Document kmNg9Q0weOkrvDraqaoNdJ4ZV

. Pharmacogenetics: Detecting Sensitive PopuIations Peter G.Shields Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland Risk assessment models strive to predict risks to humans from toxic agents. Safety factors and assumptions are incorporatedinto these models to allow a margin of error. In the case of cancer, substantial evidence shows that the carcinogenic process is a multistage process driven by the interaction of exogenous carcinogenic exposures, genetic traits, and other endogenous factors. Current risk assessment models fail to consider genetic predispositions that make people more sensitive or resistant to exogenous exposures and endogenous processes. Several cytochrome P450 enzymes, responsible for metabolically activating carcinogens and medications, express wide interindividualvariation whose genetic coding has now been identified as polymorphic and linked to cancer risk. For example, a restriction fragmentiength polymorphism for cytochrome P4501A1, which metabolizes polycyclic aromatic hydrocarbons, and cytochrome P4502E1, which metabolizes Nnitrosamines and benzene, is linked to lung cancer risk. Cytochrome P4502D6, responsible for metabolizing many clinically important medications, also is linked to lung cancer risk. The frequency for each of these genetic polymorphismsvary among different ethnic and racial groups. In addition to inherited factors for the detection of sensitive populations, determining the biologically effective doses for carcinogenic exposures also should quantitatively and qualitatively enhance the risk assessment process. Levels of carcinogen-DNA adducts reflect the net effect of exposure, absorption, metabolic activation, detoxification, and DNA repair. These effects are genetically predetermined, inducibility notwithstanding. The combination of adduct and genotyping assays pro- vide an assessment of risk that reflects recent exogenous exposure as well as one's lifetime ability to activate and detoxify carcinogens.-Environ Health Perspect 102(Suppl11k81-87 (1994) Key words: cytochrome P450, cancer, genetic polymorphisms, risk assessment ~ -- -- Introduction The protection of pcpulations from carcinogenic agents (and other toxins) generally requires a risk assessment process before regulation or remedial action. A formal risk assessment involves several steps including a hazard assessment, dose-response assessment, exposure assessment, and risk characterization ( I ) . In general, data from scientific studies are extrapolated to human experience through mathematical modeling that identifies a level of chemical exposure that might predictably result in a specific iiumber of adverse outcomes (e.g., clinical cancer). Mathematical modeling, however, essentially is a substitute for scientifically determined data and is sometimes accepted, for a variety of reasons, without adequate validation. Many risk assessment steps incorporate untested assumptions or have methodological problems such as using This article was presented at the Workshop on Pharmacokinetics Defining the Dose for Risk Assessment held 4-5 March 1992 ar the National Academy of Sciences in Washingtor DC The author is grateful to Curtis Harris for his review of this manuscript and Dorothea Dudek for her skillful editorialassistance Address corrasi;oqdence to Dr P G Shields, Laboratory of Human Carcinogenesis. Division of C o w e r Etiology. National Cancer Institute kthesda M D 20892 Telephone (301)496-1603 Fax (301)496-0497 premises that are not consistent with known scientific data (e.g., using multistage models with the number of stages less than that determined from human cancer srudies). Moreover, a fundamental limitation of risk assessment is that it usually examines risk for popularions rather than for individuals, thereby not considering interindividual variation in response to xenobiotic exposure. Individuals or groups of individuals (e.g., families or ethnicity) might be more sensitive or resistant to particular exposures based on xenobiotic activation or detoxification, DNA repair, genetic structure, etc. Thus, the risk assessment process that does not include the interaction of the environment with interindividual capacities will become increasingly limited. Carcinogenesis is a multistage process of normal growth, differentiation, and development gone awry (2,3).It is driven by spontaneous and carcinogen-induced genetic and epigenetic events. Carcinogenic agents initiate the process by causing DNA mutations and altered gene expression. These genecic effects, in concert with additional carcinogen exposure and orher genetic or epigenetic effects, lead to tumor promotion. Through these stages, cells have selective reproductive and clonal expansion capabilities. Progressive phenotypic changes and genomic instability occur (aneuploidy, mutations, and gene amplification). These genetic changes enhance the probability of initiated cells transforming into a malignancy; the odds of which are increased during repeated rounds of cell replication. Angiogenesis allows for a tumor to grow beyond 1 or 2 mm in size. Ultimately, tumor cells can disseminate through vessels invading distant tissues and establishing metastatic colonies. Each of these steps can be directly affected by carcinogen exposure. The response to these exposures, however, can vary from individual to individual (4). Note also that the current concepts of initiation, promotion, genetic, and epigenetic effects have been conceptually important but are now considered simplistic and not consistent with current human carcinogen- esis models. The role of protooncogenes and tumor suppressor genes has become increasingly apparent in the multistage model of carcinogenesis (2).Both are important to the regulatory mechanisms of growth, cell cycle control and terminal differentiation (2,5). Activation of protooncogenes enhance the probability of neoplastic transformation, which can either be an early or late event. Tumor suppressor genes code for products that, unlike protooncogenes, enhance the probability of neoplastic transformation when their activity is lost. For example, the p53 rumor suppressor gene, located on chromosome 17, is the most commonly altered suppressor gene among all tumors so far studied ( 6 ) S. ingle base substitutions 87 can result in loss of function or production this pathway, intermediates might be background. Current studies sh ofp53 proteins that either interfere with removed via conjugation [e.g., glutathione these factors are more common normal function or otherwise directly transferase), further oxidation, or reduction. fimily, ethnic, ad racial groups. enhance neoplastic transformation (7). These metabolites can then be excreted in CY? 1Al has been extensively The multistage process of carcinogenesis urine or feces. It is worthwhile noting that the metabolism of PAHs. Act is best exemplified by a model of human the activity of several enzymes involved in CYPlAl varies in lung tissues from colorectal tumorigenesis described by these activation and detoxification processes, ent persons (25.26).which is an Fearon and others (8).In the early stages, as well as D N A repair, varies markedly in trait ( 2 7 )but also can be ind loss or inactivation of APC (3.10) and individuals, so that reactions lending to the exposure to agents such as tobac MCC genes (1I), hypomethylation and formation a n d removal of BP-related (28).hducability varies among in genomic instability accompanies the pheno- adducts and mutations can occur at higher a n d is notahly higher in lun typic appearance of an adenoma. More or lower rates (4). patients than in noncancer advanced tumors involve oncogenes and O n a molecular basis, cells possess the (29,30).Levels of aryl hydrocarbon t u m o r suppressor genes, typically not ability to repair DNA damage (18).Smaller ylase activity also have been correla observed in early adenomas. The combined alkyl adducts can be excised while larger levels of DNA adducts (31)and wi events are more important than the actual adducts require the excision of several bases. nosis in l u n g cancer ( 3 2 ) .Recen order in which they occur. The genetic An extensively studied repair enzyme is the restriction fragment length PO abnormalities include Ki-rar mutations on 06-alkylguanine-DNA-alkyltransferase has been described, using Mp chromosome 12, mutation of p53 tumor (19).This enzyme repairs damage from digestion, that is reported to c suppressor genes on chromosome 17, and a alkylating agents such as tobacco-specific lung cancer risk in a Japanese deletion of DCC,the putative tumor sup- nitrosamines and other N-nitrosocom- and also found to be in genet pressor gene on chromosome 18q that may pounds. I t is a suicide protein in that it rium with a mutation in the catatylic r be involved in cell-to-cell adhesion and pos- transfers t h e alkyl group to itself and of the enzyme (34).Evidence suggests sibly metastasis. Other allelic losses can becomes inactivated. Cell cytotoxicity and the mutation results in increased metab& occur in any of the other chromosomes. tumor cell resistance are negatively corre- activation (K Kawajiri, personal commuai. Thus, it appears that at least six genetic lated with the levels of this enzyme (20), cation) and the polymorphism was corre- events occur in the development of colorec- and levels vary within organs and among lated with a 3-fold increase in lung cancer d carcinoma. people (21,ZZ). PAH-DNA adducts can b;e risk ( 3 3 ) .Further study showed that th T h e method by which carcinogenic repaired by a nucleotide excision pathway. effect was greatest in persons with s q u agents affect DNA and produce mutational A unimodal distribution of repair rates of mous cell cancer and in persons with & spectra is varied. Chemical carcinogens gen- benzo[a] pyrene diol-epoxide DNA adducts least amount of tobacco smoking history erally undergo metabolic activation to dec- has been observed using human lympho- (35).This latter point indicates that th trophilic intermediates that form D N A cytes in vitro (23).The interindividual vari- effect of this polymorphism is strongest . adducts through covalent binding (12). ation was substantially greater than the persons with less carcinogenic exposure. Promutagenic adducts can then cause muta- intraindividual variation, which suggests a The National Cancer Institum- tions through mispairing or base substitu- role for inherited kctors. University of Maryland (NCI- UMD) tions during DNA synthesis. The binding of carcinogens to DNA nucleotides is appar- Genetic hdispasitions to Cancer Case-Control Study investigated lung cer patients and controls (pulmonary dis- ently nonrandom (13-15) and has been Interindividual variation in response to ease patients and nonlung cancer patients) shown to affect protooncogenes and rumor xenobiotics and their potential carcinogenic for inherited predispositions to lung can- suppressor genes. Among the best studied effects is mediated by inherited predisposi- cer. The control groups had similar age and examples of metabolic activation is the epox- tions (Table 1). Family cancer syndromes, smoking status. The Msp 1 polymorphism idation reactions of polycyclic aromatic the most evident expression of inherited pre- of CYPlAl and lung cancer risk was stud- hydrocarbons (PAH), of which dispositions, can lead to up to a 1000-fold ied in 101 persons enrolled in this study. benzo[a]pyrene (BP) is one example increased risk of cancer in family members but no association was found with either (1617).These compounds are composed of (24).However, most individuals do not lung cancer risk or histological lung cancer fused benzene rings that are essentially water have such an obvious genetic predisposition, type. However, there was a statistically,.. insoluble but readily absorbed through the and host susceptibility relating to a specific significant difference in allelic frequencies lungs and gastrointestinal tract. They are gene is less obvious because these mutations for African Americans versus Caucasians. commonly found as combustion products of as risk factors are barely detectable above T h i s suggests that o n t h e basis of the fossil fuels (e.g., coal, diesel exhaust) and vegetable matter. Consequently, PAHs Table 1. Examples of interindividualvariation in response to xenobiotics and cancer risk or prognosis occur as environmental pollutants. BP Response becomes metabolically activated in a phase 1 reaction by forming a reactive diol epoxide Metabolic activation that can covalently bind to DNA-forming adducts. Initially, cytochrome P450 (CYF')lAl and epoxide hydroxylase catalyze Detoxification the conversion of BP to a dihydrodiol. DNA repair Then, CYP3A4 converts this product to a Protooncogene diol-epoxide (i.e., BP-7,8-diol-9,1O-epoxide) that is the reactive form. However, along Tumor suppressor gene Example Cytochrome P4501A1 Cytochrome P4502D6 Cytochrome P4502E1 MAcetyltransferase Glutathione Stransferase M1 Excision of UV damage HRAS-1 L-myc PU Human cancer type Lung (33) Lung (37.38) Lung (48) Bladder/colon (81.82) Lung/stomach/colon (87.821 Skin 163) Lung (84.85) Lung/gastric/sarcoma (86,871 Li-Fraumeni syndrome (88) i 82 DETECTlNG SENsmvE POPUlATlONS Japanese data, African Americans would be more sensitive to lung cancer. Separate analysis%y race did not reveal an associa- tion wim lung cancer risk although the numbers of each group substantially limited the statistical power of the study. Table 2 Cytochrome P4502D6 genotypes and phenotypes 142,441 ~~ Type Xba I RFLT Location Description Wild-type A B C 29 Kb 29 Kb 29 Kb 29 Kb NA~ Exon 5 Exon 3 Exon 5 Wild-type sequence Nucleotidedeletion (2737Al Splice site defect (1934G+TI Nucleotidedeletions (2702-2706 AGAI Associated phenotype Extensive Poor Poor Extensive Thus, &e study numbers are currently D 11 5 K b NA Deletionof cytochrome P4502D6 Poor being increased. In a separate Norwegian study (pd),no association with the Msp 1 'Aesults of Southernblot analysis yielding fragment length shown 'Not applicable RFLP and lung cancer was found. Importantly, this study was severely hindered by not utilizing age- and smokingmatched controls that can result in falsely negative findings. The frequency of the exon 7 mutation in cancer patients and m a t c h d controls, and its linkage with the Msp 1 W L P , in American and European ethnic groups is now required. The study of the cytochrome P4502D6 (CYP2D6, also know as the debrisoquine polymorphism) is among the best examples of inheritable interindividual differences in The D mutation is identified with the Xba 1 restriction digest and Southern blotting ( 4 0 ) .T h e A and B mutations can be identified by using a PCR mismatch assay where primers differ only by the 3-ft base that matches with either the wild-type base or the murant base (42). The assay begins with a first step that uses primers specific only for CYPZDGand not the psuedogenes C"2D7and C"2D8, which are otherwise almost 95% homologous. The second step uses rhe mismatch primers. The assay identified (47,48).O n e polymorphism includes two distinct base substitutions in the same area, which are in genetic disequilibrium so that either one can be studied (47).This area is involved inzanFciption regulation and preliminq studies indicate that one type allows for increased expression of the chloramphenicol acetyl trans- ferase gene in transfected HepG 2 cells. The frequency of the polymorphic alleles is different in Japanese and Americans (S Kato, personal communication, 1992). metabotism. This enzyme is responsible for requires careful validation to ensure correct Whether this polymorphism has a relation- metabdism of several medications includ- priming, sensitivity, and specificity. This ship to cancer risk is currently under study. ing tricyclic antidepressants, beta-blocking method can reportedly characterize over The other polymorphism is at Dral restricantihypertensives, and debrisoquine. Poor 95% of metabolic phenotypes in large num- tion enzyme site in intron 6 (M Wacanabe, metabolizers are a t risk of adverse drug bers of Europeans. T h e second method personal communication). While its bio- reactions. I n a cohort of smokers in takes advantage of a BstN 1 restriction site at logical significance is unknown, it has been London, England, a 4-fold higher risk of the B mutation and uses an altered primer reported that the distribution of genotypes lung cancer was associated with the exten- that introduces a Dra 1 restriction site at the is significantly different in lung cancer sive metabolic phenotype (37).This associ- A mutation (45)(R Wolf, personal com- cases and controls (48). ation has been confirmed in the N C I - W D lung cancer case-antrol study with an odds ratio of six (38).The exrensive metabolic phenotype also has been munication). In our laboratory, we have been genotyping individuals using a combined approach that uses the BstN I digest for the B mutation with the primers previ- Molecular D a s i m e t r y - I b ~ a CombinedcarcinogenExposwe and susceptibility shown to have an interactive effect with ously identified as nor significantly homole One indicator for the net effect of exoge- occupational exposures to asbestos and gous (42)and the mismatch assay for the A nous carcinogen exposure and inherited PAHS (39). mutation. We have found that the fre- traits for absorption, metabolism, and Gemtyping methods for W 2 D 6have quency of poor metabolizers in Caucasians DNA repair is the carcinogen- DNA been sought to avoid the requirement for by genotyping was statistically significantly adduct. Measurement of adducts can be time-consuming urinary phenotyping and higher than in African-Americans, suggest- use& for estimating a biologically effective the atrendant hypothetical risks of drug ing an inherited predisposition to lung can- dose of a carcinogen and the risk for fixed administration. These methods also would cer in African Americans (P Shields, mutations. A variety of assays are available clarifywhether the association with lung unpublished results). to identify carcinogen-DNA and protein cancermight be a result of a cancer effect Thus far,the mecbaniitic relationship of adducts. Enzyme immunoassays (49-58), on the phenotypic expression rather than CYPZD6activity to lung cancer remains 32P-postlabeling and nucleotide chro- an i n k r i t e d predisposition that predated unknown. The only known carcinogenic matography (59-61), fluorescence spec- the development of cancer. An X6a 1 RFLP substrate identified for CYP2D6 is 4- troscopy (62).synchronous fluorescence was described (40)where identifiable alleles (methyl-nitro<amino)- 1-(3-pyridyl)- 1- spectroscopy (SFS) (63-66), gas chro- were associated with the poor metabolizer butnnone ( N N K ) but not other matography and mass spectroscopy phenorype. However, it was soon found tobacco-specific nitrosamines that have been ( G U M S ) (66,67),and electrochemical that the EcoR I RFLP only correctly pre- tested (46).Thus, the lung cancer associa- detection (68)have been applied tc the dictedane-third of poor metablizers (41). tion might relate to alrered substrare analysis of human lung samples or a surro- Since.&en, it was found that most of the specificiy of the enzyme in extensive metab- gate tissue or Ceu population. poor metabolizer phenotypes could be olizers not studied, unidentified carcino- Central to the studies of DNA adducts explained by specific mutations in genic substrates, or a gene that is in linkage is the development of sensitive and specific CM-2B6 (42).These and other mutations disequilibrium with another gene related to assays that are required to detcct ferntomole have n ~ wbeen designated as A, B, C, and caner risk. and attomole levels of a d d u a s in micro- D mutetioxis (Table 2) ( 4 0 , 4 2 4 ) . CW2E1 is responsible for metabolizing gram amounts of DNA. Current methods T+ polymerase chain reaction (PCR) a number of potential human carcinogens are challenged because of the complmity assays%ave been published to determine the including benzene a n d N-nitrosamines. and multitude of possible exposures in A and B mutations in CYP2D6 (42,45). Several genetic polymorphism have been human tissues. The specificity of adduct Vdum? 102, Supplement 1 1, L)eavnber 1994 I assays, and therefore their quantitative r c l i - Tabh 3. Genetic polymorphismsand ethnicity. biliry, can be enhanced by using micro- Polymorphism preparative techniques. For example, Populations compared At riska subjecting enzymatically digested lung DNA to high-pressure liquid chromatop- phy (HPLC) followed by the 3ZP-postlabel- ing assay, which relies on three different separations (HPLC and two-dimensional thin-layer chromatography) can detect N- nitrosamine-related alkyl adducts such as 06-merhyl-2/-deoxyguanosin(e6 9 ) ,06- ethyldeoxyguanosine ( 06ethyldG) (69), N- 7-methyldeoxyguanosine (N'methyldG) genic exposures (75).Small alkyl adducts, (70)and N-7-ethyldeoxyguanosine ( S Kato, polycyclic aromatic hydrocarbons adducts, personal communiytion) at levels as low as and aromatic amines have been identified. one adduct in 10' 2'-deoxyguanosine Improved micropreparative techniques residues. O t h e r laboratories have used have led to the unambiguous identification HPLC after j2P-postlabeling (71). of specific polycyclic aromatic hydrocar- Immunoaffinity chromatography also has bons adducts in human lung (63.66).This assays are preferable to been combined with 32P-postlabeling assay finding has now been confirmed by com- because they cannot be to identify 06-methyldeoxyguanosine (D bining immunoaffinity chromatography ence of disease. For Cooper, personal communication) and with the 32P-postlabeling assay (P Shields, polycyclic aromatic hydrocarbons (P unpublished data). tion phases cannot be overstated and Shields, unpublished data). In both cases, Exposures to PAH compounds are asso- to be meticulously performed prior to && t h e level of detection was at least o n e ciated with an increased risk of lung cancer. use in large field trials. adduct in 10' unmodified deoxyguanosine. Industrial pollution, fossil fuels, a n d NOTE A DDED I N PROOF: Since tbc Another feature of assays that are dependent tobacco smoke account for the major envi- preparation of this manuscript in 1992, upon micropreparative techniques is that ronmental sources. Dietary exposures also of the data cited as personal c o m m u n i c a k they are developed using authentically syn- commonly occur because of overcooked or have now been published. There also am a thesized adduct standards, use internal stan- charcoal-broiled meats. Adduct levels have number of more recent reviews. New drp dards, a n d quantitation based upon been correlated with exposure in coke oven has identified several examples of gene+& calibration curves. workers (54,59,76), tobacco consumption ronment interactions where the effects of Organ tissue selection, multiple sources (67)a,nd urban versus rural residence metabolizing polymorphisms vary d e p e d o f exposures, and o t h e r confounders (77),but decreases during vacation from ing on exposure, further impacting u p impact upon study design and results. It occupational sources (51).Seasonal varia- risk assessment procedures and outcomes. would be optimal to use easily obtainable tion in adduct levels also has been observed body fluids such as blood or urine for risk (78).Some studies have not found correla- assessment. However, it remains to be tions with tobacco consumption but this established if blood testing will be a reliable may be because of other sources of expo- REFERENCES surrogate for other tissues. For example, sure (e.g., diet). some data suggest that the determination of PAH adducts in peripheral lymphocyte D N A reflects dietary rather than inhalational exposures (72,73).We also will need to consider cellular differences in life-span (lymphocytes and red blood cells survive longer t h a n granulocpsJ-RbUnNAlepait capacity, and metabolic capacity. It has been reported that the initial adduction of lymphocytes and granulocytes is similar but that adduct levels are more persistent in the latter (74).Our laboratory has used lymphocytes (49-5f6,3)while others have studied total white blood cells (predominantly granulocytes) (54.72).Oral mucosal cells are another relatively noninvasive source of D N A a n d can allow for t h e detection of several types of adducts (57,601. 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