DNA AND CELL BIOLOGYVolume 15, Number 4, 1996Mary Ann Liebert, Inc.Pp. 273-280

Human Drug-Metabolizing Enzyme Polymorphisms:Effects on Risk of Toxicity and Cancer

DANIEL W. NEBERT, ROSS A. McKINNON, and ALVARO PUGA

ABSTRACT

A growing number of human genetic polymorphisms in drug-metabolizing enzymes (DMEs) are being char-acterized. Some of these have been shown, quite convincingly, to be correlated with risk of toxicity or cancer,whereas others presently remain equivocal. There is good evidence that the correlation is stronger in popu-lations exposed to a variety of environmental procarcinogens; perhaps 30% of DME substrates are able to bemetabolically potentiated. Phase I DMEs, most of which represent cytochromes P450, metabolically activateprocarcinogens to genotoxic electrophilic intermediates, and Phase II DMEs conjugate the intermediates towater-soluble derivatives, completing the detoxification cycle. It follows that genetic differences in the regu-lation, expression and activity of genes coding for Phase I and Phase II DMEs would be crucial factors indefining cancer susceptibility and the toxic or carcinogenic power of environmental chemicals. Not all PhaseI and Phase II DMEs are implicated in detoxification; previous work from this and from other laboratorieshas identified candidate Phase I and Phase II genes in which certain alíeles are more likely to be associatedwith cancer susceptibility. In some cases, the allelic frequencies vary dramatically between ethnic groups. Inthis review, our current knowledge about polymorphisms in the following genes are updated: the aromatichydrocarbon receptor (AHR), the CYP1A1 structural gene (which encodes aryl hydrocarbon hydroxylase ac-

tivity), the CYP1A2 structural gene (arylamine oxidations), the CYP2C19 gene (5-mephenytoin 4-hydroxy-lase), the CYP2D6 gene (debrisoquine hydroxylase), the CYP2E1 gene (A/^V-dimethylnitrosamine TV-demeth-ylase), the null mutant for the GSTM1 gene (glutathione transferase mu), and the NAT2 gene (arylamine N-acetyltransferase). If unequivocal biomarkers of genetic susceptibility to cancer and toxicity can be developedsuccessfully, then identification of individuals at increased risk would be very helpful in the fields of publichealth and preventive medicine.

IINTRODUCTION et ai, 1994; Gonzalez and Idle, 1994). Genotoxic carcinogens

interact with DNA and form mutagenic or clastogenic DNAT has become increasingly clear that individual suscepti- adducts. Humans have multiple enzymatic metabolic pathwaysbility to adverse health effects of an environmental agent de- for activation or detoxification of DNA-reactive chemicals,

pends on the genetic makeup of the person. Differences in hu- Interindividual differences in enzymatic activities in these path-man response to carcinogens and toxicants have been linked to ways have been shown to be associated with substantial (e.g., 10-heritable differences in drug-metabolizing enzyme (DME) me- to >200-fold) increases or decreases in toxicity or cancer risk,tabolism (for review, see Pelkonen and Nebert, 1982; Nebert, in response to the same amount of exposure to an environmen-1991a,b,c). Biochemical studies have shown interindividual tal pollutant. Thus, individuals unable to detoxify adequately thevariation in the ability to metabolize procarcinogenic com- ultimate toxic or carcinogenic agent/metabolite, because of poorpounds; epidemiologic studies have revealed an association be- enzymatic activity, will suffer more cellular and genetic damagetween specific alíeles of drug- and carcinogen-metabolizing (protein and DNA adducts, genomic instability) and will be at a

genes and increased cancer risk (for review, see Idle, 1991 ; Daly greater risk of developing toxicity or cancer.

Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, OH45267-0056.

This study is dedicated to the memory of Professor Ryo Sato, who passed away January 12, 1996.

273

274 NEBERT ET AL.

PHASE I PHASE II THE AH RECEPTOR (AHR) POLYMORPHISM

DRUGS, OXYGENATEDFOODS INTERMEDIATES

CONJUGATEDINNOCUOUSPRODUCTS

I ^OXIDATIVE STRESS,L. _ ) TOXICITY,

J MUTATION,CANCER }-

FIG. 1. Diagram of the relationship between the Phase I andPhase II drug-metabolizing enzymes (DMEs) and cancer, mu-

tation, and toxicity affected by reactive oxygenated metabolites.

Drug and carcinogen metabolism is carried out by Phase I(Functionalization) and Phase II (Conjugation) reactions (Fig.1). Phase I DMEs, >95% in the cytochrome P450 superfam-ily, introduce a functional group, usually one or more hydroxylgroups, into their endogenous and exogenous substrates. Thus,a procarcinogen such as benzo[a]pyrene, found in cigarettesmoke, becomes metabolically activated to the ultimate car-

cinogen, benzo[a]pyrene fra«,y-7,8-dihydrodiol-9,10-epoxide(BPDE). There are, of course, numerous other polycyclic aro-

matic hydrocarbons in cigarette smoke. Phase II DMEs—suchas glutathione transferases, UDP glucuronosyltransferases, andN-acetyltransferases—take the P450-mediated product (or anyother endogenous or exogenous compound already having func-tional groups) and use the functional group for conjugation withsuch moieties as glutathione, glucuronic acid, sulfate, cysteine,or acetate, yielding a very hydrophilic product that can easilybe excreted. Although the combined effect of Phase I and PhaseII DME activities is the detoxification of the drug or environ-mental chemical, metabolic intermediates, such as BPDE, read-ily form protein and DNA adducts and are strong toxicants, mu-

tagens, and carcinogens, by virtue of their highly reactiveelectrophilic groups.

It is worth noting that so-called "detoxified" conjugatedproducts can also be responsible for toxic, mutagenic, or car-

cinogenic intermediates (Fig. 1). Examples include the glu-curonide conjugates of benzo[a]pyrene (Nemoto et ai, 1980),and innumerable cysteine or glutathione conjugates of halo-genated hydrocarbons, hydroquinones, isothiocyanates and ni-trosoguanidines (Monks et ai, 1990; Fritz et ai, 1991).

What is the evidence for an association between human DMEpolymorphisms and interindividual risk of toxicity or cancer?In this review, a brief update of the following DME and re-

ceptor genetic polymorphisms is discussed: the aromatic hy-drocarbon receptor (AHR), the CYP1A1 structural gene (cod-ing for aryl hydrocarbon hydroxylase activity), the CYPIA2gene (arylamine oxidations), the CYP2C19 gene (S-mepheny-toin 4'-hydroxylase), the CYP2D6 gene (debrisoquine hydrox-ylation), the CYP2E1 gene (A/,/V-dimethylnitrosamine N-demethylase), the null mutant for the GSTM1 gene (glutathionetransferase Mu), and the NAT2 gene (arylamine A/-acetyltrans-ferase).

The AHR polymorphism was originally identified from stud-ies of inducible benzo[a]pyrene metabolism among inbredmouse strains (for review, see Nebert, 1989). Aryl hydrocarbonhydroxylase (benzo[a]pyrene 3-hydroxylase; now namedCYP1A1) was found to be highly inducible and controlled bya high-affinity AHR in some strains, but not others. This dif-ference was shown not to be due to differences in the Cyplalstructural gene itself, but rather in a regulatory gene; the lackof inducibility is inherited as an autosomal recessive trait in ge-netic crosses between C57BL/6 and DBA/2 mice. The gene,and the difference in inducibility were ultimately mapped to theregulatory Ahr locus near the proximal end of mouse chromo-some 12 (Cobb et ai, 1987; Poland et ai, 1987). There is con-

vincing evidence, from inbred mouse studies, that allelic dif-ferences at the Ahr locus, which encodes the AHR, can resultin striking differences in interindividual susceptibility to can-

cer, mutagenesis, birth defects, and cell type-specific toxicityof the liver, eye, ovary, bone marrow, and immune system (forreview, see Nebert, 1989).

In humans, a correlation between genetic differences at theAHR locus and certain types of toxicity and forms of cancer

has been suggested (for review, see Nebert, 1989; Caporaso etai, 1991; Nebert et ai, 1991), but an experimental assessmentof this hypothesis has been hampered by the ethical difficulties

Table 1. Correlation of Human Malignancies with theAHRH Phenotype Among Cigarette Smokers

MalignancyCorrelation

found? ReferencesBronchogenic

carcinoma

Bronchogeniccarcinoma

Laryngealcarcinoma

Cancer of theoral cavity

Cancer of renalpelvis or ureter

Cancer of theurinary bladder

Yes Kellermann et ai (1973)Coombs et al. (1976)Guirgis et ai (1976)Emery et al. (1978)Kärki and Huhti (1978)Körsgaard and Trell (1978)Arnott et ai (1979)Gahmberg et al. (1979)McLemore et ai (1979)Kouri et ai (1982)

Noa Gurtoo et ai (1977)Jett et ai (1977)Lieberman (1978)Paigen et ai (1977)Ward et al. (1978)Paigen et al. (1979)

Yes Trell et al. (1976)

Yes Trell and Körsgaard (1978)Trell et ai (1978a)

No Trell et ai (1977)

No Trell et ai (1978b)Paigen et al. (1979)

aThese six references showing the lack of correlation repre-sent only three laboratories, whereas the 10 references indicat-ing a correlation between AHRH and lung cancer represent eightindependent laboratories.

HUMAN DME POLYMORPHISMS 275

of carrying out definitive experiments in humans. Heavy ex-

posures of human populations to dioxin, halogenated hydro-carbons, or cigarette smoking have led to the manifestations ofmalignancies, birth defects, chloracne, mental problems, earlyonset of menopause, and immunosuppression, but no cause-and-effect relationship between the AHR phenotype and thesetypes of toxicities or cancers has ever been rigorously demon-strated. Using 20-40 cc of blood from venipuncture, numerouslaboratories have studied peripheral white cell cultures in thepresence of mitogens and CYP1A1 inducer to assess the hu-man AHR phenotype and relationship to cancer (Table 1).About one-tenth of Caucasians have been found to exhibit highCYP1AI inducibility. Studies from a number of independentlaboratories have suggested that, among cigarette smokers, thehigh inducibility (AHRH) phenotype is at greater risk (estimatesrun between 3- and 20-fold) than the low inducibility (AHRL)phenotype for bronchogenic carcinoma, laryngeal carcinoma,and cancer of the oral cavity, but not cancer of the kidney,ureter, or urinary bladder. It would seem reasonable that cellsin contact with the cigarette smoke might be more prone to car-

cinogenesis than tissues distant to smoke inhalation. However,the mitogen-activated lymphocyte 3- or 4-day culture assay isnot trivial to carry out; many laboratories have experienced dif-ficulties in reproducibility of this assay, and numerous modifi-cations of the assay have been introduced during the past twodecades. Consequently, the reporting of an absence of associa-tion between the AHRH phenotype and bronchogenic cancer (asnoted in Table 1) is likely to reflect technical difficulties in thislymphocyte culture assay.

The fact that there are conflicting results from several labo-ratories underscores the importance of developing a reliable andsimple noninvasive test, such as a DNA biomarker, for deter-mining the AHR genotype in large populations. Such screeningof human populations would resolve these conflicts in the lit-erature.

The AHR is a ligand-activated basic region helix-loop-he-lix transcription factor (for review, see Swanson and Bradfield,1993). The exogenous ligands include dioxin and polycyclicaromatic hydrocarbons. How the ligand binds to the receptor,how the ligand-receptor complex becomes translocated to thenucleus, and how this complex activates transcription of theCYPIA1 gene and of various other genes has been the subjectof intensive research in this and many other laboratories (forreview, see Landers and Bunce, 1991; Whitlock, 1991;Hankinson, 1993; Nebert et ai, 1993; Swanson and Bradfield,1993). The transcriptionally active complex is a heterodimer ofAHR and ARNT, the Ah receptor nuclear translocator. All ge-netic differences in mouse toxicity, cancer, and teratogenesishave been correlated with allelic differences in the Ahr gene,and none with the Cyplal structural gene or the Arm gene(Poland and Knutson, 1982; Nebert, 1989). Because regulatorymechanisms are extremely well conserved during evolution, onewould expect the same to be true for the human AHR poly-morphism.

Mouse Hepa-1 cells, derived from a C57L mouse hepatoma,possess a high-affinity AHR responsible for high CYP1A1 in-ducibility (Nebert, 1989). AHR cDNA clones have been se-

quenced and shown to encode an 805-amino-acid protein of90,380 kD (Burbach et ai, 1992; Erna et ai, 1992). This lab-oratory has cloned and sequenced cDNAs for the C57BL/6

(Ahrbl alíele) and DBA/2 (Ahr0 alíele) mouse AHR mRNAsand shown that there are five coding polymorphisms betweenthe two alíeles (Chang et ai, 1993). Of these, the A375V changeappears to be critical for in vitro ligand affinity and possiblyforCYPlAl inducibility (Emu et ai, 1994; Poland et ai, 1994).

Using [3H]TCDD as a ligand, the human AHR has beendemonstrated in placenta, lung, heart, pancreas, kidney, tonsils,peripheral blood lymphocytes, and several tissue culture celllines. Marked AHR heterogeneity in the human population hasbeen found, with about one-tenth of the population having thehigh-affinity AHR phenotype concomitant with CYP1A1 high-inducibility (Roberts et ai, 1986; Petersen et ai, 1991; Clark etai, 1993). The human AHR cDNA has been cloned (Dolwick etai, 1993; Itoh and Kamataki, 1993; Ema et ai, 1994; Micka etai, in preparation) and localized to the short arm of chromosome7. Ligand-affinity polymorphisms similar to those described forthe murine AHR, have been demonstrated in vitro (Ema et ai,1994). We and others have found Val-381 (equivalent to mouse

Val-375) in the human low-affinity AHR; mutations that convertVal-375 to Ala also convert the low-affinity to a high-affinityAHR phenotype (Fujii-Kuriyama et ai, 1995).

An Ahr(-I-) knockout mouse line has been shown to ex-

hibit bile duct fibrosis and impaired development of the liverand immune system (Fernandez-Salguero et ai, 1995). This lineshould be very helpful in delineating the role of the AHR intoxicity and cancer.

CYP1A1 STRUCTURALGENE POLYMORPHISM

A restriction fragment length polymorphism (RFLP) affect-ing the size of Msp I fragments of the human CYP1A1 gene on

chromosome 15q22-ter was reported by this laboratory (Bale etai, 1987). In Japan, this RFLP appears to be associated with a

higher incidence of lung cancer (Kawajiri et ai, 1990) and withan I462V mutation in the heme-binding region that causes a

50% higher CYP1A1 activity (Hayashi et ai, 1991). Hayashiand colleagues (1992) then found that this RFLP, combinedwith the GSTM1 (glutathione transferase mu) null mutation, isassociated with a nine-fold increased risk of lung cancer amongcigarette smokers. From these studies, it seems plausible thatthe human CYPIA1 structural gene, or a region near this gene,might be correlated with the inducibility phenotype and withincreased risk of lung cancer.

Studies with CYPIAI in other ethnic groups do not, how-ever, support the Japanese findings. Norwegians (Tefré et ai,1991), American Caucasians and blacks (Shields et ai, 1992),Finns (Hirvönen et ai, 1992), and eastern Mediterraneans(Wedlund et ai, 1994) lack the association between the Msp IRFLP, the I462V mutation, and/or lung cancer. Thus, it now

appears that the Msp I RFLP might explain some of the genet-ically predisposed increased risk among Japanese high-in-ducibility individuals for particular types of cigarette smoke-induced cancer, but it does not hold true for other ethnic groups.

It should be noted that there are large ethnic differences inthe allelic frequencies of this polymorphism. The frequency ofthe recessive alíele is 0.31 among Japanese (Kawajiri et ai,1990) and 0.12 among Caucasians (Bale et ai, 1987; Tefré etai, 1991; Hirvönen et ai, 1992; Shields et ai, 1992), indicat-

276 NEBERT ET AL.

ing that the frequency of the recessive phenotype is seven timesmore frequent among Japanese than Caucasians. It is possiblethat, in the Japanese population, the polymorphism is in link-age disequilibrium with another mutation that is important forCYP1A1 inducibility or another gene involved in tumorigene-sis, whereas, in the Caucasian population, these mutations are

in equilibrium.

CYP1A2 (ARYLAMINE HYDROXYLASE)

CYP1A2 metabolizes aromatic amine procarcinogens, in-cluding tobacco smoke-specific nitrosamines, such as 4-(methylnitrosamino)-l-(3-pyridyl-l-butanone (NNK) (Butler er

ai, 1989; Crespi et ai, 1991). This laboratory has cloned thehuman CYPIA2 gene and reported differences in CYP1A2mRNA levels in human liver, suggesting that interindividualdifferences in CYP1A2 expression may play a role in toxicityand cancer (Ikeya et ai, 1989). Although expressed constitu-tively in liver, CYP1A2 has not been detected in human lungor in any other tissues except liver (Shimada et ai, 1992); thus,it is unclear what role CYP1A2 might play in toxicity or can-

cer. Using caffeine as a probe, a trimodal distribution of the en-

zymatic activity (consistent with high/high, high/low, andlow/low genotypes) has been detected in four separate popula-tions (Butler et ai, 1992; Tang et ai, 1994). The genetic basisof this polymorphism has been extensively searched for (Yokoiet ai, 1995), but is not yet understood.

Two Cypla2( —/—) knockout mouse lines showing strikinglydifferent phenotypes have recently been developed (Pineau etai, 1995; Liang et ai, 1996). These lines should be helpful inelucidating the role of CYP1A2 in toxicity and cancer.

CYP2C19 (S-MEPHENYTOIN4-HYDROXYLASE)

In clinical studies, poor metabolizers (PMs), of S-mepheny-toin, omeprazole, diazepam, proguanil and chlorproguanil, pro-pranolol, citalopram, imipramine, amitriptyline, mephobarbital,and hexobarbital appear to reflect a defective CYP2C19 pro-tein (for review, see Goldstein and de Moráis, 1994). The PMphenotype comprises 3-5% of Caucasians, but 18-23% ofAsians. An aberrant splice site in exon 5 accounts for 75-85%of Caucasian and Japanese PMs, and a second defect accountsfor the remaining 25% of PMs in Japanese populations(Goldstein and de Moráis, 1994). Epidemiologie studies link-ing these allelic differences with toxicity and cancer are un-

derway in numerous laboratories.

CYP2D6 (DEBRISOQUINE HYDROXYLASE)

Poor metabolizers (PMs) of the antihypertensive drug de-brisoquine were found to represent 6-10% of Caucasian popu-lations, as compared with extensive metabolizers (EMs) whohandle the drug 10-200 times more efficiently (Idle and Smith,1979). The "debrisoquine panel" now encompasses more than30 drugs and environmental chemicals, including antiarrhyth-mics, antihypertensives, /3-blockers, monoamine oxidase in-

hibitors, morphine derivatives, antipsychotics, and tricyclic an-

tidepressants (Gonzalez and Idle, 1994). Whereas more than 10alíeles attributed to the PM phenotype have been identified, themost common is the B alíele, accounting for more than 70% ofall null alíeles. The D alíele (actually deletion of the entireCYP2D6 gene) represents almost 15% of PMs. The A, E, andFálleles reflect most of the remaining 15% of PMs (for review,see Caparoso et ai, 1991; Nebert, 1991b,c; Gonzalez and Idle,1994). All of the alíeles can be determined by polymerase chainreaction (PCR) genotyping, except the D alíele, which requiresSouthern blot analysis. The gene has been localized to humanchromosome 22ql3.1 ; interestingly, an "ultrarapid metabolizer"phenotype was found to be due to amplification of the CYP2D6gene as much as 12 times, the trait being inherited in certainSwedish families (for review, see Meyer, 1994). A unifiednomenclature system for all alíeles of the human CY2D6 genehas recently been offered (Daly et ai, 1996).

The EM phenotype has been associated with increased riskof liver, gastrointestinal, and lung cancer (for review, see

Caparoso et ai, 1991; Nebert, 1991b,c; Gonzalez and Idle,1994). Innumerable epidemiologic and ethnic studies linkingthe CYP2D6 allelic differences with toxicity and cancer havebeen reported, and no doubt these studies will continue.

CYP2E1 (DIMETHYLNITROSAMINEN-DEMETHYLASE)

CYP2E1 metabolizes nitrosamines, including the procar-cinogens A'-nitrosodimethylamine and A/-nitrosopyrrolidine, as

well as alcohols, benzene, carbon tetrachloride, and the tobaccosmoke product 3-hydroxypyridine (Yang et ai, 1992). The en-

zyme is inducible by starvation and alcohol, and thus, these fac-tors might affect toxicity or carcinogenesis due to alterations inCYP2E1 activity. Although little is known about the regulationof this gene, expression is very high in human liver, where largeinterindividual differences have been observed (Guengerich et

ai, 1991). The enzyme is also expressed in rat lung, rabbit nasalepithelium, and human kidney and lymphocytes (Daly et ai,1994).

The CYP2EI gene is located on human chromosome10q24.3-qter. Two types of polymorphisms have been reportedin this gene, with regard to possible increased risk of cancer.

One of these, detectable by Rsa I digestion, alters the regula-tory region of the gene, affecting transcription levels (Hayashiet ai, 1991) and presumably decreasing metabolism of nitrosocompounds. The other is a RFLP in intron 2, detectable by DraI digestion, associated with a decreased incidence of lung can-

cer in Japanese and Swedish populations (Uematsu et ai, 1991;Persson et ai, 1993). PCR assays have been described for bothtypes of polymorphisms but have yet to be extensively appliedto studies of susceptibility to cancer and toxicity.

GSTM1 (GLUTATHIONE TRANSFERASE MU)

The glutathione-S-transferases are classified into at least fourgenetically distinct classes of enzymes (alpha, theta, mu, andpi) which conjugate xenobiotics with glutathione (Mannerviket ai, 1993). Candidate human GSTM genes have been local-

HUMAN DME POLYMORPHISMS 277

ized to several chromosomes, but, currently, the best availableevidence indicates that the highly homologous GSTMI,GSTM2, and GSTM4 genes map to chromosome 1 (Fryer et ai,1993b; Zhong et ai, 1993). Several polycyclic hydrocarbonepoxides are known substrates for the GSTM enzymes, in-cluding BPDE and other hydrocarbons in cigarette smoke, as

well as carcinogenic arylamines such as aflatoxin B) epoxide(Mannervik and Danielson, 1988). Three different allelic vari-ants have been found; two of them, GSTMI*A and GSTMI*B,encode enzymes having activity similar to that of the wild-typealíele; in contrast, a null alíele, GSTM1*0, produces no enzymeand contains a nearly complete deletion of the gene. In mostracial groups examined, 20-50% of all individuals are ho-mozygous for the null alíele (Asians are higher than Caucasians)resulting in no GSTMI enzyme (Mannervik and Danielson,1988; Fryer et ai, 1993b; Daly et ai, 1994; Nakajima et ai,1995).

Phenotypic studies among lung cancer patients have indi-cated that lack of GSTM is associated with a 2.3-fold increasedrisk of lung adenocarcinoma among heavy smokers but not

among light or non-smokers (Anttila et ai, 1994; Daly et ai,1994). Similar findings have been reported for pituitary ade-momas (Fryer et ai, 1993a) and colorectal cancer (Zhong et

ai, 1993), but not breast or bladder cancer (Zhong et ai, 1993).The role of GSTMI in cancer has become clearer with studiesthat show a nine-fold increase of squamous cell carcinoma ofthe lung, when the patient has the combination of the 1.9-kbMsp I alíele of the CYP1A1 gene and the null GSTM1*0 alíele(Hayashi et ai, 1992). More recently, the same group has foundthat, in a Japanese population stratified by the number of cig-arettes smoked during a lifetime, the odds ratio for the heaviersmokers was 41; in addition, low-level smokers contracted can-

cer sooner if they had the GSTM1*0 null alíele (Nakachi et ai,1993). Further epidemiologic studies, linking these allelic dif-ferences with toxicity and cancer, will undoubtedly continueand expand in numerous laboratories.

NAT! (ARYLAMINE A/-ACETYLTRANSFERASE)

The /V-acetylation polymorphism was originally termed the"isoniazid acetylation polymorphism" because it was recog-nized in tuberculosis patients treated with isoniazid. Individualscan be phenotyped as "slow" or "rapid" acetylators; slow acety-lators are homozygous for the slow acetylator (r) alíele, whereasrapid acetylators are either heterozygous or homozygous for therapid (R) alíele. The slow phenotype is inherited as an autoso-mal recessive trait. Frequency of the r alíele is about 0.72 inthe United States, meaning that one in every two individuals ishomozygous rlr and has the slow acetylator phenotype. Theslow acetylation frequency ranges worldwide from approxi-mately 10% in Japanese populations to more than 90% in some

Mediterranean peoples (Weber and Hein, 1985; Hickman andSim, 1991).

Two human A/-acetyltransferase functional genes (NATI,NAT2) and one pseudogene (NATP) were cloned, characterized,and localized to the same region on chromosome 8pter-qll(Blum et ai, 1990). The rapid and slow acetylator phenotypeinvolves the NAT2 gene, encoding the NAT2 enzyme whichhas a 10-times-lower Km for aromatic amines than NATI. Three

major slow acetylator alíeles (two common in Caucasians, one

common in Asians), each identifiable at a restriction endonu-clease site, have independently been identified in several hu-man populations (Blum et ai, 1990; Hickman and Sim, 1991;Vatsis et ai, 1991). The number of minor, rare NAT2 alíeles isnow greater than 20 (for review, see Vatsis et ai, 1991).

The associations between acetylation phenotypes and cancer

or toxicity has received considerable attention. The slow acetyl-ator phenotype shows a lower incidence of colorectal carcinoma(for review, Nebert, 1991 b; Gonzalez and Idle, 1994) but a higherincidence (odds ratio of 16.7) of bladder cancer (Cartwright,1984; Weber and Hein, 1985). In the latter studies, both occu-

pational exposure to arylamines and cigarette smoking were re-

quired, in conjunction with the slow acetylator phenotype, fordevelopment of bladder cancer, and no relationship was foundbetween acetylator phenotype and smoking-related bladder can-

cer in the absence of exposure to arylamines. These findings havebeen independently confirmed by Karakaya et ai (Karakaya etai, 1989) and many others. Although Hayes and co-workers(1993) surprisingly reported a lack of correlation between theslow acetylator phenotype and bladder cancer among chemicaldye workers, it should be emphasized that the workers in thisparticular study were exposed to much higher levels of chemi-cals than those in the studies of Cartwright and others (Urs A.Meyer, personal communication).

CONCLUSIONS

Many independent studies have now demonstrated the im-portance of phenotypic polymorphisms in foreign chemical me-

tabolism as risk factors in the development of cancer and otherdiseases associated with chemical exposure. In parallel withthese studies, the genetic bases of many of these polymorphismshave been elucidated, and noninvasive genotyping methods thatcan be applied to large populations have been developed. Thestudy of the relationship between genetic polymorphisms, can-

cer susceptibility, toxicity, and environmental exposure is a

new, exciting, and promising area of research. The identifica-tion of genetic factors that, acting independently or in con-

junction with other factors or with environmental exposures, in-crease the risk of toxicity or cancer will have importantimplications for the prevention, early diagnosis, and interven-tion/management of human disease.

ACKNOWLEDGMENTS

This study was supported in part by Public Health Servicegrants R01 AG09235, R01 ES06321, R01 ES06811, and P30ES06096 from the National Institutes of Health, Department ofHealth and Human Services. R.A.M. was an Australian NationalHealth and Medical Research Council C.J. Martin PostdoctoralFellow at the University of Cincinnati.

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Carcinogenesis 14, 1821-1824.

Address reprint requests to;Dr. Daniel W. Nebert

P.O. Box 670056University of Cincinnati Medical Center

Cincinnati, OH 45267-0056

Received for publication November 2, 1995; acceptedNovember 16, 1996.

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18. Cinzia Antognelli, Luigi Mearini, Vincenzo Nicola Talesa, Antonella Giannantoni, Ettore Mearini. 2005. Association ofCYP17, GSTP1, and PON1 polymorphisms with the risk of prostate cancer. The Prostate 63:3, 240-251. [CrossRef]

19. Zhengwen Jiang, Timothy P. Dalton, Li Jin, Bin Wang, Yutaka Tsuneoka, Howard G. Shertzer, Ranjan Deka, DanielW. Nebert. 2005. Toward the evaluation of function in genetic variability: Characterizing human SNP frequenciesand establishing BAC-transgenic mice carrying the human CYP1A1_CYP1A2 locus. Human Mutation 25:2, 196-206.[CrossRef]

20. Renata Canalle, Regislaine V. Burim, Luiz G. Tone, Catarina S. Takahashi. 2004. Genetic polymorphisms and susceptibilityto childhood acute lymphoblastic leukemia. Environmental and Molecular Mutagenesis 43:2, 100-109. [CrossRef]

21. Collet Dandara, Jane Sayi, Collen M. Masimirembwa, Ayoub Magimba, Sylvia Kaaya, Klerk De Sommers, Jacques R.Snyman, Julia A. Hasler. 2002. Genetic Polymorphism of Cytochrome P450 1A1 (CYP1A1) and Glutathione Transferases(M1, T1 and P1) among Africans. Clinical Chemistry and Laboratory Medicine 40:9, 952-957. [CrossRef]

22. Ian Kerridge, Lisa Lincz, Fiona Scorgie, Danica Hickey, Neil Granter, Andrew Spencer. 2002. Association betweenxenobiotic gene polymorphisms and non-Hodgkin's lymphoma risk. British Journal of Haematology 118:2, 477-481.[CrossRef]

23. Evelyne F Lhoste, Lionelle Nugon-Baudon, Sandrine Lory, Jean-Claude Meslin, Claude Andrieux. 2001. Thefermentation of lactulose in rats inoculated withClostridium paraputrificum influences the activities of liver and intestinalxenobiotic-metabolising enzymes. Journal of the Science of Food and Agriculture 81:14, 1397-1404. [CrossRef]

24. Judy Wong, Patricia Harper, Urs Meyer, Karl Bock, Klaus Morike, Jean Lagueux, Pierre Ayotte, Rachel Tyndale, EdwardSellers, David Manchester, Allan Okey. 2001. Pharmacogenetics 11:1, 85-94. [CrossRef]

25. Daniel W. Nebert. 2000. Drug-Metabolizing Enzymes, Polymorphisms and Interindividual Response to EnvironmentalToxicants. Clinical Chemistry and Laboratory Medicine 38:9, 857-861. [CrossRef]

26. H.-C. Schuppe, P. Wieneke, S. Donat, E. Fritsche, F.-M. Kohn, J. Abel. 2000. Xenobiotic metabolism, geneticpolymorphisms and male infertility. Andrologia 32:4-5, 255-262. [CrossRef]

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28. Ann G. Schwartz, Jill M. Siegfried, Linda Weiss. 1999. Familial aggregation of breast cancer with early onset lung cancer.Genetic Epidemiology 17:4, 274-284. [CrossRef]

29. Daniel W Nebert. 1999. Pharmacogenetics and pharmacogenomics: why is this relevant to the clinical geneticist?. ClinicalGenetics 56:4, 247-258. [CrossRef]

30. Glinda S. Cooper, Mary Anne Dooley, Edward L. Treadwell, E. William St. Clair, Christine G. Parks, Gary S. Gilkeson.1998. Hormonal, environmental, and infectious risk factors for developing systemic lupus erythematosus. Arthritis &Rheumatism 41:10, 1714-1724. [CrossRef]

31. Mojgan Kashani, Georg Steiner, Andrea Haitel, Katharina Schaufler, Theresia Thalhammer, Gabriele Amann, GeroKramer, Michael Marberger, Alfred Schöller. 1998. Expression of the aryl hydrocarbon receptor (AhR) and the arylhydrocarbon receptor nuclear translocator (ARNT) in fetal, benign hyperplastic, and malignant prostate. The Prostate 37:2,98-108. [CrossRef]

32. Daniel W. Nebert, Amy L. RoeEcogenetics: The Study of Gene-Environment Interactions . [CrossRef]