of the investigators, a review on cell constituents is impeded by steric hindrance arising chemical carcinogenesis along the lines of Cook and Ken through the presence of substituents in those zones. In naway's comprehensive papers seems less and less feasible addition to their K-zones, the majority of the then known within the confines of a single publication and by any single carcinogenic hydrocarbons bore a wieso-anthracenic region group of authors. And so the present paper will be limited ("L-zone"), and in those hydrocarbons bearing the two to the salient facts, interpretations, and theories concerning zones it was observed that the degree of carcinogenic the carcinogenic polycyclic hydrocarbons and their hetero- activity was in inverse proportion to the chemical re cyclic analogs which have been investigated during the activity of the L-zone (the few naphthacene hydrocar last decade. It will comprise four sections: (a) an ex bons, for instance, which had been tested, being all at that amination of the improvements in methods of assay of time found inactive). carcinogenic hydrocarbons and analogous heterocyclics; The fruitfulness of this over-all picture cannot be better (o) a survey of relationships between chemical structure illustrated than in the many instances in which theo and activity in the principal groups of newly investigated retical calculations for carcinogenic activity were subse compounds; (c) a discussion of the experimental evidence quently confirmed by the results of biological testing. thus acquired, with reference to the more recent theories Such was the case with several angular benzacridines (13) of chemical carcinogenesis; and (d) a discussion of the and, more recently, with 3,4:9,10-dibenzopyrene (17, 63). question of the endogenous biosynthesis of carcinogenic However, the intensification of research on chemical hydrocarbons. carcinogenesis that has marked the last decade, and which was due in part to the recognition of the important role of THE EARLY PICTURE OF A CARCINOGENIC polycyclic hydrocarbons in the etiology of certain human HYDROCARBON cancers, has resulted in a considerable volume of new data, From past experimental research and past interpreta in the light of which the former picture of a typical car tions there emerged a classical picture of a carcinogenic cinogenic hydrocarbon has now to be enlarged and even hydrocarbon possessing the following features: It was a revised in several respects. Advances that have been polycyclic condensed aromatic molecule, built up of at least three annelated benzene rings, the optimum number made are the outcome both of improvement in method for maximum carcinogenic activity ranging from four to ology (refinement of the routine methods of biological assay, development of new technics for investigation of the * In honor of the 80th birthday of Prof. A. Lacassagne, under physico-chemical properties of carcinogens) and of the in whose direction much of the work reviewed here has been accom crease in the number of chemical series investigatedâ€”and, plished. Received for publication April 24, 1964. within each series, in the number of substances examined. 1511

IMPROVEMENTS IN METHODOLOGY OF ASSAYS hydrocarbons which are barely active on the skin or sub Although the use of various animal species including cutaneously readily induce hepatomas when given by other rodents (rats and hamsters) is still extremely valu mouth, but also this route can often make possible the pro duction of both benign and malignant tumors of the upper able for specific experiments in carcinogenesis (induction of digestive tract; for instance, both 5,6-benzopyrido(2',3': hormone-dependent tumors, cancers of the liver, etc.), all l,2)carbazole and 5,6-benzopyrido(3',2':l,2)carbazole, the evidence points overwhelmingly to the necessity of using the mouse for the detection and evaluation of local given orally, elicit a high percentage of multiple papillomas carcinogenicity in polycyclic molecules, because in mice and epidermoid epitheliomas of the foresto mach (43). Appreciation of diffÃ©rencesin susceptibility of male and there exist strains (such as the Paris Radium Institute female mice.â€”Originally, the evaluations of carcinogenic strain XVII nc/Z) which almost never spontaneously de velop tumors of the type to be expected from the experi activity of hydrocarbons and analogs were made irrespec ments; and once these experimental tumors have been tive of the sex of the mice, and in the few cases where this induced, their growth is less likely to be influenced by factor was taken into account no differences or only extraneous factors (diet, environment, hormonal factors, negligible ones were noted (2, 7, 26). Leiter and Shear (52), however, using 3,4-benzopyrene (III) in marginal etc.)- Diversification of the tests.â€”Foran accurate evaluation doses (0.1-0.05 mg.) and a large number of animals, found of the carcinogenic activity of polycyclic hydrocarbons in that, with the strain of mice they used, there was a greater mice, it has become imperative to make use of all three of susceptibility in the males, 52 per cent of which developed the following tests: skin-painting, subcutaneous injection, tumors compared with only 36 per cent of the females. and oral administration. It has long been known that More recent research done with strain XVII nc/Z of the many hydrocarbons which readily elicit skin tumors were Radium Institute has confirmed this inequality of action inactive when administered by subcutaneous injectionâ€” of 3,4-benzpyrene (sarcoma index determined in 154 male this is in particular the case of 3,4-benzophenanthrene (I) mice, 91, and in 162 females, 47) and shown that even and its derivatives. The reverseâ€”i.e., substances active greater sex differences in susceptibility exist with many hydrocarbons tested by subcutaneous injection. Further more, in several instances it was the female mice in which cancer was more easily induced than males. Thus, 3,4:8, 9-dibenzopyrene (IV) is considerably more sarcomagenic in when given subcutaneously and inactive on skin-paintingâ€” is at least as frequent: for instance, naphtho(2',3':3,4)- pyrene, which has been found inactive on the skin, later proved quite sarcomagenic (12). An added value of the CHO subcutaneous injection procedure springs from the fact that many carcinogens can be tested on the skin only with great difficulty or even not at all, either because they are readily oxidized in the air, especially in the presence of light (e.g., the naphthacene and higher linear hydro the male than in the female mouse ; in one experiment with carbons), or because they are too poorly soluble in the this substance 34 out of 35 male animals developed a usual solvents (e.g., condensed hydrocarbons with more sarcoma in situ, whereas out of ten females, cancer was than six benzene rings, such as the tribenzopyrenes). A induced only in one (51). Similarly, with 2,3-phenylene- last important advantage is the possibility of injecting very pyrene (V), the sarcoma index1 was 27 for the males and small amounts of substance (sometimes, as with 1,2:4,5- only 5 for the females. Pronounced sex differences are dibenzopyrene (46), a single dose of 0.6 mg. is sufficient), also observed with nitrogen heterocyclic compounds: for thus permitting tests to be performed with compounds instance, with 5,6-benzopyrido(2',3':l,2)carbazole the so difficult to prepare that they are available only sarcoma index was 64 in the males as against only 7 in the in limited quantities. Indeed, it is often expedient females (47). Conversely, with 6-formyl-12-methylan- to employ minimal amounts of the substance for thanthrene (VI), the sarcomagenic effect was considerably injection, when, for instance, the compounds possess greater in the females than in the males. Wide qualitative a high general toxicity or when they tend to produce differences also exist between male and female response to ulcÃ©rationsin situ; and, further, with substances of some hydrocarbonsâ€”at least with certain strains: F-nor- very poor solubility which tend to remain in the tissue in steranthrene, given subcutaneously to mice of our strain the form of fine solid particles, the degree of dispersion (to XVII (Radium Institute), produced only sarcomas in the ensure maximum contact with the cells) is more important males, whereas the cancer the most frequently observed than the absolute amount injectedâ€”with 3,4:9,10-di- and the earliest to appear in the females was an epidermoid benzopyrene (II) Unseren and Fieser were able to recover from the tumors almost all the substance that had been 1After Iball, this has been defined as the number of animals injected (67). bearing tumors multiplied by 100, divided by the product of the number of animals alive at the time of appearance of the first As for the necessity of conducting tests by oral adminis tumor by the mean latent period in days. This formula, although tration, not only is this borne out by the fact that some arbitrary, is a convenient means of classification.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. Buu-HoÃ¯â€”ChemicalCarcinogenesis by Hydrocarbons 1513 epithelioma of the mammary gland (35). These findings cant for chemical carcinogenesis, would in the last resort point to the necessity, in future evaluations of the carcino have to give results strictly parallel to those obtained in the genic activity of compounds, by means of skin-painting or full-fledged classical tests. Thus far, no short-term test subcutaneous injections, of systematically paying attention reported satisfies this specificity requirement. to possible sex differences in responseâ€”just as in the study of chemical hepato-carcinogenesis in rats, a field in which DEVELOPMENTS IN METHODS OF PHYSICO- the importance of such differences has already been CHEMICAL INVESTIGATION OF POLYCYCLIC acknowledged. That many polycyclic hydrocarbons, and MOLECULES among them some of the most potent carcinogens found in Ample evidence exists in support of the idea that the tobacco smoke (3,4-benzopyrene and l,2:8,9-dibenzo- mode of action of carcinogenic hydrocarbons is not, at pyrene), elicit tumors more readily in male than in female least in its initial biochemical phases, basically different mice is perhaps relevant to the considerably greater in from that of other biologically active molecules such as crease in bronchial carcinoma in male smokers than in pharmacological agents. Both the existence of competi female smokers. tive inhibition of their action by means of structurally The problem of short-term tests for carcinogenesis.â€”Afew related molecules (37), and the demonstration of their procedures have been advocated for hastening the in fixation on cell proteins (31), are proofs in this direction. duction of local tumors by polycyclic molecules. Peacock Chemical carcinogenesis is therefore basically linked to recommends a technic consisting of the inclusion of small certain physical and chemical characteristics of the mole amounts of the carcinogenic hydrocarbon in embryo tissue cule of the carcinogen, and a greater understanding of the implants, which causes carcinomas in mice within 16 weeks biological action of the aromatic hydrocarbons and their (62) ; this new test for carcinogenesis is, however, more analogs can be expected to emerge from the development interesting per se than for the amount of time saved. of new methods for their physico-chemical investigation. Another possibility, advanced by the present author and The most significant progress concerning the purely Zajdela, is to hamper the metabolic deactivation of the physical methods has been in the application, to polycyclic hydrocarbon by local administration of substances which hydrocarbons, of the technic of nuclear magnetic resonance inhibit the tissue hydroxylation processes; experiments and the measurement of Rayleigh depolarized light diffu along these lines, made with very weak carcinogens of the sion. On the chemical side, it has been in the study of the benzacridine type, have not yet furnished clear-cut results. processes through which polycyclic hydrocarbons and their The above technics, however, cannot really count as heterocyclic analogs form noncovalent complexes with a short-term tests for carcinogenesis, because they still re variety of molecules, including cell proteins and nucleic quire considerably more time than is desirable for certain acids; these processes can involve "charge-transfers," van types of screeningâ€”for instance, for the study of car der Waals forces, hydrogen bonding, or a combination of cinogens in tobacco smoke and in atmospheric pollution. these factors. For expediting such screenings, several real short-term Nuclear magnetic resonance spectroscopy.â€”-Thenuclear tests have recently been proposed, of which the most fre magnetic resonance spectroscopy of the protons present in quently employed are: the molecule of a carcinogenic hydrocarbon affords im a) The tetrazolium reduction test of Iversen (33), which portant data in two domains (27). First, resonance shifts measures the increase in formazan deposition in hairless of the protons located directly on the nucleus are a func mouse epidermis after a single application of the potential tion of the 7T-electrondensities on the bearer carbon atoms, carcinogen ; and a linear relation between the "chemical" shifts (5) ob 6) The photodynamic test as performed by Epstein on served and variations in the electronic density (p)(5 = Â«Ap) Paramecium caudatum (28), which estimates the degree of has even been postulated. Their determination thus pro toxicity of the potential carcinogen toward cultures of that vides an experimental method for controlling estimations organism under irradiation with longwave ultraviolet light; of these rr-electron densities done by purely mathematical another test of this kind is the measurement of photo - computations; in fact, in certain cases (as, for instance, dynamic action on isolated mitochondria (SantamarÃa) in with the electrically dissymmetrical hydrocarbons of the which polycyclic hydrocarbons are stated to show a good azulene group) the electron density distributions deter correlation between this effect and carcinogenicity; mined experimentally are in better accord with the facts of c) The Smith "sebaceous glands test," based on the early chemistry than are the data determined mathematically, destruction of these glands following applications to the even with the most refined computation technics (e.g., the skin of mice of certain types of polycyclic carcinogens (but variable electro negativity self-consistent field method). not all) ; Table 1 gives the chemical shifts for the two protons of the d) The skin hyperplasia test, recently proposed by ??ieso-anthracenic zone (L-zone) for a series of carcinogenic GuÃ©rin,andwhich is based on the concurrent evaluation of and noncarcinogenic angular benzanthracenes and di- two parallel phenomena elicited by applications of car benzanthracenes. Angular benzacridines and dibenzac- cinogens to the mouse skin : increase in the thickness of the ridines have also been investigated in our laboratory by skin epithelium and in the number of epithelial cells; this this method. test is similar in nature to Neukomm's test, in which the Secondly, nuclear magnetic resonance spectroscopic data hyperplasia of the skin of the newt is also measured. on the protons of substituents that are important for bio Although they can furnish very useful practical indica logical activity (e.g., the methyl group) give an indication tions, these short-term tests, to be fundamentally signifi of the deformation of the skeleton of the molecule that the

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. 1514 Cancer Research Vol. 24, October 1964 steric effect exerted by the substituents brings about (for TABLE 2 instance, in the case of l'-methyl-l ,2-benzanthracene). DATAONMOLECULAROPTICALANISOTROPYOF Rayleigh depolarized light diffusion of aromatic hydro 1,2-BENZANTHRACENEDERIVATIVES(66) carbons.â€”Themeasurement of this type of light diffusion X- produced by aromatic hydrocarbons in solution is a new Compound1 5.460A1,9662,0602,0602,0602,0602,1702,3162,6452,6452,7203,7203,600E00.050.050.050.050.100.180.340.340.380.850.82CarcinogenicityMarginalWeakStrongWeakWeakStrongStrongNilWeakStrongVery technic which makes possible the direct evaluation of their molecular optical anisotropy (-y2);this value is linked to ,2-Benzanthracene3-Methyl-l the molecular polarizabilities, which in turn are an intimate 2-benzanthracene4-Methyl-l, reflection of the electronic structure. By applying this ,2-benzanthracene7-Methyl-l technic to homologs of a given basic hydrocarbon one can ,2-benzanthracene8-Methyl-l determine the relative increase in molecular optical 2-benzanthracene5-Methyl-l, anisotropy brought about by substitution; this value (E) 2-benzanthracene9-Methyl-l, is represented by the expression: E = 7i2 â€”7z2/7a2,in ,2-benzanthracene3'-Methyl-l 2-benzanthracene4'-Methyl-l,, which 7l2represents the molecular optical anisotropy of the 2-benzanthracene10-Methyl-l substituted hydrocarbon and 722stands for the molecular ,2-benzanthracene9 optical anisotropy of the basic hydrocarbon. Pacault, ,10-Dimethyl-l ,2-benz strongVery Bothorel, and UnanuÃ©(58, 66) have linked the high E anthraceneMethylcholanthrene-i'(A'); values of certain methyl homologs of 1,2-benzanthracene strong to their carcinogenic activity. Table 2 contains the data obtained on several derivatives of 1,2-benzanthracene; it can be seen that, although it is not possible to correlate E scription of them as "charge-transfer" complexes between and carcinogenicity in a quantitative way, and that al the electron acceptor A and the hydrocarbon that is acting though even qualitatively there are some exceptions, there as the electron donor D : is a striking correspondence between the extremely high E values for 9,10-dimethyl-l,2-benzanthracene and for methylcholanthrene and for their pronounced biological ac As several authors have pointed out more recently (J. C. tivity. Arcos, inter al.), the stability of these ^-complexes is en Study of complex-formation processes.â€”Ithas been known hanced by hydrogen bondings and by van der Waals for a long time that aromatic polycyclic hydrocarbons give attraction forces operating between two components, and molecular complexes with a wide variety of substances the complexes are generally considered the "sandwich" acting as electron-acceptors: metal and metalloid halo- type, the components lying in alternate parallel layers. genides and halogens themselves, polynitro compounds, Early in the studies on structure/activity relationships in quiÃ±ones, acid anhydrides, etc. The molecular com carcinogenic hydrocarbons, some kind of connection had pounds are usually highly colored and relatively stable, already been suggested by Fieser between the oncogenic and their nature has been the subject of several theories, of activity of the hydrocarbon, on the one hand, and, on the which the most generally accepted one is Mulliken's de- other, its complex-building ability and some of the proper ties of these complexes (stability and color). This paral lelism is today readily understandable, since the molecules TABLE 1 of many carcinogenic aromatic hydrocarbons (those which CHEMICALSHIFTS*FORTHE PROTONSLOCATED are "condensed" polycyclics) are known to possess struc ON THE L-ZONE (27) tures which imply the presence of both powerful electron- donating properties and strong van der Waals fieldsâ€”two â€”SHIÂ« HydrocarbonAnthracene1 genicityNilMarginalWeakStrongStrongNilNilWeakNilWeakStrongWeakSH,8.359.109.079.149.059.279.039.118.90â€”9.109.97Â«HÂ»8.358.298.188.45â€”8.278.268.338.90â€”9.108.27fe)(Â±0.003)00.8100.8840.690â€”1.0000.7700.8100+0.16701.60features that are essential to the ease of formation, sta bility, and deep color of 7r-complexes. Interest in this parallelism has been renewed in these last few years, with ,2-Benzanthracene3-Methyl-l more experimental data as well as more interpretations. ,2-benzanthracene4-Methyl-l Szent-GyÃ¶rgyi et al. (65) have re-emphasized the con ,2-benzanthracene10-Methyl-l, nection between the carcinogenicity of aromatic hydro 2-benz carbons and their capacity to form strong charge-transfer anthracenel'-Methyl-l, 2-benz complexes, and Mason (53) went as far as to postulate a anthracene3'-Methyl-l, proportionality between carcinogenic activity and the 2-benz capacity to form complexes with routine electron-acceptors anthracene4'-Methyl-l, such as picric acid. Huggins and Yang (32) consider the 2-benz coexistence of strong 7r-complex forming properties and a anthracene1,2:3 certain geometric pattern similar to that of growth-pro ,4-Dibenzanthracene5-Methyl-l, moting steroids (such as progesterone, testosterone, and 4-dibenz-anthracene1,2:5,2:3, estradici) to be an essential characteristic of mammary gland carcinogens. More recently, Allison (1) measured 6-Dibenzanthracene1,2:7, the electron-donating faculty of polycyclic carcinogens 8-DibenzanthraceneCarcino with chloranil and 1,3,5-trinitrobenzene, and inferred * Expressed as p.p.m. relative to tetramethylsilane as internal from his results that the biological activity of these carcinogens resides in their formation of "sandwich" com- reference.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. Buu-HoÃ¯â€”ChemicalCarcenogÃ©nesisbyHydrocarbons 1515 plexes with metabolic systems; in these complexes the car and theories bearing on their mechanism of action, had cinogen might act as both electron-donor and electron- until recently been drawn from the study of a few families acceptor (polycyclic aromatic hydrocarbons are known of small to medium size hydrocarbons, which included the indeed to act also as electron-acceptors with strong donors phenanthrenes, the anthracenes, the benzo- and dibenzo- like alkali metals), lying between two biochemically active anthracenes, and the benzopyrenes. However, interest sites, one electron-donating and the other electron- has since been focused on less orthodox structures which accepting. were previously neglected or among which the presence of While rightly laying stress on one salient feature com carcinogens was considered as being exceptional; and a mon to many polycyclic carcinogens, these interpretations more thorough study of these new families has proved encounter the same difficulties as most other general them to be as fertile as the ones investigated earlier. Un- theories of chemical carcinogenesis, in that each of them orthodoxy, here, refers in particular to: accounts for only a certain number of cases. First, the 1. Hydrocarbons and heterocyclic analogs whose mo complex-building capacity of polycyclic molecules is not an lecular structure is not condensed (by definition, condensed absolute property but is highly dependent on the nature of hydrocarbons are formed of an uninterrupted conjugated the partner with which the complex is to be formed. From assembly of at least three annelated rings); such com Buu-HoÃ¯and Jacquignon's systematic studies (14) on the pounds, of course, possess neither a meso-phenanthrenic behavior of a large number of electron-acceptors toward K-zone nor a wzeso-anthracenic L-zone; both carcinogenic and noncarcinogenic aromatic hydro 2. condensed hydrocarbons and heterocyclic analogs carbons and heterocyclic analogs, it is apparent that, if a without K- and L-zones, or in whose molecule the K-region sufficient number of both electron-donors and acceptors are is not free for biochemical interaction ; examined, then no correlation emerges between biological 3. hydrocarbons and heterocyclic analogs with a activity and this particular chemical property. Thus, naphthacene nucleus, which was hitherto considered as with acceptors of the cyclic anhydride type (e.g., poly- "non-carcinophilic" ; halogenated phthalic and naphthalic anhydrides), which 4. large-size, hypercondensed hydrocarbons bearing at are more selective than the usual polynitro acceptors and least six benzene rings; hence closer to biochemical systems, no complex could be 5. polycyclic nitrogen compounds containing more than prepared with some 50 carcinogenic benzacridines, whereas one N-heteroatom. stable and deep-colored complexes were easily obtained Noncondensed hydrocarbons and analogs.â€”The most with all 50 Ã•ndoles,carbazoles, and benzocarbazoles, among interesting carcinogen in this group is 1,3',5-tri-p-xenyl- which only four were carcinogenic; even with a more benzene (VII), which Dannenberg (20) reported to produce classical electron-acceptor such as picric acid, complex papillomas and epitheliomas in situ as well as hepatomas in formation occurs with difficulty with naphthacene hydro skin-painting tests in mice. Although this compound is carbons, some of which are distinctly carcinogenic. Very still the only carcinogen known in the family of polyaryls, often, the transition from a hydrocarbon to its functional its activity represents a striking exception, in view of both derivatives (e.g., from 3,4-benzophenanthrene to its its very extended molecular dimensions and its lack of any 2-acetyl derivative, or from 1,2-benzanthracene to its 10- particularly reactive zone or center. However, the car formyl derivative) sharply depresses the capacity to form cinogenicity of such a simple hydrocarbon is less surprising charge-transfer complexes but enhances carcinogenic ac tivity. Last but not least, it is probable that complex formation in biochemical systems such as those with which the polycyclic carcinogens can interact in the first place, would be due more to van der Waals forces and hydrogen bonding than to real charge-transfer processes (8). This point is evidenced by the proved possibility of inhibiting the carcinogenicity of l,2:5,6-dibenzanthracene, a strong charge-transfer complexing agent, by its decahydro deriva tive (34), whose molecule lacks the polyconjugated frame necessary for efficient charge transfer. Again, the car cinogenicity of polycyclic compounds with only one carbon- when one considers the case of 1,2-benzofluorene (VIII) carbon double bond but with pronounced capacity to form and l,2:5,6-dibenzofluorene (IX); the activity of the complexes involving van der Waals and hydrogen bond former is only marginal, but that of the latter is in forces (apocholic acid [42]) argues in favor of a possible dubitable. These two hydrocarbons are to be considered role of this latter type of complex in chemical carcino as simple polyaryls (a phenylnaphthalene in the first case, a genesis (9). dinaphthyl in the second), and not as condensed hydro carbons as usually assumed. Another simple hydrocarbon DEVELOPMENTS IN THE FIELD OF NEW CAR which had proved on one occasion to be carcinogenic (25) CINOGENIC HYDROCARBONS AND is a-ethyl-|8-sec.-butylstilbene (X), although this marginal HETEROCYCLIC ANALOGS2 activity may have been acquired only by metabolic hy- Almost all the known characteristics of carcinogenic polycyclic molecules, and consequently the interpretations in "The Ring Index" (Patterson-Capell-Walker, American Chemical Society, Washington, D. C., 1960), and has the advan 2The nomenclature/numbering adopted for polycyclic com tage of corresponding to the nomenclature and numbering used in pounds throughout this Review is the more usual of the two listed the biological papers herein cited.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. 1516 Cancer Research Vol. 24, October 1964 droxylation, since the related stilbestrol (a,0-diethyl-4,4'- TABLE 3 dihydroxystilbene) is known to produce fibromyonms oc SABCOMAOENICPOTENCIESOF FLUORANTHENEHYDROCARBONS casionally at the site of implantation. Condensed hydrocarbons and analogs devoid of K- and and la NameFluoranthene3 sex of tency sar coma L-zones or with an impeded K-zone.â€”The main hydro miceflOcf (days)â€”130comasâ€”8index050 carbon group here is that of the fluoranthenes (47). Fluor- anthene itself is inactive, but annelation with a further benzene ring leads to the highly active 3,4-benzofluoran- \109iied1\149Ã•16d" thene (XI), which is not much less carcinogenic than 3,4- benzopyrene (sarcoma index for XI : 50 in male mice and ,4-Benzofluoranthene11 62 in females; for 3,4-benzopyrene: 91 in males and 47 in 133203 108 6224 females). At a stretch, 3,4-benzofluoranthene could be considered as having a K-zone, although impeded, but the ,12-Benzofluoranthenel'-Methyl-ll,12-benzofluoranthene11 \149(UÃ 1210â€”â€”â€”â€”2655â€”â€”â€”â€”1021000027 isomerie 11,12-benzofluoranthene (XII) clearly has none,

\149fl5

,12-Tetramethylenefluoranthene3 \149ilorf1 ,4-Tetramethylene-ll ,12-tetra- methylenefluoranthene3,4:ll,12-Dibenzofluoranthene2\149Ã•16o"

(XV) \149fi4(7\H9Av. although it is distinctly sarcomagenic. Other fluoran- ,3-Phenylenepyrene*No. thene derivatives, as for instance 3,4:10,ll-dibenzofluor- 145No. 1Sar 5 anthene (XIII), are also active (71). In this group, theo * Note for this substance the considerably greater suscepti retical computations, effected by means of three different methods, failed to indicate any relationship between bility of the males. electron densities and carcinogenic activity (47) ; it is also genie potency of several fluoro derivatives of 1,2-benzan- hard to understand why annelation of a benzene ring to fluoranthene either at position 3,4 or at position 11,12 thracene led Miller and Miller (55) to the similar conclu sion that a direct involvement of the K-zone is not promotes carcinogenicity, whereas annelation in the two sites at the same time leads to an inactive hydrocarbon necessary for carcinogenesis. (3,4:ll,12-dibenzofluoranthene). Examination of the Hydrocarbons and heterocyclic analogs derived from nitrogen-containing isosters (XIV) and (XV) of hydro naphthacene.â€”Naphthacene, or linear benzanthracene, is carbons (XI) and (XII) gave even more intriguing results, not carcinogenic, and prior to our work the few naphtha compound (XIV), although isosteric with the highly car cene derivatives that had been tested were also found in cinogenic 3,4-benzofluorantheue, being totally inactive, active. From these negative results it was inferred that whereas compound (XV), although corresponding to the the presence of a reactive meso-anthracenic region, which less active 11,12-benzofluoranthene, is itself quite potent characterizes this group, precludes activity. Recently, however, we found two naphthacene hydrocarbonsâ€” (48). Table 3 summarizes the biological results obtained namely, naphtho(2',3':3,4)pyrene (XVII) and phenan- with the various fluoranthenes. thro(2',3':3,4)pyrene (XVIII)â€”to be carcinogenic; the The discrepancies between theory and experience in the fluoranthene group could perhaps be ascribed to the pres ence of a five-member ring, which creates electrical dis symmetry in these molecules, a circumstance which is known to plague the electron density computations. However, this would still not account for the carcino genicity of hydrocarbons such as 5-methyl-l,2:3,4-dibenzanthracene (XVI) (Buu-HoÃ¯and Zajdela, unpublished experiments). It is also striking to note that 1,2:3,4- dibenzanthracene itself, an inactive hydrocarbon, under goes fixation on certain cell proteins as readily as com pounds having K-zones (57). That easy protein binding through the K-zone is not a necessity for carcinogenicity is former, the more active of the two, gave sarcomas by sub also demonstrated by the many substituted 1,2-benzan- cutaneous injection in eight out of twenty mice, with a thracenes and benzacridines which, although bearing relatively short latency period (average latencies: 169 days methyl groups in that zone, are nonetheless highly active in females and 214 days in males) (41). The activity of (72). Recently, the discovery of the very strong sarcoma- these two substances is not surprising, owing to the prÃ¨s-

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. Buu-HoÃ¯â€”ChemicalCarcinogenesis by Hydrocarbons 1517 enee, in their molecule, of the pyrene arrangement known TABLE 4 to be favorable for carcinogenicity; however, they are by SABCOMAGENICPOTENCIESOFTHE FOUR ACTIVE no means exceptional cases, since the more simple naph- DIBENZOPYRENESASCOMPAREDWITH thacene hydrocarbons (XIX) and (XX), although not 3,4-BENZOPYRENE(40,51) sarcomagenic in situ, have produced leukemias and ovarian tumors (Lacassagne, Buu-HoÃ¯,and Zajdela, unpublished andsex malswithsar results). Carcinogenicity is also encountered in nitrogen Name3,4:9,10-Dibenzopyrenel,2:4,5-Dibenzopyrene3,4:8, oÃmice17o1118921 analogs of naphthacene hydrocarbons. Thus, all four sub comas171618 stances (XXI) to (XXIV) have produced sarcomas by injection (16), the cr's-bisangular compound (XXI) being distinctly more active than the Â¿raws-bisangularstructure RXXVIIIXXVIIR= R' = (XXIII). cf 14935 1434110 9-Dibenzopyrene1,2:3, rf1109led"14914c? = R' =HXXIXIIIXo. 4-Dibenzopyrene3 179129 12138 (XXI) R=H (XXIII) R= H ,4-Ben zopyreneFormulaXXVI;R 169Averagelatency(days)7581.5112111111128144160Ani (XXII) R= CH5 (XXIV) R = CH,

It is clear, therefore, that many carcinogens exist among comagenic agent known. The order of sarcomagenic hydrocarbons derived from naphthacene and their aza activity in mice (observed after three injections of 0.6 analogs and that a high degree of chemical reactivity and mg. each, given at 1-month intervals) is shown in Table 4. susceptibility to addition-reactions (of the Diels-Alder Another interesting feature of these dibenzopyrenes con diene-synthesis type) do not necessarily exclude carcino cerns the variations in the activity of their substitution- genicity. Relevant to these observations is the pro products, which run opposite to the rules governing nounced activity displayed by naphthacene itself as an structure/activity relationships for the smaller-sized hy inducer of microsomal enzyme syntheses (azo dye N-de- drocarbons. Whereas in molecules like the 1,2-benzan- methylase and zoxazolamine hydroxylase) during a study thracenes methyl substitution in the meso-anthracenic zone made recently by Arcos, Conney, and Buu-HoÃ¯(3) on a enhances the activity, 5-methyl-3,4:9,10-dibenzopyrene large number of polycyclic hydrocarbons of different (XXVI; R = CH3, R' = H) and 5-methyl-3,4:8.9-di- molecular sizes. benzopyrene (XXVII; R = CH3, R' = H) are both Large size, hypercondensed hydrocarbons.â€”Condensed considerably less active than the nonsubstituted hydro hydrocarbons bearing six and more rings had been some carbons, and a double meso-methylation, leading to 5,8- what neglected (doubtless because they were thought to dimethyl-3,4:9,10-dibenzopyrene (XXVI; R = R' = exceed the optimum size for biological activity) until CH3) and 5,10-dimethyl-3,4:8,9-dibenzopyrene (XXVII; interest in them was aroused by the advent of theories R = R' = CHa), suppresses all activity. To complicate linking carcinogenic activity with structures possessing the picture further, this by no means applies to all hydro regions with high ir-electron densities; another spur in carbons of that type, things being exactly the contrary with their direction was the demonstration of the presence of anthanthrene, a hexacyclic hydrocarbon closely related to many such hypercondensed hydrocarbons in tobacco tar the dibenzopyrenes. Anthauthrene (XXX ;R = R' = H) and atmospheric pollutants (68,70). The most interesting itself is inactive, but one meso-methylation, leading to group is that of the dibenzo- and tribenzopyrenes. Here, 6-methylanthanthrene (XXX; R = CH3, R' = H), gives in support of the Pullmans' K-region theory, from among rise to activity, this being enhanced by a second meso- the five possible dibenzopyrenes, only the one devoid of a methylation to 6,12-dimethylanthanthrene (XXX; R = inâ‚¬so-phenanthrenic zoneâ€”i.e., l,2:6,7-dibenzopyrene R' = CH3) ! Even more unexpected is the carcinogenicity (XXV), has proved inactive; the other four are all ex- of 6-formylanthanthrene (XXX; R = CHO, R' = H),

fi* this compound being still more active than the correspond ing methyl compound, despite the fact that in the electron theory of substitutions, meso-formylation deactivates the K-regions. The data concerning the relationship between structure and activity in the anthanthrene group represent, (xxv) therefore, a major difficulty for the theory of T-electron densities, since here two substituents which exert opposite electron effects (i.e., the methyl and the formyl group) confer the same enhancing properties on the carcino genicity; nor can the likelihood of an in vivo reduction of (XXVMI) (xxix) the aldehyde function to a methyl group be considered, because the activity of the formyl derivative is superior to tremely potent carcinogens, and one of them, 3,4:9,10- that of the methyl one (45). A similar situation exists dibenzopyrene, is the most powerful and fast-acting sar- with 3,4:9,10-dibenzopyrene, whose meso-aldehyde

(XXVI; R = CHO, R' = H) is distinctly more active than separate groups of workers. In Paris studies on the the corresponding methyl compound. influence of replacement, in the molecules of the carcino Hydrocarbons of the dibenzopyrene and anthanthrene genic dibenzocarbazoles, of a benzene ring by a pyridine types are not the only active compounds made up of six one, led to the discovery that many of the benzopyridocar- assembled benzene rings: 1,2-benzoperylene is a weak car bazoles thus prepared were strongly carcinogenic, several cinogen, but methylation in position 3 (or 4) leads to a of them being even more potent than the dibenzocarbazoles potent compound, which gave sarcomas in four mice out of themselves (15). Thus, 5,6-benzopyrido(2',3':l,2)car- ten given injections of 0.6 mg. subcutaneously, a fifth bazole (XXXIII) and 5,6-benzopyrido(3',2':l,2)car- animal bearing a large lung tumor (39). bazole (XXXIV) are both considerably more active than From the viewpoint of relationships between carcino- their parent l,2:5,6-dibenzocarbazole (XXXV) in the genicity and the degree of condensation of aromatic hydro carbons, it is pertinent to mention that some compounds having more than six benzene rings are nonetheless active, despite their extremely low degree of solubility. Thus, al though coronene (heptacyclic) and pyranthrene (octa- cyclic) are inactive, l,2:4,5:8,9-tribenzopyrene (XXXI; R = H) and its methyl derivative (XXXI; R = CH3) are relatively potent sarco magens. It is not yet known induction of sarcomas by subcutaneous injection and are also able, by ingestion, to elicit epitheliomas of the fore- stomach in mice; the presence of two nitrogen heteroatoms, as compared with only one in the case of 1,2:5,6-dibenzocarbazole, has deeply altered the nature of the cellular effects, since compounds (XXXIII) and (XXXIV) no CH=CH (xxxi) (xxxii) longer behave as carcinogens for the liver. In accord with the second prediction, the relative positions occupied by whether this degree of condensation represents an upper these two nitrogen atoms have a profound influence on the limit for carcinogenicity (44); insofar as molecular size is carcinogenicity, a slight shift of the pyridinic nitrogen concerned, the chances are that the threshold is far from atom resulting in a complete loss of activity, as shown in being reached, since so large a hydrocarbon as o-styryl-3,4- the case of 1,2:6, 7-dibenzo-/3-carboline (XXXVI). Simi benzopyrene (XXXII) (as judged by the computation of lar observations were made with aza analogs of 1,2:7,8- its "encumbrance area," after Arcos and Arcos [2]) has dibenzocarbazole (XXXVIII) and 3,4:5,6-dibenzocar- shown considerable carcinogenic activity with respect to bazole (XXXIX); 7,8-benzopyrido(2',3':l,2)carbazole both the number of tumors and the shortness of latency (XL) is considerably more sarcomagenic than its par period (38). Such examples are of significance when the ent compound, and 5,6-benzopyrido(3',2':3,4)carbazole theories which link carcinogenicity to the intrusion of carcinogens in molecules of nucleic acids are discussed. TABLE 5 Polycyclic compounds with several nitrogen heteroatoms.â€” That replacement of a â€”CH= group by a nitrogen atom COMPARATIVEACTIVITIES OF DIBENZOCARBAZOLESAND BENZOPYRIDOCARBAZOLES in the molecule of a carcinogenic hydrocarbon maintains the activity and may even at times enhance it was already Substance1,2:5 index patent from the example of the angular benzacridines (36), (inmice)10Ã•6401 in which family at least as many carcinogens have been found as in the 1,2-benzanthracene group; aza analogs of ,6-Dibenzocarbazole5,6-Benzopyrido 3,4-benzopyrene and of 5,6-benzochrysene are also active (48). The question whether compounds with two or more 2)carbazole5,6-Benzopyrido(3',2':l,2)carbazolel,2-Benzopyrido(3',2':5,6)carbazole1,2:6,(2', 3': 1, nitrogen heteroatoms would likewise display carcino \79ÃlOOd" genicity was of fundamental importance, since such mole cules can form complexes with appropriate cell receptors not only through their zones of high 7r-electron densities \ 739flOc?\ but also through their several nitrogen heteroatoms. If this multicentric process of attachment were to produce 79025ilOd1 ultimately the same deleterious effects as the normal forms of attachment (i.e., through zones of high ir-electron 7-Dibenzo-ÃŸ-carboline3, densities), then two important predictions could be made: first, that many polycyclic compounds bearing several 4: 5,6-Dibenzocarbazole3,4-Benzopyrido(3',2':5,6)carbazole5 nitrogen heteroatoms would be carcinogenic; and, second (concerning structure/activity relationships in this type of molecule) that the position of the various nitrogen atoms \091340" with regard to one another would be of capital importance. ,6-Benzopyrido (3' ,2' :3 ,4)carbazoleFormulaXXXVXXXIIIXXXIVXXXVIIXXXVIXXXIXXLIIXLISarcoma These predictions are entirely borne out by the results of \249 investigations carried on in the last few years by two

(XLI) is slightly more so than compound (XXXIX). The TABLE 6 SKIN CARCINOGENICITYINMICE OF ACTIVE TRICYCLOQUINAZOLINEDERIVATIVES(4)

CompoundTricycloquinazoline1 carci latency mice36204418541722No. index4417201662025 (xxxvi) (xxxvii) nomas27921851115Mean(days)186316296282277375274Iball potencies of several benzopyridocarbazoles as compared with the parent dibenzocarbazoles are given in Table 5. ycloquinazoline3-Methyl-Methyl-trie Another interesting aspect of the biological properties of -tricycloquinazoline4-Methyl -tricycloquinazoline3,8,13-Trimethyl-tricycloquinazoline3-Fluoro-tricycloquinazoline3-Bromo-tricycloquinazolineNo. this new group of benzopyridocarbazoles is the complete loss of activity brought about either by methyl substitu tion or by further annelation with a benzene ring in the vicinity of the pyridine nitrogen atom, as witnessed by the total inactivity of compounds (XLIII) and (XLIV). polycyclic hydrocarbons derived from phenanthrene can be bound to proteins by means of their K-regions. The possibility that the nuclear nitrogen atoms in carcinogenic nitrogen-containing heterocycles react with cell substrates is reinforced by the existence of a correlation between carcinogenicity and the high pK values of several angular benzacridines (59). The newer picture of a carcinogenic hydrocarbon.â€”The sum of all the newly acquired information outlined above, added to such earlier knowledge as has stood the test of time and experience, suggests now the following image for a carcinogenic hydrocarbon, which in many respects is less restricted, and perhaps more simple, than before. a) It is an aromatic molecule made up of a more or less extended conjugated system, and whose dimensions can (XLIII) (XLIV) vary within considerably wider limits than previously as sumed (compare a "small" carcinogen like 9,10-dimethyl- Research done in England on a different chemical series anthracene with a "large" one like 5-styryl-3,4-benzo- led to similar conclusions. Baldwin, Partridge et al. (4), pyrene !); the upper limit of molecular thickness compatible working on tricycloquinazoline (XLV), a hexacyclic com with activity is also higher than was thought before (wit pound containing four nitrogen heteroatoms, found for ness the activity of several dibenzacridines bearing bulky this substance a skin carcinogenicity intermediate between substituents [50]!). that of 3,4-benzopyrene and l,2:6,5-dibenzanthracene; 6) The conjugated frame offers certain sites and areas of methylated and halogenated tricycloquinazolines were also high TT-electrondensities, hence of greater covalent and active, to a degree depending on the position of the sub- noncovalent reactivity, through which the interaction with stituents. Table 6 reports the relative potencies of seven cell components is facilitated ; in many instances, there is a tricycloquinazolines tested by Baldwin et al. by skin- mÂ«so-phenauthrenic K-zone, whose involvement in the metabolic degradation of the carcinogen has been experi mentally well established (6) but whose existence is essential neither for protein-binding nor for carcinogenicity. Where meso-anthracenic L-zones are present (case of naphthacene hydrocarbons), their reactivity does not (XLV) (XLVl) necessarily preclude carcinogenicity. Replacement of â€”CH= groups by tervalent nitrogen heteroatoms may painting in mice. More recently, the same authors found have a positive or a negative effect on the carcinogenicity, compound (XLVI), which is isomerie with tricycloquin depending on the number and position of the nitrogen azoline but has a different arrangement of nitrogen atoms, atoms and on the nature of the molecule; when several to be only slightly active (60) ; skin tumors have also been nitrogen heteroatoms are present, the prime importance of induced with several phcnanthrolines, including the diaza their position in relation to one another suggests that they analog (XLVII) of 3,4-benzophenanthrene (61). None act as centers for binding with cell components, in place of, of these substances possesses a true ?Â»eso-phenanthrenic or in conjunction with, other zones of biochemical inter region. The results obtained by this group of workers are action. thus also in line with the theory that carcinogenesis can be c) Where there are substituents, these may be either associated, at least in part, with a multicentric interaction electron-donating or electron-accepting groups (except acid between a polycyclic substance containing several nitrogen functions), and their contribution to the carcinogenicity heteroatoms and cell constituents, in the same manner as may be positive or negative, depending on the type of

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. 1520 Cancer Research Vol. 24, October 1964 molecule. In the case of alkyl substituents, lengthening investigations on the possibilities of interaction between of the chain has an adverse effect on activity, owing to in polycyclic carcinogens and nucleic acids. Some fixation of crease in the encumbrance area of the carcinogen (2), the aromatic hydrocarbons, carcinogenic and noncarcinogenic, degree of loss of activity depending on the site of substi by the nucleic acids of mouse skin has in point of fact been tution. The introduction of substituents with acid hy- detected (30), though it is far less significant than for the droxyl groups (carboxyl, sulfonic acid, and phenolic func proteins (P. Daudel, experiments to be published) ; in any tions) invariably results in a sharp decrease or total loss of event, the intimate nature of this binding is not known, carcinogenicityâ€”an effect which must be due to a de although a direct intrusion, suggested by some authors, of parture from the "normal" molecular orientation of the the polycyclic carcinogen into the spaces between the base- carcinogen within cellular lipid structures, produced by the pairs in nucleic acid structures appears very unlikely for strong hydrophilic radical. both steric and energetic reasons, even taking into con These, then, are the basic physico-chemical character sideration (as did Dannenberg recently [22]) the property istics which are to be borne in mind when formulating or of the chromosomes to swell up in the sites of gene-ac assessing general theories on the mode of interaction of tivity; nor would it be easy to explain the carcinogenic polycyclic aromatic hydrocarbons and their heterocyclic process (which can be induced by many substances that analogs with cell components. The electronic theory of are nonmutagenic) on the basis of a mere tampering with carcinogens which has proved of such great value in the the genetic code by such intrusion. Indeed, carcinogenesis past, as a guide in the search for active compounds through a direct tampering with the genetic code seems less through the maze of organic chemistry, can probably con likely than through a perturbation in the transmission of tinue to play that role if through adequate refinements this code, perhaps through a combination of RNA particles and/or modifications it can integrate the new experi with proteins that have been modified by the hydro mental data. carbons; such "masking" phenomena are known to exist Although there is now ample evidence that polycyclic when protein antigens are fixed on RNA (Campbell and carcinogens can interact with cell proteins, the intimate Garwey). nature of the receptors involved and of the binding proc In addition to proteins, and perhaps to nucleic acids, the esses at work is not known, any more than is the relation of lipid structures also play an essential role in hydrocarbon these biochemical transformations to the biological event carcinogenesis, if only by virtue of their part in the trans of carcinogenesis. Although a statistical study of the port of the carcinogen to its target. This role is evidenced affinity of hydrocarbons, carcinogenic or not, for proteins by the strikingly universal decrease in carcinogenicity of the mouse skin, indicates, grosso modo, a greater binding which results from substitution with hydrophilic acid facility among the active compounds (23), it is not yet groups. clear whether there is specificity in respect to the loci occupied by carcinogens and noncarcinogens on the pro THE QUESTION OF POLYCYCLIC HYDRO tein structures. Recent work by Heidelberger et al. (24) CARBONS AS POSSIBLE ENDOGENOUS does suggest that the active 1,2:5,6-dibenzanthracene is CARCINOGENS bound by some water-soluble protein fractions (similar The possibility that carcinogens may be formed within to those in the rat liver which bind 2-acetaminofluorene the body as a result of some metabolic deviation is a ques (64, 69) to a greater extent than is the noncarcinogenic tion that has excited interest ever since methylcholanthrene l,2:3,4-isomer; this again, however, would still be a was prepared from a bile acid and then recognized as a matter of only qualitative differentiation. As concerns powerful carcinogen. Although there is not yet any proof the nature of the linkages involved in the interaction be that living organisms are able to synthesize polycyclic tween carcinogen and protein, the wide variety of carcino aromatic hydrocarbons, carcinogenic or not, most of the genic structures, the presence of strong van der Waals and reactions through which known metabolites of the cell hydrogen-bonding forces on both sides, and the existence could in theory be converted into such hydrocarbons are of polycyclic carcinogens that are chemically inert save for already known to occur endogenously, and others are mild their non-covalent complex formation capacity (deoxy- enough to be conceivable. One substrate often considered cholic and apocholic acid), all speak in favor of a primary as a possible source of carcinogen endosynthesis is cho non-covalent, "hole-plugging" process (formation of an in lesterol (XLVIII). Following Bergmann's suggestion that clusion compound), the covalent complex formation experi cyclization, dehydration, and dehydrogenation of this sub mentally detected being but a second stageâ€”i.e., the me- stance could give rise to the 1,2-benzanthracene derivative tabolization of the hydrocarbon. Indeed, in one theory (XLIX) which he termed "steranthrene" (5), now called on the mode of action of polycyclic carcinogens, it is the 3,6-dimethylsteranthrene, Dannenberg prepared a closely "hole-plugging" process, considered as competitive with related hydrocarbon, the so-called angular steranthrene the normal protein-binding procedure of chemical informa (L), which proved to be highly carcinogenic both on skin- tion substances, that is regarded as essential for carcino painting and on injection (19). Another conceivable genicity (10); charge-transfer and ionization potentials, cyclization of cholesterol, suggested by Buchta, would lead characteristic of the highly conjugated frame which poly to a different type of hydrocarbon, represented by formula cyclic hydrocarbons possess, come next as agents of the (LI), and whose basic skeleton is known as F-nor-steran- ensuing nonlethal deterioration of the host-protein, along threne. Recently, F-nor-steranthrene itself (LII), tested lines similar to Miller's protein-deletion hypothesis (54). in our laboratory, proved to be an extremely potent car Recent progress in biochemical genetics has prompted cinogen which, given in subcutaneous injections, produced

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. Buu-HoÃ¯â€”ChemicalCarcinogenesis by Hydrocarbons 1521 a large number of sarcomas in male mice (Radium Insti nous formation of carcinogenic hydrocarbons may be from tute strain XVII nc/z) and, in the females, both sarcomas the theoretical viewpoint, it must be recognized that it is and epidermoid parakeratotic mammary epitheliomas highly speculative. As a "referee" of this review has which started in the excretory ducts (35). A methyl pointed out, known biochemical reactions in animals tend homolog of F-nor-steranthrenc, tested more recently, also to be degradative: thus, ring hydroxylation, side-chain proved actively carcinogenic. oxidation, conjugation with glutathione, etc., would lead in a direction opposite to the building up of a condensed CH. aromatic hydrocarbon. Furthermore, it does not seem necessary to explain spontaneous cancer in terms of en dogenous hydrocarbon synthesis when other endogenous

(XLVIIl) (hormones) or exogenous factors can account more simply for the induction of many neoplasms.

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N. P. Buu-Hoï

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