Slow Acetylation of N-Acetyltransferase 2 and the Risk of Bladder Cancer
Slow Acetylation of N-Acetyltransferase 2 and the Risk of Bladder Cancer
By Stewart M. Polsky, MD, and David A. Corral, MD
Arylamines are a group of aromatic organic chemicals that have been widely used in the dye industry and are also found in cigarette smoke. The carcinogenic potential of these substances is well recognized, and a role in the etiology of urothelial transitional cell carcinoma (bladder, ureter, and renal pelvis) has been hypothesized.1 The N-acetylation of arylamines and acetoxy esters (constituents of tobacco carcinogens), a process carried out by the enzyme N-acetyltransferase, is considered to be a major detoxification step of the arylamine carcinogens in humans.2 N-acetyltransferase is functionally expressed by two polymorphic isoenzymes: N-acetyltansferase 1 (NAT1) and N-acetyltransferase 2 (NAT2), with each gene located on chromosome 8. NAT1 is expressed in many tissues, including urothelial epithelium.3 In contrast, NAT2 is mainly found in the liver.4
Individual differences in human susceptibility to carcinogenic agents may depend on an individual’s genetic makeup. Several polymorphisms of human drug-metabolizing enzymes influence an individual’s susceptibility for chemical carcinogenesis. Polymorphisms are defined as less frequent phenotypes, which occur in at least 1% of a population and are caused by gene mutations, specifically point mutations, frame shift mutations, or gene deletions resulting in most cases (but not always) in enzymes with reduced or no activity. Large interethnic differences in the frequency of gene mutations responsible for polymorphisms have been observed.5
The "isoniazid acetylation polymorphism" (N-acetylation) was first identified in the late 1940s when isoniazid was in widespread use for the treatment of tuberculosis. A high incidence of peripheral neuropathy was encountered and found to represent the unusually slow clearance of the toxic parent compound, isoniazid. Subsequently, individuals were phenotyped as "slow" or "rapid" acetylators based on the relative activity of NAT. The slow acetylator phenotype was found to be inherited as an autosomal recessive trait, and 40-60% of Caucasians are homozygous for the slow acetylator alleles.6 Worldwide, the frequency of the slow acetylator phenotype ranges from about 10% in the Japanese to greater than 90% in some Mediterranean populations.
Two human N-acetyltransferase functional genes (NAT1 and NAT2) and one pseudogene (NATP) have been cloned and localized to chromosome 8. The rapid and slow acetylator phenotype was found to involve the NAT2 gene principally, encoding the NAT2 enzyme, which has a 10-fold lower affinity for aromatic amines than does NAT1.7 Three major slow acetylator NAT2 alleles (two common in Caucasians and one common in Asians) have been identified, and the number of minor, rare NAT2 alleles is now greater than 20.8
It has been shown that NAT2 mutations do not lead to a total absence, but rather a decrease in the amount of activity of the enzyme. In contrast to fast acetylators who have the ability to detoxify arylamines, slow acetylators tend to form N-hydroxy-derivatives (proximate bladder carcinogens) via N-hydroxylation. The N-hydroxy-derivatives are subsequently O-esterified by NATs of the bladder epithelium (predominantly NAT1) to N-acetoxyesters that can easily form DNA adducts in vivo (ultimate bladder carcinogens). Further metabolism of N-acetoxyesters via heterocyclic cleavage produces arylnitrenium ions, which have been suggested to be very potent carcinogens.9 Both acetoxyester and arylnitrenium ions are highly mutagenic and, given their accumulation in the bladder epithelium after transportation through the circulation, have the ability to cause irreversible changes to DNA of urothelium cells and, therefore, tumor initiation. The possible site of DNA damage in urothelial cells has been proposed to be guanine residues.10
As stated previously, NAT2 polymorphism affects approximately 50% of Caucasians, and the majority of their slow acetylation alleles are associated with three point mutations: 590A, 341C, and 857A of the NAT2 gene on chromosome 8 (all 3 mutations accounting for the slow acetylating phenotype in more than 95% of Caucasian slow acetylators).11 Early on, various studies trying to demonstrate an association between NAT2 genetic polymorphisms and chemically induced bladder cancer either yielded conflicting results or did not suggest the existence of specific genotypes among slow acetylators that may carry a high risk for bladder cancer.12 Recent studies have altered that previous trend.13
Lolis et al performed a case-control study involving 89 patients with transitional cell carcinoma and 147 controls.13 NAT2 genotypes were identified by allele specific polymerase chain reaction (PCR) on peripheral blood DNA samples. A statistically significant difference in frequency of genotypes was found between the two groups. The transitional cell carcinoma patient group had the higher frequency of slow acetylation genotypes. Among slow acetylators, homozygotes 341C/341C and compound heterozygotes 341C/857A had the most excessive risk for bladder cancer, regardless of the type of exposure to arylamines. The 341C/341C genotype was found to be associated with more aggressive disease, in terms of tumor grade at presentation.
A study by Bell et al examined the polymorphisms of the NAT1 or NAT2 genes among 116 urothelial cancer patients (90 male, 26 female; 67.5 ± 11.2 years; bladder, 96; ureter, 13; renal pelvis, 7) with urothelial transitional cell carcinoma, and 122 healthy control individuals (72 male, 50 female; 62.4 ± 16.5 years) in a Japanese population.14 PCR-restriction fragment length polymorphism technique was used to determine the NAT1 (NAT1*4 [wild-type], NAT1*10, NAT1*11) and NAT2 (NAT2*4, low activity alleles NAT2*5, NAT2*6, and NAT2*7) genotypes. Alleles occurring at less than 1% frequency among Asians were not considered (NAT1*14, NAT1*15, and NAT1*17). Frequency of NAT1*10 genotype was higher among urothelial transitional cell carcinoma cases vs. controls; however, the differences were not statistically significant. The frequency of the slow NAT2 genotype in patients with urothelial cancer showed a statistically significant increase compared with controls. Individuals with combined NAT1*10 and NAT2 slow genotypes were at a 7.3-fold higher risk compared with the low risk group with NAT1*4 and rapid NAT2 genotypes. Their findings suggest that individuals with high activity for NAT1 and slow NAT2 genotype may have a higher risk of urothelial transitional cell carcinoma compared with individuals with other combinations.
A significant percentage of human urinary bladder carcinomas can be attributed to environmental exposure to various arylamines—recent data estimate 19% among men and 6% among women.15 Of the non-dietary factors, tobacco smoking is regarded as the major cause of bladder cancer, accounting for at least 40-50% of all cases; whereas substances such as 4-aminobiphenyl and beta-naphtylamine, which are also NAT2 substrates, represent established human carcinogens.16 Cigarette smoking and occupational exposure to arylamines are important factors, in conjunction with the slow acetylator phenotype, for development of bladder cancer. No relationship was found between acetylator phenotype and smoking-related bladder cancer in the absence of exposure to arylamines. NAT2 slow acetylators were found to have an increased risk of bladder cancer among cigarette smokers. The NAT1*10 allele alone was reported to be a risk factor in one study, but not in another. In a study by Chen et al, 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 was observed among those with NAT1*10 or NAT2 slow acetylators, but not among NAT2 rapid acetylators without NAT1*10 allele.17
In addition to bladder cancer, the N-acetyltransferase polymorphisms have been associated with other carcinomas. Rapid acetylators (with respect to N-acetyltransferase NAT2) were shown to have an increased risk of colon cancer but a decreased risk of bladder cancer.9,18 Conversely, slow acetylators were found to exhibit less risk of colorectal cancer but a higher risk of bladder cancer. An association between a NAT1 variant allele (NAT*10, due to mutations in the polyadenylation site causing an approximately two-fold higher activity) and colorectal cancer among NAT2 rapid acetylators was observed, suggesting a possible interaction between NAT1 and NAT2.5 Glutathione S-transferase M1 and T1 are polymorphic due to large deletions in the structural gene. Others have shown an increased risk of oral cancer in those with the NAT1*10 allele. Combination of GST M1-O and NAT2 slow acetylation was associated with a markedly increased risk of lung cancer.19 Some investigators observed associations between the acetylator phenotype and cancer of organs other than colon and bladder. An increase of slow acetylator phenotypes (NAT2) were observed among patients with hepatocellular cancer.20 In postmenopausal women, NAT2 modified the association of smoking with risk of breast cancer.21 For slow acetylators, smoking increased breast cancer risk in a dose-dependent manner; whereas for rapid acetylators, smoking was not associated with increased breast cancer risk. Very recently, cigarette-smoking postmenopausal women with the slow acetylator phenotype have been shown to be as much as four-fold more likely to develop breast cancer than those with the rapid acetylator allele.21 Toxic nitrosamines in tobacco smoke, which are degraded by NAT2 more slowly over many years, might cause the individual to be more susceptible to breast cancer.
Transitional cell carcinoma is characterized by great differences in behavior, suggesting a heterogeneous disorder. Despite this heterogeneity, tumor grade and stage remain the most useful prognostic variables in bladder cancer.22 Epidemiological studies have shown that the phenotype of low activity NAT2 is associated with an increased risk of urothelial cancer among occupationally exposed dye workers.23 The genetic alterations underlying the NAT2 polymorphism have been determined and correlated with NAT2 enzyme activities. Fast acetylators seem to have an advantage in terms of a decreased risk of bladder cancer, reinforcing the concept of the protective role of fast acetylation in the detoxification of the arylamines and their carcinogenic derivatives. Although NAT2 polymorphism has been extensively studied, the importance of NAT1 polymorphism remains to be established.13 (Dr. Polsky is a Urology resident at the State University of New York at Buffalo.)
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