The Role of Analytical Chemistry in Carcinogenesis by Waiter 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 t o 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
EFORE AXALYTICAL CHEMISTS
can
B get to work assaying man’s hos-
tile environment for the cause of cancer, they need a working hypothesis for the mechanism of chemicals as cancer agents. T h e suspicion t h a t definite chemical substances may be the major cause of cancer originated with the observation by Sir Percival Pott in 1 7 i 5 ( 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 Ichika\va ( 2 ) and then the mouse skin by Kennaway in 1959 (3). T h e chemicals inyolved appeared to lie polynuclear hydrocarbons such as benzo ( a ) pyrene and 3-niethylcliolanthrene. 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 that 2-naph22A
ANALYTICAL CHEMISTRY
thylamine causes bladder cancer in the dog ( 6 ) . T h e 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 that 2naphthylamine has t o be metabolized in a specific manner only known to be present in man 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 ;V-hydroxy-acetylaminofluorene as the proximal carcinogens (Figure 1) ( 7 ) ,while with dialkylnitroamines the proximal carcinogcn appears t o be related t o 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 p-propiolactone (Figure 1) ( 9 ) . Initiators and Promoters
T o attempt to classify carcinogens into chemically related species is further complicated by the observation that the production of cancer depends on a variety of factors acting independently of each other and presumably acting on distinct biological targets. The clearest demonstration of this came out of the experiments of Berenblum and his associates with mouse skin ( I O ) , 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 ) ( 1 1 , 12) ; phorbol itself, as n-ell as phorbol acetate, is essentially without activity. Thus, the
REPORT FOR ANALYTICAL CHEMISTS chemical specificity of promoting agents also appears to be a requirement 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 the experiment, the initiator, and another, the promoter, that is applied thrice weekly until the appearance of papillomas or cancers. The action resulting from the initiator has been called “initiation” and it can be shown to be irreversible in its biological response. An initiatoi can be applied to mouse skin followed in ten months by the promoter and the same number of tumors will appear as when promotion treatment was started after only two weeks (1.2). On the other hand, promotion appears t o be a reversible phenomenon since application three weeks apart is ineffective. It is not necessary to apply the initiating agent and the promoting agent to the sarnc spot to prociuce a tumor. K h e n urethane or 2-aminofluorene
”,
are fed t o mice the tumor will appear where the promoting agent is applied ( 1 3 ) . The evidence for the case of chemical carcinogenesis can be summarized: 1 ) . Chemicals from our environment are capable of causing cancer. 2 ) . They can perform this feat either directly-e.g., p-propiolactone, a biological alkylating agent, or after metabolism to an activated form-e.g., 2-naphthylamine to bis-2-aminonaphthyl phosphate. 3 ) . The place where cancer occurs can be determined by the interaction of a different reversibly active promoting agent. Mechanism of Carcinogenesis
For the chcmist t o he of any service, a general hypothesis for the mechanism by which these chemicals interact with biological targets is required. Modern molecular biology proritics a framework for the mechanism of carcinogenesis which is the denionstration t h a t all biological information is contained in
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the master tape of DSA in a code form. The code is the order of its constituents, adenine, guanine, thymidine, and cytosine which is transcribed by R S h polymerase into a working tape of RXA which in turn is translated into specific amino acid corporation into proteins. Thus, a change in the master tape DKA will result in a protein of different structure (Figure 3 ) . The active initiating carcinogens can he shown to modify DNA by, for example, substitution on one of the constituents ( 1 4 ) . On replication of such a substituted DNA a wrong base niay be incorporated producing a somatic mutation. The promoting agent niay act by permitting the mutation to be expressed (15). The major portion of the genetic information is covered by a curtain of repressive histones so that errors behind the curtain do not do harm until they are permitted to see the light of day. Several mechanisms have been proposed for gene activation such as the acetylation of lysine leading
“2
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Figure 2. Structure of purified principle responsible for the Drornotion of carcinogenesis observed’with croton oil on mouse skin
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B-Propiolactone Figure 1. Proximal and direct carcinogens VOL. 41, NO. 3, MARCH 1969
23A
Report
G
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.. ,. RNA from DNA,
t o the discharge of the histone (16). The one we favor is selective hydrolysis of the histones by proteolytic enzyme ( 1 5 ) . V a t s o n and Crick established t h a t DIYA exists in the form of a double helix ( 1 7 ) . T h e helix is held together by specific forces between the bases adenine and thymine and bet\Yeen guanine and cytosine. If an initiating carcinogen combines with one of these bases the structure is disturbed. T h e disturbance can be measured by changes in two physical criteria, T m and buoyant density, and with greater sensitivity by the lowering of the priming action of the modified DNA with RNA polymerase. T m is a measure of the heat stability of the DNA, s-hich is determined by noting the rise of UV absorption due to the separation of the two strands, and is defined as the temperature a t the midpoint of the total rise of CV absorption obtained b y heating the D N A solution. A substitution by BPL on N-7 of guanine, or of acetoxy-AXF on C-8 of gdanine lowers the attraction between G and C as indicated by a lowered T m ( 1 8 ) . another physical criterion of D N A , 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 substitution by these two carcinogens (18). DX-1 exerts its biological power by interacting with two enzymes, DSA and RNA polymerase (see Figure 31. T h e precise structure of DYa4 or R S A formed with these t\Yo enzymes is dictated by the 24A
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 DNAis an altered protein containing a different amino acid sequence
priming DNA. T h e precursors of the polymer are the purine and pyrimidine deoxyribose or ribose triphosphates in the presence of primer DXA and the enzyme lines up the purine and pyrimidine bases on a phosphate backbone and releases pyrophosphate. Carcinogen modified D N A is a poor primer for R S X polymerase. This can be demonstrated by reacting DKA with carcinogens in vitro (18, 19) or isolating DSA from carcinogen treated animals and using it as primer with RN,4 polymerase (20). D S h 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 120). T h e inhibition of RNA polymerase priming by carcinogens may he responsible for some of their early toxicity, but the lack of inhibition of DN.4 polymerase allows for the occurrence of mutations, since the substituted DNA m a y incorrectly or ambiguously direct the incorporation of a base into its daughter DXA ( 2 1 ) . T h e ability of initiating carcinogens t o cause mutations in bacteria has heen correlated among 10 epoxides; six which are known to be carcinogenic were mutagenic in bacteria and four were inactive in both system3 . Acetoxy-AAF was positive in both systems while the parent XXF and its metabolite -Y-hydroxy-XAF' was negative in both qystems ( 2 2 ) . All these criteria wpporl the notion that carc in o gens cap ab le of initiating can cers in mousc skin combine with the DSrl causing misreading and EOinatic 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 concept to be explored with other cancer test systems. Repressed vs. Expressed Genetic information
The mechanism of promotion involves the expression of genetic information which is repressed in the chromosomes. According t o Jacob and Nonod, "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). I n relation to cancer the lack of expression of all the errors introduced into the genome is fortunate and can be considered an evolutionary safeguard. This is not the only biological protection built into the system, since repair systems which operate by cutting out pieces of the D K h modified by a substituent, which are then replaced using the undamaged second strand as primer ( 2 4 ) .If this repair is carried out before the D S h strand has had a chance t o replicate or be expressed in any way, no noticeable damage will have occurred. The most obvious materials involved in repression of genetic information in mammalian systems are a group of basic proteins called histones. The total information of the genome is only required during c>mhryonicdewlopment after which a great tical of the information is permunmtly 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. The repressor material in bacteria appears to be a specific protein ( 2 5 ) but 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 ( 1 5 ) . This would support the notion t h a t the mechanism of these agents is through the removal of histones by hydrolytic enzymes. Vnfortunately, this kind of thesis does not lead to a generalization useful to the analytical chemist, hut does provide the enzymologist Lyith some clues. Thus, the capability t o 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, that is, the individual giving up smoking returns to the normal risk after 10 years, supports the promotional thesis ( 2 9 ) . The chemical nature of promoting agents defies generalization. I n addition to the highly specific phorbol ester, many other purified materials of known structure such as the esters of sorbitol. the tweens, as well as many 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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Report weaker than 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 t h a t 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. The 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). H o w v e r , 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 than his nonsmoking coworker ( 3 2 ) . The air we breathe, contaminated with cigarettc smoke or not, is clearly not the only source of carcinogens; the food we eat and the drugs we take may 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 aninials (33, 34'1. The chemical appears t o 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 l r a k e n fern and extracts from a variety of fungi (35373. .Motoxin B,, a liver carcinogen in r a t , combines with DNA, reducing its priming activity with RNA polymerase: it fits well into the category of initiating chemicals described above. The cycasins are naturally occurring nitroso amines and appear t o be metabolized t o a proximal carcinogen methylazoxymethanol. Thus, the properties of these naturally occurring carcinogens are similar to those introduced hy man.
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M a n y drugs modify the enzyme pattern of the host and may affect the response of carcinogens from other sources either by accelerating its metabolism t o a proximal carcinogen or by gene activation. Neither of these unpleasant prospects have been clearly translated into the production of tumors, but the iiiductioii of many enzymes resembling t h a t produced by known carcinogens has been demonstrated with drugs such as phenobarbitol (38). Epidyoniological 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 t o 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. H e identified it as the onset of heavy cigarette smoking and found no reason t o blame the sudden appearaiicc of an air pollutant for the increased Occurrence of lung cancer. T h e increase in lung cancer incidcnce in the United States became clinically of importance in 1920 and as pointed out by Dorii (40) followed a similar pattern. The competition for etiological agent most responsible for the appearance of lung cancer betmcen 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 ( 3 1 ) ) but the cigarette smoker shows the highest rate of lung cancer in all circumstances.
~
A latent period for the onset of the clinical rnanifestation of the disease appears t o be the one uncontroversial sobering fact which has appeared from these studies, as ~ l asl from the experience with aromatic amines in bladder cancer and from data of scrotal tumors in chimney sweepers. T h e fact is cer-
tainly discouraging, since even if me knew the nature of all the carcinogenic materials \ye daily consume in one form or another, and removed them immediately, clear results of this would not be evident for twenty years. T h e main task for the realistic experimenter in this area is to shortcut this twenty years. T h e biologist looks for species which show faster responses to carcinogenic agents. The niodern 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 t h a t has been proposed. Literature Cited
(1) P. Pott, “Chirurigical Observations Relative to the Cataract, the Polypous of the Sose, The Cancer of the Scrotum, The Different Kinds of Ruptures, and the Mortification of the Toes and Feet.” Printed by T. J. Carnegie, 1775 Hawcs, Clarice and Collins, London, 1775. (2) K. Yamagiwa and I
