GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis.

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case-control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the 'null' genotype) were 1.26 (0.85-1.88) and 1.54 (1.05-2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71-1.51), former (0.95; 0.75-1.20) or current smokers (1.33; 0.91-1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14-2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22-8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

Skin metabolism of aminophenols: human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo.

4-Amino-2-hydroxytolune (AHT) is an aromatic amine ingredient in oxidative hair colouring products. As skin contact occurs during hair dyeing, characterisation of dermal metabolism is important for the safety assessment of this chemical class. We have compared the metabolism of AHT in the human keratinocyte cell line HaCaT with that observed ex-vivo in human skin and in vivo (topical application versus oral (p.o.) and intravenous (i.v.) route). Three major metabolites of AHT were excreted, i.e. N-acetyl-AHT, AHT-sulfate and AHT-glucuronide. When 12.5 mg/kg AHT was applied topically, the relative amounts of each metabolite were altered such that N-acetyl-AHT product was the major metabolite (66% of the dose in comparison with 37% and 32% of the same applied dose after i.v. and p.o. administration, respectively). N-acetylated products were the only metabolites detected in HaCaT cells and ex-vivo whole human skin discs for AHT and p-aminophenol (PAP), an aromatic amine known to undergo N-acetylation in vivo. Since N-acetyltransferase 1 (NAT1) is the responsible enzyme, kinetics of AHT was further compared to the standard NAT1 substrate p-aminobenzoic acid (PABA) in the HaCaT model revealing similar values for K(m) and V(max). In conclusion NAT1 dependent dermal N-acetylation of AHT represents a 'first-pass' metabolism effect in the skin prior to entering the systemic circulation. Since the HaCaT cell model represents a suitable in vitro assay for addressing the qualitative contribution of the skin to the metabolism of topically-applied aromatic amines it may contribute to a reduction in animal testing.

Para-phenylenediamine and allergic sensitization: risk modification by N-acetyltransferase 1 and 2 genotypes.

BACKGROUND: Para-phenylenediamine (PPD) is a common contact sensitizer causing allergic contact dermatitis, a major skin problem. As PPD may need activation to become immunogenic, the balance between activation and/or detoxification processes may influence an individual's susceptibility. PPD is acetylated and the metabolites do not activate dendritic-like cells and T cells of PPD-sensitized individuals. OBJECTIVES: To investigate whether PPD can be acetylated in vitro by the two N-acetyltransferases 1 (NAT1) and 2 (NAT2). Based on the assumption that N-acetylation by NAT1 or NAT2 is a detoxification reaction with respect to sensitization, we examined whether NAT1 and NAT2 genotypes are different between PPD-sensitized individuals and matched controls. METHODS: Genotyping for NAT1 and NAT2 polymorphisms was performed in 147 PPD-sensitized individuals and 200 age- and gender-matched controls. Results Both PPD and monoacetyl-PPD were N-acetylated in vitro by recombinant human NAT1 and to a lesser extent by NAT2. Genotyping for NAT1*3, NAT1*4, NAT1*10, NAT1*11 and NAT1*14 showed that genotypes containing the rapid acetylator NAT1*10 allele were under-represented in PPD-sensitized cases (adjusted odds ratio 0.72, 95% confidence interval 0.45-1.16). For NAT2, NAT2*4, NAT2*5AB, NAT2*5C, NAT2*6A and NAT2*7B alleles were genotyped. Individuals homozygous for the rapid acetylator allele NAT2*4 were under-represented in cases compared with controls (4.3% vs. 9.4%), but this trend was not significant. CONCLUSIONS: With respect to data indicating that NAT1 but not NAT2 is present in human skin, we conclude that NAT1 genotypes containing the rapid acetylator NAT1*10 allele are potentially associated with reduced susceptibility to PPD sensitization.

Role of human CYP1A1 and NAT2 in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mutagenicity and DNA adducts.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in multiple organs and numerous species. Bioactivation of PhIP is initiated by PhIP N(2)-hydroxylation catalysed by cytochrome P450s. Following N-hydroxylation, O-acetylation catalysed by N-acetyltransferase 2 (NAT2) is considered a further possible activation pathway. Genetic polymorphisms in NAT2 may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were used to test the effect of CYP1A1 and NAT2 polymorphism on PhIP genotoxicity. Cells transfected with NAT2*4 had significantly higher levels of N-hydroxy-PhIP O-acetyltransferase (p = 0.0150) activity than cells transfected with NAT2*5B. Following PhIP treatment, CHO cell lines transfected with CYP1A1, CYP1A1/NAT2*4 and CYP1A1/NAT2*5B each showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis not observed in untransfected CHO cells. dG-C8-PhIP was the primary DNA adduct formed and levels were dose dependent in transfected CHO cells in the order: CYP1A1 < CYP1A1 and NAT2*5B < CYP1A1 and NAT2*4, although levels did not differ significantly (p > 0.05) following one-way analysis of variance. These results strongly support activation of PhIP by CYP1A1 with little effect of human NAT2 genetic polymorphism on mutagenesis and DNA damage.

Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1.

Genetic variants of human N-acetyltransferase 1 (NAT1) are associated with cancer and birth defects. N- and O-acetyltransferase catalytic activities, Michaelis-Menten kinetic constants (K(m) and V(max)) and steady-state expression levels of NAT1-specific mRNA and protein were determined for the reference NAT1*4 and variant human NAT1 haplotypes possessing single nucleotide polymorphisms (SNPs) in the open reading frame. Although none of the SNPs caused a significant effect on steady-state levels of NAT1-specific mRNA, C97T(R33stop), C190T(R64W), C559T (R187stop) and A752T(D251V) each reduced NAT1 protein level and/or N- and O-acetyltransferase catalytic activities to levels below detection. G560A(R187Q) substantially reduced NAT1 protein level and catalytic activities and increased substrate K(m). The G445A(V149I), G459A(synonymous) and T640G(S214A) haplotype present in NAT1*11 significantly (P<0.05) increased NAT1 protein level and catalytic activity. Neither T21G(synonymous), T402C(synonymous), A613G(M205V), T777C(synonymous), G781A(E261K) nor A787G(I263V) significantly affected K(m), catalytic activity, mRNA or protein level. These results suggest heterogeneity among slow NAT1 acetylator phenotypes.

N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplotype on urinary bladder cancer risk.

A role for the N-acetyltransferase 2 (NAT2) genetic polymorphism in cancer risk has been the subject of numerous studies. Although comprehensive reviews of the NAT2 acetylation polymorphism have been published elsewhere, the objective of this paper is to briefly highlight some important features of the NAT2 acetylation polymorphism that are not universally accepted to better understand the role of NAT2 polymorphism in carcinogenic risk assessment. NAT2 slow acetylator phenotype(s) infer a consistent and robust increase in urinary bladder cancer risk following exposures to aromatic amine carcinogens. However, identification of specific carcinogens is important as the effect of NAT2 polymorphism on urinary bladder cancer differs dramatically between monoarylamines and diarylamines. Misclassifications of carcinogen exposure and NAT2 genotype/phenotype confound evidence for a real biological effect. Functional understanding of the effects of NAT2 genetic polymorphisms on metabolism and genotoxicity, tissue-specific expression and the elucidation of the molecular mechanisms responsible are critical for the interpretation of previous and future human molecular epidemiology investigations into the role of NAT2 polymorphism on cancer risk. Although associations have been reported for various cancers, this paper focuses on urinary bladder cancer, a cancer in which a role for NAT2 polymorphism was first proposed and for which evidence is accumulating that the effect is biologically significant with important public health implications.

Metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by 16 recombinant human NAT2 allozymes: effects of 7 specific NAT2 nucleic acid substitutions.

Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylation of arylamine carcinogens and the metabolic activation of N-hydroxyarylamine and N-hydroxyarylamide carcinogens by O- and N,O-acetylation, respectively. Rapid and slow acetylator phenotype is regulated at the NAT2 locus, and each has been associated with differential risk to certain cancers relating to carcinogenic arylamine exposures. We examined arylamine N-acetylation, N-hydroxyarylamine O-acetylation, and N-hydroxyarylamide N,O-acetylation catalytic activities of 16 different recombinant human NAT2 alleles expressed in an Escherichia coli JM105 expression system. NAT2 alleles contained nucleic acid substitutions at G191A (Arg64-->Gln), C282T (silent), T341C (Ile114-->Thr), C481T (silent), G590A (Arg197-->Gln), A803G (Lys268-->Arg), G857A (Gly286-->Glu), and various combinations of substitutions in the 870-bp NAT2-coding region. Expression of each NAT2 allele produced equivalent amounts of immunoreactive recombinant NAT2 protein with differential levels of N-, O-, and N,O-acetylation activity. Catalytic activities of each of the recombinant human NAT2 allozymes followed the relative order N-acetylation > O-acetylation > N,O-acetylation. Catalytic activation rates for the metabolic activation of N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl by O-acetylation and N-hydroxy-2-acetylaminofluorene by N,O-acetylation showed very strong correlations to the N-acetylation of 2-aminofluorene. NAT2 alleles with nucleic acid substitution T341C (NAT2*5A,*5B,*5C) expressed recombinant NAT2 allozymes, with the greatest reductions in metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by O- and N,O-acetylation, respectively. NAT2 alleles with nucleic acid substitutions G191A (NAT2*14A,*14B) and G590A (NAT2*6A,*6B) expressed recombinant NAT2 allozymes with more moderate reductions. NAT2 alleles with nucleic acid substitution G857A (NAT2*7A,*7B) expressed recombinant NAT2 allozymes with the smallest but yet significant reductions. NAT2 alleles with nucleic acid substitutions C282T (silent), C481T (silent), and A803G (Lys268-->Arg) expressed recombinant NAT2 allozymes that did not have significant reductions in the metabolic activations of N-hydroxyarylamines and N-hydroxyarylamides. The differential capacity for the metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by recombinant human NAT2 allozymes encoded by polymorphic NAT2 alleles supports the hypothesis that acetylator phenotype may predispose to cancers related to activation of N-hydroxy-arylamine and N-hydroxyarylamide carcinogens.

Metabolic activation of N-hydroxy-2-aminofluorene and N-hydroxy-2-acetylaminofluorene by monomorphic N-acetyltransferase (NAT1) and polymorphic N-acetyltransferase (NAT2) in colon cytosols of Syrian hamsters congenic at the NAT2 locus.

Acetylator genotype is regulated at the polymorphic acetyltransferase (NAT2) gene locus in humans and other mammals such as Syrian hamsters. Human slow acetylator phenotypes have been associated with increased incidences of urinary bladder cancers, whereas rapid acetylators have been associated with increased incidences of colorectal cancers. The genetic predisposition of rapid acetylators to colorectal cancers suggests localized metabolic activation of arylamine carcinogen metabolites by polymorphic N-acetyltransferase (NAT2) in colon tissues. We tested this hypothesis in Bio. 82.73/H Syrian hamster lines which are congenic at the NAT2 gene locus. Congenic Bio. 82.73/H Syrian hamsters expressed acetylator genotype-dependent N-acetyltransferase activity in colon cytosols toward arylamine carcinogens such as 2-aminofluorene and 4-aminobiphenyl. Partial purification of the hamster colon cytosol by anion exchange chromatography identified two N-acetyltransferase isozymes analogous to those previously described in liver and urinary bladder. One of the isozymes (NAT2) exhibited acetylator genotype-dependent expression for the N-acetylation of each arylamine tested: p-aminophenol; 2-aminofluorene; 4-aminobiphenyl; 3,2'-dimethyl-4-aminobiphenyl; and 2-amino-dipyrido[1,2-a:3',2'd]imidazole as well as for the metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to form DNA adducts. Although NAT2 catalyzed the metabolic activation of N-hydroxy-2-acetyl-aminofluorene to DNA adducts, the rates were lower, were paraoxon-sensitive, and did not reflect acetylator genotype. A second isozyme (NAT1) also catalyzed the N-acetylation of each arylamine as well as the metabolic activation of N-hydroxy-2-aminofluorene and N-hydroxy-2-acetylaminofluorene to DNA adducts at rates that were independent of acetylator genotype. Metabolic activation of N-hydroxy-2-aminofluorene catalyzed by both NAT1 and NAT2 was resistant to 100 microM paraoxon, an inhibitor of microsomal deacetylases. Metabolic activation of N-hydroxy-2-acetylaminofluorene by NAT1 and NAT2 was partially sensitive to 100 microM paraoxon. Michaelis-Menten kinetic constants were determined for the colon NAT1 and NAT2 isozymes and compared to previous determinations for liver NAT1 and NAT2. For each of the arylamines tested, both apparent Km and apparent Vmax were higher for NAT2 than NAT1. In rapid acetylator hamster colon, NAT2/NAT1 activity ratios were 18 and 13 for the N-acetylation of 2-aminofluorene and 4-aminobiphenyl and 28 for the O-acetylation of N-hydroxy-2-aminofluorene. These results strongly support the role of the polymorphic NAT2 gene locus in the local metabolic activation of N-hydroxyarylamine carcinogens in colon and provide mechanistic support for human epidemiological studies suggesting a predisposition of rapid acetylators to colorectal cancer.

Acetylator genotype (NAT2)-dependent formation of aberrant crypts in congenic Syrian hamsters administered 3,2'-dimethyl-4-aminobiphenyl.

Some but not all human epidemiological studies suggest a higher incidence of colon cancer in rapid acetylator individuals. Aberrant crypts, the earliest morphologically evident preneoplastic lesions in chemical colon carcinogenesis, were measured in rapid and slow acetylator congenic Syrian hamsters administered 3,2' -dimethyl-4-aminobiphenyl, an aromatic amine colon carcinogen, to investigate the specific role of the acetylator genotype (NAT2) in colon carcinogenesis. Age-matched rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/ H-Pat(s) acetylator female Syrian hamsters congenic at the NAT2 locus received a s.c. injection of 3,2' -dimethyl-4-aminobiphenyl (100 mg/kg) at the start of weeks 1 and 2. After 10 and 14 weeks, the hamsters were sacrificed, and each whole cecum, colon, and rectum was stained with 0.2% methylene blue, fixed in 4% paraformaldehyde, and examined under a dissecting microscope for the presence of aberrant crypts. Aberrant crypts were identified in the cecums and colons of both rapid and slow acetylator congenic hamsters treated with 3,2' -dimethyl-4-aminobiphenyl but not in vehicle controls. The size of the aberrant crypt foci was larger in the colon than in the cecum, and the highest frequency of aberrant crypt foci was observed in the cecum. No aberrant crypts were detected in the rectum. The frequency of aberrant crypt foci was significantly higher (2-3-fold) in rapid versus slow acetylator congenic hamsters in both cecum (P = 0.0352) and colon (P = 0.0006). These results support human epidemiological studies that suggest the rapid acetylator genotype is associated with higher risk of colon cancer induced by aromatic amines.

Acetylator genotype-dependent expression of arylamine N-acetyltransferase in human colon cytosol from non-cancer and colorectal cancer patients.

Human epidemiological studies suggest an association between rapid acetylator phenotype and colorectal cancer. Acetylator genotype-dependent expression by the human colon of arylamine N-acetylation capacity, catalyzed by acetyl coenzyme A-dependent N-acetyltransferase(s) (EC 2.3.1.5) (NAT), may be an important risk factor in the initiation of colorectal cancer. Human colon cytosols from 48 fresh surgical samples were investigated for NAT activity toward p-aminobenzoic acid and the arylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Apparent Vmax determinations of NAT activity toward these substrates indicated that 40 of these colons segregated into 3 distinct phenotypes. The distribution of the patients into rapid (5), intermediate (18), or slow (17) acetylators is a ratio that is not significantly different from the expected Hardy-Weinberg distribution of 3:16:21 (chi 2 = 2.206, P = 0.363). Significantly greater mean apparent Vmax levels were found in colons from rapid as compared to intermediate acetylators (1.5-3-fold) (P less than 0.001) and intermediate as compared to slow (2.5-3-fold) (P less than 0.005) acetylator phenotypes for the four arylamine substrates. Apparent Km determinations indicated that human colon NAT from rapid acetylators had a significantly lower affinity for the arylamine substrates (P less than 0.05) compared to intermediate or slow acetylator groups. No difference in apparent Km was detected for the cofactor acetyl coenzyme A between the three acetylator phenotypes. The colon samples were also tested for cytosolic N-hydroxy-2-acetylaminofluorene sulfotransferase activity and found to be monomorphically distributed for this enzyme activity. Of the 40 colon samples, 37 were from individuals of known pathology, 25 with colorectal cancer and 12 with no diagnosed neoplasia. Comparisons between mean apparent Vmax and mean apparent Km levels for each of the acetylator phenotypes indicated no significant differences between non-cancer and colorectal cancer patients. The distribution of rapid, intermediate, and slow acetylator phenotypes among the colon samples derived from colorectal cancer patients was precisely that predicted from published frequencies for the rapid and slow acetylator allele in Americans of African and European ancestry.

Polymorphic expression of acetyl coenzyme A-dependent arylamine N-acetyltransferase and acetyl coenzyme A-dependent O-acetyltransferase-mediated activation of N-hydroxyarylamines by human bladder cytosol.

Human epidemiological studies suggest a genetic predisposition to bladder cancer among slow N-acetylators. The capacity of human bladder to N-acetylate arylamines, catalyzed by acetyl coenzyme A-dependent N-acetyltransferase(s) (EC 2.3.1.5) (NAT), may be an important step in the activation and/or deactivation of arylamines in the pathways leading to the initiation of bladder cancer. Another possible activation step is the direct O-acetylation of N-hydroxyarylamines via O-acetyltransferase(s) (OAT) to DNA-binding electrophiles. Human bladder cytosol from nine fresh autopsy specimens were investigated for NAT activity towards p-aminobenzoic acid, and the arylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Apparent Km determinations indicated little difference in NAT affinity (100-300 microM) for any of the substrates between the nine individual bladders. However, the apparent Vmax determinations indicated that the bladders could be classified into rapid or slow acetylator phenotypes based on their NAT activity towards 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Four of the bladder cytosols had mean activities significantly (P less than 0.01) higher (approximately 10-fold) than the mean NAT activities of the other five bladder cytosols towards each arylamine carcinogen. However, no significant difference was detected in their NAT activities using p-aminobenzoic acid as a substrate. The human bladder cytosols were also tested for their capacity to activate N-hydroxy-3,2'-dimethyl-4-aminobiphenyl to a DNA-binding electrophile through a direct OAT-mediated catalysis. The N-hydroxyarylamine OAT activity also discriminated between two levels of activation, being significantly (P = 0.0002) higher (about twofold) in the rapid N-acetylator bladder cytosols, that correlated (r = 0.94) with the measured levels of NAT activity in each bladder cytosol. These results suggest that NAT activity and OAT activity of the human bladder vary concordantly with N-acetylator phenotype. The polymorphic expression of these acetylation activities may be important risk factors in human susceptibility to bladder cancer from arylamine carcinogens.

Purification of hepatic polymorphic arylamine N-acetyltransferase from homozygous rapid acetylator inbred hamster: identity with polymorphic N-hydroxyarylamine-O-acetyltransferase.

The polymorphic acetyltransferase isozyme expressed in homozygous rapid acetylator inbred hamster liver cytosol was purified over 2000-fold by sequential Q-Sepharose fast-flow anion-exchange chromatography, Sephacryl S-200 high-resolution size-exclusion chromatography, Mono Q anion-exchange fast-protein liquid chromatography, and preparative polyacrylamide gel electrophoresis. The isozyme migrated as a single homogeneous monomer following both preparative and sodium dodecyl sulfate-polyacrylamide electrophoresis. The molecular weight was estimated at 34,170 following elution via size-exclusion chromatography and 35,467 following migration via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneous polymorphic acetyltransferase exhibited a broad substrate specificity; it catalyzed the acetyl coenzyme A-dependent N-acetylation of p-aminobenzoic acid, carbocyclic arylamine carcinogens such as 2-aminofluorene, 4-aminobiphenyl and beta-naphthylamine, and heterocyclic arylamine carcinogens such as 2-aminodipyrido[1,2-a:3'2'd]imidazole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. It also readily catalyzed the acetyl coenzyme A-dependent metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to DNA adducts but not the metabolic activation (via intramolecular, N,O-acetyltransfer) of N-hydroxy-2-acetylaminofluorene or N-hydroxy-4-acetylaminobiphenyl to DNA adducts. Conversely, the partially purified monomorphic acetyltransferase isozyme from the same hamsters readily catalyzed the metabolic activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl, and rates of metabolic activation of these substrates did not differ between homozygous rapid and slow acetylator liver, intestine, kidney, and lung cytosols. Heat inactivation rates for the purified polymorphic acetyltransferase isozyme were first order and indistinguishable for the acetyl coenzyme A-dependent N-acetylation and O-acetylation activities. The results strongly suggest the expression of a single polymorphic acetyltransferase product of the hamster polymorphic acetyltransferase gene that catalyzes both acetyl coenzyme A-dependent N-acetylation and O-acetylation of arylamine and N-hydroxyarylamine carcinogens but not the metabolic activation of N-hydroxy-N-acetylarylamines (arylhydroxamic acids) via intramolecular N,O-acetyltransfer. Consequently, acetylator genotype-dependent metabolic activation of N-hydroxyarylamines to a DNA adduct in hamster is catalyzed by direct O-acetylation of the hydroxyl group and not via sequential N-acetylation followed by N,O-acetyltransfer.

Human N-acetylation of benzidine: role of NAT1 and NAT2.

These studies were designed to assess metabolism of benzidine and N-acetylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism was assessed using human recombinant NAT1 and NAT2 and human liver slices. For benzidine and N-acetylbenzidine, Km and Vmax values were higher for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzidine and N-acetylbenzidine were 54 and 535, respectively, suggesting that N-acetylbenzidine is a preferred substrate for NAT1. The much higher NAT1 and NAT2 Km values for N-acetylbenzidine (1380 +/- 90 and 471 +/- 23 microM, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 microM, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic parameters over a 20-fold range of acetyl-CoA concentrations demonstrated that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intact liver tissue metabolism using human liver slices. Samples incubated with either [3H]benzidine or [3H]N-acetylbenzidine had a similar ratio of N-acetylated benzidines (N-acetylbenzidine + N',N'-diacetylbenzidine/ benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N'-diacetylbenzidine. With [3H]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreased the amount of N-acetylbenzidine produced, resulting in a decreased ratio of acetylated products. This is consistent with benzidine being a NAT1 substrate. N-Acetylation of benzidine or N-acetylbenzidine by human liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices possessing the NAT1*10 compared to the NAT1*4 allele. Thus, a combination of human recombinant NAT and liver slice experiments has demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorphic expression in human liver.

Acetylator genotype and arylamine-induced carcinogenesis.

A diverse array of arylamine chemicals derived from industry, diet, cigarette smoke and other environmental sources are carcinogenic. These chemicals require metabolic activation by host enzymes to chemically reactive electrophiles to initiate the carcinogenic response. Genetic regulation of activation and/or deactivation pathways are thought to account in large measure for corresponding differences in tumor incidence from these chemicals between tissues, between species, or between individuals within a species. Various acetyltransfer reactions are involved in arylamine metabolism and much has been learned regarding their enzymology, genetic regulation, and toxicological significance. The small amount of human data are supported by systematic investigations carried out in animal models characterized with respect to the acetylation polymorphism. Enzymological and genetic investigations suggest that common enzymes encoded by the acetyltransferase gene carry out a diverse set of acetyltransferase reactions. Thus, the acetylation polymorphism can influence both activation and deactivation pathways in arylamine metabolism. Of particular significance recently have been reports documenting the O-acetylation of N-hydroxyarylamine carcinogens and its genetic coregulation with the well-characterized arylamine N-acetylation polymorphism. The toxicological consequences of this polymorphic pathway have yet to be fully explored. Epidemiological investigations show associations between acetylator phenotype and the incidence and/or severity of tumors in the urinary bladder, colon and larynx. Associations between acetylator phenotype and breast cancer are more equivocal and require further study. The divergent influence of acetylator phenotype on the incidence of tumors in different organ sites suggests an important role for extrahepatic acetyltransferases, and further characterization of them in human and animal tissues is needed. The advent of newer methodologies to monitor chemical exposures and to measure acetylator phenotype (rapid, intermediate and slow) using less invasive and more standardized protocols should soon result in a much more definitive understanding regarding the role of acetylator status in arylamine-induced carcinogenesis.

A role for bioactivation and covalent binding within epidermal keratinocytes in sulfonamide-induced cutaneous drug reactions.

Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Cloning, expression, and functional characterization of rapid and slow acetylator polymorphic N-acetyl-transferase encoding genes of the Syrian hamster.

Syrian hamster acetylation capacity is catalysed by two N-acetyltransferase isozymes (NAT1 and NAT2). Hamster NAT2 (polymorphic) displays acetylator-genotype dependent activity resulting in high, intermediate, and low activity levels in homozygous rapid, heterozygous and homozygous slow acetylators, respectively. A lambda gt10 size-selected genomic library was constructed from Eco RI-digested homozygous slow acetylator Bio. 82.73/H-Pats congenic hamster DNA and screened with a hamster NAT1 probe. A 4.2 kb Eco RI insert from a positive clone was subcloned into pUC18 and the intron-free NAT2 coding region was sequenced. The NAT2 coding regions from genomic templates of other homozygous rapid and slow acetylator congenic and inbred hamster lines were amplified by the polymerase chain reaction, cloned, and sequenced. Two NAT2 alleles were found, one (NAT2*15) from each homozygous rapid acetylator line and one (NAT2*16A) from each homozygous slow acetylator line. NAT2*15 contained an 870 bp open reading frame encoding a 290 amino acid protein. NAT2*16A was similar except for two silent (T36C and A633G) and one nonsense (C727T) substitutions yielding a 242 amino acid open reading frame. The NAT2*15 and NAT2*16A alleles were expressed in Escherichia coli JM105 and the recombinant proteins were characterized. Electrophoretic mobilities of the NAT2 15 and NAT2 16A recombinant hamster proteins differed and correlated with the theoretical molecular weights calculated from their respective open reading frames. NAT2 16A exhibited 500-to 1000-fold lower maximum velocities compared to NAT2 15 for N-acetylation of all arylamine and hydrazine substrates tested. NAT2 16A also catalysed the metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides at rates 33- and 23-fold lower than NAT2 15. Intrinsic clearance (Vmax/Km) calculations suggest that N-acetylation of p-aminobenzoic acid and 2-aminofluorene in Syrian hamsters is catalysed primarily by NAT2 (NAT2 15) in rapid acetylators but by NAT1 (NAT1 9) in slow acetylators. These results provide a molecular basis for rapid and slow acetylator phenotype in the Syrian hamster.

Syrian hamster monomorphic N-acetyltransferase (NAT1) alleles: amplification, cloning, sequencing, and expression in E. coli.

N-acetyltransferases have an important role in the metabolism of arylamine and hydrazine drugs and carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-induced toxicities and carcinogenesis. Syrian hamsters express two N-acetyltransferase isozymes; one varies with acetylator genotype (polymorphic) and has been termed NAT2; the other does not (monomorphic) and has been termed NAT1. The intronless NAT1 coding region was cloned via the polymerase chain reaction from homozygous rapid acetylator and homozygous slow acetylator congenic and inbred hamster genomic DNA templates and sequenced. The NAT1 alleles from the homozygous rapid (NAT1) and homozygous slow (NAT1s) acetylator hamsters differed in one nucleotide, but the mutation is silent with no change in deduced amino acid sequence. To characterize the enzyme products of the NAT1 alleles, we developed a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based plasmid vector pKK223-3 for over-production of recombinant NAT1 in E. coli strain JM105. Induced cultures from selected NAT1-inserted transformants yielded high levels of soluble protein capable of N-acetylation, O-acetylation, and N,O-acetylation. The recombinant NAT1r and NAT1s proteins did not differ in substrate specificity, specific activity, Michaelis-Menten kinetic properties, intrinsic stability, and electrophoretic mobility. Also, the over-expressed NAT1 proteins displayed substrate-specificity and electrophoretic mobilities characteristic of NAT1 isolated from Syrian hamster liver and colon cytosols.

Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms.

N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues. Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood. To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T). Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A. Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A. No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles. These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity.

Functional characterization of nucleotide polymorphisms in the coding region of N-acetyltransferase 1.

N-acetyltransferase 1 (NAT1) catalyses the activation and/or deactivation of aromatic and heterocyclic amine carcinogens. A genetic polymorphism in NAT1 is associated with an increased risk of various cancers and drug toxicities, but epidemiological investigations are severely compromised by a poor understanding of the relationship between NAT1 genotype and phenotype. Human reference NAT1*4 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Large reductions in N- and O-acetyltransferase catalytic activities were observed for recombinant NAT1 allozymes encoded by NAT1*14B, NAT1*15, NAT1*17, NAT1*19 and NAT1*22. Each of these alleles exhibited NAT1 protein expression levels below the limit of detection as measured by Western blot. No differences between high and low activity NAT1 alleles were observed in relative mRNA expression or relative transformation efficiency. The recombinant NAT1 17 and NAT1 22 allozymes showed reduced intrinsic stability when compared with NAT1 4. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) N-acetylation was not catalysed by any of the NAT1 allozymes. Large differences in the metabolic activation via O-acetylation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) were noted for NAT1 allelic variants. The results of these studies suggest an important role for the NAT1 genetic polymorphism in metabolism of aromatic and heterocyclic amine carcinogens. Furthermore, these results suggest that low NAT1 phenotype results from NAT1 allelic variants that encode reduced expression of NAT1 and/or less-stable NAT1 protein.

Glutathione S-transferase genotypes and stomach cancer in a population-based case-control study in Warsaw, Poland.

Glutathione S-transferases are important in the detoxification of a wide range of human carcinogens. Previous studies have shown inconsistent associations between the GSTT1 and GSTM1 null genotypes and stomach cancer risk. We investigated the relationship between these and related genotypes and stomach cancer risk in a population-based case-control study in Warsaw, Poland, where stomach cancer incidence and mortality rates are among the highest in Europe. DNA from blood samples was available for 304 stomach cancer patients and 427 control subjects. We observed a 1.48-fold increased risk for stomach cancer (95% confidence interval 0.97-2.25) in patients with the GSTT1 null genotype but no evidence of increased risk associated with the GSTM1, GSTM3 or GSTP1 genotypes. Furthermore, the stomach cancer risk associated with the GSTT1 null genotype varied by age at diagnosis, with odds ratios of 3.85, 1.91, 1.78 and 0.59 for those diagnosed at ages less than 50, 50-59, 60-69 and 70 years or older, respectively (P trend = 0.01). This was due to a shift in the GSTT1 genotype distribution across age groups among stomach cancer patients only. These results suggest that the GSTT1 null genotype may be associated with increased risk of stomach cancer.