Elsevier

Biochemical Pharmacology

Volume 76, Issue 7, 1 October 2008, Pages 929-937
Biochemical Pharmacology

Comparison of CYP1A2 and NAT2 phenotypes between black and white smokers

https://doi.org/10.1016/j.bcp.2008.07.024Get rights and content

Abstract

The lower incidence rate of transitional cell carcinoma of the urinary bladder in blacks than in whites may be due to racial differences in the catalytic activity of enzymes that metabolize carcinogenic arylamines in tobacco smoke. To examine this, we compared cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 activities (NAT2) in black and white smokers using urinary caffeine metabolites as a probe for enzyme activity in a community-based study of 165 black and 183 white cigarette smokers. The paraxanthine (1,7-dimethylxanthine, 17X)/caffeine (trimethylxanthine, 137X) ratio or [17X + 1,7-dimethyluric acid (17U)]/137X ratio was used as an indicator of CYP1A2 activity. The 5-acetyl-amino-6-formylamino-3-methyluracil (AFMU)/1-methylxanthine (1X) ratio indicated NAT2 activity. The odds ratio for the slow NAT2 phenotype associated with black race was 0.4; 95% confidence intervals 0.2–0.7. The putative combined low risk phenotype (slow CYP1A2/rapid NAT2) was more common in blacks than in whites (25% vs. 15%, P < 0.02). There were no significant racial differences in slow and rapid CYP1A2 phenotypes, and in the combined slow NAT2/rapid CYP1A2 phenotype. Age, education, cigarette smoking amount, body mass index, GSTM1 and GSTM3 genotypes were unrelated to CYP1A2 and NAT2 activity. Intake of cruciferous vegetables (primarily broccoli), red meat, carrots, grapefruit and onions predicted CYP1A2 activity either for all subjects or in race-specific analyses. Carrot and grapefruit consumption was related to NAT2 activity. Collectively, these results indicated that phenotypic differences in NAT2 alone or in combination with CYP1A2 might help explain the higher incidence rates of transitional cell bladder cancer in whites.

Introduction

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth most common in women in the United States. The incidence rates of transitional cell carcinoma, which account for the majority of bladder tumors, are approximately twice as high in white men vs. black men and 1.5 times higher in white women than in black women [1]. Squamous cell carcinoma of the bladder is rare but higher in blacks than in whites [2]. Tobacco smoke contains arylamines, which are a known class of bladder carcinogens and thought to be the likely causative agent responsible for the increased risk of bladder cancer in cigarette smokers [3], [4]. The major route for arylamine activation to reactive electrophiles involves N-hydroxylation by cytochrome P4501A2 (CYP1A2) [5]. The detoxification pathway occurs via N-acetylation by N-acetyltransferase-2 (NAT2) [6]. Differences in the activity of these enzymatic pathways may affect the risk for bladder cancer as the slow acetylation phenotype and polymorphisms in NAT2 genes have been linked with increased levels of 4-aminobiphenyl (4-ABP)–hemoglobin adducts in smokers and with increased risk for bladder cancer [7], [8].

The urinary ratios of caffeine metabolites (Fig. 1) are commonly used as a probe for the in vivo phenotyping of CYP1A2 [5]. Approximately 95% of the primary systematic clearance of caffeine has been attributed to CYP1A2 activity [9]. Twin studies indicate that most of the wide variation in CYP1A2 activity is determined by genetic factors [10]. However among the >25 single nucleotide polymorphisms in CYP1A2 that have been identified (www.imm.ki.se/CYPalleles), few have been associated with altered function. The CYP1A2*1C and CYP1A2*1F polymorphisms have been associated with decreased enzyme inducibility in smokers [11], [12]. CYP1A2 activity is also induced or affected by cigarette smoking, diet, and environmental factors [13], [14], [15]. Some data indicate that activity levels may vary by race or ethnicity. In children, mean urinary caffeine metabolite ratios were higher in whites than in blacks [16]. In contrast theophylline clearance was higher in blacks than in whites and latinos [17], and propranolol 4-hydroxylase activity in liver microsomal specimens was higher in samples obtained from black surgical patients than from white patients [18].

Measurement of caffeine metabolites has also been used for the assessment of NAT2 activity (acetylation phenotype), as the formation of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) from caffeine is principally catalyzed by the non-inducible NAT2 (Fig. 1) [19], [20]. Genetic polymorphisms in the NAT2 gene are predictive of bladder cancer [21]. They are also correlated with the NAT2 activity, explaining about half of the variance in the distribution of the AFMU/1X phenotype [22], [23], [24]. However there is little data on racial differences in the NAT2 phenotype. The slow isoniazid acetylation phenotype was more common in black American and African tuberculosis patients than in white patients [25], [26]. Other data indicate similar isoniazid acetylation phenotype prevalences between white and black Americans [27], [28]. The slow sulphamethazine acetylation phenotype was more prevalent in Nigerian than in various Caucasian subjects [29], [30], [31], [32], [33], [34], [35]. The slow NAT2 genotype was reported to be more common in whites than in black South Africans [36].

Utilizing the measurement of urinary caffeine metabolites to assess metabolic phenotype for CYP1A2 activation and NAT2 detoxification pathways [37], [38], [39], we sought to determine if racial differences in the biochemical activation or detoxification of arylamines might contribute to the higher incidence rates of bladder cancer in whites than in blacks. Thus, the current study compares the CYP1A2, NAT2 and their combined phenotypes in a large group of black and white adult smokers. Since polymorphisms in glutathione transferases (GST), including GSTM1-null, together with slow acetylation phenotype have been linked with increased risk of bladder cancer [40], [41], [42], [43] and GSTM1 null genotype was associated with enhanced 4-ABP hemoglobin adducts [44], we also examined the effects of GSTM polymorphisms on CYP1A2 and NAT2 activity.

Section snippets

Materials

Caffeine (137X) and its metabolites 1,7-dimethylxanthine (17X), 1-methylxanthine (1X), 1,7-dimethyluric acid (17U), and 1-methyluric acid (1U) were obtained from Sigma Chemical Co. (St. Louis, MO). 5-Acetylamino-6-formylamino-3-methyluracil was obtained from Dr. B.K. Tang at the University of Toronto. All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.

Subjects and study design

There were 348 (165 non-Hispanic black and 183 white) current cigarette smokers who participated

Results

The subjects included 82 black men, 83 black women, 89 white men and 94 white women. The mean age was 34.8 in blacks and 33.2 in whites, and the age of smoking onset and total years of smoking were similar between blacks and whites (Table 1). The mean daily cigarette intake was 14.2 in blacks and 22.3 in whites. More blacks than whites smoked mentholated cigarettes (83.6% vs. 23.0%, P < 0.01). AFMU/1X values were determined for 330 subjects.

The correlation coefficients for age, body mass index

Discussion

The phenotypic characterization of CYP1A2 can be subjective, with as many as 50% of subjects classified as rapid activators. Butler et al. [20] reported a trimodal distribution (17X + 17U)/137X in a number of different populations of 12–13% slow, 51–67% intermediate, and 20–37% rapid. This was similar to our trimodal distribution (10%, 54% and 37%). The bimodal distribution of 17X/137X indicated that about 30% of our subjects were rapid activators, which was similar to findings reported elsewhere

Acknowledgements

We thank Julie Cox and Daniella Scott for their efforts in subject recruiting and Steven Colosimo, Alex Fair and Faith Azuogu for their technical assistance. This work was supported by NIH grants CA68384 and K07 CA104231.

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