In the century since Rehn (1895) first observed a high incidence of bladder cancer in workers employed in the dye manufacturing industry, epidemiological studies have established a clear causal link between environmental exposure to arylamine procarcinogens such as 2-naphthylamine and 4-aminobiphenyl and the incidence of human bladder cancer (Cartwright et al., 1982;Cohen & Johannson, 1992;Silverman et al., 1992). Arylamines are not carcinogenic in the parent form, but require metabolic activation to reactive electrophiles in order to exert their carcinogenic effects (Guengerich, 1992;Grant et al., 1997). The enzyme pathways which may be involved in arylamine biotransformation are complex (for a review, see Grant et al., 1997). Among the enzymes believed to be central to this process for a number of arylamines are cytochrome P4501A2 (CYP1A2) and two acetyltransferases, NAT1 and NAT2, which take part in competing detoxification and activation steps. Each of these enzymes possesses a characteristic pattern of interindividual variation in human populations.
Hepatic CYP1A2 has been proposed to mediate a critical first step in the bioactivation of certain arylamine procarcinogens via the formation of hydroxylamines (Kadlubar et al., 1977;Guengerich & Shimada, 1991). Interindividual variations in CYP1A2 activity have been attributed largely to the effects of environmental inducers such as the polycyclic aromatic hydrocarbons (PAHs) found in cigarette smoke (Kadlubar et al., 1992). Evidence linking elevated CYP1A2 activity with increased bladder cancer risk has been reported (Kaderlik & Kadlubar, 1995). In the liver, NAT2 competes with CYP1A2 for arylamine substrates, catalysing an N-acetylation reaction that produces non-toxic arylamides. NAT2 displays a liver- and gut-selective pattern of expression, and is also known to be genetically polymorphic, with over 50% of Caucasian individuals being identified as phenotypically ‘slow acetylators'. Thus, individuals with the slow acetylator phenotype would be deficient in this liver detoxification pathway, and the activation pathway via hydroxylamine formation could therefore predominate (Grant et al., 1997). Early evidence linking the ‘slow acetylator’ phenotype with the incidence of bladder cancer has been provided by several epidemiological studies, the association being strongest in subjects with documented exposure to arylamines (Cartwright et al., 1982;Vineis, 1992;Ritsch et al., 1995). Hydroxylamines, once formed, may either be transported to the bladder via the bloodstream or could be further metabolized in the liver by other conjugating enzymes, such as UDP-glucuronosyltransferase (UGT). Under the more acidic conditions of the bladder lumen, such conjugates may spontaneously hydrolyse to regenerate the hydroxylamine. To account for the observed bladder selectivity of arylamine carcinogens, it is also plausible that further bioactivation step(s) may occur locally in the bladder itself. In this regard, in addition to the detoxifying N-acetylation reactions already described, NAT1 and NAT2 have also been shown to participate in metabolic activation reactions via O-acetylation and N, O-acetyltransfer of N-hydroxy arylamine derivatives. The products of such reactions are unstable acetoxy esters which break down to form electrophilic, DNA- reactive nitrenium ions.
Since NAT1 has been shown to be expressed in most tissues, including the luminal epithelial cells of the bladder (Hein et al., 1992;Bell et al., 1995a), and NAT2 expression in bladder remains to be confirmed, it has been speculated that high NAT1-mediated O-acetylation activity in this tissue may be a factor for increased bladder cancer risk. In recent years, the search for a genetic basis for variations in NAT1 activity has led to the discovery of a number of NAT1 allelic variants (Vatsis & Weber, 1993;Weber & Vatsis, 1993;Hughes et al., 1998). Evidence has suggested that one such variant, NAT1*10, may be associated with elevated NAT1 activity (Badawi et al., 1995;Bell et al., 1995a) and an increased risk for cancers of the colon (Bell et al., 1995b) and bladder (Taylor et al., 1998), with these associations being strongest among smokers.
The first objective of the present study was to compare the catalytic activities of NAT1, NAT2 and CYP1A2 in normal human bladder epithelium. The bladder, although being a relatively selective target organ for carcinogens of the arylamine class, is distal to initial sites of exposure to these chemicals. Thus, it would follow that one or more localized bioactivation steps within the bladder epithelium could partly account for this tissue selectivity. The distinct kinetic selectivities of NAT1 and NAT2 for the N- versus O-acetylation of different homo- and heterocyclic amines (Hein et al., 1993a,b;Wild et al., 1995), coupled with differences in the levels of expression of these two enzymes in the liver and bladder, could explain the observation that increased bladder cancer risk is associated with decreased NAT2 but increased NAT1 activity. Previous studies have shown that N- and O-acetylation of arylamine procarcinogens can occur in human bladder cytosol (Kirlin et al., 1989;Land et al., 1989) and in bladder epithelial cells in culture (Frederickson et al., 1992;Swaminathan & Reznikoff, 1992), although it is generally unclear from these earlier studies whether the detected activities are mediated by NAT1 or the classically polymorphic NAT2. NAT2 activity has so far been detected predominantly in the liver (Grant et al., 1991) and gut (Kirlin et al., 1991), while NAT1 may have a more ubiquitous expression (Hanna, 1994), including in blood (Weber & Vatsis, 1993). CYP1A2 is generally considered to be expressed predominantly in the liver, both constitutively and as a result of induction (McKinnon et al., 1991;Schweikl et al., 1993). Although, previous immunoblotting assays have demonstrated no CYP1A2 protein expression in bladder (Kadlubar & Guengerich, 1992), the presence of any low levels of CYP1A2 activity remains to be determined.
Our second objective was to test the hypothesis that bladder cancer patients display high CYP1A2, low NAT2 and high NAT1 activities compared to control subjects. We undertook a case–control study of 53 patients diagnosed with transitional cell carcinoma of the bladder and 96 cancer-free control subjects matched for age, gender and smoking status. Subjects were administered the in-vivo probe drugs caffeine, to provide indices of CYP1A2 and NAT2 function, and PAS to provide indices of NAT1 function. NAT1 and NAT2 genotypes were also determined. A conditional logistic regression model was used to compare measures of enzyme activity.
Materials and methods
All procedures were approved by Research Ethics Boards of the Hospital for Sick Children, the Toronto General Hospital, and the University of Toronto, and all participants gave their informed consent to participate. Patients diagnosed with new or recurrent transitional cell carcinoma (TCC) were recruited in Toronto at the Division of Urology, Toronto General Hospital and at the Princess Margaret Hospital. Control subjects were recruited primarily through advertisements placed in seniors’ newspapers and hospital bulletins. Patients had a positive diagnosis of TCC of the bladder and no other cancer, while control subjects had no self-reported history of any cancer. Normal bladder epithelium was obtained from a total of 103 TCC patients. A subset of 53 patients and 96 matched control subjects were enrolled in the probe drug study. Prior to participation in the drug study, subjects were informed of the dietary restrictions which included abstention from alcohol, methylxanthine-containing products and grapefruit products [which contain potent CYP1A2 inhibitors (Fuhr et al., 1993)] from 16 h before the study until the end of the 6-h study period. Questionnaires were administered to all subjects to record gender, age, ethnic background, occupational and smoking histories. Smoking status was coded as a 3-level variable, with individuals categorized as nonsmokers, current smokers or former smokers. Non-smokers were defined as subjects who were not currently smoking and had smoked less than 100 cigarettes in their lifetime. Former smokers were defined as subjects who had stopped smoking at least 2 weeks prior to the study. Occupational risk for exposure to arylamine carcinogens was coded as a 2-level variable: little or no risk, and high risk. High risk occupations were defined a priori on the basis of earlier epidemiological data (Hein et al., 1992;Silverman et al., 1992). Subjects were considered to have previous occupational risk if they had occupied one or more high risk occupations for more than 1 year. Matched sets of patients and control subjects were formed based on gender, age (within 5 years) and smoking status.
TISSUE COLLECTION AND PROCESSING
Samples of bladder epithelium, determined as normal by a urological surgeon, were obtained by transurethral cold-cup biopsy from 103 patients diagnosed with transitional cell carcinoma of the bladder. Samples were promptly immersed in liquid nitrogen and stored at –80 °C until use. Frozen tissue samples (50–200 mg) were powdered with a liquid nitrogen-cooled shock gun and homogenized in 600 μl of TEDK buffer (10 mm triethanolamine; 1 mm EDTA; 50 mm KCl; 1 mm DTT; pH 7.0). Homogenates were centrifuged at 4 °C at 9000 g for 20 min. Freshly prepared supernatants were used as the source of enzymes for NAT1 and NAT2 activity assays. A sufficient quantity of bladder tissue was obtained from six patients undergoing total or partial cystectomies to enable the isolation of microsomal fractions. In this case, frozen samples of bladder tissue (approximately 2 g) were powdered, homogenized in two volumes of 100 mm Tris-KCl, pH 7.4, and 9000 g supernatants were further centrifuged at 100 000 g for 1 h. Microsomal pellets were resuspended in Tris-KCl and recentrifuged under the same conditions. The final microsomal pellets were resuspended in the Tris-KCl buffer. Freshly prepared microsomal fractions were used for CYP1A2 activity assays and for Western blotting procedures. Recombinant wild-type NAT1 and NAT2 as positive controls were expressed in E. coli and prepared as previously described (Dupret & Grant, 1992). Protein contents were determined using a dye-binding method (Bradford, 1976).
IN-VITRO ENZYME ASSAYS
HPLC was used to quantify product formation by NAT1, NAT2 and CYP1A2. All HPLC analyses were performed using a Beckman Ultrasphere ODS 5 μm column (150 × 4.6 mm) (Beckman Instruments Inc., Fullerton, CA, USA) with a Shimadzu system, consisting of two LC6A pumps (Shimadzu Scientific Instruments Inc., Columbia, MD, USA), a SIL6B autosampler (Shimadzu Scientific Instruments Inc.), and a SPD6A variable-wavelength ultraviolet absorbance detector (Shimadzu Scientific Instruments Inc.).
Bladder 9000 g supernatant fractions (diluted 50-fold in TEDK buffer) were assayed for NAT1 activity using PAS as an acceptor substrate as described elsewhere (Hughes et al., 1998), using PAS concentrations of 5, 10, 15, 20, 50, 100 and 200 μm, and an acetyl CoA cofactor concentration of 100 μm, maintained at a constant level by a cofactor regenerating system. Apparent Michaelis kinetic constants (Km) and maximal velocities (Vmax) were estimated by non-linear regression using a curve-fitting software program (K CatTM, Biosoft, Cambridge, UK).
Incubations for NAT2 activity measurements were performed essentially as described for the NAT1 assays except that samples of undiluted bladder 9000 g supernatant were used, and SMZ was used as a NAT2-selective substrate. Because of the generally low rate of turnover of this substrate, sufficient quantities of tissue were available to determine SMZ acetylation at no more than 1 substrate concentration (200 μm). Thus, enzyme velocities were compared using this substrate concentration in the 94 individuals from whom this measurement could be performed. Reactions were carried out for 2 h at 37 °C and were stopped with the addition of 10 μl of 15% perchloric acid. Supernatants were analysed by isocratic HPLC at a flow rate of 2 ml/min and detection of product formation by ultraviolet absorbance at 254 nm. The mobile phase was 90% solvent A : 10% solvent B (w/w), where solvent A consisted of 23.1 mm sodium perchlorate, and 19.1 μm perchloric acid and solvent B was 100% methanol.
The O-deethylation of phenacetin was used as a selective and sensitive measure of CYP1A2 activity to test for its presence in bladder microsomal fractions, and was performed essentially as previously described (Butler et al., 1989). Reactions were carried out for 1 h at 37 °C, with a single substrate concentration of 100 μm phenacetin. Phenacetin and its metabolite, acetaminophen, were analysed by gradient HPLC, with a flow rate of 1 ml/min and a ultraviolet absorbance detector setting of 254 nm. Solvent A was 0.5% acetic acid (v/v) and solvent B was 100% methanol. The gradient program was 6% B for 0–12 min; 6–80% B for 12–14 min; 80% B for 14–18 min; 80–6% B for 18–20 min; 6% B for 20–22 min. The retention times of acetaminophen and phenacetin were 10.9 min and 19.3 min, respectively. Human liver microsomes were used as a positive control for this assay, since CYP1A2 is known to be expressed in this tissue.
DETECTION OF IMMUNOREACTIVE CYP1A2 PROTEIN BY WESTERN BLOTTING
Bladder or liver microsomal protein (40 μg/lane) or recombinant CYP1A2 (20 μg/lane) were separated electrophoretically on 9% sodium dodecyl sulfate-polyacrylamide gels as described previously (Butler et al., 1992). Proteins were transferred to nitrocellulose membranes and incubated with rabbit anti-human CYP1A2 antiserum (a gift from Dr J.S. Leeder). An enhanced chemiluminescence detection system (Amersham, Bucks, UK) was used to visualize immunoreactive protein bands.
IN-VIVO PROBE DRUG STUDY
On the morning of the study day, each subject was given 200 mg of caffeine (Wake-UpsTM, Adrem Ltd., Ontario, Canada) at t = 0 h, and 500 mg of Nemasol Sodium (ICN Pharmaceuticals, Montreal, Canada), equivalent to 365 mg of PAS, at t = 4 h in a hospital setting by a registered nurse. Venous blood was drawn at t = 1 h and t = 5 h and plasma was prepared. Two additional aliquots of blood were taken from each subject at t = 1 h. From one of these aliquots (12 ml), genomic DNA was isolated (Miller et al., 1988), and the second (1 ml) was immediately shock-frozen in liquid nitrogen and stored at –80 °C for later analysis of whole blood NAT1 activity. Each subject also provided a 2-h urine sample for the interval from 0 to 2 h following PAS administration (4–6 h after caffeine administration). Urine samples were acidified to pH 3.5, and urine and plasma samples were stored at –20 °C until the time of analysis.
Urine samples were processed for caffeine and its metabolites using a modification of the method described by Grant et al. (1984). Briefly, 40 μl aliquots of urine, saturated with ammonium sulfate, were spiked with 40 μl of acetaminophen (120 μg/ml; internal standard) and extracted with 1 ml of chloroform/isopropanol (90 : 10, v/v). Organic phases were dried under a stream of nitrogen and residues were resuspended in 100 μl of mobile phase. Caffeine metabolites were analysed by gradient HPLC using a Beckman Ultrasphere ODS 5 μm column (250 × 4.6 mm) with a flow rate of 1.1 ml/min and ultraviolet absorbance setting at 280 nm. Solvent A consisted of 0.05% acetic acid (v/v) and solvent B consisted of 100% methanol. The gradient program was 0–5 min, 8% B; 5–15 min, 8–10% B; 15–18 min, 10–20% B; 18–30 min, 20% B; 30–32 min, 20–70% B; 32–35 min, 70% B; 35–37 min, 70–8% B; 37–40 min, 8% B. Concentrations of caffeine and its metabolites were quantified by comparing their peak heights, relative to the internal standard, with that of a sample of blank urine spiked with known amounts of caffeine and each of its metabolites. The urinary index used to determine in-vivo CYP1A2 activity was the molar ratio (AFMU + 1U + 1X)/17U.
Aliquots of plasma were also analysed for caffeine and its dimethylxanthine metabolites as described for urine, except that extractions were carried out using 100% chloroform. The plasma index used to determine in-vivo CYP1A2 activity was the molar ratio of paraxanthine/caffeine (17X/137X) in the 5-h plasma sample. Caffeine plasma clearance was also estimated from the area under the concentration–time curve derived using the plasma samples taken 1 and 5 h after caffeine administration.
The molar ratio of AcPAS/PAS in urine and plasma, and the N-acetylation of PAS by a sample of whole blood lysate were determined as described previously (Hughes et al., 1998).
Urine samples were analysed for caffeine metabolites as described for the CYP1A2 phenotyping. The urinary indices used to determine in-vivo NAT2 phenotypes were the molar ratios AFMU/1X and AFMU/(AFMU + 1X + 1U).
The method described by Hughes et al. (1998) was used to detect mutations in the following NAT1 alleles:NAT1*10 (T1088A), NAT1*14A (T1088A, G560A), NAT1*14B (G560A), NAT1*11 (T640G, Δ1075), and NAT1*15 (C559T). To determine the presence of the A752T mutation (present in NAT1*22), the NAT1 gene was amplified using N2PC5 and N1PC3 primers as described previously (Lin et al., 1998). An aliquot of the amplified fragment was digested with Bgl II and restriction fragments were separated on a 2% agarose gel.
NAT2 genotyping was performed as previously described (Deloménie et al., 1996) to detect mutations in the following NAT2 alleles:NAT2*5A (T341C, C481T), NAT2*5B (T341C, C481T, A803G), NAT2*6A (C282T, G590A) NAT2*6B (G590A), NAT2*7A (G857A), NAT2*7B (C282T, G857A), NAT2*12A (A803G), NAT2*12B (C282T, A803G), NAT2*13 (C282T), NAT2*14B (G191A, C282T).
Conditional logistic regression (CLR) analysis using the EGRETTM program (Cytel Software Corp., Cambridge, MA, USA) was used to compute an odds ratio estimate (OR) in order to compare activities of each of the enzyme phenotyping parameters between patients and control subjects. ANOVA was used to compare the CYP1A2 enzyme activity among the three smoking status levels. Correlation tests were performed using the Pearson moment correlation coefficient. Differences in mean in-vitro NAT1 enzyme activities across NAT1 genotypes were determined using the Mann–Whitney U-test.
IN-VITRO BLADDER ENZYME ACTIVITIES
NAT1 activity was detected in all 103 bladder samples, with a mean apparent Vmax of 1.93 ± 2.03 nmol AcPAS formed/min/mg protein and a range of 0.01–13.31 nmol/min/mg protein. NAT1 genotyping information was available on 44 patients. In agreement with recent data reported elsewhere on blood NAT1 activity (Hughes et al., 1998), we found no association between the presence of the NAT1*10 allele and elevated NAT1 bladder enzyme activity, except for one sample with elevated NAT1 activity. (Fig. 1). Samples harbouring the NAT1*14A and NAT1*22 alleles displayed NAT1 activities that were lower than the mean activities associated with the NAT1*4/NAT1*4 genotype (Fig. 1). We found no correlation between bladder cytosols and blood lysates for either Vmax (r = 0.01, P > 0.1, n = 44) or Km (r = 0.04, P > 0.1, n = 44) values for PAS acetylation (data not shown). The sample with elevated NAT1 bladder activity showed blood NAT1 activity within normal range (97.3 pmol/min/mg protein;Fig. 2).
Sufficient tissue was obtained from 94 of the 103 patients for determination of SMZ acetylating activity as an indicator of NAT2 function. SMZ acetylation was detected in 86 of the samples. NAT2 genotyping information was available on 44 of these patients. The mean rate of AcSMZ formation at a substrate concentration of 200 μm was 4.27 ± 3.89 pmol/min/mg, with a range of 0.1–18.8 pmol/min/mg. The mean activity in rapid acetylators (4.58 ± 3.91 pmol/min/mg;n = 21) was higher than that measured in slow acetylators (2.97 ± 2.78 pmol/min/mg;n = 23), but this difference was not statistically significant (P > 0.1). Since NAT1 can also metabolize SMZ, although with a 50-fold lower affinity for SMZ than NAT2 (Dupret et al., 1994;Deloménie et al., 1997), we correlated bladder PAS and SMZ acetylation rates to determine whether the observed SMZ acetylation activities were mediated by NAT2 or by NAT1. PAS and SMZ acetylation activities in bladder did not correlate significantly (r = 0.11;P > 0.1, data not shown). Although this may partly reflect the extremely low levels of AcSMZ detectable in some cases, the lack of correlation also supports the suggestion that NAT2 contributes to SMZ acetylation in bladder.
We found no evidence for CYP1A2 activity using microsomal preparations from bladder mucosa using the phenacetin-O-deethylase assay, even after prolonged incubation times with high microsomal protein concentrations. Human liver microsomes were used as a positive control and, under the same conditions displayed an activity of 40 pmol/min/mg protein. This was 100-fold higher than the limit of detection (0.4 pmol/min/mg protein). There was also no evidence of CYP1A2 immunoreactive protein in bladder on Western blots (data not shown). Both the lack of detectable CYP1A2 catalytic activity and the lack of immunoreactive CYP1A2 protein in bladder suggest that CYP1A2 is not likely to be expressed in human bladder tissue to any significant degree.
Patient and control profiles
A summary of patient and control profiles is presented in Table 1. As expected, there was a higher frequency of male bladder cancer cases than female cases, since studies have demonstrated that bladder cancer occurs more frequently in men (Cohen & Johannsson, 1992;Silverman et al., 1992). Due to matching, the mean ages for both patients (65.8 years) and control subjects (64.2 years) were similar. Similarly, the frequency of subjects, both patients and control subjects, who had formerly smoked was the highest, at 66.0% and 64.6%, respectively. The control group had a slightly lower frequency of individuals who had ever worked in occupations associated with possible exposure to arylamine carcinogens (43.7%) compared with the patients (50.9%). Caucasian was the most prevalent ethnicity in both groups.
NAT1 acetylator phenotypes and genotypes
NAT1 activity was determined using three indices: (i) the plasma ratio AcPAS/PAS, measured 1 h after PAS administration; (ii) the urinary ratio AcPAS/PAS, determined in urine samples collected for the 0–2 h interval following PAS administration; and (iii) NAT1 enzyme activity determined directly in whole blood lysates in vitro. There were no statistically significant differences in predicted NAT1 activity between patients and control subjects using the in-vivo plasma index (P = 0.08) or the in-vitro NAT1 activity blood assay (P = 0.25;Table 2). There was, however, a higher urinary ratio index in the patient population than in control subjects (P = 0.04;Table 3), which was found to be largely due to lower levels of PAS among patients compared to control subjects [patients: 155.5 ± 229.8 nmol/ml, n = 47; control subjects 257.5 ± 299.4 nmol/ml, n = 88; OR = 1.23; 95% confidence interval (CI) = 0.94, 1.41;P = 0.06] and not higher levels of AcPAS (patients: 6109.4 ± 9656.8; control subjects: 5801.0 ± 5721.2; OR = 1.01; 95% CI = 0.92, 1.10;P = 0.45). Although this decrease in urinary PAS among patients compared to control subjects was of borderline significance, it may have contributed to the elevated urinary AcPAS/PAS ratio observed in the patients. This effect may indicate an as yet unknown alternative pathway of PAS metabolism which could be more significant in patients than in control subjects. The indices of NAT1 function were poorly correlated with each other (data not shown). In order to determine the most valid index of NAT1 activity, correlations with NAT1 genotypes were carried out.
The distribution of NAT1 alleles was similar between patients and control subjects (Table 3). There was no association between the presence of any of the NAT1 alleles and NAT1 activity as predicted using either the plasma or urinary indices (data not shown). However, the in-vitro NAT1 blood assay did show a phenotype–genotype association, since NAT1 activity using this test was found to be lowest among subjects with the NAT1*4/14A, NAT1*10/14A and NAT1*4/22 genotypes (Fig. 2). NAT1*14A and NAT1*22 have been previously demonstrated to encode proteins with defective activity (Hughes et al., 1998;Lin et al., 1998). Thus, the in-vitro blood assay was selected to be the most accurate predictor of NAT1 activity.
NAT2 phenotypes and genotypes
In-vivo NAT2 activity was determined using two indices: (i) the urinary molar ratio AFMU/1X and (ii) the urinary molar ratio AFMU/(AFMU + 1X + 1U), each determined in a pooled urine sample for the interval from 4 to 6 h after caffeine administration. A bimodal distribution of rapid and slow NAT2 acetylators was observed using both ratios. The AFMU/1X and AFMU/(AFMU + 1X + 1U) ratios were highly correlated; however, two subjects were phenotyped as rapid acetylators using the AFMU/1X ratio but were phenotypically (and genotypically) slow acetylators using the AFMU/(AFMU + 1X + 1U) ratio. The reason for this phenotyping discrepancy was found to be due to elevated xanthine oxidase activity (decreased 1X levels and increased 1U levels) in these two subjects (data not shown). The AFMU/(AFMU + 1X + 1U) urinary index was therefore selected as the best index of NAT2 activity for this study because it is not affected by variations in xanthine oxidase activity. This index lacked statistical power to detect differences less than 15%. Nevertheless, we found no statistically significant difference in the frequency of NAT2 slow acetylators between patients (60.4%;n = 53) and control subjects (58.3%;n = 96; OR = 0.98; 95% CI = 0.49, 1.96;P = 0.96). At the genetic level, the distribution of NAT2 allelic variants was similar for patients and control subjects except for a higher frequency of the NAT2*5A allele in patients than in control subjects (P = 0.02;Table 4).
CYP1A2 oxidizer phenotypes
In-vivo CYP1A2 activity was determined using three indices: (i) caffeine plasma clearance determined using samples collected 1 h and 5 h after caffeine administration; (ii) the plasma ratio 17X/137X measured 5 h after caffeine administration; and (iii) the urinary molar ratio (AFMU + 1X + 1U)/17U determined in pooled urine for the 4–6 h time interval after caffeine administration. There were no statistically significant differences between patients and control subjects with respect to any of these indices of CYP1A2 activity (Table 5). The three indices were highly correlated with each other (data not shown).
It has been previously demonstrated that cigarette smoking induces CYP1A2 activity (Kadlubar et al., 1992). In this study, we determined whether any of the three indices for CYP1A2 activity was elevated in smokers. Each of the CYP1A2 indices was significantly higher in smokers than in nonsmokers or former smokers (data not shown). It was not possible to determine values for caffeine clearance in approximately 20% of the subjects, due to the observation of higher plasma caffeine levels at 5 h than at 1 h after drug administration. This could have been a result of either delayed caffeine absorption or non-compliance with dietary caffeine abstention during the course of the study period. The urinary ratio, which is less sensitive to variations in caffeine absorption than the plasma indices (Grant et al., 1984), was therefore selected as the most robust index of CYP1A2 activity.
This study demonstrates that variable levels of NAT1 and NAT2 arylamine acetyltransferase activity are expressed in normal human bladder epithelium. We found that NAT1 is expressed in human bladder, with activity levels comparable with, if not slightly higher than, those reported previously in liver (Grant et al., 1991). This suggests that NAT1-mediated N- and O-acetylation of arylamines and their hydroxylamine metabolites may occur locally in the bladder. Comparisons between bladder NAT1 activity and genotype were possible in 44 of the 103 subjects. These results confirm those of our phenotyping study (Hughes et al., 1998), indicating that, except for one outlier, there are no differences in NAT1 activity between subjects with the NAT1*4 and NAT1*10 genotypes (Fig. 1). This is in contrast to a previous study which had indicated that NAT1 activity was significantly elevated in bladder and colon among individuals possessing the NAT1*10 variant (Bell et al., 1995a). One possible reason for this discrepancy may be the smaller sample size in the previous study.
Little is known of the relative levels of NAT1 expression in various tissues. Here we report an approximately 10-fold higher expression of NAT1 in bladder than in blood lysates among the 44 patients from whom both blood and bladder samples were obtained. However, we found no significant correlation between NAT1 activity in bladder and that determined using whole blood lysates. This could be due to differences in the tissue-selective pattern of expression of NAT1 activity. The range of activities may also preclude any correlative associations between the tissues other than large variations associated with specific genotypes. For example, although the sample size was small, the NAT1*14 allele was associated with decreased activity in both bladder and blood activity assays (Figs 1 and 2).
To date, it has not been clearly established whether NAT2 is expressed in bladder tissue. We have recently found evidence of NAT2 mRNA expression in bladder using hybridization histochemistry methods (Windmill et al., 2000). Although previous Western blotting analyses failed to detect NAT2 immunoreactive protein in bladder (Stanley et al., 1996), the authors indicated that the antibodies may have been of insufficient affinity to detect low NAT2 levels. Badawi et al. (1995) did not detect the acetylation of NAT2-selective SMZ in bladder. We detected SMZ N-acetylation in 80% of the bladder samples we analysed, since the detection limit of our assay (0.05 pmol AcSMZ/min/mg protein) is 20-fold lower than that reported by Badawi and coworkers. Further evidence for NAT2 expression in bladder comes from the following observations. First, the lack of correlation between PAS and SMZ N-acetylation rates in bladder suggests that NAT1 is not the only enzyme mediating SMZ N-acetylation. Second, the trend towards higher SMZ N-acetylation rates in subjects who were rapid acetylators (4.58 ± 3.91 pmol/min/mg protein) compared to the slow acetylators (2.97 ± 2.78 pmol/min/mg protein), although not statistically significant, suggests that NAT2 may be expressed at low levels in the bladder. This difference would likely be more marked if it were not for the significant contribution of NAT1 to the acetylation of SMZ.
The possibility of NAT2 expression in the bladder has obvious potential toxicological significance. Both NAT1 and NAT2 can mediate the O-acetylation of N-hydroxylamines to proximate acetoxy ester carcinogens (Hein et al., 1993a,b). However, until recently, variation in O-acetyltransferase (OAT) activity in the bladder was believed to be attributed to variation in the expression of NAT1 alone. If NAT2 is also expressed in bladder, then variation in its expression likely also contributes to variation in OAT activity. Indeed, although Badawi et al. (1995) did not detect NAT2-mediated SMZ N-acetylation in bladder, the lack of correlation they observed between OAT and NAT1 activity led them also to the conclusion that some OAT activity may be mediated by low levels of NAT2 expressed in bladder. In light of the distinct substrate selectivities of NAT1 and NAT2 for N- and O-acetylation reactions, expression of both of these enzymes in bladder may have important implications for the risk of cancers in this organ due to the competition between these detoxifying and bioactivating pathways, which will also depend upon the particular arylamine to which the organ is exposed.
We did not detect CYP1A2 activity in bladder using either functional or immunoblotting assays. Although this is not an unexpected finding, since CYP1A2 is generally considered to be expressed in a liver-specific fashion, it nonetheless has important implications for pathways of arylamine metabolic activation and the tissue-selectivity of cancers associated with arylamine exposures. CYP1A2-mediated N-hydroxylation is considered to be a prerequisite first step in the bioactivation of many homo- and heterocyclic amines. If this is indeed so, our finding implies that this reaction must take place at a site distant from the bladder. Thus, a mechanism for arylamine-induced bladder cancer must include transport of the hydroxylamine from its site of formation to the bladder lumen where the initiation of epithelial cell transformation takes place.
In addition to determining the activity of these enzymes in human bladder, we investigated phenotypic and genotypic profiles in CYP1A2, NAT2 and NAT1 among bladder cancer patients and cancer-free control subjects using a case–control study design. A positive association between elevated CYP1A2 activity and bladder cancer risk has been previously suggested (Beland & Kadlubar, 1986;Kaderlik & Kadlubar, 1995). After controlling for smoking status, we found no differences in CYP1A2 activity between TCC patients and control subjects. Cigarette smoke is a source of arylamine carcinogens which are bioactivated by CYP1A2 (Kadlubar et al., 1988) but also contains PAHs which induce CYP1A2 activity (Campbell et al., 1987a). In our study, current smokers were found to have significantly higher CYP1A2 activity than nonsmokers or former smokers, consistent with results from other studies (Campbell et al., 1987b;Kadlubar et al., 1992). However, since CYP1A2 is an inducible enzyme, it is likely that CYP1A2 activity at the time of initial carcinogen exposure was different from that predicted from our probe drug study. Thus interpretation of the information obtained from the CYP1A2 index as it relates to bladder cancer risk must be made cautiously.
In this study, we tested three indices of NAT1 activity to predict NAT1 phenotype. Of these, we selected the in-vitro whole blood PAS acetylation assay as the most appropriate for two reasons. First, the in-vitro whole blood assay is not susceptible to variability due to interpatient variations in competing pathways for PAS biotransformation (Mandell & Sande, 1985), flow-dependent excretion kinetics, and renal function which likely contribute to variations in the in-vivo plasma and urinary indices. The second reason is that the in-vitro whole blood assay showed the strongest association with activities predicted from NAT1 genotyping. NAT1*14A and NAT1*22 alleles have been previously shown to express proteins with defective NAT1 activity (Butcher et al., 1998;Hughes et al., 1998;Lin et al., 1998;Payton & Sim, 1998).
There has been considerable interest in the NAT1 allelic variant, NAT1*10 (Badawi et al., 1995;Bell et al., 1995a;Taylor et al., 1998), which has been reported to be associated with elevated levels of NAT1 activity. This NAT1 variant was shown to occur more frequently among colorectal cancer patients than in control subjects (Bell et al., 1995b). Taylor et al. (1998) reported a significant association between the NAT1*10 allele and increased bladder cancer risk among heavy smokers (approximately 50 pack years). However, Probst-Hensch et al. (1996) reported no association between the NAT1*10 variant and colon cancer. Our study showed that the NAT1*10 allele did not occur more frequently in bladder cancer patients compared to control subjects (Table 4). This was also reported by Okkels et al. (1997). We also found no association between the presence of the NAT1*10 allele and elevated NAT1 activity using any in-vivo or in-vitro index of activity in our probe drug study, or by analysis of the activity directly in bladder tissue. This is in agreement with results reported previously by our laboratory (Hughes et al., 1998) and by Bruhn et al. (1999). Overall, neither our NAT1 phenotyping nor genotyping analyses revealed any differences between patients and control subjects. One limitation to the NAT1*10 genotyping assay in our study is that the test we employed does not distinguish the NAT1*10 allele from that of the NAT1*18A or the NAT1*29 (NAT database: http://www.louisville.edu/medschool/pharmacology/NAT. html). These variants have additional mutations in the 3′ untranslated region of NAT1 and have been reported to occur infrequently in the Caucasian and Japanese populations (Lo-Guidice et al., 2000;Yang et al., 2000).
We also investigated the association between the NAT2 acetylator phenotype and bladder cancer incidence. We observed no significant differences in the proportion of rapid and slow acetylators between the patient and control groups. This is consistent with a previous study that reported similar NAT2 phenotype frequencies among 374 bladder cancer patients and 372 controls (Brockmöller et al., 1996). This is in contrast to several other studies that had suggested a significant association between the slow NAT2 phenotype and bladder cancer risk (Lower et al., 1979;Cartwright et al., 1982;Ritsch et al., 1995). These inconsistencies highlight the need for large multicentre studies to investigate the extent to which variations in the phenotype of modifying enzymes affect cancer risk. Meta-analysis studies of multiple epidemiological reports combining a large number of bladder cancer cases indicate that NAT2 does confer a modest risk for bladder cancer risk (d'Errico et al., 1996;Marcus et al., 2000a) particularly among smokers (Marcus et al., 2000b). Data collection from multiple current studies is in progress to perform additional analyses of NAT2 activity and that of other biotransforming enzymes with respect to cancer risk (Taioli, 1999).
The association of specific NAT2 genotypes and bladder cancer risk has not been extensively reported. In our study, the NAT2*5A genotype occurred more frequently among the patients compared to the controls. Okkels et al. (1997) had also reported that, among smokers, the NAT2*5 alleles occurred more frequently among bladder cancer patients compared to controls. The significance of this is at present unknown. Interestingly, the recombinant human NAT2*5 alleles, particularly NAT2*5A, confer the lowest NAT, OAT and N,O-AT activities compared to the NAT2*6, 7, 12, 13 and 14 alleles (Hein et al., 1995).
Variations in the activity of CYP1A2, NAT1 and NAT2 have been previously suggested to be associated with bladder cancer risk, but it should be emphasized that any effect is strongly dependent on exposure (Caporaso & Goldstein, 1995). With no exposure to arylamine carcinogens, there is no increased risk for bladder cancer regardless of the activity of the activating enzymes. Moreover, at extremely high exposure levels (either through occupational exposure or cigarette smoking, two common but distinct sources of arylamine exposure) bladder cancer may occur regardless of variation in the activity of activating enzymes (Hayes et al., 1993;Hirvonen et al., 1994). The activities of the many other competing biotransforming enzymes that can metabolize arylamine procarcinogens will also significantly impact the extent to which CYP1A2, NAT1 and NAT2 will metabolize arylamine procarcinogens. Variation in the activity of such other enzymes has also been suggested to mediate risk for bladder cancer development, including glutathione-S-transferases (Brockmöller et al., 1994), sulfotransferases (Meerman et al., 1994), glucuronosyltransferases (Orzechowski et al., 1994), CYP2C19, CYP2D6, CYP3A4 (Branch et al., 1995) and PHS (Zenzer & Davis, 1988).
This research was supported by the National Cancer Institute of Canada (Research Grant 046) with funds from the Canadian Cancer Society, by a post-doctoral Fellowship (N.C.H) from the Research Institute, Hospital for Sick Children, and by studentships (S.A.J.V) from the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Health. D.M.G. is a Scientist of the Medical Research Council of Canada.
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