N-acetyltransferase 2 Phenotype, Occupation, and Bladder Cancer Risk: Results from the EPIC Cohort

Discussion

When reassessing the role of metabolic genes as susceptibility factors, it has been concluded that NAT2 might be one of the few examples of a consistent association with cancer (23). The study of NAT2 polymorphisms has been supported by the mechanistic assumption that slow acetylation could be associated with increased bladder cancer risk, given the role of NAT2 in the metabolism of aromatic amines (24). Here, we took advantage of the large database and biorepositories of the EPIC to explore NAT2 slow acetylation. However, we found no main effect and could not observe support for an effect modification of the association of bladder cancer with exposure to tobacco smoke, aromatic amines, or PAH. An analysis of high-grade bladder cancers indicated a lower risk in dyestuff-exposed subjects, but the statistical power was limited for exploring subgroups.

NAT2 slow acetylation was proposed for genetic testing for purposes of compensation or preventive activities among workers with exposure to aromatic amines. Few studies have explored the interaction of the NAT2 acetylation status with occupational exposure to aromatic amines (24). Our results do not provide evidence for a strong interaction, but EPIC is a population-based cohort with a low prevalence of rare occupations like coke-oven works that are known to entail a bladder cancer risk. The statistical power of this analysis as well as of other population-based studies conducted so far is not sufficient to exclude the presence of interactions in rare at-risk occupations.

Although many studies reported on the main effect of slow acetylation, only two case–control studies had enough statistical power to assess the role of NAT2 in bladder cancer. This is the first report on NAT2 slow acetylation from a large prospective study that had adequate statistical power to evaluate a 1.4-fold main effect for the association with bladder cancer (7). On the basis of fewer cases, an OR of 1.33 (95% CI, 0.77–2.31) was estimated in women from the Nurses' Health Cohort and of 0.78 (95% CI, 0.53–1.15) in men from the Health Professional's Follow-up Study (15). Previous meta-analyses included small historical case–control studies that used a variety of methods to deduce NAT2 acetylation (6–10). Concerns can be raised about the phenotyping of cases with bladder cancer because both the disease and the treatment may influence urinary metabolite concentrations. Only two recent case–control studies were powerful enough to evaluate NAT2 slow acetylation (7, 11). The Spanish study was hospital-based and reported an OR of 1.4 (95% CI, 1.2–1.7) and the population-based New England study estimated an OR of 1.04 (95% CI, 0.82–1.28) for the association of NAT2 slow acetylation with bladder cancer. The summary OR of 1.11 (95% CI, 0.89–1.38) from the two large case–control studies and three prospective studies is lower than previously estimated with historical studies (6–10). There is still heterogeneity between these recent studies due to a higher OR in the Spanish study. The difference is at least partially explained by a 10% lower proportion of slow acetylators in Spanish hospital controls than in Spanish population controls from EPIC.

Several methodologic reasons can be hypothesized that might explain the observed differences in the proportion of NAT2 slow acetylators, which in turn can contribute to the heterogeneity between studies. NATs are phylogenetically unstable genes with a large genetic diversity of NAT2 alleles in humans. The slow variants of the NAT2 gene may reflect a selective advantage in populations practicing farming (25). Although genomic features suggest a relatively homogenous European population, migration and isolation may result in genetic diversity (26). For example, Spanish Basques carry markers that indicate an isolated subpopulation (27). Population stratification may lead to false-positive associations of genetic variants with the disease if cases and controls include subjects who differ in geographic origin and may have a discordant ancestry level. Case–control studies are likely more prone to this specific bias than are prospective studies where both study groups are originating from the same target population.

Besides population stratification, minor uncertainties along the line from SNP selection to haplotype reconstruction might occur or even accumulate. Genotyping problems cannot be fully ruled out for this prominent locus (28). Part of the presumed evidence for slow acetylation has been derived from historical studies where quality control measures have been rarely reported. A visual inspection of the putative genotype can be prone to observer bias, whereas MALDI TOF MS calls genotypes from mass differences in the amplicons (29). It was frequently not possible to compare our genotype distributions with published data, because most NAT2 reports show only the distribution of the deduced phenotypes. HWE deviations are supportive for genotype misclassification and can bias the risk estimates in meta-analyses (30). Our genotype distributions were in HWE and in good agreement with other large control populations in Europeans (31). Although PHASE can deduce alleles with incomplete data, missing calls can be biased toward a certain genotype (32). We obtained a full 6-SNP genotype for 91% of the subjects. Notably, we observed a slight difference in the genotype distribution at nt 341 compared with the New England study. This SNP is located in a CG-rich region and can pose genotyping problems. We found relatively more missing calls than for other SNPs, and most discordant pairs were related to the heterozygous genotype. CC and TT calls were determined as CT at the other Sequenom platform and vice versa, but not as TT or CC, respectively.

Another source of some uncertainty is the deduction of NAT2 alleles from few SNPs because most Caucasians carry mutations. An allele is presented by a diploid DNA sequence of the gene. If short amplicons are analyzed, alleles can only be deduced for subjects carrying more than one heterozygous SNP. Computational algorithms like PHASE have been applied only recently to infer NAT2 haplotypes (33). Despite an overall good performance, some subjects can be misclassified (34). Furthermore, the SNPs selected for genotyping should capture the genetic variation at this locus according to the underlying linkage disequilibrium structure. A good performance has also been shown for a tagging SNP (35, 36). The fraction of subjects with slow variants usually increases with an increasing number of SNPs. Many studies in Caucasians genotyped a 6-SNP set, whereas the studies nested in other large cohorts used a 3-SNP genotype (15), which is assumed to capture the acetylation status with high sensitivity and specificity (28). Cascorbi and Roots considered 282C>T and 341T>C to be sufficient for predicting the acetylation phenotype in non-Africans (37). We achieved a coverage of 97% for 481C>T, 590G>A, and 857G>A and 92% for 282C>T and 341T>C.

Variation in risk estimates of NAT2 slow acetylation may also arise from the crude categorization of subjects as slow and fast, or intermediate and rapid acetylators. Because of the small fraction of about 5% rapid acetylators (7, 11, 38), we refrained from a more extensive stratification of fast acetylators. Functional characterization of NAT2 SNPs revealed multiple mechanisms conferring acetylation capacity, for example, by changing protein activity, thermostability, or substrate specificity (39). The NAT2 polymorphism can be shown with a bimodal distribution of the ratio of urinary concentrations of two metabolites after administering caffeine (38). The variation of this ratio within groups of slow or fast acetylators is very large in common allele combinations, and phenotype data for rare allele combinations are yet inconclusive (40).

The established bladder cancer risk associated with aromatic amines and the observed differences in metabolic activity associated with NAT2 variants have lent theoretical plausibility to the hypothesis that NAT2 slow acetylation may modify the bladder cancer risk (41). The minor or even null main effect of NAT2 slow acetylation on bladder cancer risk in our study also needs to be discussed with regard to the complexity of the metabolism. It is important to note that canids even lack NAT expression (42), and NAT knockout mice do not exhibit a different phenotype (43). Human NATs form a cluster of three genes, comprising a pseudogene and the functional genes NAT1 and NAT2 that share 81% of their sequence (44). Both isoforms catalyze N- and O-acetylation reactions but vary in substrate specificity (45). Caffeine, which is not an aromatic amine, has been administered to deduce the acetylation status based on a ratio of metabolites that are excreted into urine following N-acetylation as detoxifying mechanism of arylamines. However, less is known about the influence of NAT2 variants on the O-acetylation due to a limited access to the target tissue in healthy humans. In addition, other highly polymorphic enzymes like P4501A2 contribute to the metabolism of arylamines (46). A pathway analysis will be subject of another report.

An accurate exposure assessment is important for exploring gene–environment interactions (47). The assessment of occupational exposure poses various challenges in population-based studies. Job-exposure matrices are a general tool of population-based studies to estimate exposure probability and intensity associated with job titles (48). Combining different dimensions into an exposure score may partially misclassify some subjects, in addition to differing evaluations of various experts when generating JEMs. Aromatic amines have been rarely assessed with a JEM. On the basis of a semiquantitative assessment of occupational exposure, we could confirm the bladder cancer risk associated with aromatic amines and dyestuffs (49). However, the risk estimates are lower than in older studies, in particular, for occupations in the European chemical and rubber industry or in asphalt workers (50, 51). This is likely due to the ban of carcinogenic aromatic amines from many industrial processes and the substitution of tar by bitumen. Concern has been raised about the use of hair dyes, as hairdressers were among the occupations with an elevated bladder cancer incidence, for example in Northern Europeans (51). We also found an excess risk of bladder cancer, but it was not significant likely due to small numbers.

We could further provide support for an increased bladder cancer risk associated with PAH exposure as previously shown in a pooled analysis of European case–control studies (50). Combustion of organic matter is a common source of PAH and occurs at many workplaces. This has been frequently addressed before, also in combination with aromatic amines during the hot application of tar or in coke production and coal gasification (52). The cancer risk for agents that occur jointly at the same workplaces can hardly be disentangled by statistical means. Mutual adjustment attenuates the risk estimates and may underestimate the effect of the driving factor with a higher carcinogenic potency. We found higher risks in smokers with substantial occupational exposure as compared with smokers who were never exposed to aromatic amines or PAH.

However, we found no support for an effect modification by NAT2 slow acetylation. EPIC was not aimed at investigating occupational exposures, and due to its population-based design, the prevalence of certain high-exposed occupations was low. Risk estimates for single occupations are additionally impaired by inflation of type I error. Also lacking information on the occupational history and duration of exposure weakens the detection of work-related bladder cancer risks. Additional analyses provided no evidence for a residual confounding by smoking.

We confirmed tobacco consumption as a major risk factor for bladder cancer, where arylamines are suspected to be the primary causative agents (53). Certain PAH compounds are another group of carcinogens in tobacco smoke. A comprehensive analysis of smoking and bladder cancer in EPIC has been reported elsewhere (54). Reviews and meta-analyses supported a joint effect of NAT2 slow acetylation with tobacco smoking (7, 10, 11). We found no interaction with smoking or a difference of the relative risk estimates of slow acetylation in smokers compared with never-smokers. However, the statistical power was not sufficient to rule out a weak effect modification in heavy smokers.

A striking feature of bladder cancer is its heterogeneous clinical behaviour, likely due to different molecular pathways (55). We therefore explored NAT2 slow acetylation also in high-grade bladder cancer. We estimated similar risks of slow acetylation except a slightly lower risk in dyestuff-exposed subjects that is in line with a lower risk in Chinese benzidine-exposed workers (12). We further found a lower risk in women, though not significant. Gender differences are known in bladder cancer risk, recurrence, and survival (56). A survival analysis will be subject of another report.

In conclusion, the findings from this prospective investigation are compatible with previous evidence showing excess risks of bladder cancer associated with occupational exposure to aromatic amines and are supportive for a role of PAHs in the development of bladder cancer. However, neither found an excess risk of bladder cancer associated with NAT2 slow acetylation, nor did we find that NAT2 slow acetylation modified the relative risk associated with exposure to aromatic amines, PAH, or tobacco smoke. NAT2 acetylation status is unlikely to be a useful marker for predicting bladder cancer in the population at large, or among workers.