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Genetic Polymorphism of Cytochrome P450 as a Biomarker of Susceptibility to Environmental ToxicRy Jun-Yan Hong and Chung S. Yang College of Pharmacy, Rutgers University, Piscataway, New Jersey Cytochrome P4~O {CYP) enzymes are responsible for the metabolism of numerous xenobiotics and endogenous compounds, inc{uding the metabolic activation of most environmental toxic chemicals and carcinogens.. Both metabolic and genetic polymorphisms have been identified for human CYP enzymes. The association of CYP genetic potymorphism and human cancer risk, and susceptibility to environmental hazards, have received increasing attention. This article briefly reviews the approaches and methods currently used in CYP genetic polymorphism studies. In add~on, the current status and perspe~ves of using CYP genetic polymorphism as a btomarker of individual suscapffoility .'co cancer and erMronmamal toxicity are discussed. -- Environ Health Perspect 105(Suppl 4}:759762 (1997) Key words: cytochrome P450, metabolism, genetic polymorphisms, biomarker, susceptibility, cancer risk, environmental toxicity CytodLrom¢ PZ'~Os (CYgs) ~ a supetfa.~y of hemoproteins that ~¢ ~¢ bio=~- ~rmadon 6£~ x~oblod~ ~d ~d~ b~ (1,2). ~o~h ~em ~ ~hpp~g sube~rate specificides, in~vidual GYP £or~ ha~ si~E~c ~ren~ in m~tc pr~c=. On ~, ~¢dv¢ ~vi~ ~cnc sub~ hme been ~ed co ~h It ~ w~ ~lbh~ ~ most ~v~n- ment~ to~c ~e~s and carcinogens. ne~ m be metabo~y a~vat~ • ~r to~c or carcinogenic effects. major e~¢ ~m~ ~ xenobintic o~m, ~s play a ¢id~ mle ~ ~e meta- bolic ~vation of maa7 enviro~ent~ ~e~. In some ~=, This paper was prepared as background for the Workshop on Susce~tlb~y to ErMr~nmerdal Hazarc{s ~e~ ~ ~e S~enSfic Group on Me~odci~[es ~ ~e Safe~ E~lua~on d Chem~al$ (SGOMSEC} held I7~ Mar~ 1~ In ~ Ruled. Ma~ receded at EHP 5 N~ember 1998; accepted 18 ~b~ 1 ~ We thank T.J. Sm~ for helpful discussion and Y. Wa~ ~d J. ~ f~ ~ in ~) ~u~t Address correspondence to Dr. J-Y. Hong, ~bo~tow for Cancer Research, College ~ ~a~aw, Ru~em Un~rs;~, ~s~, ~ ~ Telep~ne: (~8) ~5358. F~ (90~ ~687. ~ ~bm~a~ used: cD~ ~plemen=w DN~ ~en; RF~, ~n f~me~ I~ ~h~sm; ~R, ~er~ ~on ~I~ P~. metabolism leads to the detoxiflcation of toxic chemic=/s. Of 15 human GYP enzyraes so far char- =u'~erJzed, 8 fortns ofCYP (CYP1AI, ~ 2A6, 2(29, 2C19, 2D6, 2E1, and 3A4) have been shown to be polymorphic at the phenotypin or geaotypic level, or both (3-5). In addition to metabolizing various drugs, these polymorphic CYP enzyme~ are involved in metabolizing =. large number of envh'onmental carcinogens and toxic com- pound= (1). Because an individuz['s capa- bility to metabolize these toxicaa= can be altered by" carrying the variant alleles, generic polymorphlsms of CYP enzymes have been proposed as a biomarker of ~.~- ceptibility to envirorunent~l carcirmgenesis and toxicity. This ~per b~iefly review the methodologies for CYP polymorphism stud2es and emphasizes the comparison between the genotyping and phenotyping approache,. The use of genetic polymor- phlsms of human CYP enzymes as a suscep- tibility biomarker and the direction of future ~esearth will be discumed. Phenotypic Determination of CYP Polymorphisms It is well known that there are large intezL,~lividual variations in CYP-catalyzed drug biotramformation (1). The CYP poly- morpl~sr~ were initially identified by deter- mining the metaboEc ratio, i.e., the ratio of the blood or urinary amount of the parent drug over its metabolke, in ihdividua~ to whom the probe drug was administered. Metabolic poiymorphism is usually indicated in a population where the fre- quency distribution of the metabolic ratio is shown re be bimodal or trimodal. The bimodal distribution i~ due to the existence of poor and extensive metabollzets, whereto the trimodal distribution is caused by the Fresence of additional k~termediate metab- olizets. Examples for bimodal distribution include debrisoquine 4-hydroxylation (catalyzed bT CYP2D6) and coumarin 7-b.ydroxylarion (catalyzed by CYP2A6) (6"). The 3-demethylation of caffeine is ~m activity marker of CYPIA2, znd a trimodal distribution for caffeine metabolism was observed in nonsmokers (7). Be~ides the metabolism in rive, the polymorphisms of CYP enzymes cart be determined at other pheuotypic levels. These include enzyme activity determina- tions for metabolism in vitro with micro- seines, CYP enzyme protein levels detected by immunological method= such as immu- nobler and immunohistochemica[ ana/yses, and CYP mRNA levels menaced by differ- ent nucleic acid hybridization techniques (Northern and ~lot blotting, RNase protec- tion, and in sltu hybridization). In recent years researchers have deveJoped reverse transcription coupled with polymerase chain reaction (RT-PCP0 to detect CYP mRNA in a sma//amount of tissue sample. If an appropriate internal standard is included in the eDNA synthesis reaction, the RT-PCR can be quantitative in assess- ing the amount ofCYP mRNA in the sam- ples. Expression of CYPIAI mRNA [n human lymphocytes and its reg~*lation by 2,3,7,8 -terrachlorodibenzo-~-diox~n have been successfully determined by this method (8). The phenotyping approach, especially" with a~tivity-related assays such as metabo- lism in vivo and in vitro, dizectly finks the expression of a given CYP form re the metabolism oflts probe substrata. However, a critical issue is the dif~cttlty in finding the right probe drug~ that are specific for diagnosing a particular CYP form and sa/'e enough for in rive metabolism studies. Another major concern with the pheno- typing approach is the confounding effects of dietary factors or coadministration of drug~ that could affect expression of the metabolizing CYP enzymes (9). This could, in tara, aEer dee metabo/~m of ~e probe drug. Induction or suppression of CY~ enzyme activities by xenobiotics, including dlerary compounds, has been .Environmental Healeh Persp~ • V~ 105, Sup~ement 4 • June 1997 759 This art[cie is for individual use only and may not be further reproduced or stored electronically without wdtten permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) US DEPT HEALTH HUMAN SERVICES PUBLIC HEALTH SERVICE USA

HONG AND YANG well documented in animals (9)i In humans, induction of CYP2EI by alcohol &inking (I0) and of CYPIA2 by ingestion of charbroiled meats or cruciferous vegeta- bles have been observed (11). In addition, human CYPIA2 is induced by cigarette smoking (12,13). Finally, the availability of substantial amounts of quality-assured human tissues (except peripheral blood cells) could be a limiting factor for deter- mination of CYP enzyme activity in vitro, as well as for determination of GYP protein and mRNA levels. Genetic Basis of Metabolic Polymorphisms The genetic basis of several CYP-involved metabolic polymorphisms has been eluci- dated. Mutations in the GYP genes are believed to bc a major mechanism for alter- ing enzyme expression and/or catalytic activities. Both point mutations and dele- tions are observed in the polymorphic CYP genes. Depending on their locations in the gene sequence, polymorphic changes could have either two general effects or none at all Mutations in the coding region of a CYP gent that cause amino acid substitu- tions could alter the catalytic activity by causing a direct change in the protein structure. Mutations in the noncoding region, on the other side, may alter the level of mRNA expression by influencing transcription, mRNA stabilization, or pm- mRNA splicing. All of the mutations with functional significance ~ believed to have a remarkable impact on an individual's capability to metabolize certain drugs and environmental chemicals. However, the functional sigr,Ailcance of a majority of the polymorphic changes in CYP genes so far identified is still not known. Once the metabolic polymorphism involving a particular CYP enzyme is demonstrated, various molecular biology approaches and techniques can be used to look for possible genetic cha~ges. A success- fi~ example of this approach is the discovery of CYP2D6genetic polymorphism by Goazales et al. (14). A~er eDNA cloning and DNA sequencing, they demonstrated that a mutant 2D6"allele is responsible for the majority of "poor metabolizers." Further work established that a mutation at a splic- ing sire caused the production of defective 2D6 mRNA and a total absence of 2D6 protein (15). Prior to labor-intenslve DNA sequencing work, the possible DNA sequence alterations can now be screened by single-strand conformation polymorphism analysis that detects the mutatlon-caused mobility skirl of the DNA fragments on gel electrophoresis (16) or by other methods such as denaturing gradient gel dec- trophoresis (17). For functional analysis, the catalTdc activity of CYP enzymes can be studied by expressing different variant CYP proteins with various eDNA expression systems if the polymorphic changes are localized in the coding region. If the poly- morphic loci am in the noncoding region, thek effects on the transcriptional regulation can be studied by linking the mutated sequence with a reporter gene. Genotyping Approach As long as the polymorphic sites ofa CYP gene are clearly identified, it is simple to determine an individual's genotype by cur- rent molecular biology techniques. If a polymorphic site in a CYP gene changes the recognition sequence of a restriction enzyme, or if the genetic polymorphism involves a large deletion, the genetic poly- morphism can be identified by restriction fragment length polymorphism (RFLP) analysis, in which DNA is subjected to Southern blotting after digestion with appro- priate restriction enzymes and hybridized with specific probes. Recent advances in PCR technology have greatly increased our capability to detect genetic polymorphisms of CYP enzymes. With small amounts of human tissue or cell samples, DNA amplification can be carried out with proper PCR primers for any parricular sequence of a poIymorphic CYP gene. The DNA source co~a be from blood leukocytes, buccal epithelial cells, hair roots, or normally exfo- fluted cells, such as bladder epithelium in the urine. DNA can also be obtained from stored pathological tissue sections, which provide great advantage .for retrospective studies. It is now feasible to determine sev- era CYP genetic polymorphlsms with less than 10 pl of blood collected from the fin- ger dp. The PCR-amplified DNA sequence containing the polymorphie sites can then be analyzed by RFLP with restriction diges- tion and visualized on a stained gel after elecrrophoresis. Comparing the wild-type samples, if the genetic potymorphlsm results in a loss--or in some cases a gain-- of a restriction site, the band pattern ca the gel will be different. If the PCR primers are designed to be within the missing sequence of a ddetion polymorphism, there will be no PCR product formed. Obviously, the RFLP method cannot be used to screen the CYP genetic poly- morphisms La which the DNA sequence alterations cause no changes at suitable restriction sites. In this case, genotyping can be carried out by allele-specific PCR with a set of mutation-specific primers for amplification. Several polymorphisms of CYP1A1, 2,46, and 2D6 have been idenri- fled by the allele-speciflc PCR method (18-20). If necessary, the results from PCR-RFLP and allele-specific PCR can be confirmed by PCR-direct sequencing. La contrast to the phenotyping approach, genotyping is not affected by drugs or dietary factors that might modulate the metabolic activity of CYP enzymes. As mentioned previously, the PCR-based genotyping techniques require only a small amount of DNA, which can be obtained by less invasive or noninvasive means or from longdme stored pathological samples. The genotyping approach also allows accurate prediction of the homozygous or hererozy- gous status of an individual who carries the variant allele. All of these are particularly usfful for large population studies in which genedc polymorphisms of CYP enzymes may be susceptibility markers. CYP Genetic Polymorphisms and Cancer Risk The association of CYP genetic polymor- phisms and human cancer risk has received increasing attention. Examples include CYP1A1 with lung and breast cancers (21-23), CYP2D6with different types of cancer (24), and CYP2E2 with lung, liver, and naropharyngeal cancers (21,25-28). However, many reports are controversial. One important factor in interpreting these results is that there are significant ethnic differences in frequency distribution of the CYP genetic polymorphisms. For example, an association of CYP2E1 DraI genetic polymorphism and susceptibility to tung cancer was suggested in a study of a Japanese population (29) but was not observed in Caucasians (26,27,30). This discrepancy was believed to be caused by a significantly low distribution frequency of CYP2E1 DraI polymorphism in Caucasian populations (27). Current Problems and Perspectives Research on genetic polymorphisms of CYP enzymes can provide a molecular basis for interindividual variations in metabolizing drugs and environmental toxic chemicals. In addition to this mechanistic information, the studies hold great promise/a identifying susceptible individuals and protecting them from environmental toxicity. If a given 760 Environmental Health Perspectives • Vol I 0~, S.upplement ~ • June 1997 T~is article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial end other penalties. (c) US DEPT HEALTH HUMAN SERVICES PUBLIC HEALTH SERVICE USA

POL~f, ORPH|$19~ OF CVTOCHROME P450 AND SUSCEPTIBILITY CYP polymotphic genotype causes enhance- ment in the metabolic activation of the rdated substrata toxicants, individuals with such variant alleles should avoid exposure to those toxic compounds. Knowing the identity of the chemical to which one would be exposed and the polymorphic CYP form involved could be particularly useful in preventing chemical toxicity from occupational exposure. Studies on GYP genetic poiymorphism and environmental toxicity could be very rewarding, and teseaw, A activities in this fidd are expected to increase in the near future~ However, we are facing the following challenges: a) With current PCR-based t~chniqaes and rapid development of molecular biol- ogy approaches, identification of CYP genetic polymorphisms and genotypic screening ofa subpopulation are nor diffl- cuk. More new CYP genetic polymor- phisms are expected to be discovered. The mos.t critical challenge, however, is to. establlsh the functional importance of dif- ferent polymorphic variants, espedally for those polymorphic sites locared in the noncoding regions of the CYP genes. Using an in vitro uansfecr.ion system with CATas a reporter gene, it has been observed that the RsaI polymorphic site in the 5'-flanking sequence of the CYP2E2 gene caused a 10-fnid increase ia transcrip- tional activity in comparison with the wild-type sequence (31). It is important to demonstrate whether such extent of regulation occurs in viv#. Similarly, the results from the in vitro activity assays with the expressed variant CYP proteins need to be verified in phenotypic studies with human populations. b) Our current efforts have been focused oa the association of CYP generic polymorphisms with cancer risks and [ass on the occupational toxicity in which the biological end points are not cancer occur- mace. Human carcinogenesis is a long-term, muitistep process, Although metabolic activation by CYP enzymes is known to constitute the first and critical step in envi- ronmental carcinogens, there use many other important steps involved, such as phase II enzyme detoxiflcation, DNA repair, and immunosurveiilance, as well as many modulating factors such as dieta~/ components. In addition, it is difficult or impossible to know the number and idea- tity of the carcinogens involved or the expo- sure levels. Assessing the role of genetic polymorphism ofa pa_~cular CYP form by using cancer occurrence ~ a biological end point is therefore a very" difficult task. We believe that at th/s stage it is mote legible to determine the role of CYP genetic poly- morphisms in the susceptibility of workers to chemical toxicity ia occupational expo- sures in which cancer occurrence is not an end point. An advantage in using worker populations is that the identity and the exposure levels of the toxic chemicals are in general clearly known. In addition, the workers are probably mote homogeneous than the gene~l populadun and more easily accessible for follow-up studies. c) More research is needed for charac- terization of the substrata specificity and the enzyme kinetics of human CYP enzymes. Knowledge of which eavizon- mental toxicants and carcinogens are the subsuates of the polymorphic CYP enzymes is important, but that knowledge is not enough in designing z population study. Although it is known that CYP enzymes have overlapp/ng substrata spec~ficities, the CYP form with a high Vm~ v'due and the lowest K~, value is usually the prindpal one involved in the metabolism and is therefore believed to be the one most relevant to the sittmdun in viva. Obviously, if a particu- lar CYP form is found to be able to acti- wte a toxic chemical bur the K~, value is much higher than the physiological con- centmtion of that chemical after expostue, that CYP form.may be of little relevance to the real in vivo situation. Studies in this direction will also hdp in devdoping more selective ~probe~ drugs for different CYP forms, which is inevitably required in establishing the relationship between CYP genetic polymophisms and metabolic activity in rico. d) More information is needed to understand the regulation of CY'P enzymes in humans. The studies should include the effect of dietary" compounds on the expres- . sioa of CYP enzymes and the expression profile of CYP enzymes ia the tissues that are targets for chemical toxicity or genesis. Without this information, the role of CYP genetic polymorphism in the biological consequences may not be accurately assessed. e) Humans are exposed to numerous environmental toxicants. For example, more than 40 cardnngens have been found. in tobacco products and tobacco smoke (32). Multiple pulymurphic CYP enzymes are involved in metabolizing these cardno- yeas. Even for a given subpopulatioa mainly exposed to a single toxic chemical, it is possible that more than one form of CYP enzyme is involved in the activation or detoxiflcation. Therefore, it is recom- mended that whenever possible a combina- tional polymorphism analysis ott all the involved CYP forms be carried out to obtain a complete picture of the role of CYP enzymes in susceptibility to cancer and toxicity. The availability of current PCR technology allows'us to use DNA samples stored for extensive periods (e.g., blood dotted on f~ter paper or pathological tissue sections) for the analysis of different CYP genetic polymorphisms. 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Supplement 4 • June 1997 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) US DEPT HEALTH HUMAN SERVICES PUBLIC HEALTH SERVICE USA