2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome P4501A1 and rapid or slow acetylator N-acetyltransferase 2.

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MeIQx-induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiologic studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators.     

DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.     

Distribution and concordance of N-acetyltransferase genotype and phenotype in an American population.

Polymorphic arylamine N-acetyltransferase 2 (NAT2) status varies widely between individuals and ethnic groups and has been associated with susceptibility to several cancers. Few studies have reported the distribution of NAT2 status for Caucasian-American populations or evaluated the concordance between methods of assessment for cancer cases and controls. In our study, distribution of NAT2 status was classified by genotype and phenotype measurements in PANCAN, a population-based case-control study of pancreatic cancer, and concordance between measurements was evaluated for 33 cases and 222 controls. Major genotypes and alleles among controls were *5B/*6A, *5B/*5B, *4/*6A, and *5B/*4. One putative new allele was found in a single individual. Genotypes and phenotypes were classified as rapid or slow, according to a bimodal model. Presence of the *4 (wild-type) allele defined a NAT2 genotype as rapid. The NAT2 phenotype was analyzed by the caffeine assay. Ratios of 5-acetylamino-6-formylamino-3-methyluracil to 1-methylxanthine were determined, and individuals with values of > or =0.66 were identified as having a rapid phenotype. In our population, 58.1 and 59.5% of control subjects were classified as slow acetylators by phenotype and genotype, respectively. Concordance of NAT2 genotype and phenotype classification was 97.8% in the bimodal model. A similar analysis was completed for a trimodal model. Concordance of genotype and phenotype was high in cases (90.9%) and similar to controls; genotyping alone provided an efficient, accurate method of analysis for acetylator status. A comparison with two previous reports revealed subtle differences in genotype and allele distribution but exhibited overall similarity with other Caucasian-American populations.     

Determinants of 4-aminobiphenyl-DNA adducts in bladder cancer biopsies

Exposure to 4-aminobiphenyl (4-ABP) is an important determinant of urinary bladder cancer in humans. We have analyzed by gas chromatography-mass spectrometry the DNA adducts of 4-ABP in 75 bladder cancer biopsies. The purpose was to understand whether smoking, N-acetyltransferase 2 (NAT2) polymorphism, diet or tumor grade were determinants of 4-ABP-DNA levels. 4-ABP-DNA adducts were above the detection limit of 0.1 fmol/microg DNA for 37/75 patients. Overall the level of adducts was 2.7 +/- 0.7 (mean +/- SE) fmol/microg DNA (86 +/- 22 adducts/10(8) normal nucleotides, mean +/- SE). A strong association with grade was observed. In the group of patients with detectable 4-ABP-DNA adducts the odds ratio for having a tumor grade of 2 or 3 was respectively 4.3 (95% CI 0.8-21.9) and 6 (1.3-27.5), compared with grade 1. A non-statistically significant association was found between adduct levels and the deduced slow acetylator phenotype in grades 2 and 3. The intake of fruit and vegetables produced a lower frequency of detectable adducts, though the association was not statistically significant. Detectable 4-ABP-DNA adducts were clearly associated with current smoking in higher tumor grades (grade 3 versus grades 1 + 2, odds ratios 10.4; 95% CI 1.7-63.1). Overall, our findings indicate that higher levels of DNA adducts characterize more invasive tumors (higher tumor grades). This seems to be facilitated by smoking and contrasted by the intake of fruit and vegetables.     

Modifying Effect of Smoking on GSTM1 and NAT2 in Relation to the Risk of Bladder Cancer in Mongolian Population: A Case-Control Study

Objectives: Tobacco smoking is the predominant risk factor for bladder cancer as it contains cancer-causing chemicals. However, genetic factors may play important role in response towards chemical carcinogens. In this study we aim to investigate genetic polymorphisms of glutathione S-transferase M1 (GSTM1) and N-acetyltransferase 2 (NAT2) as determinants of bladder cancer risk, independently and in combination with tobacco use in the Mongolian population. Materials and Methods: The current study was a hospital-based case-control study including 60 histologically confirmed bladder cancer patients and 60 cancer-free controls. PCR-RFLP assay was used to determine the presence of GSTM1 and NAT2 polymorphisms in bladder cancer patients and controls. GSTM1 and NAT2 were tested using binary logistical regression analysis with adjustment or stratification according to the smoking. Results: There were 46 men and 14 women diagnosed with bladder cancer, with mean age was 58±4. The controls included 37 men and 23 women with a mean age of 57±3. The frequency of GSTM1 null genotype was higher in controls (71.67%) than in bladder cancer patients (58.33%) without statistical significance (OR=0.5534; 95% CI=0.2586-1.1843), (p=0.128). The NAT2 low acetylator phenotype was more common in patients with bladder cancer (15%) than in controls (5%). Furthermore, individuals with NAT2 low acetylator phenotype had a nearly 3.35-fold increased risk to develop bladder cancer (OR=3.35; 95% CI=0.8604-13.0657), (p=0.081) while the risk was even higher when combined with null GSTM1 genotype (OR=4; 95% CI=0.4459-37.5308), (p=0.213) but there was no statistical significance. Prevalence of smoking in bladder cancer patients was higher than controls and increased significantly the risk of bladder cancer (OR=8.31; 95% CI=3.66-18.88). Smokers with GSTM1 null genotype were at 5-fold higher risk of bladder cancer (OR=5.0; 95% CI=1.55-16.16), (p=0.007) while NAT2 low acetylator phenotype increased bladder cancer risk by 20-fold (OR=20.5; 95% CI=2.33-80.86), (p=0.006). Conclusion: The current study shows that tobacco smokers with the NAT2 low acetylator phenotype and GSTM1 null genotype have the highest risk of bladder cancer in the Mongolian population.     

2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced DNA adducts and genotoxicity in chinese hamster ovary (CHO) cells expressing human CYP1A2 and rapid or slow acetylator N-acetyltransferase 2

Heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) are present in diet and cigarette smoke. Bioactivation in humans includes N-hydroxylation catalyzed by cytochrome P4501A2 possibly followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A2 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A2 catalytic activity levels did not differ significantly (P > 0.05) among the CYP1A2-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of N-acetyltransferase (P = 0.0001) and N-hydroxy-PhIP O-acetyltransferase (P = 0.0170) catalytic activity than cells transfected with NAT2*5B. PhIP caused dose-dependent decreases in cell survival and significant (P < 0.001) increases in mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in all the CYP1A2-transfected cell lines. Transfection with NAT2*4 or NAT2*5B did not further increase hprt mutagenesis. PhIP-induced hprt mutant cDNAs were sequenced, and 80% of the mutations were single base substitutions at G:C base pairs. dG-C8-PhIP DNA adduct levels were dose-dependent in the order: untransfected < transfected with CYP1A2 < transfected with CYP1A2 and NAT2*5B < transfected with CYP1A2 and NAT2*4. Following incubation with 1.2 microM PhIP, DNA adduct levels were significantly (P < 0.05) higher in CHO cells transfected with CYP1A2/NAT2*4 versus CYP1A2/NAT2*5B. These results strongly support an activation role for CYP1A2 in PhIP-induced mutagenesis and DNA damage and suggest a modest effect of human NAT2 and its genetic polymorphism on PhIP DNA adduct levels.     

2-Aminofluorene-DNA adducts in mouse urinary bladder: effect of age, sex and acetylator phenotype.

Formation of urinary bladder DNA-2-aminofluorene adducts in inbred and acetylator congenic mice was measured 3 h after a 60 mg/kg dose of the arylamine carcinogen. The sensitivity of 32P-postlabeling with HPLC analysis permitted quantitation of adducts in individual mouse bladders. Acetylator phenotype was a significant determinant of DNA damage in female mice as slow acetylators had higher levels of bladder DNA adducts than rapids. This correlation is the reverse of that seen with hepatic DNA. Age was also a significant determinant of DNA damage as older mice (20-23 weeks) formed more bladder DNA adducts than young (7 week) mice. The age-related increase in bladder adduct formation was seen in both sexes of all mouse lines. Male B6 mice exposed to 2-aminofluorene at 20-23 weeks of age showed a 26-fold higher level of bladder DNA adducts than males exposed at 7 weeks. In addition to the large increase in total adduct level, the older male B6 mice produced significant amounts of an unidentified, early-eluting adduct peak that had chromatographic properties similar to an aminofluorene-DNA adduct produced through peroxidative activation. These results indicate that age, sex and acetylator phenotype are all important determinants of aromatic amine-bladder DNA adduct formation in mice.     

Acetylation phenotype, carcinogen-hemoglobin adducts, and cigarette smoking

Levels of 4-aminobiphenyl-hemoglobin adducts in smokers of blonde (flue-cured) and black (air-cured) tobacco have been found to be proportional to bladder cancer risk. In addition, risk of bladder cancer due to exposure to occupational carcinogens is elevated in genetically determined slow acetylators. In this study of normal male volunteers, 4-aminobiphenyl-hemoglobin adducts were found to be related to both the quantity and the type of tobacco smoked, as well as to the acetylator phenotype (independently of smoking habits). The demonstration that both the genetically determined slow acetylator phenotype and tobacco smoking are independently associated with levels of the carcinogen 4-aminobiphenyl in adducted hemoglobin suggests a single mechanism to explain the contribution of genetic susceptibility and environmental exposure in bladder carcinogenesis.     

Permanent hair dyes and bladder cancer: risk modification by cytochrome P4501A2 and N-acetyltransferases 1 and 2

We have previously reported permanent hair dye use to be a significant risk factor for bladder cancer in US women. We also have examined N-acetyltransferase-2 (NAT2) phenotype in relation to the hair dye-bladder cancer relationship, and found that the association is principally confined to NAT2 slow acetylators. In the present study, we assessed the possible modifying effects of a series of potential arylamine-metabolizing genotypes/phenotypes (GSTM1, GSTT1, GSTP1, NAT1, NAT2, CYP1A2) on the permanent hair dye-bladder cancer association, among female participants (159 cases, 164 controls) of the Los Angeles Bladder Cancer Study. Among NAT2 slow acetylators, exclusive permanent hair dye use was associated with a 2.9-fold increased risk of bladder cancer (95% CI = 1.2-7.5). The corresponding relative risk in NAT2 rapid acetylators was 1.3 (95% CI = 0.6-2.8). Frequency- and duration-related dose-response relationships confined to NAT2 slow acetylators were all positive and statistically significant. No such associations were noted among NAT2 rapid acetylators. Among CYP1A2 'slow' individuals, exclusive permanent hair dye use was associated with a 2.5-fold increased risk of bladder cancer (95% CI = 1.04-6.1). The corresponding risk in CYP1A2 'rapid' individuals was 1.3 (95% CI = 0.6-2.7). Frequency- and duration-related dose-response relationships confined to CYP1A2 'slow' individuals were all positive and statistically significant. No such associations were noted among CYP1A2 'rapid' individuals. Among lifelong non-smoking women, individuals exhibiting the non-NAT1*10 genotype showed a statistically significant increase in bladder cancer risk associated with exclusive permanent hair dye use (OR = 6.8, 95% CI = 1.7-27.4). The comparable OR in individuals with the NAT1*10 genotype was 1.0 (95%CI = 0.2-4.3). Similarly, all frequency- and duration-related dose-response relationships confined to individuals possessing the non-NAT1*10 genotype were positive and statistically significant. On the other hand, individuals of NAT1*10 genotype exhibited no such associations.     

Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N- acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino- 3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N- hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N- acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.      

Distribution and Role of N-acetyltransferase 2 Genetic Polymorphisms in Bladder Cancer Risk in a Lebanese Population

Background: In Lebanon, bladder cancer (BC) has an unusually high prevalence. Individuals who are exposed to aromatic amines from smoking or certain occupations and carrying the slow N-acetyl transferase 2 (NAT2) acetylator’ phenotype may be at a higher risk. Methods: Data and DNA from 115 Lebanese BC cases and 306 controls were examined. Ten NAT2 single nucleotide polymorphisms were genotyped, seven of which were then included in haplotype and phenotype analysis. Results: BC patients were more likely to be males (87.8% vs. 54.9%) and current smokers (60.9% vs. 26.5%) when compared to controls. In both groups, most participants had the slow NAT2 acetylator phenotype (66.1% of BC cases vs 62.7% of controls; P=0.302) with the NAT2*5B and *6A haplotypes being the most common. The odds ratio (95%CI) of having BC among slow NAT2 acetylators was 1.157 (0.738-1.815) and remained non-significant after adjustment [1.097 (0.666-1.806)]. Sensitivity analysis with a subgroup of 113 cases and 84 controls for which occupational history was available revealed a statistically significant association between slow NAT2 acetylators and BC in females only. The sample size was however very small and the CI quite wide. Conclusions: This is the first study to evaluate the distribution of NAT2 haplotypes and their potential role in BC in a Lebanese population. The absence of any significant association may be due to the relatively small sample size, the unavailability of matching by gender, and the lack of evaluation of genetic interactions with extent of active and passive smoking, exposure to environmental pollutants, diet, and other genes. The potential association limited to females needs further evaluation.     

N-Acetyltransferase 2 polymorphisms, cigarette smoking and alcohol consumption, and oral squamous cell cancer risk.

The risk of squamous cell cancers of the oral cavity (OSCC) is strongly related to the use of tobacco and alcohol. N-Acetyl transferases 1 and 2 (NAT2) metabolize aryl- and heterocyclic amines that are present in tobacco smoke. NAT2 slow acetylator phenotype or genotype is related to reduced ability to detoxify these xenobiotics that are carcinogenic in tissues in which smoking-related cancers develop (e.g. bladder). We studied the association between the deduced NAT2 acetylator phenotypes and OSCC risk in a population-based study of 341 cases and 552 controls. In-person interviews provided information on tobacco use and alcohol consumption. Nucleotide substitutions at position 191, 341, 590, 803 and 857 were determined by a combination of oligonucleotide ligation assays and PCR/RFLP assays. There was no overall association between acetylator status with OSCC risk; the odds ratios for slow and intermediate acetylators, as compared with the rapid acetylators, were 1.2 (95% CI 0.7-2.2) and 1.1 (95% CI 0.6-2.0), respectively. The percent increase in risk of OSCC per pack-year cigarette smoking was similar among slow acetylators (3.0%, 95% CI 2.1-4.0) and the combined intermediate and rapid acetylators (3.5%, 95% CI 2.4-5.0). In contrast, the risk of OSCC per weekly alcoholic drink was stronger among the combined rapid and intermediate acetylators (3.3%, 95% CI 1.8-4.9) compared with slow acetylators (1.6%, 95% CI 0.6-2.7) (interaction P = 0.055). These data raise the possibility that NAT2 may be involved in the activation of one or more pro-carcinogens associated with alcohol consumption.     

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.     

NAT2 slow acetylation and bladder cancer in workers exposed to benzidine

This study expands a previous study of NAT2 polymorphisms and bladder cancer in male subjects occupationally exposed only to benzidine. The combined analysis of 68 cases and 107 controls from a cohort of production workers in China exposed to benzidine included 30 new cases and 67 controls not previously studied. NAT2 enzymatic activity phenotype was characterized by measuring urinary caffeine metabolite ratios. PCR-based methods identified genotypes for NAT2, NAT1 and GSTM1. NAT2 phenotype and genotype data were consistent. A protective association was observed for the slow NAT2 genotype (bladder cancer OR = 0.3; 95% CI = 0.1 = 1.0) after adjustment for cumulative benzidine exposure and lifetime smoking. Individuals carrying NAT1wt/*10 and NAT1*10/*10 showed higher relative risks of bladder cancer (OR = 2.8, 95% CI = 0.8-10.1 and OR = 2.2, 95% CI = 0.6-8.3, respectively). No association was found between GSTM1 null and bladder cancer. A metaanalysis risk estimate of case-control studies of NAT2 acetylation and bladder cancer in Asian populations without occupational arylamine exposures showed an increased risk for slow acetylators. The lower limit of the confidence interval (OR = 1.4; 95% CI = 1.0-2.0) approximated the upper confidence interval for the estimate obtained in our analysis. These results support the earlier finding of a protective association between slow acetylation and bladder cancer in benzidine-exposed workers, in contrast to its established link as a risk factor for bladder cancer in people exposed to 2-naphthylamine and 4-aminobiphenyl. Study findings suggest the existence of key differences in the metabolism of mono- and diarylamines.     

N-acetyltransferase 2 influences cancer prevalence in hMLH1/hMSH2 mutation carriers.

Hereditary nonpolyposis colorectal cancer (HNPCC), an inherited cancer predisposition syndrome, has been associated with germline mutations in DNA mismatch repair (MMR) genes. Because a deficiency in MMR does not predict a specific cancer phenotype, modifying genes may account in part for the variation in disease expression. We determined the N-acetyltransferase 2 (NAT2) genotype in 26 unaffected and 52 cancer-affected hMLH1/hMSH2 mutation carriers coming from 21 Swiss HNPCC families. Slow acetylators were found to be significantly (P < 0.03) more prevalent in the group of affected mutation carriers. Our results suggest a protective effect of the NAT2 rapid acetylator phenotype, an observation that could have implications for genetic counseling and management of MMR gene mutation carriers.     

Tissue distribution of N-acetyltransferase 1 and 2 catalyzing the N-acetylation of 4-aminobiphenyl and O-acetylation of N-hydroxy-4-aminobiphenyl in the congenic rapid and slow acetylator Syrian hamster

N-acetyltransferase 1 (NAT1) and 2 (NAT2) enzymes catalyzing both deactivation (N-acetylation) and activation (O-acetylation) of arylamine carcinogens such as 4-aminobiphenyl (ABP) were investigated in a Syrian hamster model congenic at the NAT2 locus. NAT2 catalytic activities (measured with p-aminobenzoic acid) were significantly (P < 0.001) higher in rapid than slow acetylators in all tissues (except heart and prostate where activity was undetectable in slow acetylators). NAT1 catalytic activities (measured with sulfamethazine) were low but detectable in most tissues tested and did not differ significantly between rapid and slow acetylators. ABP N-acetyltransferase activity was detected in all tissues of rapid acetylators but was below the limit of detection in all tissues of slow acetylators except liver where it was about 15-fold lower than rapid acetylators. ABP N-acetyltransferase activities correlated with NAT2 activities (r2 = 0.871; P < 0.0001) but not with NAT1 activities (r2 = 0.132; P > 0.05). Levels of N-hydroxy-ABP O-acetyltransferase activities were significantly (P < 0.05) higher in rapid than slow acetylator cytosols for many but not all tissues. The N-hydroxy-ABP O-acetyltransferase activities correlated with ABP N-acetyltransferase activities (r2 = 0.695; P < 0.0001) and NAT2 activities (r2 = 0.521, P < 0.0001) but not with NAT1 activities (r2 = 0.115; P > 0.05). The results suggest widespread tissue distribution of both NAT1 and NAT2, which catalyzes both N- and O-acetylation. These conclusions are important for interpretation of molecular epidemiological investigations into the role of N-acetyltransferase polymorphisms in various diseases including cancer.     

Aromatic DNA adducts and polymorphisms of CYP1A1, NAT2, and GSTM1 in breast cancer

Previous studies by us and others have shown a significantly higher level of aromatic DNA adducts in normal adjacent breast tissue samples obtained from breast cancer patients than in those obtained from non-cancerous controls. The increased amount of DNA damage could be related to excess environmental carcinogen exposure and/or genetic susceptibility to such exposure. In the current study, we investigated the relationship between the levels of aromatic DNA adducts in breast tissues and polymorphisms of the drug-metabolizing genes cytochrome P4501A1 (CYP1A1), N-acetyltransferase-2 (NAT2), and glutathione S-transferase M1 (GSTM1), in 166 women having breast cancer. DNA adducts were measured using (32)P-postlabeling and information on smoking status was obtained from medical records. When pooled data of smokers and non-smokers were analyzed by multiple regression analyses, no significant correlation was found between the level of total DNA adducts and age, race, or polymorphisms of CYP1A1, GSTM1, and NAT2. The only significant predictor of the level of DNA adducts in breast tissues was smoking (P = 0.008). When data were analyzed separately in smokers and non-smokers, however, a significant gene-environment interaction was observed. Smokers with CYP1A1*1/*2 or *2/*2 genotypes had a significantly higher level of DNA adducts than those with the CYP1A1*1/*1 genotype. This effect was not seen among non-smokers. There was also a gene-gene interaction, as smokers with combined CYP1A1*1/*2 or CYP1A1*2/*2 genotypes and GSTM1 null had a much higher level of adducts than those with either CYP1A1 or GSTM1 polymorphism. Genetic polymorphisms of CYP1A1 and NAT2 were also significantly correlated with the frequency of certain types of DNA adducts. For example, a bulky benzo[a]pyrene (B[a]P)-like adduct was detected in 26% of the samples, the presence of which was not related to age, race, smoking status, or GSTM1 and NAT2 genotype. However, a significantly higher frequency of the B[a[P-like adduct was found in individuals having CYP1A1*1/*2 or *2/*2 genotypes than in those having the *1/*1 genotype (P = 0.04). In addition, individuals having slow NAT2 alleles had a significantly higher frequency of the typical smoking-related DNA adduct pattern, i.e. a diagonal radioactive zone (DRZ), than others did (P = 0.008). These findings suggest that polymorphisms of CYP1A1, GSTM1, and NAT2 significantly affect either the frequency or the level of DNA adducts in normal breast tissues of women having breast cancer, especially in smokers. Further large-scale studies are required to determine the exact role of these polymorphisms and types of DNA damage in breast cancer susceptibility.     

Modulation of benzo[a]pyrene diolepoxide-DNA adduct levels in human white blood cells by CYP1A1, GSTM1 and GSTT1 polymorphism

The modulation of benzo[a]pyrene diolepoxide (BPDE)-DNA adduct levels by polymorphisms in the CYP1A1, GSTM1 and GSTT1 genes was assessed in leukocytes of Caucasian males. Eighty-nine coke oven workers (35 smokers, 36 ex-smokers and 18 non-smokers) were recruited from job categories with different exposure levels to polycyclic aromatic hydrocarbons (PAH), together with 44 power plant workers (all smokers) not exposed to PAH. BPDE-DNA adducts were detected in 69 of 133 (52%) DNA samples with a 100-fold variation (range 0.2-44 adducts/10(8) nt) and a median of 1.6 adducts/10(8) nt. All samples with the GSTM1 active genotype (n = 59) and five out of 74 samples with GSTM1*0/*0 (7%) showed non-detectable adducts (<0.2 adducts/10(8) nt) and 69 of 74 subjects with GSTM1*0/*0 (93%) had detectable adducts (>0.2 adducts/10(8) nt). The difference in adduct level between the GSTM1*0/*0 and GSTM1 active genotypes was highly significant (P < 0.0001). No significant difference in adduct level between the GSTT1*0/*0 and GSTT1 active genotypes was seen. All heterozygotes (CYP1A1*1/*2) from subjects of GSTM1 active type did not have detectable adducts. Among the GSTM1-deficient individuals (n = 69), 42 with the CYP1A1*1/*1 genotype showed a lower adduct level (median 1.3, range 0.2-4.1 adducts/10(8) nt) compared with 26 individuals with heterozygous mutated CYP1A1*1/*2 genotypes (median 2.5, range 0.4-6.1 adducts/10(8) nt, P < 0.015). One individual with low PAH exposure and the rare combination CYP1A1*2A/*2A-GSTM1*0/*0 showed an extremely high level of 44 adducts/10(8) nt. Significant differences in detectable adduct levels were found between the CYP1A1*1/*1 and CYP1A1*1/*2 genotypes in the exposed group low + medium (P = 0.01) and for all adduct levels, detectable and non-detectable (set at a fixed value), in highly exposed individuals and in ex-smokers (P = 0.03), whereas no such differences were observed in the control group. Mutated CYP1A1*1/*2 increased the adduct level in non-smokers from the exposed group (1.4 versus 2.2 adducts/10(8) nt), but had no effect on the smokers from the exposed group (2.3 versus 2.8 adducts/10(8) nt). When all variables were dichotomized, statistical evaluation showed that CYP1A1 status (P = 0.015), PAH exposure (P = 0.003) and smoking (P = 0.006) had significant effects on adduct levels which increased in the order: CYP1A1*1/*1 < CYP1A1(*1/*2 or *2A/*2A); environmental exposure < occupational exposure; non-smokers < smokers, whereby adducts increased with cigarette dose and the duration of smoking. Higher levels of BPDE-DNA adducts in individuals with the combined CYP1A1(1/*2 or *2A/*2A)-GSTM1*0/*0 genotype suggest that these genotype combinations are at increased risk for contracting lung cancer when exposed to PAH.     

Sequence variants of NAT1 and NAT2 and other xenometabolic genes and risk of lung and aerodigestive tract cancers in Central Europe.

Tobacco smoke contains an extensive cocktail of highly carcinogenic chemicals. Individuals with a slower elimination rate of the chemicals in tobacco smoke may have increased exposure to their carcinogenic properties compared with those with a faster rate. Polymorphisms that alter the function of the genes involved in the activation or the detoxification of the chemical carcinogens in tobacco smoke can potentially influence an individual's risk of developing a tobacco-related cancer. To test this hypothesis, we have genotyped polymorphisms in 16 genes involved in metabolism of chemical carcinogens in a Central and Eastern European case-control study comprising 2,250 lung cases, 811 upper aerodigestive cancer (UADT) cases, and 2,704 controls. The N-acetyltransferase (NAT) genes were the most implicated in risk, with the NAT1*10 haplotype showing an inverse association in lung cancer, in both heterozygote carriers [odds ratio (OR), 0.81; 95% confidence interval (95% CI), 0.70-0.93] and homozygote carriers (OR, 0.70; 95% CI, 0.48-1.01), suggesting a genotype dose response (P < 0.001). In UADT cancer, a similar inverse association was noted in NAT1*10 although only in heterozygotes (OR, 0.78; 95%CI, 0.65-0.95). In NAT2, when considering the individuals inferred acetylator phenotypes based on their NAT2 diplotype, "slow" acetylators compared with intermediate or fast acetylators showed no association with risk. None of the other 14 genes provided robust evidence of an association for either lung or UADT cancer. We therefore conclude that, of the genetic variation studied, NAT1 gene was the most likely candidate to influence the risk of developing a tobacco-related cancer.     

N-acetyltransferase 2 (NAT2) genotypes, cigarette smoking, and the risk of breast cancer

N-acetyltransferases (NATs) are important catalytic enzymes that metabolize carcinogenic arylamines. NAT2 genotype might modify the role of cigarette smoking, a source of arylamine exposure, in breast cancer. We conducted a nested case-control study to investigate the association between NAT2 genotype, smoking and breast cancer risk among women (110 cases, 113 matched controls) from the CLUE II cohort in Washington County, MD. Compared to women with the slow acetylator genotype, the main effects odds ratios (OR) for NAT2 were 1.4 for the intermediate acetylator genotype (95% confidence limits (CL) 0.7, 2.7) and 3.6 for the homozygous rapid acetylator genotype (95% CL 1.1, 11.4) (P for trend = 0.05). Smoking was associated in the direction of increased breast cancer risk in slow acetylators (e.g., >15 pack-years versus never smokers OR 2.0; 95% CL 0.7, 5.8) but not in rapid acetylators. These associations were not statistically significant in the total study population, but a statistically significant interaction between smoking and NAT2 acetylator status was present in postmenopausal women. The main effect of NAT2 in the direction of increased risk suggests that exposures to NAT2-activated carcinogens other than cigarette smoke may be important in this study population. The results for smoking were consistent with an inactivation role for NAT2 in breast cancer.