Genetic polymorphisms of N-acetyltransferase 1 and 2 and risk of cigarette smoking-related bladder cancer

Aromatic amines from cigarette smoking or occupational exposure, recognized risk factors for bladder cancer, are metabolized by N-acetyltransferases (NAT). This study examined the association of (NAT) 1 and 2 genotypes with the risk of smoking-related bladder cancer. A total of 74 pathologically confirmed bladder cancer patients and 184 controls were serially recruited from the National Taiwan University Hospital. History of cigarette smoking and other risk factors for bladder cancer was obtained through standardized questionnaire interview. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 and NAT2 by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Allele frequency distributions of NAT1 and NAT2 were similar between cases and controls. There was a significant dose–response relationship between the risk of bladder cancer and the quantity and duration of cigarette smoking. The biological gradients were significant among subjects carrying NAT1*10 allele or NAT2 slow acetylators, but not among NAT2 rapid acetylators without NAT1*10 allele. The results are consistent with the hypothesis that NAT1 and NAT2 might modulate the susceptibility to bladder cancer associated with cigarette smoking. © 1999 Cancer Research Campaign

, there were inconsistent findings regarding the effect of NAT1 genetic polymorphism on bladder cancer (Okkels et al, 1997;Taylor et al, 1998). The specific aim of this study is to examine the effects of NAT1 and NAT2 on cigarette smoking-related bladder cancer through a case control comparison design.

Study subjects
In this case control study, 74 patients affected with urinary bladder cancer were serially recruited from National Taiwan University Hospital. All of them were diagnosed histologically to be affected with transitional cell carcinoma. A total of 184 control subjects within the same age range of cases were recruited from health examination clinic (77.8%) and urology clinic (22.2%) in the same hospital. They were not affected with any cancer. Subjects who had lived in the arseniasis-endemic area were excluded from this study. The proportion of males was 78.4% for cases and 77.7% for controls. Cases and controls had similar frequency for distribution of age. There were 44.6% cases and 54.3% controls with an age below 65 years old. Because there were insufficient male elderly subjects to be selected as controls from the health examination clinic, several male controls were recruited from the urology clinic among patients with benign prostatic hypertrophy. Benign prostatic hypertrophy is a very common condition among elderly males in Taiwan, and no association between NAT and benign prostatic hypertrophy has ever been documented.
Standardized personal interview based on a structured questionnaire was carried out to collect information on risk factors, including sociodemographic characteristics, residential and occupational history, habits of cigarette smoking and consumption of Genetic polymorphisms of N-acetyltransferase 1 and 2 and risk of cigarette smoking-related bladder cancer alcohol, tea and coffee, dietary habits, personal and family history of urinary disease and cancers. Duration and quantity of cigarette smoking was inquired in detail. Subjects who had smoked cigarettes more than 3 days a week for more than 6 months were classified as cigarette smokers and the others as non-smokers. A peripheral blood sample was collected from each subject using disposable vacuum syringe containing heparin. DNA was extracted from peripheral lymphocytes by Genomix DNA extraction kit (Talent, Follatoio, Italy) resuspended in deionized distilled water, and stored at -20°C until genotyping.

Genotypes of NAT1 and NAT2
NAT1 was amplified by polymerase chain reaction (PCR) using the primers N1-OA (5′-GCTCACCAGTTATCAACTGAC) and N1-OB (5′-AACCAACATTAAAAGCTTTCT) and resulted in a 233-base pair amplificate. The PCR mixture was composed of 1000-2000 ng DNA, 20 pmole of each NAT1 primers, 1 U Taq polymerase (Takara Taq, Takara Shuzo, Japan), 5 µl 10 × PCR buffer (100 mM Tris-HCl (pH 8.3), 500 mM potassium chloride (KCl), 15 mM magnesium chloride (MgCl 2 )) and 4 µl 2.5 mM deoxynucleoside triphosphates in a final volume of 50 µl. The reaction mixture was placed for 5 min at 94°C, and then subjected to 30 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 45 s. This was followed by a final step at 72°C for 10 min. Negative control was included in each set of PCR analyses. The PCR products were electrophoresed in 1.5% agarose gel (FMC, Rockland, ME, USA) and then extracted the DNA by QIAquick gel extraction kit (QIAGEN, Germany). The product was processed with a Taq-dye terminator cycle sequencing ready reaction kit (Applied Biosystems Inc., Foster City, CA, USA). The fragments were then analysed with an Applied Biosystems 373A automated sequencer with a denaturing 6% polyacrylamide gel.

Statistical analysis
The differences in frequency distributions of bladder cancer risk factors between cases and controls were tested for their statistical significance by χ 2 tests. In the univariate analysis, odds ratios (OR) with a 95% confidence interval (CI) for each risk factor were calculated after the adjustment for age (continuous variable), sex and educational level (categorized variable) through logistic regression analysis. Cases had a slightly older mean age at recruitment, and lower educational levels than controls.

RESULTS
While fewer cases (10%) than controls (20%) were habitual coffee drinkers, there was no significant association between coffee drinking and bladder cancer after adjustment for age, sex and educational level (OR = 0.6, 95% CI = 0.2-1.4). The proportions of habitual alcohol drinking were 30% for cases and 24% for controls, a multivariate-adjusted OR of 1.1 (95% CI 0.6-2.2). Forty-five per cent cases and 48% controls were tea drinkers with a multivariate-adjusted OR of 0.9 (95% CI 0.5-1.7). Few study subjects had well documented occupational exposures related to bladder cancer. Only one case and one control were engaged in printing and painting industry, and three cases and two controls were professional drivers. The consumption of coffee, alcohol and tea, as well as the above occupational exposures, were not considered as confounding factors. Table 1 compares the cigarette smoking habit between bladder cancer cases and healthy controls. Cases had a higher percentage of cigarette smokers than controls (0.05 < P < 0.10) showing an OR of developing bladder cancer of 1.90 (95% CI 0.99-3.64) after adjustment for age, sex and educational level. There were significant dose-response relationships between the risk of bladder cancer and the duration and quantity of cigarette smoking. The frequency distributions of NAT1 and NAT2 alleles and genotypes in cases and controls are compared in Table 2. Cases and controls had similar allele frequency distributions of NAT1 and NAT2. A total of 61.5% cases and 64.9% controls had one or two alleles of NAT1*10; 20.6% cases and 24.0% controls were NAT2 slow acetylators. The genotype frequency distributions of NAT1 and NAT2 were alike in cases and controls. The combined frequency distribution of NAT1 and NAT2 was also similar in cases and controls.
As shown in Table 3, the OR of developing bladder cancer for cigarette smoking was further analysed by stratifying study subjects according to NAT1 and NAT2 genotypes. Among cases, those subjects carrying NAT1*10 allele and NAT2 slow acetylators had an increased bladder cancer risk associated with cigarette smoking, they were classified as one group to be compared with cases with NAT2 rapid acetylators and without NAT1*10 allele as another group. A significant association between bladder cancer and cigarette smoking was observed among subjects carrying NAT1*10 allele or NAT2 slow acetylators showing an OR of 2.34 (95% CI 1.03-5.31), but not among NAT2 rapid acetylators without NAT1*10 allele (OR 2.07, 95% CI 0.32-13.30). The dose-response relationships between the risk of bladder cancer and the quantity and duration of cigarette smoking were also statistically significant among those who had NAT1*10 allele or NAT2 slow acetylators, but not for cases with NAT2 rapid acetylators but without NAT1*10 allele.

DISCUSSION
As in previous studies of cigarette smoking and bladder cancer, we also observed an increased risk of bladder cancer among cigarette smokers in this study. The metabolism of carcinogenic arylamines in tobacco smoke is mediated by enzymes including NAT1 and NAT2. Both NAT1 and NAT2 are genotypically and phenotypically polymorphic with variable genotype frequencies in NAT genetic polymorphism and bladder cancer 539 British Journal of Cancer (1999) 81(3), 537-541 © 1999 Cancer Research Campaign  different ethnic groups. The allele frequency of NAT1*10 in controls of this study was 41.8%, which is significantly higher (χ 2 = 4.00, P < 0.05) than the 30.0% observed in Caucasians (Bell et al, 1995b). The frequency of NAT2 slow acetylator in controls of this study was 24.0%, which is similar to that observed among Chinese in Hong Kong (27.0%) (Lin et al, 1993) and lower than that among American Caucasians (55.0%) (Bell et al, 1993). The percentage of NAT2 slow acetylator is significantly lower in Taiwanese than in American Caucasians (χ 2 = 20.14, P < 0.001). NAT2 slow acetylators were found to have an increased risk of bladder cancer among cigarette smokers (Risch et al, 1995;Brockmoller et al, 1996). The NAT1*10 allele alone was reported to be a risk factor in one study (Taylor et al, 1998), but not in another (Okkels et al, 1997). In this study, the genetic polymorphism of neither NAT1 nor NAT2 was significantly associated with the development of bladder cancer. However, the significant dose-response relationships between cigarette smoking and bladder cancer were observed in this study among those with NAT1*10 or NAT2 slow acetylators, but not among NAT2 rapid acetylators without NAT1*10 allele.
Some aromatic amines are metabolically inactivated through acetylation in the liver mainly by NAT2 (Kadlubar and Badawi, 1995). Rapid NAT2 acetylators are considered to have a higher level of non-toxic metabolites and a lower risk of bladder cancer than slow NAT2 acetylators. Some other arylamines were catalysed by cytochrome P450 1A2 in the liver, and the N-hydroxy arylamine metabolites can then enter the blood stream. The N-hydroxy arylamine metabolites are further metabolized into highly electrophilic N-acetoxy derivatives by NAT1, which is expressed mainly in urinary bladder epithelium (Kirlin et al, 1989;Frederickson et al, 1994). This will lead to an elevated level of arylamine-DNA adducts and an increased risk of bladder cancer. The dose-response relationship between cigarette smoking and bladder cancer is thus significant among cigarette smokers who have NAT1*10 allele and/or cigarette-smoking NAT2 slow acetylators.
In addition to NAT1 and NAT2, the metabolism of arylamines also involves a number of other enzymes including sulphotransferases (Kato and Yamazoe, 1994), prostaglandin H synthase (Smith et al, 1992), cytochrome P450 1A2 (Butler et al, 1989;Fleming et al, 1994) and glucuronyltransferases. These enzymes may also contribute to the metabolic steps of arylamines relevant to the development of bladder cancer. Further examination of effects of these enzymes on the cigarette smoking-related bladder cancer in humans is noteworthy.