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Mutation Research, 247 (1991) 283-291 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100087W MUT 00061
Future research directions in cancer ecogenetics Gilbert S. Omenn School of Public Health and Community Medicine, University of Washington SC-30, Seattle, WA 98195 (U.S.A.)
Keywords: Cancer ecogenetics; Ecogenetics, cancer; Polymorphisms; Risk characterization; Biotransformation
Summary There are many productive directions for future research in cancer ecogenetics. Genetic variation in susceptibility to chemicals and other carcinogenic agents has been neglected in most epidemiologic and rodent investigations of cancer etiology. Genetic variation is important to characterization of risks for population subgroups. Genetic investigations also may enhance inquiries into the underlying mechanisms of carcinogenesis and of cancer prevention. Polymorphisms of cytochrome P450 mono-oxygenases, epoxide hydrolase, glutathione-S-transferases, and N-acetyltransferase offer important windows on biotransformation of pro-carcinogens. Assays in peripheral blood cells need to be related closely to variation in activity in target organs. Tumor suppressor genes, signal transduction pathways, and cell surface receptors are additional sites where genetic variation would be highly important to cancer risks.
Carcinogenic and other risks from exposures to exogenous chemicals depend not only on the intrinsic properties and dose of the chemical, but also on target sites in the host and on biotransformation and repair responses in the host. These host responses and target molecules are specified genetically and often have significant genetically determined variation. Other authors in this Colloquium have explored in detail relevant concepts and techniques of molecular biology and cytogenetics and especially their intersection in gene mapping, the activation of proto-oncogenes, and the progressive steps of carcinogenesis. Inherited cancer syndromes and chromosome breakage syndromes offer 'experi-
Correspondence: Gilbert S. Ornenn, M.D., Ph.D., School of Public Health and Community Medicine, University of Washington SC-30, 1959 N.E. Pacific Street, Seattle, WA 98195 (U.S.A.).
ments of Nature' that may reveal important aspects of carcinogenesis, particularly in humans. Thus, humans become a favorable species for studies of cancer biology.
The framework for decision making: a pervasive influence of genetic variation When approaching questions about the carcinogenicity or potential carcinogenicity of chemicals (or physical or biological agents), it is useful to have a framework for organizing the scientific work, the review of scientific evidence, and the subsequent decision-making process of regulators and other interested parties. Such a framework, based upon reports from the Office of Science and Technology Policy (Calkins et al., 1980) and the National Research Council (1983), is shown in Fig. 1.
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epidemiology H A Z A R D IDENTIFICATION~
lifetime rodent bioassay short-term tests
//dose/response RISK C H A R A C T E R I Z A T I O N ~
(potency)
exposures v a r i a t i o n in s u s c e p t i b i l i t y
j RISK R E D U C T I O N
--
information substitution restriction/ban
Fig. 1. Framework for identification,characterization,and control of environmental carcinogens.
Concepts and techniques of genetics are important in many parts of this framework and need to be better understood and more widely applied. We need to stimulate molecular biologists, toxicologists, and epidemiologists to recognize and investigate genetic variation.
The hazard identification stage Many of the short-term tests employ specific mutational or cytogenetic endpoints as markers for cancer-related processes. However, the cell lines and the animals used in in vivo/in vitro tests need to be characterized more extensively for special properties reflecting their origins and any subsequent selection. The rodent bioassay increasingly is being investigated for comparative toxicokinetics, which should include explicit attention to known variation in biotransformation and repair among the rodents and among humans. In fact, the regulatory policy of relying upon the results from sensitive species, strains, and sex is based in part on the recognition that humans are far more outbred than the rodents used in these laboratory tests. Similarities and differences in specific proto-oncogene activation and suppression mechanisms between rodents (Beer and Pitot, 1989) and humans (Yuspa and Poirier, 1988) should be a fertile field, as well. Finally, the field of epidemiology is undergoing a pair of major changes that should facilitate attention to genetic variation. The move beyond
statistical associations to seek evidence of 'biological plausibility' about underlying mechanisms of effect puts a high value on elucidating the molecular and cellular actions of test chemicals and understanding the basis of differences in susceptibility in distinguishable human subpopulations. Specific assays of exposures and of their presumed effects, utilizing monoclonal antibodies to detect and quantify D N A adducts (e.g. B P D E D N A adducts in coke oven or foundry workers (Harris et al., 1985; Perera et al., 1988), or oncogene proteins [e.g. fes and H-ras p21 proteins in PCB-exposed workers (Brandt-Rauf, 1988)), can advance epidemiologic studies beyond crude estimates of exposure based upon job descriptions. Likewise, attempts to test the generally tentative conclusions of observational studies through randomized, double-blind prevention trials or well-phased risk-reduction implementation programs may, themselves, be dependent upon the differential responses of different human subpopulations.
The risk characterization stage The relative potency of agents may be modified notably by biotransformation pathways, as well as by differences at the target sites of action and repair of and compensation for initial effects. It is puzzling that the many abnormalities in DNA repair of lesions induced by radiation of various kinds and by chemicals of various classes, as pre-
285 sented in this Colloquium, have not yet led us to recognize less severe deficiencies of the same enzymes that may contribute to differential susceptibility to these same exogenous agents in much larger numbers of people. Also, one may hope that the investigation of both radiation and chemical exposures in D N A repair studies might lead others to examine the similarities and differences, and interactions, of radiation and chemical exposures. Exposures to the chemical of concern must be translated into target-cell concentrations. Measuring tissue-level concentrations of specific chemical adducts with D N A holds promise for dosimetry, as has been emphasized in reports published in 1989 by the N A S / N R C Board on Environmental Studies and Toxicology: Biologic Markers in Reproductive Toxicology and Biologic Markers in Pulmonary Toxicology. At target sites, a set of interactions will occur with other agents whose biotransformation and effects are subject to genetically determined variation, as well. These agents include other carcinogens, anti-carcinogens, and modifiers of various host responses. The multifaceted role of variation in susceptibility is highlighted with an explicit line in the risk characterization stage of this framework (Fig. 1).
The risk reduction stage There may be multiple ways to accomplish risk reduction objectives, potentially including ways to identify individuals who seem to be especially susceptible to a particular chemical. These individuals may be motivated to take special steps, beyond those to be implemented to protect every"5~ one, or take personal protective steps earlier. Further discussion about variation in biotransformarion
Known genetic variation in susceptibility to environmental agents - - drugs, inhaled pollutants, pesticides, foods, food additives, infectious agents, and physical agents - - can be classified as differences in metabolism of the agent or differences in susceptibility at the cellular level to the action of the agent (Omenn and Motulsky, 1978; Omenn and Gelboin, 1984).
Many chemicals, to become carcinogenic, must be activated enzymatically, usually by microsomal cytochrome P450 mono-oxygenases, to genotoxic electrophilic intermediates. Other enzyme systems detoxify carcinogenic chemicals by hydrolysis of epoxides, conjugation with glutathione, or acetylation. Extending the presentations in the previous papers about the P450 gene superfamily, the carbon oxidation polymorphism, and other metabolic steps, we cite here some evidence of genetically determined variation in 4 key biotransformation pathways: N-acetyltransferases, cytochrome P450 monooxygenases, epoxide hydrolase, and glutathione-S-transferases. These ecogenetic polymorphisms may put humans at differential risk from similar environmental exposures. Our group at the University of Washington Sehool of Public Health, supported by the National Institute of Environmental Health Sciences and the Charles A. Dana Foundation, is especially interested in mechanisms underlying risk of lung cancer, since lung cancers account for 6% of all deaths and 25% of all cancer deaths in this country and represent an epidemic in b o t h men and women. The causes are well known: 85% of cases are due to carcinogens in cigarette smoke; most of the rest are due to environmental exposures to asbestos, arsenic, radon gas, and a few other chemicals. Cigarette smoking and chemicals also are important causes of urinary bladder cancers. Finally, we are much interested in assessing the risk to humans from aflatoxin, in light of the markedly different responses of rats and mice to this pro-carcinogen encountered as a fungal contaminant in our diet.
Polymorphism of N-acetyltransferase (Hein, 1988) More than 30 years ago, a polymorphism of this liver enzyme was discovered to account for marked differences in blood levels of the anti-TB drug isoniazid after a standard dose. The blood levels fit a bimodal distribution. The difference was shown by family studies to be due to a single gene, with the recessive slow acetylator phenotype associated with higher levels of active drug and, therefore, propensity to adverse effects. It is logical to inquire which environmentally encountered arylamines and hydrazines are metabolized by the same drug-metabolizing system. In fact, the potent
286 human bladder carcinogens /3-naphthylamine, benzidine, and 4-aminobiphenyl require acetylation to be inactivated. The hypothesis that slow acetylators would be at higher risk for bladder cancers than are rapid acetylators has been tested, as discussed earlier in this Colloquium. The first study comparing bladder cancer cases in Copenhagen with noncancer controls for acetylator phenotype found an excess of slow acetylators, as predicted. With a 50-70% prevalence of slow acetylators in the general Caucasian population, however, the difference was not statistically significant. Two improvements in experimental power may be pursued. The first is to choose a population with much lower prevalence of slow acetylators: only 10-15% of Japanese are slow acetylators, and a group of retired workers with definite history of working with these bladder carcinogens is under clinical and urinary tract cytogenetic surveillance. Unfortunately, acetylator phenotyping is not yet being done in this population, primarily because of reluctance to administer a drug as part of the phenotyping (Matsushima, personal communication). The alternative is to know that the bladder cancer cases really were exposed to the suspect carcinogens. Such a population was studied in Huddersfield (U.K.), where 22/23 cases were slow acetylators, the 23rd case was atypical, and there was no association between acetylator phenotype and cigarette smoking history. The enhanced risk for bladder cancer in slow acetylators has been confirmed in several additional studies (Hein, 1988). At the biochemical level, N-acetyltransferase (NAT) has been demonstrated in bladder epithelia; the associated hydroxamic O-acetyl transferase activity (OAT) is very low. In contrast, OAT, which generates DNA-adduct-forming intermediates, predominates in colon, where heterocyclic arylamines may be found as pyrolysis products from char-broiled meat or fish. It is possible that the rapid acetylator phenotype thereby predisposes to colon cancer. Such is the specificity of ecogenetic interactions. Wendell Weber of the University of Michigan has prepared a D N A probe based upon the unusual amino acid sequence TyrGlnMetTrpGlnPro; a probe produced to react with rabbit NAT has
been shown by Blum in Zurich to cross-react with human N A T (Blum, personal communication). Such probes are needed to develop a convenient assay with peripheral blood cells, and to investigate tissue-specific differences in gene expression. Meanwhile, in vivo phenotyping has been facilitated by the administration of coffee or tea and then measurement of an acetylated metabolite of caffeine (Tang et al., 1989), rather than utilizing a sulfa drug (sulfamethazine) unapproved for clinical use.
Polymorphisms of cytochrome P450 monooxygenases (see Idle and Nebert papers, this volume) These microsomal enzymes are a complex set of gene products with overlapping substrate specificity, but differential responses to inducers, susceptibility to inhibitors, and pattern of expression in different tissues of the same organism. The important procarcinogen, benzo[a]pyrene, is activated by P450s and epoxide hydrolase to the highly carcinogenic electrophile BP-7,8-diol-9,10-epoxide, and is detoxified by pathways utilizing glutathione transferase or epoxide hydrolase. A recent revision of the nomenclature shows some 70 different P450s in several evolutionarily related families for which at least some sequence data are available (Nebert et al., 1989). Despite such complexity, several specific P450s have been identified that are expressed polymorphically in humans. These include P450 isozymes that catalyze the metabolism of the drugs debrisoquine, mephenytoin, and nifedipine, respectively. The mutant analysis so successfully applied to other biological processes is proving valuable here. The debrisoquine or carbon oxidation polymorphism. This notable drug-metabolizing polymorphism was described 10 years ago (Idle and Smith, 1979; Inaba et al., 1980). Alicyclic hydroxylation of debrisoquine, aromatic hydroxylation of guanoxan, oxidative dealkylation of phenacetin, and N-oxidation of sparteine all are affected by a single-gene difference in activity of a minor P450 (now designated P450IID1). Two aspects of this polymorphism command attention. First, the activity difference is large, with about a 200-fold difference in debrisoquine hydroxylation between the 7 9% of the Caucasian population who are
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poor metabolizers and the majority who are extensive metabolizers. Second, the range of enzymatic conversions is broad, with more than 30 drugs now known to be subject to oxidation by this P450. From the occupational and environmental point of view, one may inquire what other chemical conversions might be tied to the debrisoquine metabolism polymorphism. The first claim was that Nigerians with liver cancers probably induced by aflatoxin exposures have a lower-than-control frequency of poor metabolizers; this finding fit the hypothesis that poor metabolizers would be less capable of metabolic activation of the promutagen/procarcinogen aflatoxin (Idle et al., 1981). The status of this finding is uncertain, since in vitro and in vivo studies in rat strains (DA vs. Lewis) suggest that the DB P450 is not a key determinant of aflatoxin activation, at least in the rat. Another fungal toxin, ochratoxin A, is known to be toxic to the kidney and lead to urinary bladder cancers. This toxin has been found in cereals produced and stored in areas of Bulgaria with a high incidence of Balkan endemic nephropathy and urinary tract tumors. Like debrisoquine, ochratoxin A undergoes 4-hydroxylation to become activated. A research team from the International Agency for Research on Cancer reported a modest deficiency of poor metabolizers among affected persons (Castegnaro et al., 1970). Ochratoxin A caused marked increases in lipid peroxidation (measured by ethane exhalation) and in kidney D N A content of 8-hydroxyguanosine in Lewis rats, but not in DA rats, which had only 25-50% of the ochratoxin 4-hydroxylase activity of Lewis rats. Finally, without regard to specific carcinogens, but presumably related to polycyclic aromatic hydrocarbons (and possibly nitrosamines), Ayesh et al. (1984) found a significant excess of extensive metabolizers among lung cancer patients in Britain. A National Cancer Institute group has confirmed those conclusions and shown an enhanced risk of extensive metabolizers for occupational exposures to asbestos and PAHs. There is considerable uncertainty about whether or not the intermediate phenotype (based on urinary metabolite ratios) enhances the cancer risk; if not, inclusion of the intermediate (heterozygous) persons would
bias against detecting an association. Molecular genotyping will be needed to settle this open question. Molecular techniques have been applied to the debrisoquine P450 system. Distlerath et al. (1985) isolated the specific P450. Gonzalez et al. (1988) showed immunochemically that poor metabolizers have negligible amounts of the P450 enzyme protein and then cloned and sequenced cDNAs from livers of several extensive metabolizers and several poor metabolizers. All extensive metabolizers had the same c D N A (1568 bp) on partial sequence analysis, indicating that there is only 1 active hepatic P450 gene in the human l i D subfamily (unlike the rat). The P450IID1 gene has been mapped to chromosome 22 and has 9 exons. 3 variant mRNAs were identified as products from 3 mutant genes producing incorrectly spliced mRNA. 2 m R N A variants contained sequences of intron 5 or of intron 6, while the third was a deletion variant, with transcription probably initiated at a cryptic promoter within intron 3. In each case the truncated protein would likely be an unstable, rapidly degraded, undetectable version of the normal protein of 497 amino acids. At least 1 case was probably a heterozygote, since there was a small amount of detectable activity. Based upon the Hardy-Weinberg relationship, a population frequency of 9% homozygotes would correspond to a heterozygote frequency of 42%. Subsequently, Skoda et al. (1988a) analyzed white blood cell genomic D N A from 24 unrelated poor metabolizers (PMs) and 29 unrelated extensive metabolizers (EMs) for restriction fragment length polymorphisms. Using XbaI, 75% of PMs had 1 or both of 2 polymorphic fragments, shown by segregation in several families to be allelic with a common fragment in EM individuals. These 2 fragments were in linkage disequilibrium with RFLPs generated by 4 and 5 additional restriction endonucleases, respectively. Nevertheless, onefourth of the PMs were not identified with these endonucleases and probes. No individuals doubly heterozygous for these 2 RFLPs were identified and no individuals homozygous for the 44-kb allele (58% of PMs) were identified. The locus is highly polymorphic; in all, 14 RFLPs were found, but only 2 appear to be associated with the PM phenotype.
288 Thus, at least 3 central problems remain. First, there appear to be multiple mutant alleles producing the poor metabolizer phenotype; DNA probe diagnosis is, therefore, complicated and will require multiple sequence probes a n d / o r RFLPs. Second, no candidate carcinogenic chemical in cigarette smoke has been identified as a substrate for the debrisoquine P450. Finally, for cancer susceptibility, it is necessary to clarify whether the polymorphic enzyme deficiency in peripheral white blood cells is expressed also in the target tissue, such as lung, for each particular carcinogenic agent. Redlich and Omiecinski (1989) in our group are addressing the third question. They have successfully utilized the polymerase chain reaction and oligomer probes for selected regions of the P450IID1 gene to demonstrate gene expression both in peripheral lymphocytes and in lung tissues of humans. Northern blots were not sufficiently sensitive to detect expression of the gene products. Phenotyping may be feasible by molecular sizing gel experiments with the PCR products.
Epoxide hydrolase (EH) Only 1 isozyme of epoxide hydrolase acts on xenobiotic chemicals, a microsomal enzyme whose gene has been mapped to chromosome l q (Skoda et al., 1988b). Thus, EH should be more amenable to investigation than the P450s (above) or the glutathione S-transferases (below). This EH can be induced by phenobarbital, trans-stilbene oxide, and acetylaminofluorene. Typically, it inactivates electrophilic epoxides to inactive dihydrodiols, but in the case of BP the 7,8-diol is a substrate for a P450 which produces a new highly carcinogenic epoxide. Omiecinski, Wilson and Eaton have found considerable variation among 188 adult humans in white blood cell microsomal EH activity with BP 4,5-oxide as substrate (Omenn et al., 1990). There are a few outliers with high activity. They and others have isolated and sequenced the human EH cDNA, which demonstrates extensive homology with both rat and rabbit microsomal EH (Hassett et al., 1990). In addition, they have isolated the human EH gene and have sequenced the 5'-end and flanking regions. Variants have been identified among fetal liver specimens. Studies are in
progress examining the association between EH gene expression in lymphocytes, lung and fiver from the same individuals (Redlich and Omiecinski, 1989). Remarkable technical advances now permit comparisons of tissue-specific expression at the level of mRNAs. Many were discussed at a recent UCLA Symposium on Biotechnology and Human Genetic Predisposition to Disease (Cantor et al., 1990). Many improvements on the widely used polymerase chain reaction are being reported, including R N A amplification with transcript sequencing (RAWTS) (Sarkar and Sommer, 1989). This technique incorporates a phase promoter into a PCR oligonucleotide primer, allowing an abundance of transcript to be made after amplification of mRNA by PCR.
Glutathione-S-transferases (GST) These detoxifying enzymes form inactive conjugates with glutathione. At least 8 different isozymes are known in mammalian species, probably including man, and some of them are inducible, further complicating analysis. Nevertheless, Seidegard et al. (1985) identified a very common polymorphism of lymphocyte GST activity; about 45% of Caucasians are essentially deficient in GST activity against the substrate trans-stilbene oxide (TSO). Mendelian inheritance of this trait has been confirmed by Motulsky and colleagues in our Dana Program. If TSO is a surrogate for carcinogenic substrates, these individuals might be more susceptible than others to resulting cancers. Pero's group in Sweden and Mantle in Dublin have found an increased prevalence of deficient individuals among lung cancer cases. Although epoxides of styrene and of benzo[a]pyrene were proposed as potential substrates, the key in vivo substrates for this polymorphic GST isozyme remain unknown. Heckbert, Eaton, and Motulsky have tested lymphocytes from individuals with cancers related to cigarette smoking, those with cancers unrelated to smoking, and non-cancer controls. BP 4,5-oxide as substrate gives a correlation coefficient of 0.640.72 with G S T against TSO, but the trimodality associated with TSO could not be discerned. PNSO and CNDB as substrates are poorly correlated with TSO activity and are unimodal. Although the
289 results are not fully analyzed, there appears to be little or no evidence for higher prevalence of the deficient allele among cancer cases in this large study. An additional study is under way (Nazar, Weiss, Eaton, et al.), analyzing GST and EH in lung cancer and normal lung tissues. Meanwhile, Eaton, Ramsdell and Monroe have explored the puzzhng huge difference in susceptibility of rats and mice to liver cancer from aflatoxin B1. As little as 15 ppb AFB in the diet produces liver cancers in rats, with 100% tumor incidence at 100 ppb. Yet no such tumors are observed in mice even up to 150000 ppb AFB dose. P450s convert AFB 1 to the 8,9-epoxide which forms adducts with N-7 of guanine nucleotides, or to much less hazardous metabohtes AFM, AFQ and AFP. GST is important in inactivating the 8,9-epoxide. It is convenient to estimate an 'index of susceptibility' from the ratios of activation rates (AFB-epoxide f o r m a t i o n / t o t a l oxidative biotransformation) to inactivation rates (AFB-epoxide GSH conjugation/AFB-epoxide formation). The activation ratio is just as great in mice as in rats; in fact, the component of epoxide formation is 3 times greater in mice. However, the GST-mediated inactivation ratio is far greater in mice. A F B - D N A adduct formation in mice was detected at a level 1.2% of that in rats for the same dose, corresponding closely to a ratio of activation/inactivation rates 2% of that in the rats (Omenn et al., 1990). On the same basis, humans and monkeys appear to have intermediate susceptibility, but more closely resemble the rat. Moreover, at low AFB concentrations the activation ratio increases in humans and rats (but not in mice), so there is no comfort in low-dose exposures. Thus far, assays of microsomes prepared from 14 human hvers obtained at organ donation have shown 2-3-fold interindividual variation in biotransformation to form AFB-epoxide and the AFQ and A F M metabolites. No family studies or genetic analyses have been feasible to date, and no extremely lowor high-activity individuals have been encountered. Agents thought to be chemopreventive against cancers - - such as r-carotene-rich vegetables, cruciferous vegetables, and the antioxidant BHA - - increase GST activity and decrease both adduct formation and tumor incidence in rats
(Ramsdell and Eaton, 1988). We are engaged in a major chemoprevention trial with a combination of r-carotene and retinol in heavy smokers and in asbestos-exposed workers, populations at high risk for lung cancer (Omenn, 1989). The scientific basis for the trial includes an association of low dietary intake or blood levels of r-carotene or vitamin A with high lung cancer risk; animal and cell culture studies showing prevention or reversal of late stages of carcinogenesis with the agents; and postulated mechanisms that r-carotene acts as an electron scavenger, while retinoic acid and retinol are ligands for nuclear zinc-finger proteins coded by genes on chromosomes 3p and 17 (where ' t u m o r suppression' genes have been localized). No studies have yet been undertaken to detect polymorphic or other genetic variation in these genes and proteins.
Analogous approaches to signal transduction pathways Weinstein (1988a,b), Ali et al. (1989), Weston et al. (1989), and many others have begun to explore the many interrelationships between the molecular biology of growth and differentiation and the aberrations that occur in tumorigenesis. The search for genetically determined variation in the genes and the gene products of metabolic pathways discussed above should have a direct parallel in studies of oncogenes, specific protein kinases, growth factors, tumor inhibitory factors, and their receptor molecules. We urge that those carrying out research on the 3 dimensions of structure, function, and regulation of expression of oncogenes add a fourth dimension to their studies: individual variation in these genes. For example, a c-mos variant identified as an E c o R I restriction fragment length polymorphism occurs in many patients with breast cancers both in their tumors and in their normal lymphocyte DNA, but in none of the 70 non-cancer patients thus far studied (Lidereau et al., 1987). Transformation of cells also may produce effects mediated through the phase II detoxification pathways discussed above, as in the case of v-Hras- or v-raf-transformed rat liver epithehal cells, which have resistance to cytotoxic chemicals in the
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form of multidrug resistance, in part due to marked increase in GST-P activity (Burr et al., 1988).
Concluding comments The major biotransformation pathways in humans for activation and detoxification of potentially carcinogenic chemicals encountered in our working and general living environment have notable genetic variation. Much work needs to be done to characterize the functional significance of genetic variation already known and genetic variation likely to be found. Identifying the relevant xenobiotic substrates will be important in evaluating genetic predispositions to cancers, birth defects, and organ-system effects. Always it must be emphasized that predisposition or susceptibility applies selectively to specific chemicals and specific enzymatic or receptor interactions; the same individuals may be less susceptible to many other exposures. Such selectivity is apparent also among rodents tested in the lifetime bioassay. For example, while mice are remarkably resistant, compared with rats, to aflatoxin, mice are more sensitive to polycyclic aromatic hydrocarbons. The tools of biotechnology make feasible many direct studies of enzymes, mRNA, and D N A for the steps of biotransformation. Of course, there are many other important variables in the mediation of effects of environmental chemical exposures, including the modifying influences of diet and physiology, DNA repair, immune surveillance, tumor promotion, tumor progression, and tumor suppression. Individual behavior will greatly i~afluence levels of exposure from occupational, recreational, and general activities. Thus, one must be cautious in recommending genetic testing for predisposing traits, proceeding first to anticipate the potential desirable and undesirable uses of such information, then to research carefully the relative risk and attributable risk of any specific genetic trait for any specific exposure and disease outcome before considering the ramifications of routine screening (Omenn, 1982). The multifactorial interactions of ecogenetics also place a high priority on advances in epidemiologic and biostatistical methods for design and analysis of such complex phenomena as carcinogenesis. Given the rapid pace of technical advances in
genetics and the great public interest in effective identification, characterization, and reduction of risks to humans from environmental chemicals, we may predict that cancer ecogenetics will emerge as a significant subfield of the environmental health sciences during the next few years.
References Ali, l.U., G. Merlo, R. Callahan and R. Lidereau (1989) The amplification unit on chromosome 11q13 in aggressive primary human breast tumors entails the bcl-1, int-2 and hst loci, Oncogene, 4, 89-92. Ayesh, R., J.R. Idle, J.C. Ritchie, M.J. Crothers and M.R. Hetzel (1984) Metabolic oxidation phenotypes as markers for susceptibility to lung cancer, Nature (London), 312, 169-170. Beer, D.B., and H.C. Pitot (1989) Proto-oncogene activation during chemically induced hepatocarcinogenesis in rodents, Mutation Res., 220, 1-10. Brandt-Rauf, P.W., and H.L. Niman (1988) Serum screening for oncogene proteins in workers exposed to PCBs, Br. J. Indust. Med., 45, 689-693. Burt, R.K., S. Garfield, K. Johnson and S.S. Thorgeirsson (1988) Transformation of rat liver epithelial cells with v-Hras or v-raf causes expression of MDR-1, glutathione-Stransferase-P and increased resistance to cytotoxic chemicals, Carcinogenesis, 9, 2329-2332. Calkins, D.R., R.L. Dixon, C.R. Gerber, D. Zarin and G.S. Omenn (1980) Identification, characterization and control of potential human carcinogens: a framework for federal decision-making, J. Natl. Cancer Inst., 64, 169-175. Cantor, C., C.T. Caskey, L. Hood, D. Kamely and G.S. Omenn (Eds.) (1990) Biotechnology and Human Genetic Predisposition to Disease, Wiley-Liss, New York. Castegnaro, M., J.C. Bereziat, J. Michelon et al. (1970) Ochratoxin A in relation to nephropathy and bladder cancer, World Health Organization International Agency for Research on Cancer, Biennial Report, 1986-87, IARC, Lyon, pp. 35-37. Distlerath, I.M., P.E.B. Reilly, M.V. Martin, G.G. Davis, G.R. Wilkinson and F.P. Guengerich (1985) Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin o-deethylation, Two prototypes for genetic polymorphism in oxidative drug metabolism, J. Biol. Chem.~ 260, 90579067. Gonzalez, F.J., R.C. Skoda, S. Kimura et al. (1988) Characterization of the common genetic defect in humans deficient in debrisoquine metabolism, Nature (London), 331,442-446. Harris, C.C., K. Vahakangas, M.J. Newman, G.E. Trivers, A. Shamsuddin, N. Sinopoli, D.L. Mann and W.E. Wright (1985) Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral blood lymphoeytes and antibodies to the adducts in serum from coke oven workers, Proc. Natl. Acad. Sci. (U.S.A.), 82, 6672-6676.
291 Hassett, C.M., S.T. Turnblom, A. DeAngeles and C.J. Omiecinski (1989) Rabbit microsomal epoxide hydrolase: isolation and characterization of a cDNA clone for the xenobiotic metabolizing enzyme, Arch. Biochem. Biophys., 271,380-389. Hein, D.W. (1988) Acetylator genotype and arylamine-induced carcinogenesis, Biochim. Biophys. Acta, 948, 37-66. Idle, J.R., and R.L. Smith (1979) Polymorphisms of oxidation at carbon centers of drugs and their clinical significance, Drug Metab. Rev., 9, 301-317. Idle, J.R., A. Mahgoub, T.P. Sloan et al. (1981) Some observations on the oxidation phenotype status of Nigerian patients presenting with cancer, Cancer Lett., 11, 331-338. Inaba, T., S.V. Otton and W. Kalow (1980) Deficient metabolism of debrisoquine and sparteine, Clin. Pharmacol. Ther., 27, 547-549. Lidereau, R., S.T. Cole, C.J. Larsen and D. Mathieu-Mahul (1987) A single point mutation responsible for c-mos polymorphism in cancer patients, Oncogene, 1, 235-237. National Research Council (1983) Risk Assessment in the Federal Government, Managing the Process, National Academy Press, Washington, DC. Nebert, D.W., D.R. Nelson, M. Adesnik, M.I. Coon, R.W. Estabrook et al. (1989) The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci, DNA, 8, 1-13. Omenn, G.S. (1982) Predictive identification of hyper-susceptible individuals, J. Occup. Med., 24, 369-374. Omenn, G.S. (1989) Chemoprevention of occupationally related lung cancer, Wash. Publ. Health, 7, 35-36. Omenn, G.S., and H.V. Gelboin (Eds.) (1984) Genetic Variability in Responses to Chemical Exposure, Banbury Report 16, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Omenn, G.S., and A.G. Motulsky (1978) Ecogenetics: genetic variation in susceptibility to environmental agents, in: B.H. Cohen, A.M. Lilienfeld and P.C. Huang (Eds.), Genetic Issues in Public Health and Medicine, Thomas, Springfield, IL, pp. 83-111. Omenn, G.S., C.J. Omiecinski and D.L. Eaton (1990) Ecogenetics of chemical carcinogens, in: C. Cantor, C.T. Caskey, L. Hood, D. Kamely and G.S. Omenn (Eds.), Biotechnology and Human Genetic Predisposition to Disease, Wiley-Liss, New York. Perera, F.P., K. Hemminki, T.L. Young, D. Brenner, G. Kelly and R.M. Santella (1988) Detection of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers, Cancer Res., 48, 2288-2291.
Ramsdell, H.W., and D.L. Eaton (1988) Modification of aflatoxin B1 biotransformation in vitro and DNA binding in vivo by dietary broccoli in rats, Toxicol. Environ. Health, 25, 269-284. Redlich, G.A., and C.J. Omiecinski (1989) Genetic expression of cytochrome P450s and epoxide hydrolase in human lung, American Thoracic Society, 1989 Annual Meeting abstracts. Sarkar, G., and S.S. Sommer (1989) Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity, Science, 244, 331-334. Seidegard, J., J.W. DePierre and R.W. Pero (1985) Hereditary interindividual differences in the glutathione transferase activity towards trans-stilbene oxide in resting human mononuclear leukocytes are due to a particular isozyme(s), Carcinogenesis, 6, 1211-1216. Skoda, R.C., F.J. Gonzalez, A. Demierre et al. (1988a) Two mutant aUeles of the human cytochrome P450dbl gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs, Proc. Natl. Acad. Sci. (U.S.A.), 85, 5240-5243. Skoda, R.C., A. Demierre, O.W. McBride, F.J. Gonzalez and U.A. Meyer (1988b) Human microsomal xenobiotic epoxide hydrolase, J. Biol. Chem., 263, 1549-1554. Tang, B.K., D. Kadar and W. Kalow (1987) An alternative test for acetylator phenotyping with caffeine, Clin. Pharmacol. Ther., 42, 509-513. Weinstein, I.B. (1988a) Strategies for inhibiting multistage carcinogenesis based on signal transduction pathways, Mutation Res., 202, 413-420. Weinstein, I.B. (1988b) The origins of human cancer: Molecular mechanisms of carcinogenesis and their implications for cancer prevention and treatment - - twenty-seventh G.H.A. Clowes Memorial Award Lecture, Cancer Res., 48, 41354143. Weston, A., J.C. Willey, R. Modali, H. Sugimura, F.M. McDowell, J. Resau, B. Light, A. Haugen, D.L. Mann, B.F. Trump and C.C. Harris (1989) Differential loss of genes from chromosomes 3, 11, 13 and 17 in squamous cell carcinoma and adenocarcinoma of the human lung, Proc. Natl. Acad. Sci. (U.S.A.), 86, 5099-5103. Yuspa, S.H., and M.C. Poirier (1988) Chemical carcinogenesis: from animal models to molecular models in one decade, Adv. Cancer Res., 50, 25-70.
Mutation Research, 247 (1991) 283-291 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100087W MUT 00061
Future research directions in cancer ecogenetics Gilbert S. Omenn School of Public Health and Community Medicine, University of Washington SC-30, Seattle, WA 98195 (U.S.A.)
Keywords: Cancer ecogenetics; Ecogenetics, cancer; Polymorphisms; Risk characterization; Biotransformation
Summary There are many productive directions for future research in cancer ecogenetics. Genetic variation in susceptibility to chemicals and other carcinogenic agents has been neglected in most epidemiologic and rodent investigations of cancer etiology. Genetic variation is important to characterization of risks for population subgroups. Genetic investigations also may enhance inquiries into the underlying mechanisms of carcinogenesis and of cancer prevention. Polymorphisms of cytochrome P450 mono-oxygenases, epoxide hydrolase, glutathione-S-transferases, and N-acetyltransferase offer important windows on biotransformation of pro-carcinogens. Assays in peripheral blood cells need to be related closely to variation in activity in target organs. Tumor suppressor genes, signal transduction pathways, and cell surface receptors are additional sites where genetic variation would be highly important to cancer risks.
Carcinogenic and other risks from exposures to exogenous chemicals depend not only on the intrinsic properties and dose of the chemical, but also on target sites in the host and on biotransformation and repair responses in the host. These host responses and target molecules are specified genetically and often have significant genetically determined variation. Other authors in this Colloquium have explored in detail relevant concepts and techniques of molecular biology and cytogenetics and especially their intersection in gene mapping, the activation of proto-oncogenes, and the progressive steps of carcinogenesis. Inherited cancer syndromes and chromosome breakage syndromes offer 'experi-
Correspondence: Gilbert S. Ornenn, M.D., Ph.D., School of Public Health and Community Medicine, University of Washington SC-30, 1959 N.E. Pacific Street, Seattle, WA 98195 (U.S.A.).
ments of Nature' that may reveal important aspects of carcinogenesis, particularly in humans. Thus, humans become a favorable species for studies of cancer biology.
The framework for decision making: a pervasive influence of genetic variation When approaching questions about the carcinogenicity or potential carcinogenicity of chemicals (or physical or biological agents), it is useful to have a framework for organizing the scientific work, the review of scientific evidence, and the subsequent decision-making process of regulators and other interested parties. Such a framework, based upon reports from the Office of Science and Technology Policy (Calkins et al., 1980) and the National Research Council (1983), is shown in Fig. 1.
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epidemiology H A Z A R D IDENTIFICATION~
lifetime rodent bioassay short-term tests
//dose/response RISK C H A R A C T E R I Z A T I O N ~
(potency)
exposures v a r i a t i o n in s u s c e p t i b i l i t y
j RISK R E D U C T I O N
--
information substitution restriction/ban
Fig. 1. Framework for identification,characterization,and control of environmental carcinogens.
Concepts and techniques of genetics are important in many parts of this framework and need to be better understood and more widely applied. We need to stimulate molecular biologists, toxicologists, and epidemiologists to recognize and investigate genetic variation.
The hazard identification stage Many of the short-term tests employ specific mutational or cytogenetic endpoints as markers for cancer-related processes. However, the cell lines and the animals used in in vivo/in vitro tests need to be characterized more extensively for special properties reflecting their origins and any subsequent selection. The rodent bioassay increasingly is being investigated for comparative toxicokinetics, which should include explicit attention to known variation in biotransformation and repair among the rodents and among humans. In fact, the regulatory policy of relying upon the results from sensitive species, strains, and sex is based in part on the recognition that humans are far more outbred than the rodents used in these laboratory tests. Similarities and differences in specific proto-oncogene activation and suppression mechanisms between rodents (Beer and Pitot, 1989) and humans (Yuspa and Poirier, 1988) should be a fertile field, as well. Finally, the field of epidemiology is undergoing a pair of major changes that should facilitate attention to genetic variation. The move beyond
statistical associations to seek evidence of 'biological plausibility' about underlying mechanisms of effect puts a high value on elucidating the molecular and cellular actions of test chemicals and understanding the basis of differences in susceptibility in distinguishable human subpopulations. Specific assays of exposures and of their presumed effects, utilizing monoclonal antibodies to detect and quantify D N A adducts (e.g. B P D E D N A adducts in coke oven or foundry workers (Harris et al., 1985; Perera et al., 1988), or oncogene proteins [e.g. fes and H-ras p21 proteins in PCB-exposed workers (Brandt-Rauf, 1988)), can advance epidemiologic studies beyond crude estimates of exposure based upon job descriptions. Likewise, attempts to test the generally tentative conclusions of observational studies through randomized, double-blind prevention trials or well-phased risk-reduction implementation programs may, themselves, be dependent upon the differential responses of different human subpopulations.
The risk characterization stage The relative potency of agents may be modified notably by biotransformation pathways, as well as by differences at the target sites of action and repair of and compensation for initial effects. It is puzzling that the many abnormalities in DNA repair of lesions induced by radiation of various kinds and by chemicals of various classes, as pre-
285 sented in this Colloquium, have not yet led us to recognize less severe deficiencies of the same enzymes that may contribute to differential susceptibility to these same exogenous agents in much larger numbers of people. Also, one may hope that the investigation of both radiation and chemical exposures in D N A repair studies might lead others to examine the similarities and differences, and interactions, of radiation and chemical exposures. Exposures to the chemical of concern must be translated into target-cell concentrations. Measuring tissue-level concentrations of specific chemical adducts with D N A holds promise for dosimetry, as has been emphasized in reports published in 1989 by the N A S / N R C Board on Environmental Studies and Toxicology: Biologic Markers in Reproductive Toxicology and Biologic Markers in Pulmonary Toxicology. At target sites, a set of interactions will occur with other agents whose biotransformation and effects are subject to genetically determined variation, as well. These agents include other carcinogens, anti-carcinogens, and modifiers of various host responses. The multifaceted role of variation in susceptibility is highlighted with an explicit line in the risk characterization stage of this framework (Fig. 1).
The risk reduction stage There may be multiple ways to accomplish risk reduction objectives, potentially including ways to identify individuals who seem to be especially susceptible to a particular chemical. These individuals may be motivated to take special steps, beyond those to be implemented to protect every"5~ one, or take personal protective steps earlier. Further discussion about variation in biotransformarion
Known genetic variation in susceptibility to environmental agents - - drugs, inhaled pollutants, pesticides, foods, food additives, infectious agents, and physical agents - - can be classified as differences in metabolism of the agent or differences in susceptibility at the cellular level to the action of the agent (Omenn and Motulsky, 1978; Omenn and Gelboin, 1984).
Many chemicals, to become carcinogenic, must be activated enzymatically, usually by microsomal cytochrome P450 mono-oxygenases, to genotoxic electrophilic intermediates. Other enzyme systems detoxify carcinogenic chemicals by hydrolysis of epoxides, conjugation with glutathione, or acetylation. Extending the presentations in the previous papers about the P450 gene superfamily, the carbon oxidation polymorphism, and other metabolic steps, we cite here some evidence of genetically determined variation in 4 key biotransformation pathways: N-acetyltransferases, cytochrome P450 monooxygenases, epoxide hydrolase, and glutathione-S-transferases. These ecogenetic polymorphisms may put humans at differential risk from similar environmental exposures. Our group at the University of Washington Sehool of Public Health, supported by the National Institute of Environmental Health Sciences and the Charles A. Dana Foundation, is especially interested in mechanisms underlying risk of lung cancer, since lung cancers account for 6% of all deaths and 25% of all cancer deaths in this country and represent an epidemic in b o t h men and women. The causes are well known: 85% of cases are due to carcinogens in cigarette smoke; most of the rest are due to environmental exposures to asbestos, arsenic, radon gas, and a few other chemicals. Cigarette smoking and chemicals also are important causes of urinary bladder cancers. Finally, we are much interested in assessing the risk to humans from aflatoxin, in light of the markedly different responses of rats and mice to this pro-carcinogen encountered as a fungal contaminant in our diet.
Polymorphism of N-acetyltransferase (Hein, 1988) More than 30 years ago, a polymorphism of this liver enzyme was discovered to account for marked differences in blood levels of the anti-TB drug isoniazid after a standard dose. The blood levels fit a bimodal distribution. The difference was shown by family studies to be due to a single gene, with the recessive slow acetylator phenotype associated with higher levels of active drug and, therefore, propensity to adverse effects. It is logical to inquire which environmentally encountered arylamines and hydrazines are metabolized by the same drug-metabolizing system. In fact, the potent
286 human bladder carcinogens /3-naphthylamine, benzidine, and 4-aminobiphenyl require acetylation to be inactivated. The hypothesis that slow acetylators would be at higher risk for bladder cancers than are rapid acetylators has been tested, as discussed earlier in this Colloquium. The first study comparing bladder cancer cases in Copenhagen with noncancer controls for acetylator phenotype found an excess of slow acetylators, as predicted. With a 50-70% prevalence of slow acetylators in the general Caucasian population, however, the difference was not statistically significant. Two improvements in experimental power may be pursued. The first is to choose a population with much lower prevalence of slow acetylators: only 10-15% of Japanese are slow acetylators, and a group of retired workers with definite history of working with these bladder carcinogens is under clinical and urinary tract cytogenetic surveillance. Unfortunately, acetylator phenotyping is not yet being done in this population, primarily because of reluctance to administer a drug as part of the phenotyping (Matsushima, personal communication). The alternative is to know that the bladder cancer cases really were exposed to the suspect carcinogens. Such a population was studied in Huddersfield (U.K.), where 22/23 cases were slow acetylators, the 23rd case was atypical, and there was no association between acetylator phenotype and cigarette smoking history. The enhanced risk for bladder cancer in slow acetylators has been confirmed in several additional studies (Hein, 1988). At the biochemical level, N-acetyltransferase (NAT) has been demonstrated in bladder epithelia; the associated hydroxamic O-acetyl transferase activity (OAT) is very low. In contrast, OAT, which generates DNA-adduct-forming intermediates, predominates in colon, where heterocyclic arylamines may be found as pyrolysis products from char-broiled meat or fish. It is possible that the rapid acetylator phenotype thereby predisposes to colon cancer. Such is the specificity of ecogenetic interactions. Wendell Weber of the University of Michigan has prepared a D N A probe based upon the unusual amino acid sequence TyrGlnMetTrpGlnPro; a probe produced to react with rabbit NAT has
been shown by Blum in Zurich to cross-react with human N A T (Blum, personal communication). Such probes are needed to develop a convenient assay with peripheral blood cells, and to investigate tissue-specific differences in gene expression. Meanwhile, in vivo phenotyping has been facilitated by the administration of coffee or tea and then measurement of an acetylated metabolite of caffeine (Tang et al., 1989), rather than utilizing a sulfa drug (sulfamethazine) unapproved for clinical use.
Polymorphisms of cytochrome P450 monooxygenases (see Idle and Nebert papers, this volume) These microsomal enzymes are a complex set of gene products with overlapping substrate specificity, but differential responses to inducers, susceptibility to inhibitors, and pattern of expression in different tissues of the same organism. The important procarcinogen, benzo[a]pyrene, is activated by P450s and epoxide hydrolase to the highly carcinogenic electrophile BP-7,8-diol-9,10-epoxide, and is detoxified by pathways utilizing glutathione transferase or epoxide hydrolase. A recent revision of the nomenclature shows some 70 different P450s in several evolutionarily related families for which at least some sequence data are available (Nebert et al., 1989). Despite such complexity, several specific P450s have been identified that are expressed polymorphically in humans. These include P450 isozymes that catalyze the metabolism of the drugs debrisoquine, mephenytoin, and nifedipine, respectively. The mutant analysis so successfully applied to other biological processes is proving valuable here. The debrisoquine or carbon oxidation polymorphism. This notable drug-metabolizing polymorphism was described 10 years ago (Idle and Smith, 1979; Inaba et al., 1980). Alicyclic hydroxylation of debrisoquine, aromatic hydroxylation of guanoxan, oxidative dealkylation of phenacetin, and N-oxidation of sparteine all are affected by a single-gene difference in activity of a minor P450 (now designated P450IID1). Two aspects of this polymorphism command attention. First, the activity difference is large, with about a 200-fold difference in debrisoquine hydroxylation between the 7 9% of the Caucasian population who are
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poor metabolizers and the majority who are extensive metabolizers. Second, the range of enzymatic conversions is broad, with more than 30 drugs now known to be subject to oxidation by this P450. From the occupational and environmental point of view, one may inquire what other chemical conversions might be tied to the debrisoquine metabolism polymorphism. The first claim was that Nigerians with liver cancers probably induced by aflatoxin exposures have a lower-than-control frequency of poor metabolizers; this finding fit the hypothesis that poor metabolizers would be less capable of metabolic activation of the promutagen/procarcinogen aflatoxin (Idle et al., 1981). The status of this finding is uncertain, since in vitro and in vivo studies in rat strains (DA vs. Lewis) suggest that the DB P450 is not a key determinant of aflatoxin activation, at least in the rat. Another fungal toxin, ochratoxin A, is known to be toxic to the kidney and lead to urinary bladder cancers. This toxin has been found in cereals produced and stored in areas of Bulgaria with a high incidence of Balkan endemic nephropathy and urinary tract tumors. Like debrisoquine, ochratoxin A undergoes 4-hydroxylation to become activated. A research team from the International Agency for Research on Cancer reported a modest deficiency of poor metabolizers among affected persons (Castegnaro et al., 1970). Ochratoxin A caused marked increases in lipid peroxidation (measured by ethane exhalation) and in kidney D N A content of 8-hydroxyguanosine in Lewis rats, but not in DA rats, which had only 25-50% of the ochratoxin 4-hydroxylase activity of Lewis rats. Finally, without regard to specific carcinogens, but presumably related to polycyclic aromatic hydrocarbons (and possibly nitrosamines), Ayesh et al. (1984) found a significant excess of extensive metabolizers among lung cancer patients in Britain. A National Cancer Institute group has confirmed those conclusions and shown an enhanced risk of extensive metabolizers for occupational exposures to asbestos and PAHs. There is considerable uncertainty about whether or not the intermediate phenotype (based on urinary metabolite ratios) enhances the cancer risk; if not, inclusion of the intermediate (heterozygous) persons would
bias against detecting an association. Molecular genotyping will be needed to settle this open question. Molecular techniques have been applied to the debrisoquine P450 system. Distlerath et al. (1985) isolated the specific P450. Gonzalez et al. (1988) showed immunochemically that poor metabolizers have negligible amounts of the P450 enzyme protein and then cloned and sequenced cDNAs from livers of several extensive metabolizers and several poor metabolizers. All extensive metabolizers had the same c D N A (1568 bp) on partial sequence analysis, indicating that there is only 1 active hepatic P450 gene in the human l i D subfamily (unlike the rat). The P450IID1 gene has been mapped to chromosome 22 and has 9 exons. 3 variant mRNAs were identified as products from 3 mutant genes producing incorrectly spliced mRNA. 2 m R N A variants contained sequences of intron 5 or of intron 6, while the third was a deletion variant, with transcription probably initiated at a cryptic promoter within intron 3. In each case the truncated protein would likely be an unstable, rapidly degraded, undetectable version of the normal protein of 497 amino acids. At least 1 case was probably a heterozygote, since there was a small amount of detectable activity. Based upon the Hardy-Weinberg relationship, a population frequency of 9% homozygotes would correspond to a heterozygote frequency of 42%. Subsequently, Skoda et al. (1988a) analyzed white blood cell genomic D N A from 24 unrelated poor metabolizers (PMs) and 29 unrelated extensive metabolizers (EMs) for restriction fragment length polymorphisms. Using XbaI, 75% of PMs had 1 or both of 2 polymorphic fragments, shown by segregation in several families to be allelic with a common fragment in EM individuals. These 2 fragments were in linkage disequilibrium with RFLPs generated by 4 and 5 additional restriction endonucleases, respectively. Nevertheless, onefourth of the PMs were not identified with these endonucleases and probes. No individuals doubly heterozygous for these 2 RFLPs were identified and no individuals homozygous for the 44-kb allele (58% of PMs) were identified. The locus is highly polymorphic; in all, 14 RFLPs were found, but only 2 appear to be associated with the PM phenotype.
288 Thus, at least 3 central problems remain. First, there appear to be multiple mutant alleles producing the poor metabolizer phenotype; DNA probe diagnosis is, therefore, complicated and will require multiple sequence probes a n d / o r RFLPs. Second, no candidate carcinogenic chemical in cigarette smoke has been identified as a substrate for the debrisoquine P450. Finally, for cancer susceptibility, it is necessary to clarify whether the polymorphic enzyme deficiency in peripheral white blood cells is expressed also in the target tissue, such as lung, for each particular carcinogenic agent. Redlich and Omiecinski (1989) in our group are addressing the third question. They have successfully utilized the polymerase chain reaction and oligomer probes for selected regions of the P450IID1 gene to demonstrate gene expression both in peripheral lymphocytes and in lung tissues of humans. Northern blots were not sufficiently sensitive to detect expression of the gene products. Phenotyping may be feasible by molecular sizing gel experiments with the PCR products.
Epoxide hydrolase (EH) Only 1 isozyme of epoxide hydrolase acts on xenobiotic chemicals, a microsomal enzyme whose gene has been mapped to chromosome l q (Skoda et al., 1988b). Thus, EH should be more amenable to investigation than the P450s (above) or the glutathione S-transferases (below). This EH can be induced by phenobarbital, trans-stilbene oxide, and acetylaminofluorene. Typically, it inactivates electrophilic epoxides to inactive dihydrodiols, but in the case of BP the 7,8-diol is a substrate for a P450 which produces a new highly carcinogenic epoxide. Omiecinski, Wilson and Eaton have found considerable variation among 188 adult humans in white blood cell microsomal EH activity with BP 4,5-oxide as substrate (Omenn et al., 1990). There are a few outliers with high activity. They and others have isolated and sequenced the human EH cDNA, which demonstrates extensive homology with both rat and rabbit microsomal EH (Hassett et al., 1990). In addition, they have isolated the human EH gene and have sequenced the 5'-end and flanking regions. Variants have been identified among fetal liver specimens. Studies are in
progress examining the association between EH gene expression in lymphocytes, lung and fiver from the same individuals (Redlich and Omiecinski, 1989). Remarkable technical advances now permit comparisons of tissue-specific expression at the level of mRNAs. Many were discussed at a recent UCLA Symposium on Biotechnology and Human Genetic Predisposition to Disease (Cantor et al., 1990). Many improvements on the widely used polymerase chain reaction are being reported, including R N A amplification with transcript sequencing (RAWTS) (Sarkar and Sommer, 1989). This technique incorporates a phase promoter into a PCR oligonucleotide primer, allowing an abundance of transcript to be made after amplification of mRNA by PCR.
Glutathione-S-transferases (GST) These detoxifying enzymes form inactive conjugates with glutathione. At least 8 different isozymes are known in mammalian species, probably including man, and some of them are inducible, further complicating analysis. Nevertheless, Seidegard et al. (1985) identified a very common polymorphism of lymphocyte GST activity; about 45% of Caucasians are essentially deficient in GST activity against the substrate trans-stilbene oxide (TSO). Mendelian inheritance of this trait has been confirmed by Motulsky and colleagues in our Dana Program. If TSO is a surrogate for carcinogenic substrates, these individuals might be more susceptible than others to resulting cancers. Pero's group in Sweden and Mantle in Dublin have found an increased prevalence of deficient individuals among lung cancer cases. Although epoxides of styrene and of benzo[a]pyrene were proposed as potential substrates, the key in vivo substrates for this polymorphic GST isozyme remain unknown. Heckbert, Eaton, and Motulsky have tested lymphocytes from individuals with cancers related to cigarette smoking, those with cancers unrelated to smoking, and non-cancer controls. BP 4,5-oxide as substrate gives a correlation coefficient of 0.640.72 with G S T against TSO, but the trimodality associated with TSO could not be discerned. PNSO and CNDB as substrates are poorly correlated with TSO activity and are unimodal. Although the
289 results are not fully analyzed, there appears to be little or no evidence for higher prevalence of the deficient allele among cancer cases in this large study. An additional study is under way (Nazar, Weiss, Eaton, et al.), analyzing GST and EH in lung cancer and normal lung tissues. Meanwhile, Eaton, Ramsdell and Monroe have explored the puzzhng huge difference in susceptibility of rats and mice to liver cancer from aflatoxin B1. As little as 15 ppb AFB in the diet produces liver cancers in rats, with 100% tumor incidence at 100 ppb. Yet no such tumors are observed in mice even up to 150000 ppb AFB dose. P450s convert AFB 1 to the 8,9-epoxide which forms adducts with N-7 of guanine nucleotides, or to much less hazardous metabohtes AFM, AFQ and AFP. GST is important in inactivating the 8,9-epoxide. It is convenient to estimate an 'index of susceptibility' from the ratios of activation rates (AFB-epoxide f o r m a t i o n / t o t a l oxidative biotransformation) to inactivation rates (AFB-epoxide GSH conjugation/AFB-epoxide formation). The activation ratio is just as great in mice as in rats; in fact, the component of epoxide formation is 3 times greater in mice. However, the GST-mediated inactivation ratio is far greater in mice. A F B - D N A adduct formation in mice was detected at a level 1.2% of that in rats for the same dose, corresponding closely to a ratio of activation/inactivation rates 2% of that in the rats (Omenn et al., 1990). On the same basis, humans and monkeys appear to have intermediate susceptibility, but more closely resemble the rat. Moreover, at low AFB concentrations the activation ratio increases in humans and rats (but not in mice), so there is no comfort in low-dose exposures. Thus far, assays of microsomes prepared from 14 human hvers obtained at organ donation have shown 2-3-fold interindividual variation in biotransformation to form AFB-epoxide and the AFQ and A F M metabolites. No family studies or genetic analyses have been feasible to date, and no extremely lowor high-activity individuals have been encountered. Agents thought to be chemopreventive against cancers - - such as r-carotene-rich vegetables, cruciferous vegetables, and the antioxidant BHA - - increase GST activity and decrease both adduct formation and tumor incidence in rats
(Ramsdell and Eaton, 1988). We are engaged in a major chemoprevention trial with a combination of r-carotene and retinol in heavy smokers and in asbestos-exposed workers, populations at high risk for lung cancer (Omenn, 1989). The scientific basis for the trial includes an association of low dietary intake or blood levels of r-carotene or vitamin A with high lung cancer risk; animal and cell culture studies showing prevention or reversal of late stages of carcinogenesis with the agents; and postulated mechanisms that r-carotene acts as an electron scavenger, while retinoic acid and retinol are ligands for nuclear zinc-finger proteins coded by genes on chromosomes 3p and 17 (where ' t u m o r suppression' genes have been localized). No studies have yet been undertaken to detect polymorphic or other genetic variation in these genes and proteins.
Analogous approaches to signal transduction pathways Weinstein (1988a,b), Ali et al. (1989), Weston et al. (1989), and many others have begun to explore the many interrelationships between the molecular biology of growth and differentiation and the aberrations that occur in tumorigenesis. The search for genetically determined variation in the genes and the gene products of metabolic pathways discussed above should have a direct parallel in studies of oncogenes, specific protein kinases, growth factors, tumor inhibitory factors, and their receptor molecules. We urge that those carrying out research on the 3 dimensions of structure, function, and regulation of expression of oncogenes add a fourth dimension to their studies: individual variation in these genes. For example, a c-mos variant identified as an E c o R I restriction fragment length polymorphism occurs in many patients with breast cancers both in their tumors and in their normal lymphocyte DNA, but in none of the 70 non-cancer patients thus far studied (Lidereau et al., 1987). Transformation of cells also may produce effects mediated through the phase II detoxification pathways discussed above, as in the case of v-Hras- or v-raf-transformed rat liver epithehal cells, which have resistance to cytotoxic chemicals in the
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form of multidrug resistance, in part due to marked increase in GST-P activity (Burr et al., 1988).
Concluding comments The major biotransformation pathways in humans for activation and detoxification of potentially carcinogenic chemicals encountered in our working and general living environment have notable genetic variation. Much work needs to be done to characterize the functional significance of genetic variation already known and genetic variation likely to be found. Identifying the relevant xenobiotic substrates will be important in evaluating genetic predispositions to cancers, birth defects, and organ-system effects. Always it must be emphasized that predisposition or susceptibility applies selectively to specific chemicals and specific enzymatic or receptor interactions; the same individuals may be less susceptible to many other exposures. Such selectivity is apparent also among rodents tested in the lifetime bioassay. For example, while mice are remarkably resistant, compared with rats, to aflatoxin, mice are more sensitive to polycyclic aromatic hydrocarbons. The tools of biotechnology make feasible many direct studies of enzymes, mRNA, and D N A for the steps of biotransformation. Of course, there are many other important variables in the mediation of effects of environmental chemical exposures, including the modifying influences of diet and physiology, DNA repair, immune surveillance, tumor promotion, tumor progression, and tumor suppression. Individual behavior will greatly i~afluence levels of exposure from occupational, recreational, and general activities. Thus, one must be cautious in recommending genetic testing for predisposing traits, proceeding first to anticipate the potential desirable and undesirable uses of such information, then to research carefully the relative risk and attributable risk of any specific genetic trait for any specific exposure and disease outcome before considering the ramifications of routine screening (Omenn, 1982). The multifactorial interactions of ecogenetics also place a high priority on advances in epidemiologic and biostatistical methods for design and analysis of such complex phenomena as carcinogenesis. Given the rapid pace of technical advances in
genetics and the great public interest in effective identification, characterization, and reduction of risks to humans from environmental chemicals, we may predict that cancer ecogenetics will emerge as a significant subfield of the environmental health sciences during the next few years.
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