The Role of Analytical Chemistry in Carcinogenesis Studies by Walter Troll New York University Medical Center New York City
In chemical carcinogenesis studies, biologists hope to find species with fast responses to carcinogenic agents; molecular biologists (modern chemists) hope to find a mechanism for the disease to clarify what materials are dangerous; the analytical chemist hopes to measure these materials and support the proposed mechanism
B EFORE
ANALYTICAL CHEMISTS can get to work assaying man's hostile environment for the cause of cancer, they need a working h y pothesis for the mechanism of chemicals as cancer agents. The suspicion t h a t definite chemical substances m a y be the major cause of cancer originated with the observation by Sir Percival Pott in 1775 (1) t h a t chimney sweepers have an unusually high incidence of cancer of the scrotum, which was ascribed to their occupational exposure to soot. The nature of the chemicals directly responsible was not uncovered until many years later with the help of experimental animals, the rabbit ear in 1915 by Yamagiwa and Ichikawa (2) and then the mouse skin by Kennaway in 1959 (3). The chemicals involved appeared to be polynuclear hydrocarbons such as b e n z o ( a ) pyrene and 3-methylcholanthrene. Another chemical criminal of this type was apprehended by the initial observation of Rehn in 1815 (4) t h a t workers in dye manufacture had an unusually high incidence of bladder cancer, followed in 1938 by the observation of Heuper t h a t 2-naph-
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ANALYTICAL CHEMISTRY
thylamine causes bladder cancer in the dog (5). The observation of bladder cancer in the dog followed many failures to produce any kind of cancer in many species by this agent. One reason for this failure we know now, is the fact t h a t 2naphthylamine has to be m e t a b olized in a specific manner only known to be present in m a n and dog (6). T h e proximal carcinogen, as this type of biologically made carcinogen came to be known, is a bis-2-aminonaphthyl phosphate (Figure 1). Biological activation of apparently harmless compounds appears to be one of the general rules of the production of cancer by chemicals. Thus, the Millers and their associates working with 2-acetylaminofluorene (the proposed insecticide turned rat liver carcinogen) identified esters of JV-hydroxy-acctylaminofluorene as the proximal carcinogens (Figure 1) (7), while with dialkylnitroamines the proximal carcinogen appears to be related to diazomethane (Figure 1) (8). T h e number of direct carcinogens not requiring metabolic activation has been shrinking but the group rela-
tively safe from this type of erosion are alkylating carcinogens such as (8-propiolactone (Figure 1) (9). Initiators and Promoters
To a t t e m p t to classify carcinogens into chemically related species is further complicated by the observation t h a t the production of cancer depends on a variety of factors acting independently of each other and presumably acting on distinct biological targets. T h e clearest demonstration of this came out of the experiments of Berenblum and his associates with mouse skin (10). When the skin of mice was painted with a low dose of benzo(a)pyrene, no significant tumor production occurred unless the original painting was followed by repeated regime of painting with a "promoting agent." If the procedure was reversed by adding the promoting agent first, no tumors resulted. The chemical principle of croton oil has been purified and it turns out to be a highly specific ester of the alcohol phorbol (Figure 2) (11, 12) ; phorbol itself, as well as phorbol acetate, is essentially without activity. Thus, the
REPORT FOR ANALYTICAL CHEMISTS are fed t o mice t h e tumor will a p pear where the promoting agent is applied (13). T h e evidence for t h e case of chemical carcinogenesis can be summarized: 1 ) . Chemicals from our environment are capable of causing cancer. 2 ) . T h e y can per form this feat either directly—e.g., (8-propiolactone, a biological al kylating agent, or after metabolism to an activated form—e.g., 2-naphthylamine t o bis-2-aminonaphthyl phosphate. 3 ) . T h e place where cancer occurs can be determined by the interaction of a different r e versibly active promoting agent.
chemical specificity of promoting agents also appears to be a require ment for cancer production. T h e appearance of cancer in mouse skin depends on a t least two factors: one t h a t is applied a t the beginning of t h e experiment, t h e initiator, a n d another, t h e pro moter, t h a t is applied thrice weekly until the appearance of papillomas or cancers. T h e action resulting from the initiator has been called "initiation" and it can be shown to be irreversible in its biological r e sponse. An initiator can be applied to mouse skin followed in t e n months by t h e promoter a n d t h e same number of tumors will appear as when promotion treatment was started after only two weeks (12). On the other hand, promotion a p pears to be a reversible phenom enon since application three weeks apart is ineffective. I t is not neces sary to apply the initiating agent and t h e promoting agent t o t h e same spot to produce a tumor. When urethane or 2-aminofluorene
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Mechanism of Carcinogenesis
F o r the chemist to be of any ser vice, a general hypothesis for t h e mechanism by which these chemi cals interact with biological targets is required. Modern molecular biol ogy provides a framework for t h e mechanism of carcinogenesis which is t h e demonstration t h a t all bio logical information is contained in
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the master t a p e of D N A in a code form. T h e code is the order of its constituents, adenine, guanine, thymidine, and cytosine which is transcribed by R N A polymerase into a working tape of R N A which in turn is translated into specific amino acid corporation into pro teins. Thus, a change in the master tape D N A will result in a protein of different structure (Figure 3 ) . T h e active initiating carcinogens can be shown t o modify D N A by, for example, substitution on one of the constituents (14). On replica tion of such a substituted D N A a wrong base m a y be incorporated producing a somatic mutation. T h e promoting agent m a y act by per mitting t h e mutation to be ex pressed (15). The major portion of the genetic information is covered by a curtain of repressive histones so t h a t errors behind t h e curtain do not do harm until they are per mitted t o see the light of day. Several mechanisms have been proposed for gene activation such as the acetylation of lysine leading
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to the discharge of the histone (16). The one we favor is selective hy drolysis of the histoires by proteo lytic enzyme (15). Watson and Crick established that D N A exists in the form of a double helix (17). The helix is held together by specific forces between the bases adenine and thymine and between guanine and cytosine. If an initiating carcinogen combines with one of these bases the struc ture is disturbed. The disturbance can be measured by changes in two physical criteria, T m and buoyant density, and with greater sensitiv ity by the lowering of the priming action of the modified D N A with R N A polymerase. T m is a measure of the heat stability of the DNA, which is determined by noting the rise of UV absorption due to the separation of the two strands, and is defined as the temperature at the midpoint of the total rise of UV absorption obtained by heating the D N A solution. A substitution by B P L on N-7 of guanine, or of acetoxy-AAF on C-8 of guanine lowers the attraction between G and C as indicated by a lowered T m (18). Another physical cri terion of DNA, depending in part on the double stranded nature of this polymer, is the buoyant density in cesium chloride observed by ultracentrifugation in an analytical ultracentrifuge. This criterion is similarly depressed after substitu tion by these two carcinogens (18). D N A exerts its biological power by interacting with two enzymes, D N A and RNA polymerase (see Figure 3). The precise structure of D N A or R N A formed with these two enzymes is dictated by the 24 A .
ANALYTICAL CHEMISTRY
Figure 3. The role of DNA in transcription and the translation of RNA into a specific protein. The final result of a mutation—modification of DNA— is an altered protein containing a dif ferent amino acid sequence
priming D N A . The precursors of the polymer are the purine and pyrimidine deoxyribose or ribose triphosphates in the presence of primer D N A and the enzyme lines up the purine and pyrimidine bases on a phosphate backbone and re leases pyrophosphate. Carcinogen modified D N A is a poor primer for R N A polymerase. This can be demonstrated by reacting D N A with carcinogens in vitro (18, 19) or isolating D N A from carcinogen treated animals and using it as primer with R N A polymerase (20). D N A polymerase priming, on the other hand, is only slightly affected by combination with carcinogens, and in fact, increases in efficiency after the appearance of tumors (20). The inhibition of R N A poly merase priming by carcinogens may be responsible for some of their early toxicity, but the lack of in hibition of D N A polymerase allows for the occurrence of mutations, since the substituted D N A may in correctly or ambiguously direct the incorporation of a base into its daughter D N A (21). The ability of initiating carcino gens to cause mutations in bacteria has been correlated among 10 ep oxides ; six which are known to be carcinogenic were mutagenic in bacteria and four were inactive in both systems. Acetoxy-AAF was positive in both systems while the parent AAF and its metabolite V-hydroxy-AAF was negative in both systems (22). All these cri teria support the notion t h a t car cinogens capable of initiating can cers in mouse skin combine with the D N A causing misreading and so matic mutation. Unfortunately,
this is probably less than half the story of carcinogenesis. Promotion, as noted above with mouse skin, may present the other half for t h a t system but surely remains a con cept to be explored with other can cer test systems. Repressed vs. Expressed Genetic Information
The mechanism of promotion in volves the expression of genetic in formation which is repressed in the chromosomes. According to Jacob and Monod, "The fundamental problem of chemical physiology and embryology is to understand why tissue cells do not express all the time the potentialities inherent in their genome" (23). In relation to cancer the lack of expression of all the errors introduced into the genome is fortunate and can be con sidered an evolutionary safeguard. This is not the only biological pro tection built into the system, since there are D N A repair systems which operate by cutting out pieces of the D N A modified by a substi tuent, which are then replaced using the undamaged second strand as primer (24). If this repair is carried out before the D N A strand has had a chance to replicate or be ex pressed in any way, no noticeable damage will have occurred. The most obvious materials in volved in repression of genetic in formation in mammalian systems are a group of basic proteins called histones. The total information of the genome is only required during embryonic development after which a great deal of the information is permanently lost or unavailable. This is different in bacteria where
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specific loci, such as the ability to synthesize galactosidase, need to be induced or repressed but where most of the genetic information is available to the organism. T h e repressor material in bacteria appears to be a specific protein (25) b u t the cloud of protecting histones is characteristic of the multicellular differentiated organism. T h e precise biological mechanism by which histones are removed to bring about embryonic development is not known, but evidence for the involvement of proteases resembling trypsin has come from work with eggs of echinoderms (26-28). Promoting agents have been shown to release lysosomal enzymes in direct proportion to their activity (15). This would support the notion t h a t the mechanism of these agents is through the removal of histones by hydrolytic enzymes. Unfortunately, this kind of thesis does not lead to a generalization useful to the analytical chemist, but does provide the enzymologist with some clues. T h u s , the capability to liberate lysosomal enzymes from a preparation of rabbit liver lysosomes has become the first independent guide post for assessing promoting activity of suspected chemical offenders (15). This technique needs to be expanded until it can replace the more laborious mouse skin test. Lung Cancer
The importance of promoting agents for the production of lung cancer as a result of cigarette smoking has been pointed out by a number of investigators (29, 30). T h e fact t h a t the insult appears to be reversible, t h a t is, the individual giving up smoking returns to the normal risk after 10 years, supports the promotional thesis (29). T h e chemical nature of promoting agents defies generalization. I n addition to the highly specific phorbol ester, m a n y other purified materials of known structure such as the esters of sorbitol, the tweens, as well as m a n y fractions from cigarette smoke condensates containing phenols or acids have been shown to be active as promoters in mouse skin (29). T h e promoting activity of these agents is considerably
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VOL. 4 1 , NO. 3, MARCH 1969
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26A · ANALYTICAL CHEMISTRY
Report weaker t h a n t h a t noted with croton oil and its purification products. When we move to the area of air pollution, we observe a family of compounds containing both initiators and promoters, such as phenols of unknown constitution, the question of their direct role remains open (31). There is however, no question that the cigarette smokers or urban populations exposed to automobile exhaust products have a significantly higher rate of lung cancer, but the correlation of all the different kind of exposures in relation to production of cancer remains extremely difficult. T h e situation is clearer when we have a starting point, for example, with uranium miners or chromium workers where the product of the radioactive metal radon, or the chromium metal are the obvious prime suspects (32). However, all the other known agents which increase lung cancer in the rest of the population serve to confuse the picture. For example, the smoking miner has twenty times the incidence of lung cancer t h a n his nonsmoking coworker (32). The air we breathe, contaminated with cigarette smoke or not, is clearly not the only source of carcinogens ; the food we eat and the drugs we take m a y make a sizable contribution. Prominent among the materials under suspicion are the aflatoxins, which appear to be the active carcinogens present in certain batches of peanut meal fed to a variety of domestic animals (33, 34). The chemical appears to arise from a fungus growing on the nut and is, perhaps, the most studied of the larger group of plant carcinogens which includes the cycasins, extracts of braken fern and extracts from a variety of fungi (3537). Aflotoxin Blt a liver carcinogen in rat, combines with D N A , reducing its priming activity with R N A polymerase; it fits well into the category of initiating chemicals described above. T h e cycasins are naturally occurring nitroso amines and appear to be metabolized to a proximal carcinogen methylazoxymethanol. Thus, the properties of these naturally occurring carcinogens are similar to those introduced by man.
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Many drugs modify the enzyme pattern of the host and may affect the response of carcinogens from other sources either by accelerating its metabolism to a proximal carcinogen or by gene activation. Neither of these unpleasant prospects have been clearly translated into the production of tumors, but the induction of many enzymes resembling that produced by known carcinogens has been demonstrated with drugs such as phenobarbitol (38). Epidyomological investigation of the appearance of lung cancer has succeeded in delineating a latent period of two decades for the appearance of the clinically recognizable disease from initial exposure to the carcinogenic material. These data were obtained by Clemmessen and his coworkers studying the increase of mortality from lung cancer beginning approximately in 1931 in Copenhagen (39). Clemmessen placed the introduction of the etiological agent during the period between 1900 and 1910. He identified it as the onset of heavy cigarette smoking and found no reason to blame the sudden appearance of an air pollutant for the increased occurrence of lung cancer. The increase in lung cancer incidence in the United States became clinically of importance in 1920 and as pointed out by Dorn (Jfi) followed a similar pattern. The competition for etiological agent most responsible for the appearance of lung cancer between products from cigarette smoke and atmospheric pollutants has remained with the main support for the air pollutant being the increased lung cancer noted in urban regions compared to country areas (31), but the cigarette smoker shows the highest rate of lung cancer in all circumstances.
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ANALYTICAL CHEMISTRY
A latent period for the onset of the clinical manifestation of the disease appears to be the one uncontroversial sobering fact which has appeared from these studies, as well as from the experience with aromatic amines in bladder cancer and from data of scrotal tumors in chimney sweepers. The fact is cer-
tainly discouraging, since even if we knew the nature of all the carcinogenic materials we daily consume in one form or another, and removed them immediately, clear results of this would not be evident for twenty years. The main task for the realistic experimenter in this area is to shortcut this twenty years. The biologist looks for species which show faster responses to carcinogenic agents. The modern successor to the chemist—the molecular biologist—attempts to formulate a mechanism for the disease which will give him immediate insight into what materials are dangerous, and finally, the analytical chemist sweeps up by measuring these materials with a fervent hope of supporting the mechanism that has been proposed. Literature Cited (1) P . Pott, "Chirurigical Observations Relative to the Cataract, the Polypous of the Nose, The Cancer of the Scrotum, The Different Kinds of Ruptures, and the Mortification of the Toes and Feet," Printed by T. J. Carnegie, 1775 Hawes, Clarice and Collins, London, 1775. (2) K. Yamagiwa and K. Ichikawa, Mitt. Med. Fan Tokio, 1 5 , 295 (1915). (3) A. Haddow in "The Physiopathology of Cancer" (Homburger Ed) 2nd ed. Chapter 14, Harper, New York, 1959. (4) L. Rehn, Arch. Klin, Chir. 50, 588 (1895). (5) W. C. Hueper, F . H . Wiley, and H . Wolfe, Ind. Ayg., 20, 46 (1938). (6) W. Troll and S. Belman, In '^Bladder Cancer," Aesculapius Publishing Co., Birmingham, Ala., U.S.A., Chapter 2, 1967. (7) E. C. Miller and J. A. Miller, Pharmacol. Rev., 18, 805 (1966). (8) P. N . Magee and J. M. Barnes, Advan. Cancer Res., Vol. 10, 164 (1967). (9) E. D. Palmes, L. Orris, and N . Nelson, Am. Ind. Hyg. Assoc. J., 2 3 , 257 (1962). (10) I. Berenblum and P. Shubik, Brit. J. Cancer, 3, 109 (1949). (11) E . Hecker, Cancer Res., 28, 2338 (1968). (12) B. L. Van Duuren, "Progress in Experimental Tumor Research" 1 1 , 31 (1968). (13) J. H. Weisburger and E . K. Weisburger, "Methods in Cancer Research" 1,307 (1967). (14) P. Brookes and P . P. Lawley, Brit. Med. Bull, 2 0 , 91 (1964). (15) G. Weissmann, W. Troll, B . L. Van Duuren, and G. Sessa, Biochem. Pharmacol., 17, 2421 (1968). (Continued
on page 30 A)
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(16) V. G. Allfrey, Cancer Res., 26, 2026 (1966). (17) F. H . C. Crick and J. D. Watson, Proc. Roy. Soc. (London), 223, 80 (1954). (18) W. Troll, E. Rinde, and P. Day. Biochim. Biophys. Acta., 174, 211 (1969). (19) S. Belman, T. Huang, E. Levine, and W. Troll, Biochem. Biophys. Res. Comm., 14, 463 (1964). (20) W. Troll, S. Belman, E. Berkowitz, Z. F . Chmielewicz, J. L. Ambrus, and T. J. Bardos, Biochim. Biophys. Acta, 157, 16 (1968). (21) W. Troll and E. Berkowitz, Symposium of Physical Chemistry of Carcinogenicity, Hebrew University Press, 1969 (In Press). (22) F . Mukai and W. Troll, N.Y. Acad, of Sciences, (1969) In Press. (23) J. Monod and F. Jacob, Cold Spring Harbor Symp. Quart. Biol., 26, 389 (1967). (24) P. D. Lawley, Progress Nucleic Acid Res. Mol. Biol, 5, 89 (1966). (25) M. Ptashnc, Nature, 214, 5085 (1967). (26) Y. Mano, Biochem. Biophys. Res. Commun., 2 5 , 216 (1966). (27) A. Monroy, R. Maggio, and A. M. Rinaldi, Proc. Natl. Acad. Sci. U.S., 54, 101 (1965). (28) W. Troll, A. Grossman, and S. Chasis, Biol. Bull., 135, No. 2, p. 440 (1968). (29) E. L. Wynder and D. Hoffmann, Science, 162, 862 (1968). (30) B. L: Van Duuren, Cancer Res., 2 8 , 2357 (1968). (31) P. Kotin, Cancer Res., 16, 375 (1956). (32) W. C. Hueper, "Occupational and Environmental Cancers of the Respiratory System," Springer Verlag, New York, 1966. (33) M. C. Lancaster, Cancer Res., 2 8 , 2288 (1968). (34) G. N . Wogan, ibid, 28, 2282 (1968). (35) G. L. Laquer and M. Spatz, ibid, 28,2262 (1968). (36) J. M. Price and M. Pamukcu, ibid, 28,2247 (1968). (37) F. Blank, O. Cain, G. Just, D. R. Mcranze, B. Shimkin. and R. Wicder, ibid, 28, 2276 (1968). ' (38) A. V. Gelboin, Advan. Cancer Res., 10, 1 (1967). (39) J. Clemmessen, A. Nielsen, and E. Jensen, Brit. J. Cancer 5, 159 (1951). (40) H. F. Dorn, Acta Union. Internat Contre CE Cancer, 9, 126 (1953).
Research supported by Project Grants from the N I H (USPHS R e search Grants Nos. CA-09568 and CA-08491) and a grant from Allied Chemical Corp., and is part of a Core Program supported by the U S P H S , Bureau of State Services Grant ES-00014, and the National Cancer Institute Grant CA-069S9.
Walter Troll, Associate Professor at the New York University Medical Center, Institute of Environmental Medicine and Dept. of Physiological Sciences, was born in Vienna, Austria. He earned his B.S. degree in 1944 from the University of Illinois, his M. S. degree in 191fi at the Pennsylvania State University, and his Ph.D. degree in 1951 at New York University. Before returning to NYU in 1956, Dr. Troll held positions with the May Institute for Medical Research in Cincinnati. Ohio, and the Cancer Research Institute, New England Deaconess Hospital in Boston, Mass. His main research interests have been in colorimetric assay and amino acid analysis, blood clotting, and the mechanism of carcinogenesis. Dr. Troll is a member of the American Society of Biological Chemists, the ACS, and the New York Academy of Sciences. Dr. Troll presided over a symposium on "Analysis in Carcinogenesis" at the 1968 Eastern Analytical Symposium, New York City, Nov. 13. This report is based in part on his presentation at this meeting.
