Toxicology Letters 120 (2001) 269 280 www.elsevier.

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Genetic variability in susceptibility and response to toxicants

Merrill C. Miller, III a, Harvey W. Mohrenweiser b, Douglas A. Bell a,*
a

National Institute of En6ironmental Health Sciences, 111 Alexander Dri6e, Building 101, Room B323, P.O. Box 12233, C3 -03, Research Triangle Park, NC 27709, USA b Biology and Biotechnology Research Program, Lawrence Li6ermore National Laboratory, Li6ermore, CA, USA

Abstract Everyone has a unique combination of polymorphic traits that modify susceptibility and response to drugs, chemicals and carcinogenic exposures. The metabolism of exogenous and endogenous chemical toxins may be modied by inherited and induced variation in CYP (P450), acetyltransferase (NAT) and glutathione S -transferase (GST) genes. We observe that specic at risk genotypes for GSTM1 and NAT1/2 increase risk for bladder cancer among smokers. Genotypic and phenotypic variation in DNA repair may affect risk of somatic mutation and cancer. Variants of base excision and nucleotide excision repair genes (XRCC1 and XPD ) appear to modify exposure-induced damage from cigarette smoke and radiation. We are currently engaged in discovering genetic variation in environmental response genes and determining if this variation has any effect on gene function or if it is associated with disease risk. These and other results are discussed in the context of evaluating inherited or acquired susceptibility risk factors for environmentally caused disease. 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Xenobiotics; Variability; Polymorphisms; DNA adducts; DNA repair; Genetic risk

1. Introduction Every individual is unique. This includes not only personal appearance and preferences but, most importantly, responses to the environment and susceptibility to diseases. Genetic variability gives a species the ability to adapt to the environment over time and, at this point in human evolution, has resulted in various environmental response phenotypes. For example, some individuals may not tolerate alcohol while others show
* Corresponding author. Tel.: + 1-919-5417686; fax: + 1919-5411479. E -mail address: bell1@niehs.nih.gov (D.A. Bell).

little effect. Some individuals may not respond to pain medications while others fall into a deep sleep at a similar dose. Some people may develop cancer after exposure to a hazardous chemical while co-workers with similar exposures never develop tumors. These susceptibilities and responses can often be linked to an individuals ability to metabolize toxicants or repair damage from toxicants encountered in the environment. An individuals response to environmental chemicals will vary depending on the presence of DNA sequence variations (polymorphisms) within critical genes in that individual. These genetic polymorphisms may affect the level of expression, the structure, or the catalytic activity of metabolic or

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DNA repair enzymes, thereby inuencing toxicant susceptibility. Some specic examples include the following: (1) the relationship between enzymes that activate (e.g. CYP, N -acetyltransferases) or detoxicate (e.g. glutathione S -transferases) carcinogens found in cigarette smoke and the risk of lung and bladder cancer; (2) the susceptibility of individuals with XRCC1 or XPD polymorphisms to various types of genotoxic damage. A general view is emerging that there are dozens of genes that modulate response to environmental exposures. The analysis of polymorphism in these genes may be helpful in understanding the distribution of exposure risk in human populations, in estimating risks due to exposure in susceptible subpopulations and, possibly, in predicting individual risks due to exposure. This work briey reviews polymorphisms in environmentally relevant genes and discusses the direction of future work in this area.

synonymous amino acid substitutions arising from single nucleotide polymorphisms (SNPs). Here synonymous substitution is dened as replacement of one amino acid by a different, but similar, amino acid. Because similar amino acids tend to be represented by related codons, this means that random synonymous base changes tend to produce minimal mutational effects on a protein (Lewin, 1994). For example, AGT codes for serine, while ACT codes for the structurally similar amino acid threonine. Synonymous SNPs are unlikely to change protein activity, whereas non-synonymous SNPs (substitution with an amino acid having substantially different chemical properties) may have detrimental effects. Changes in protein function may have profound effects on phenotype or have more subtle effects that are observed only under specic conditions, such as exposure or stress.

3. Drug response 2. Polymorphism Polymorphisms are sequence variations such as nucleotide substitutions, deletions/insertions, and gene duplications/deletions. By denition, polymorphisms occur at a population frequency of at least 1%, although this arbitrary value is loosely interpreted in practice. These variations may or may not cause alterations in protein function and phenotype. Most polymorphisms are located outside gene boundaries and have no apparent effect on anything. If a polymorphism is within a genes coding region, in an exon, amino acid substitution may occur at that position resulting in changes in protein activity ranging from slight to signicant (Wormhoudt et al., 1999). A polymorphism in the promoter could alter the rate of transcription. A polymorphism located at an intron/exon boundary in a gene may produce incomplete or inactive proteins as a result of incorrect mRNA splicing. Polymorphisms characterized by whole gene deletions, such as the GSTM1 null allele, clearly eliminate any functional enzyme activity, while polymorphisms which are duplications of the entire gene may result in higher levels of activity. There may also be synonymous or nonPolymorphism can affect an individuals susceptibility to infection, response to drug therapy or alcohol consumption, metabolism of xenobiotics, etc. There are many reports of subpopulations experiencing a greater therapeutic effect from standard drug dosages due to their reduced capacity to inactivate the drug molecules (e.g. slow acetylators). Conversely, higher drug dosages may be required to observe the desired therapeutic response for individuals capable of rapidly eliminating drugs (e.g. fast acetylators) (Zacest and Koch-Weser, 1972; Forstrom et al., 1974; Jounela et al., 1975; Ramsay et al., 1984; Wormhoudt et al., 1999). Similarly, individuals may experience side effects depending on their ability to metabolize therapeutic agents. A specic example involves the increased myelosuppression experienced by fast acetylators undergoing amonade therapy (Ratain et al., 1991).

4. Chemical/carcinogen exposure and response We are regularly exposed to a variety of carcinogenic polycyclic aromatic hydrocarbons and

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aromatic amines in tobacco smoke, automotive exhaust, and cooked foods (Felton et al., 1994; Vineis, 1994). It is widely accepted that exposure to these environmental chemicals is associated with elevated risk of cancer (Doll and Peto, 1981). It is also evident that an individuals risk of developing a chemical-induced cancer depends on the chemical dosage and characteristics, an individuals sensitivity to the chemical carcinogen, and other components of lifestyle such as nutrition (Lazarus et al., 1998; Sato et al., 1999). Lung cancer mortality is related to the amount and duration of smoking (Doll and Peto, 1978), yet only a modest proportion of smokers actually suffer from smoking-related cancers. Tobacco smoke contains thousands of toxic organic compounds formed during combustion, including polycyclic aromatic hydrocarbons, aromatic amines and N -nitrosamines (Vineis, 1994). For these compounds to produce carcinogenic effects, they must be distributed to the tissues, be metabolized (either detoxied or activated to DNA reactive species) and the DNA damage must persist to produce mutations despite the efforts of several DNA repair systems (Pelkonen and Nebert, 1982; Kadlubar and Badawi, 1995; Oude Ophuis et al., 1998). However, it is apparent that humans exhibit variability in each of these processes and this affects an individuals susceptibility to DNA damage and risk of disease (Fraumeni, 1975; Guengerich, 1988; Guengerich and Shimada, 1991; Idle, 1991; Kawajiri and Fujii-Kuriyama, 1991; Nebert, 1991; Omenn, 1991; Soucek et al., 1994; Gut et al., 1996; Oude Ophuis et al., 1998; Sato et al., 1999).

adducts which, if not repaired, may eventually produce somatic mutations and cancer. Individuals with genetically determined high phase I activity and low phase II activity would presumably produce higher quantities of reactive intermediates and suffer from higher levels of DNA damage (Rojas et al., 2000). Conversely, individuals with low phase I activity and high phase II may have a lower risk of DNA damage. Kellermann and others observed that individuals with high aryl-hydrocarbon-hydroxylase activity (CYP1A1) were at higher risk for smoking-induced cancers (Kellermann et al., 1980). CYP2E1 also plays an important role in activating many occupationally relevant carcinogens such as vinyl chloride, benzene, acrylonitrile and butadiene (Lieber, 1997). Differences in CYP2E1 inducibility and sequence have been reported, and high CYP2E1 activity has been associated with benzene poisoning (Rothman et al., 1997; Fritsche et al., 2000). Numerous epidemiological studies suggest that polymorphisms in activation and detoxication are associated with increased risk of developing a variety of cancers, including cancers of the lung (Kellermann et al., 1980; Hirvonen et al., 1993; Nakachi et al., 1993; Kihara and Noda, 1994; Deakin et al., 1996; Sato et al., 1999), colon, bladder (Bell et al., 1993a,b; Brockmoller et al., 1996), mouth (Muscat et al., 1996; Park et al., 1997; Sato et al., 1999) and head and neck (Trizna et al., 1995).

5.1. N -Acetyltransferases
N -Acetyltransferases are enzymes that may either activate or inactivate aromatic and heterocyclic amine carcinogens depending on the specic type of acetylation that occurs. N-Acetylation of molecules is typically a detoxifying reaction, while O-acetylation can produce reactive species. Sequence polymorphisms in NAT1 and NAT2 genes may cause variation in an individuals capacity for N-acetylation, thus affecting susceptibility of that individual to environmental toxins (Blum et al., 1991; Deguchi, 1992; Hickman et al., 1992; Bell et al., 1993a,b, 1995a,b). Both high and low NAT1 activity (Weber and Vatsis, 1993) and fast and slow NAT2 acetylator phenotypes have been re-

5. Variation in carcinogen metabolism In general, carcinogens are oxidized to reactive intermediates by phase I enzymes (e.g. CYPs), while phase II enzymes (e.g. glutathione S -transferases, N -acetyltransferases, sulfotransferases) generally mediate the conjugation of water-soluble moieties (such as glutathione) to these reactive metabolites, rendering them harmless (Ernster et al., 1991). Reactive metabolites that are not detoxied may react with DNA to form DNA

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ported. Approximately one-half of those of European ancestry inherit two NAT2 alleles that are characterized by reduced acetylation activity (Bell et al., 1993a,b; Lin, 1996). Polymorphisms in NAT2 (or NAT1) have been associated with an increased risk of bladder and colorectal cancer (Ilett et al., 1987; Wohlleb et al., 1990; Lang et al., 1994; Bell et al., 1995a,b; Wormhoudt et al., 1999). Interestingly, because bladder and colorectal carcinogens reach the target organs via different physiological and metabolic pathways, individuals with slow acetylation genotypes appear to be at higher risk for bladder cancer but at lower risk for colon cancer (Mommsen et al., 1985; Yu et al., 1994; Risch et al., 1995; Wormhoudt et al., 1999) (for a review of NAT2 and bladder cancer, see Marcus et al. (2000)).

5.2. Glutathione S -transferases

The glutathione S -transferases conjugate glutathione to various potentially carcinogenic compounds, which facilitates their elimination from the body (Mannervik and Danielson, 1988; Board et al., 1990; Beckett and Hayes, 1993; Hayes and Pulford, 1995; Oude Ophuis et al., 1998). Five classes of soluble GSTs are known in humans, including alpha (A), mu (M), pi (P), theta (T) (Mannervik et al., 1992; Hayes and Pulford, 1995; Coggan et al., 1998) and zeta (Z) (Board et al., 1997) with various subfamilies for each class (i.e. A1 A4, M1 M5, P1, T1 T2 and Z1) (for a review, see Eaton and Bammler (1999)). Numerous polymorphisms in M1, M3, P1, T1, T2, and Z1 have been identied (Ali-Osman et al., 1997; Watson et al., 1998). Among these are polymorphisms that result in nonfunctional, deleted alleles (e.g. GSTM1 and GSTT1) (Seidegard et al., 1988; Pemble et al., 1994). It is estimated that 45% of whites lack a functional GSTM1 allele while 20% of whites lack a functional GSTT1 allele. One of the consequences of inheriting a nonfunctional GSTM1 allele appears to be an increased risk for cancer, particularly of the bladder and lung (Bell et al., 1993a,b; Brockmoller et al., 1996). There is some evidence that polymorphisms in activating and detoxifying enzymes may interact to modulate the level of reactive species that form

DNA adducts, the precursors to somatic mutation. Combinations of CYP and GST genotypes may increase benzo[a ]pyrene diolepoxide (BPDE)DNA adduct formation and thereby increase the risk of tumor formation. Rojas et al. (2000) demonstrated that the combination of CYP1A1 (*2A/*2A or *2B) and GSTM1 (*0/*0) genotypes affects BPDE-DNA adduct levels in human leukocytes. Their studies suggest that individuals with combined CYP1A1 (*1/*2 or *2A/*2A) GSTM1 (*0/*0) genotype may be at a higher risk for developing lung cancer following tobacco smoke exposure. This information provides an indication of the complexity involved in chemical carcinogenesis and the possible roles of multiple activating/detoxicating gene products in this process. Our understanding of how genetic variation affects metabolic enzymes has provided insight into how chemical carcinogens are able to damage DNA. These mechanisms are thoroughly reviewed by Meyer and Zanger (1997). Many studies have established that carcinogen metabolism polymorphisms are important determinants of cancer risk at the population level, and it is likely that new metabolism polymorphisms will be discovered in other genes that modulate response to environmental exposures. Chemical and endogenous DNA adduct formation occurs constantly despite detoxication mechanisms. A complex system of DNA repair is in place in mammals to correct DNA adducts, damaged bases or other mutagenic lesions before DNA replication sets the change permanently. As illustrated in Fig. 1, it is the interplay of all of these protective factors that determines the degree to which damage is translated into mutation and disease. Recently a large effort has gone into characterizing variation in DNA repair genes.

6. DNA repair variation If DNA repair systems can correct carcinogeninduced or endogenous DNA damage that escape detoxication systems, then the consequences of high risk metabolic genotypes may be less signicant. DNA repair enzymes maintain the integrity

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of the genetic code by minimizing replication errors caused by damaged or rearranged DNA templates and by removing damaged DNA segments. DNA damage may be a consequence of normal cellular function (e.g. replication errors, oxidative metabolism) or of environmental factors such as radiation or xenobiotic chemicals (Mohrenweiser and Jones, 1998). Thus, DNA repair genes have a critical role in protecting the genome from mutations (Bohr, 1995; Chu and Mayne, 1996; Mohrenweiser and Jones, 1998). Because DNA repair is a multi-path, multi-step, multi-enzyme process, and routine functional assays are not available, understanding how polymorphism in repair genes affects repair function and disease is a difcult technical problem. A large number of these enzymes depend on direct protein protein interaction to form large repair complexes. Therefore, the delity of these systems can potentially be affected by polymorphisms that

alter either the enzymatic activity or the ability of a component protein to bind a necessary protein partner. It is known that individuals lacking in various elements of DNA repair are predisposed to tumors (Bohr, 1995; Ma et al., 1995; Tlsty et al., 1995), and changes in DNA repair activity may also affect a tumors sensitivity to alkylating agents or cisplatin (Zamble and Lippard, 1995; Chaney and Sancar, 1996; Damia et al., 1998). DNA repair may take place by one of several pathways, depending on the type of damage, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair, or recombinatorial repair. Base excision repair enzymes remove the damaged region of DNA and then ll in the missing bases using the opposite strand as the template. BER involves multiple enzymes that work in a stepwise fashion. In brief, when nonbulky DNA base adducts are formed (often oxidized bases), DNA glycosylases catalyze their removal, leaving an abasic site. This abasic site is then modied by apurinic/apyrimidinic endonucleases which incise the DNA 3% and 5% to the abasic site. The resulting gap is then lled by DNA polymerase and the remaining nicked DNA is sealed by DNA ligase. For a more thorough review, see Wilson (1998). Nucleotide excision repair enzymes are responsible for removing bulky DNA damage that distorts the DNA helix such as UV photoproducts (thymine dimers) and bulky chemical adducts (e.g. benzo[a ]pyrene diolepoxide-guanine adducts). Similar to BER, NER has four steps involving damage recognition, incision, gap-lling and ligation. It is more complex than BER in that the excinuclease that drives the process is a complex of at least 16 proteins (Sancar, 1996). Defects in NER are associated with the inherited condition xeroderma pigmentosum, which is characterized by photosensitivity and a predisposition to cancer and neurological degeneration (Scriver, 1989). Mismatch repair enzymes repair base mismatches and small loop-outs arising from misincorporation or slippage along the template that occur during replication. Recombinational repair involves enzymes that correct double strand breaks and interstrand cross-links and operates primarily via nonhomologous recombination in mammals (Mohrenweiser and Jones, 1998).

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Multiple polymorphisms have been identied during the resequencing of the exons and adjacent intronic regions of several base and nucleotide excision repair genes in healthy individuals (Broughton et al., 1996; Shen et al., 1998; Fan et al., 1999; Mohrenweiser, unpublished data). It is apparent that the coding regions of many DNA repair genes can withstand some variability without lethal effects although it is not clear how polymorphic changes in the amino acid sequences of these proteins affect their participation in DNA repair. Of interest, four of seven amino acid substitutions reported for APEX, an endonuclease responsible for initiating repair of apurinic/apyrimidinic sites, (also known as Ref1, Hap1, or APE1) are associated with reduced function (Hadi et al., 2000). The impact of these variants on cellular responses, such as G2 delay, is still unknown. To date, however, defects in APEX have been associated only with amyotrophic lateral sclerosis, (Kisby et al., 1997) and its role in this process is unclear (Olkowski, 1998). Similarly, XPF polymorphisms, such as those indicated that result in the amino acid changes shown in Table 1, can be considered only as theoretically capable of affecting cancer susceptibility based on biochemical studies (Fan et al., 1999). It is clear that specic mutations in XPD are associated with xeroderma pigmentosum, trichoth-

iodystrophy, and xeroderma pigmentosum with Cockayne syndrome due to a defect in NER (Lehmann, 1995; Evans et al., 1997; Wood, 1997; Coin et al., 1998). Polymorphic variants of this gene have been associated with differences in DNA repair capacity in vitro but to date have not been linked to any specic disease susceptibility (Lunn et al., 2000). A deciency in XRCC1, an enzyme involved in BER, has been associated with increased sensitivity of EM9 cells to camptothecin, suggesting its importance in DNA repair (Barrows et al., 1998). Furthermore, a polymorphism in XRCC1 (Arg399Gln) is associated with higher levels of somatic mutations in the glycophorin A locus among smokers. This may indicate that this polymorphism reduces DNA repair function in some way, possibly by affecting PARP binding and BRCT domains of the XRCC1 gene (Lunn et al., 1999). With regard to disease risk, other polymorphisms in XRCC1 have been linked to squamous cell carcinoma of the head and neck, with a more dramatic affect occurring in smokers and drinkers (Sturgis et al., 1999) although a recent report describes a study with the opposite effect for XRCC1 in head and neck cancer (Watson et al., 2000). Less is known about the effects of SNPs on other genes involved in DNA repair. Although these initial studies are consistent with an impact

Table 1 Occurrence of single nucleotide polymorphisms in base and nucleotide excision repair enzymes Gene Base excision repair a APEX FEN1 LIG1 LIG3 PCNA POLB XRCC1 No. variants (total) Amino acid changes Chromosomes analyzed

13 0 60 34 5 7 45

4 0 10 3 0 2 8 0 1 1 5 2

256 144 184 184 144 72 256 72 72 72 72 72

Nucleotide excision repair ERCC1 10 RAD23A 8 XPA 4 XPD 22 XPF 16

a

Data obtained from the NIEHS-DIR Environmental Genome Project/LLNL project, DOE /LLNL database (Shen et al., 1998; Mohrenweiser, unpublished data).

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of common variants impacting on protein function, additional biochemical, population and clinical studies will be needed to determine the role of variation in DNA repair genes as a risk factor in human disease.

7. Environmental genomics It is the goal of environmental genomics to understand how genetic variability inuences individual responses to environmental effects, based on the assumption that high risk genotypes accumulate more damage and therefore are at greater risk of developing exposure-related diseases. To accomplish this goal, it is important to discover novel polymorphisms in environmentally relevant genes and to evaluate the functional impact and disease risks associated with these polymorphisms. Deciding how to proceed with such projects presents many difcult issues. At the outset one must make decisions about: What candidate genes should be selected for in-depth variation analysis? What populations should be studied? Should the focus be on disease phenotypes or healthy individuals? Should the process be phenotype- or genotype-directed? Following the discovery phase, more difcult issues arise involving how to evaluate the functional relationship between genotype and phenotype at both the biochemical and whole organism level. Determining how genetic variation impacts on complex multigene interactions involving numerous parallel and overlapping biological pathways will present an enormous scientic challenge. The application of new information to risk assessment and disease prevention efforts will be technically difcult but, perhaps, is even more challenging from an ethical, legal and social point of view. In the past, discovery of genetic variation has been phenotype-directed. That is, individuals with adverse drug response or clinical disease phenotypes were identied rst and various genetic mapping approaches, including segregation analysis and positional cloning, were used to identify the gene and the variations producing the phenotype. This laborious and time consuming approach has been highly effective for identifying genes that cause rather dramatic phenotypes, such as cystic

brosis. With the advent of modern genomic technologies we now have the ability to characterize sequence variation in a large number of candidate genes rst and then analyze the association of these polymorphisms with genetically complex diseases. The incorporation of highly parallel, high throughput analysis allows scaling efciencies to be applied to these difcult problems. In their attempt to identify SNPs in candidate genes for essential hypertension, Halushka et al. (1999) used high-density variation detection arrays to study 75 genes encoding proteins known or suspected to be involved in blood pressure homeostasis. By this method they were able to screen samples for SNPs in all 75 genes simultaneously across a population with diverse ethnicity. This project generated a database describing variation in the normal population and also uncovered a large number of new candidate disease polymorphisms. This type of high-throughput method is facilitating rapid discovery of SNPs involved in genetically complex diseases and moving the eld forward. SNP discovery projects include government supported projects at the National Institute of Environmental Health Sciences (NIEHS), National Institute of General Medical Sciences (NIGMS), National Human Genome Research Institute (NHGRI), National Heart Lung and Blood Institute (NHLBI), Department of Energy Lawrence Livermore National Laboratory (DOE LLNL), and pharmaceutical company projects such as the SNPs Consortium (TSC), Celera Genomics and many others. By all estimates, these projects will generate millions of SNPs and a very high density genetic map in the next few years. To deal with the ever-increasing numbers of polymorphisms produced by these projects, various databases are, or will soon be, available to researchers. These include databases supported by the NIEHS Environmental Genome Project and University of Utah (http:// www.niehs.nih.gov/envgenom/; http://www.genome.utab.edu/genesnps), NIEHS-DIR/Lawrence Livermore National Laboratory project (web address not available), the National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov/SNP), The SNP Consortium (http://snp.cshl. org/), and a European Consortium (http://hgbase.cgr.ki.se/). Currently these databases are not

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integrated, containing divergent information from a wide variety of different population sources, and they are missing data on many important known functional polymorphisms. In addition, information about projects and annotation of the data are currently sparse but it is hoped that these situations will improve very soon. The NIEHS/Utah GeneSNPs website is linked to NCBIs dbSNP database and resequencing projects funded by NIEHS (University of Utah, University of Washington) are depositing their data in dbSNPs. The SNPs Consortium has recently deposited 300 000 SNPs in the dbSNP database. The other databases contain varying amounts of data from a wide variety of sources. For example, for LIG1, 60 variants have been identied in resequencing the exons and adjacent intronic regions of 184 chromosomes (Table 1, DOE/LLNL, Mohrenweiser, unpublished data), while at the time of this writing (October, 2000), NCBIs dbSNPs and the European consortiums HGBASE databases contain 77 (90 chromosomes sampled) and three vari-ants (no information on chromosomes) for this gene, respectively. For XPD, DOE/LLNL reports 22 variants identied from resequencing 72 chromosomes, while HGBASE reports 17 variants and dbSNPs reports no variants. The differences are due to the number of chromosomes sampled, the ethnic background of samples, the method for ascertaining variants and whether the project focused on resequencing coding region only, anking and introns as well, or random genomic regions. The selection of regions for resequencing usually reects the goal of a given project, that is, some focus on functionally signicant variation or development of high density genome wide genetic maps. Presumably databases will eventually provide annotation on these points.

8. Variation in environmental response genes Natural selection provides a means for life to adapt to the environment (e.g. climate, food, predation) and therefore to survive. The very large

number of metabolism genes exhibiting functional polymorphism suggests that the presence of this degree of variation was advantageous to human populations in the past. While xenobiotic metabolizing enzymes convert many toxic components in food (especially plants) to nontoxic metabolites, it is evident that some nontoxic chemicals are rendered toxic by the same enzymes. The high frequency of nonfunctional alleles, and also highly inducible (or ultrahigh activity) alleles among the xenobiotic metabolizing enzymes suggests that selection pressure has maintained these polymorphisms in the human population. That is, at some point in time there was a survival advantage in having blocked metabolism, while at some other time or place it was advantageous to have ultrahigh metabolism. Presumably this has led to the situation where both types of alleles are relatively common in human populations. Finally, we suggest that variation in genes involved in detection and repair of DNA damage will result in measurable impact on repair phenotype and subsequently the prevalence of cells carrying somatic mutations. DNA repair deciencies are known to produce both neurological disease and higher rates of cancer, yet it is very difcult to predict the effects of newly discovered variants. Early reports suggesting links between common polymorphisms and cancer are intriguing but are far from denitive. Analysis of disease-causing mutations in the XPD gene indicates there is a broad spectrum of phenotypic effects that depend upon the location of the mutation in the protein. As we begin to analyze the role of genetic variation in complex biological processes and complex disease phenotypes it is likely to be difcult to disentangle the relationship between genotype and phenotype. However, the enormous commitment of resources combined with the rapid rate of technology development, and easy access to the exponentially increasing amount of sequence data, provides the opportunity for truly dramatic future discoveries. In addition, as our knowledge of inherited susceptibility to environmentally caused disease grows and matures, it is our hope that we can develop prevention strategies which will serve to protect susceptible populations.

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Acknowledgements Resequencing of DNA repair genes was carried out under the auspices of the US DOE at Lawrence Livermore National Laboratory; contract No. W-7405-ENG-48 and an Interagency Agreement Y1-ES-8054-05 from NIEHS.

References
Ali-Osman, F., Akande, O., Antoun, G., Mao, J.X., Buolamwini, J., 1997. Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S -transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins. J. Biol. Chem. 272, 1000410012. Barrows, L.R., Holden, J.A., Anderson, M., DArpa, P., 1998. The CHO XRCC1 mutant, EM9, decient in DNA ligase III activity, exhibits hypersensitivity to camptothecin independent of DNA replication. Mutat. Res. 408, 103110. Beckett, G.J., Hayes, J.D., 1993. Glutathione S -transferases: biomedical applications. Adv. Clin. Chem. 30, 281380. Bell, D.A., Taylor, J.A., Butler, M.A., Stephens, E.A., Wiest, J., Brubaker, L.H., Kadlubar, F.F., Lucier, G.W., 1993a. Genotype/phenotype discordance for human arylamine Nacetyltransferase (NAT2) reveals a new slow-acetylator allele common in African-Americans. Carcinogenesis 14, 1689 1692. Bell, D.A., Taylor, J.A., Paulson, D.F., Robertson, C.N., Mohler, J.L., Lucier, G.W., 1993b. Genetic risk and carcinogen exposure: a common inherited defect of the carcinogenmetabolism gene glutathione S -transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J. Natl. Cancer Inst. 85, 11591164. Bell, D.A., Badawi, A.F., Lang, N.P., Ilett, K.F., Kadlubar, F.F., Hirvonen, A., 1995a. Polymorphism in the N-acetyltransferase 1 (NAT1) polyadenylation signal: association of NAT1*10 allele with higher N-acetylation activity in bladder and colon tissue. Cancer Res. 55, 52265229. Bell, D.A., Stephens, E.A., Castranio, T., Umbach, D.M., Watson, M., Deakin, M., Elder, J., Hendrickse, C., Duncan, H., Strange, R.C., 1995b. Polyadenylation polymorphism in the acetyltransferase 1 gene (NAT1) increases risk of colorectal cancer. Cancer Res. 55, 35373542. Blum, M., Demierre, A., Grant, D.M., Heim, M., Meyer, U.A., 1991. Molecular mechanism of slow acetylation of drugs and carcinogens in humans. Proc. Natl. Acad. Sci. USA 88, 5237 5241. Board, P., Coggan, M., Johnston, P., Ross, V., Suzuki, T., Webb, G., 1990. Genetic heterogeneity of the human glutathione transferases: a complex of gene families. Pharmacol. Ther. 48, 357 369. Board, P.G., Baker, R.T., Chelvanayagam, G., Jermiin, L.S., 1997. Zeta, a novel class of glutathione transferases in a

range of species from plants to humans. Biochem. J. 328, 929 935. Bohr, V.A., 1995. DNA repair ne structure and its relations to genomic instability. Carcinogenesis 16, 2885 2892. Brockmoller, J., Cascorbi, I., Kerb, R., Roots, I., 1996. Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 2, glutathione S -transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res. 56, 3915 3925. Broughton, B.C., Steingrimsdottir, H., Lehmann, A.R., 1996. Five polymorphisms in the coding sequence of the xeroderma pigmentosum group D gene. Mutat. Res. 362, 209 211. Chaney, S.G., Sancar, A., 1996. DNA repair: enzymatic mechanisms and relevance to drug response. J. Natl. Cancer Inst. 88, 1346 1360. Chu, G., Mayne, L., 1996. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy: do the genes explain the diseases? Trends Genet. 12, 187 192. Coggan, M., Whitbread, L., Whittington, A., Board, P., 1998. Structure and organization of the human theta-class glutathione S -transferase and D-dopachrome tautomerase gene complex. Biochem. J. 334, 617 623. Coin, F., Marinoni, J.C., Rodolfo, C., Fribourg, S., Pedrini, A.M., Egly, J.M., 1998. Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH. Nat. Genet. 20, 184 188. Damia, G., Guidi, G., DIncalci, M., 1998. Expression of genes involved in nucleotide excision repair and sensitivity to cisplatin and melphalan in human cancer cell lines. Eur. J. Cancer 34, 1783 1788. Deakin, M., Elder, J., Hendrickse, C., Peckham, D., Baldwin, D., Pantin, C., Wild, N., Leopard, P., Bell, D.A., Jones, P., Duncan, H., Brannigan, K., Alldersea, J., Fryer, A.A., Strange, R.C., 1996. Glutathione S -transferase GSTT1 genotypes and susceptibility to cancer: studies of interactions with GSTM1 in lung, oral, gastric and colorectal cancers. Carcinogenesis 17, 881 884. Deguchi, T., 1992. Sequences and expression of alleles of polymorphic arylamine N-acetyltransferase of human liver. J. Biol. Chem. 267, 18140 18147. Doll, R., Peto, R., 1978. Cigarette smoking and bronchial carcinoma: dose and time relationships among regular smokers and lifelong non-smokers. J. Epidemiol. Commun. Health 32, 303 313. Doll, R., Peto, R., 1981. The Causes of Cancer: Quantitative Estimates of Avoidable Risks of Cancer in the United States Today. Oxford University Press, Oxford. Eaton, D.L., Bammler, T.K., 1999. Concise review of the glutathione S -transferases and their signicance to toxicology. Toxicol. Sci. 49, 156 164. Ernster, L., Esumi, H., Fujii, Y., Gelboin, H.V., Kato, R., Sugimura, T., 1991. Xenobiotics and cancer: implications for chemical carcinogenesis and cancer chemotherapy. In: Proceedings of the 21st International Symposium of the Princess Takamatsu Cancer Research Fund, Tokyo, 1990. Jpn. Sci. Soc. Press, Tokyo.

278

M.C. Miller, III et al. / Toxicology Letters 120 (2001) 269280 Jounela, A.J., Pasanen, M., Mattila, M.J., 1975. Acetylator phenotype and the antihypertensive response to hydralazine. Acta Med. Scand. 197, 303 306. Kadlubar, F.F., Badawi, A.F., 1995. Genetic susceptibility and carcinogen-DNA adduct formation in human urinary bladder carcinogenesis. Toxicol. Lett. 82-83, 627 632. Kawajiri, K., Fujii-Kuriyama, Y., 1991. P450 and human cancer. Jpn. J. Cancer Res. 82, 1325 1335. Kellermann, G., Jett, J.R., Luyten-Kellermann, M., Moses, H.L., Fontana, R.S., 1980. Variation of microsomal mixed function oxidase(s) and human lung cancer. Cancer 45, 1438 1442. Kihara, M., Noda, K., 1994. Lung cancer risk of GSTM1 null genotype is dependent on the extent of tobacco smoke exposure. Carcinogenesis 15, 415 418. Kisby, G.E., Milne, J., Sweatt, C., 1997. Evidence of reduced DNA repair in amyotrophic lateral sclerosis brain tissue. Neuroreport 8, 1337 1340. Lang, N.P., Butler, M.A., Massengill, J., Lawson, M., Stotts, R.C., Hauer-Jensen, M., Kadlubar, F.F., 1994. Rapid metabolic phenotypes for acetyltransferase and cytochrome P4501A2 and putative exposure to food-borne heterocyclic amines increase the risk for colorectal cancer or polyps. Cancer Epidemiol. Biomarkers Prev. 3, 675 682. Lazarus, P., Sheikh, S.N., Ren, Q., Schantz, S.P., Stern, J.C., Richie, J.P., Jr, Park, J.Y., 1998. p53, but not p16 mutations in oral squamous cell carcinomas are associated with specic CYP1A1 and GSTM1 polymorphic genotypes and patient tobacco use. Carcinogenesis 19, 509 514. Lehmann, A.R., 1995. Nucleotide excision repair and the link with transcription. Trends Biochem. Sci. 20, 402 405. Lewin, B., 1994. Genes V. Oxford University Press, Oxford. Lieber, C.S., 1997. Cytochrome P-4502E1: its physiological and pathological role. Physiol. Rev. 77, 517 544. Lin, H.J., 1996. Smokers and breast cancer. Chemical individuality and cancer predisposition. J. Am. Med. Assoc. 276, 1511 1512. Lunn, R.M., Langlois, R.G., Hsieh, L.L., Thompson, C.L., Bell, D.A., 1999. XRCC1 polymorphisms: effects on aatoxin B1-DNA adducts and glycophorin A variant frequency. Cancer Res. 59, 2557 2561. Lunn, R.M., Helzlsouer, K.J., Parshad, R., Umbach, D.M., Harris, E.L., Sanford, K.K., Bell, D.A., 2000. XPD polymorphisms: effects on DNA repair prociency. Carcinogenesis 21, 551 555. Ma, L., Hoeijmakers, J.H., van der Eb, A.J., 1995. Mammalian nucleotide excision repair. Biochim. Biophys. Acta 1242, 137 163. Mannervik, B., Danielson, U.H., 1988. Glutathione transferases structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283 337. Mannervik, B., Awasthi, Y.C., Board, P.G., Hayes, J.D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M., Pearson, W.R., et al., 1992. Nomenclature for human glutathione transferases. Biochem. J. 282, 305 306. Marcus, P.M., Vineis, P., Rothman, N., 2000. NAT2 slow acetylation and bladder cancer risk: a meta-analysis of 22 case-control studies conducted in the general population. Pharmacogenetics 10, 115 122.

Evans, E., Moggs, J.G., Hwang, J.R., Egly, J.M., Wood, R.D., 1997. Mechanism of open complex and dual incision formation by human nucleotide excision repair factors. EMBO J. 16, 6559 6573. Fan, F., Liu, C., Tavare, S., Arnheim, N., 1999. Polymorphisms in the human DNA repair gene XPF. Mutat. Res. 406, 115 120. Felton, J.S., Knize, M.G., Dolbeare, F.A., Wu, R., 1994. Mutagenic activity of heterocyclic amines in cooked foods. Environ. Health Perspect. 102 (Suppl. 6), 201204. Forstrom, L., Mattila, M.J., Mustakallio, K.K., 1974. Acetylator phenotype, minimal maintenance dose and haemolytic effect of dapsone in dermatitis herpetiformis. Ann. Clin. Res. 6, 308 310. Fraumeni, J.F., Jr, 1975. Respiratory carcinogenesis: an epidemiologic appraisal. J. Natl. Cancer Inst. 55, 10391046. Fritsche, E., Pittman, G.S., Bell, D.A., 2000. Localization, sequence analysis, and ethnic distribution of a 96-bp insertion in the promoter of the human CYP2E1 gene. Mutat. Res. 432, 1 5. Guengerich, F.P., 1988. Roles of cytochrome P-450 enzymes in chemical carcinogenesis and cancer chemotherapy. Cancer Res. 48, 2946 2954. Guengerich, F.P., Shimada, T., 1991. Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem. Res. Toxicol. 4, 391407. Gut, I., Nedelcheva, V., Soucek, P., Stopka, P., Tichavska, B., 1996. Cytochromes P450 in benzene metabolism and involvement of their metabolites and reactive oxygen species in toxicity. Environ. Health Perspect. 104 (Suppl. 6), 1211 1218. Hadi, M.Z., Coleman, M.A., Fidelis, K., Mohrenweiser, H.W., Wilson, I.D., 2000. Functional characterization of ape1 variants identied in the human population. Nucleic Acids Res. 28, 3871 3879. Halushka, M.K., Fan, J.B., Bentley, K., Hsie, L., Shen, N., Weder, A., Cooper, R., Lipshutz, R., Chakravarti, A., 1999. Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat. Genet. 22, 239 247. Hayes, J.D., Pulford, D.J., 1995. The glutathione S -transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30, 445600. Hickman, D., Risch, A., Camilleri, J.P., Sim, E., 1992. Genotyping human polymorphic arylamine N-acetyltransferase: identication of new slow allotypic variants. Pharmacogenetics 2, 217 226. Hirvonen, A., Husgafvel-Pursiainen, K., Anttila, S., Vainio, H., 1993. The GSTM1 null genotype as a potential risk modier for squamous cell carcinoma of the lung. Carcinogenesis 14, 1479 1481. Idle, J.R., 1991. Is environmental carcinogenesis modulated by host polymorphism? Mutat. Res. 247, 259266. Ilett, K.F., David, B.M., Detchon, P., Castleden, W.M., Kwa, R., 1987. Acetylation phenotype in colorectal carcinoma. Cancer Res. 47, 14661469.

M.C. Miller, III et al. / Toxicology Letters 120 (2001) 269280 Meyer, U.A., Zanger, U.M., 1997. Molecular mechanisms of genetic polymorphisms of drug metabolism. Annu. Rev. Pharmacol. Toxicol. 37, 269296. Mohrenweiser, H.W., Jones, I.M., 1998. Variation in DNA repair is a factor in cancer susceptibility: a paradigm for the promises and perils of individual and population risk estimation? Mutat. Res. 400, 1524. Mommsen, S., Barfod, N.M., Aagaard, J., 1985. N-Acetyltransferase phenotypes in the urinary bladder carcinogenesis of a low-risk population. Carcinogenesis 6, 199201. Muscat, J.E., Richie, J.P., Jr, Thompson, S., Wynder, E.L., 1996. Gender differences in smoking and risk for oral cancer. Cancer Res. 56, 51925197. Nakachi, K., Imai, K., Hayashi, S., Kawajiri, K., 1993. Polymorphisms of the CYP1A1 and glutathione S -transferase genes associated with susceptibility to lung cancer in relation to cigarette dose in a Japanese population. Cancer Res. 53, 2994 2999. Nebert, D.W., 1991. Role of genetics and drug metabolism in human cancer risk. Mutat. Res. 247, 267281. Olkowski, Z.L., 1998. Mutant AP endonuclease in patients with amyotrophic lateral sclerosis. Neuroreport 9, 239242. Omenn, G.S., 1991. Future research directions in cancer ecogenetics. Mutat. Res. 247, 283291. Oude Ophuis, M.B., van Lieshout, E.M., Roelofs, H.M., Peters, W.H., Manni, J.J., 1998. Glutathione S -transferase M1 and T1 and cytochrome P4501A1 polymorphisms in relation to the risk for benign and malignant head and neck lesions. Cancer 82, 936 943. Park, J.Y., Muscat, J.E., Ren, Q., Schantz, S.P., Harwick, R.D., Stern, J.C., Pike, V., Richie, J.P., Jr, Lazarus, P., 1997. CYP1A1 and GSTM1 polymorphisms and oral cancer risk. Cancer Epidemiol. Biomarkers Prev. 6, 791797. Pelkonen, O., Nebert, D.W., 1982. Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol. Rev. 34, 189222. Pemble, S., Schroeder, K.R., Spencer, S.R., Meyer, D.J., Hallier, E., Bolt, H.M., Ketterer, B., Taylor, J.B., 1994. Human glutathione S -transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. Biochem. J. 300, 271276. Ramsay, L.E., Silas, J.H., Ollerenshaw, J.D., Tucker, G.T., Phillips, F.C., Freestone, S., 1984. Should the acetylator phenotype be determined when prescribing hydralazine for hypertension? Eur. J. Clin. Pharmacol. 26, 3942. Ratain, M.J., Mick, R., Berezin, F., Janisch, L., Schilsky, R.L., Williams, S.F., Smiddy, J., 1991. Paradoxical relationship between acetylator phenotype and amonade toxicity. Clin. Pharmacol. Ther. 50, 573579. Risch, A., Wallace, D.M., Bathers, S., Sim, E., 1995. Slow N-acetylation genotype is a susceptibility factor in occupational and smoking related bladder cancer. Hum. Mol. Genet. 4, 231 236. Rojas, M., Cascorbi, I., Alexandrov, K., Kriek, E., Auburtin, G., Mayer, L., Kopp-Schneider, A., Roots, I., Bartsch, H., 2000. Modulation of benzo[a ]pyrene diolepoxide-DNA ad-

279

duct levels in human white blood cells by CYP1A1, GSTM1 and GSTT1 polymorphism. Carcinogenesis 21, 35 41. Rothman, N., Smith, M.T., Hayes, R.B., Traver, R.D., Hoener, B., Campleman, S., Li, G.L., Dosemeci, M., Linet, M., Zhang, L., Xi, L., Wacholder, S., Lu, W., Meyer, K.B., Titenko-Holland, N., Stewart, J.T., Yin, S., Ross, D., 1997. Benzene poisoning, a risk factor for hematological malignancy, is associated with the NQO1 609C T mutation and rapid fractional excretion of chlorzoxazone. Cancer Res. 57, 2839 2842. Sancar, A., 1996. DNA excision repair. Annu. Rev. Biochem. 65, 43 81. Sato, M., Sato, T., Izumo, T., Amagasa, T., 1999. Genetic polymorphism of drug-metabolizing enzymes and susceptibility to oral cancer. Carcinogenesis 20, 1927 1931. Scriver, C.R., 1989. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York. Seidegard, J., Vorachek, W.R., Pero, R.W., Pearson, W.R., 1988. Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion. Proc. Natl. Acad. Sci. USA 85, 7293 7297. Shen, M.R., Jones, I.M., Mohrenweiser, H., 1998. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res. 58, 604 608. Soucek, P., Filipcova, B., Gut, I., 1994. Cytochrome P450 destruction and radical scavenging by benzene and its metabolites. Evidence for the key role of quinones. Biochem. Pharmacol. 47, 2233 2242. Sturgis, E.M., Castillo, E.J., Li, L., Zheng, R., Eicher, S.A., Clayman, G.L., Strom, S.S., Spitz, M.R., Wei, Q., 1999. Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck. Carcinogenesis 20, 2125 2129. Tlsty, T.D., Briot, A., Gualberto, A., Hall, I., Hess, S., Hixon, M., Kuppuswamy, D., Romanov, S., Sage, M., White, A., 1995. Genomic instability and cancer. Mutat. Res. 337, 1 7. Trizna, Z., Clayman, G.L., Spitz, M.R., Briggs, K.L., Goepfert, H., 1995. Glutathione S -transferase genotypes as risk factors for head and neck cancer. Am. J. Surg. 170, 499 501. Vineis, P., 1994. Epidemiology of cancer from exposure to arylamines. Environ. Health Perspect. 102 (Suppl. 6), 7 10. Watson, M.A., Stewart, R.K., Smith, G.B., Massey, T.E., Bell, D.A., 1998. Human glutathione S -transferase P1 polymorphisms: relationship to lung tissue enzyme activity and population frequency distribution. Carcinogenesis 19, 275 280. Watson, M.A., Olshan, A.F., Weissler, M.C., Bell, D.A., 2000. XRCC1 polymorphisms (Arg194Trp and Arg399Gln) and risk for head and neck cancer. Proc. Am. Assoc. Cancer Res. Annu. Mtg. 41, 557. Weber, W.W., Vatsis, K.P., 1993. Individual variability in p-aminobenzoic acid N-acetylation by human N-acetyltransferase (NAT1) of peripheral blood. Pharmacogenetics 3, 209 212. Wilson, S.H., 1998. Mammalian base excision repair and DNA polymerase beta. Mutat. Res. 407, 203 215.

280

M.C. Miller, III et al. / Toxicology Letters 120 (2001) 269280 Yu, M.C., Skipper, P.L., Taghizadeh, K., Tannenbaum, S.R., Chan, K.K., Henderson, B.E., Ross, R.K., 1994. Acetylator phenotype, aminobiphenyl-hemoglobin adduct levels, and bladder cancer risk in white, black, and Asian men in Los Angeles, California. J. Natl. Cancer Inst. 86, 712 716. Zacest, R., Koch-Weser, J., 1972. Relation of hydralazine plasma concentration to dosage and hypotensive action. Clin. Pharmacol. Ther. 13, 420 425. Zamble, D.B., Lippard, S.J., 1995. Cisplatin and DNA repair in cancer chemotherapy. Trends Biochem. Sci. 20, 435 439.

Wohlleb, J.C., Hunter, C.F., Blass, B., Kadlubar, F.F., Chu, D.Z., Lang, N.P., 1990. Aromatic amine acetyltransferase as a marker for colorectal cancer: environmental and demographic associations. Int. J. Cancer 46, 2230. Wood, R.D., 1997. Nucleotide excision repair in mammalian cells. J. Biol. Chem. 272, 2346523468. Wormhoudt, L.W., Commandeur, J.N., Vermeulen, N.P., 1999. Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione S -transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity. Crit. Rev. Toxicol. 29, 59124.