NAT2 slow acetylator genotype is associated with increased risk of lung cancer among non-smoking Chinese women in Singapore

Adeline Seow7, Bin Zhao1, Wee-Teng Poh4, Ming Teh2, Philip Eng5, Yee-Tang Wang6, Wan-Cheng Tan3, Edmund J.D. Lee1 and Hin-Peng Lee

Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, 16 Medical Drive, Singapore, 117597,
1 Department of Pharmacology,
2 Department of Pathology and
3 Department of Medicine, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore, 119260,
4 Department of Pathology and
5 Department of Respiratory and Critical Care Medicine, Singapore General Hospital, Outram Road, Singapore, 169608 and
6 Department of Respiratory Medicine, Tan Tock Seng Hospital, Moulmeri Road, Singapore 308433


    Abstract
 Top
 Abstract
 Introduction
 References
 
Among non-smokers, the factors resulting in lung carcinogenesis are poorly understood. We conducted a hospital-based case-control analysis of 294 Chinese women, of whom 217 were non-smokers, to evaluate the role of polymorphic N-acetyltransferase (NAT2) as a susceptibility factor for the disease. The proportion of slow acetylator genotypes among non-smoking cases (n = 92) and controls (n = 125) was 38.0 and 24.0%, respectively [odds ratio (OR) 2.0, 95% confidence interval (CI) 1.1–3.7]. No effect of NAT2 genotype was seen among smokers. Among non-smokers, the effect was marked for adenocarcinomas (OR 2.1, 95% CI 1.1–4.0). As NAT2 activity is known to modify risk of arylamine-induced carcinogenesis, our results suggest that exposure to arylamines in the environment may play a role in risk of lung cancer among non-smokers.

Abbreviations: 4-ABP, 4-aminobiphenyl; NAT2, N-acetyltransferase-2.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Polymorphic N-acetyltransferase-2 (NAT2), one of the two NAT isoenzymes in humans, is involved in the metabolic activation of various therapeutic agents and environmental agents with carcinogenic potential (1,2). This enzyme catalyzes the acetylation of arylamines which are ubiquitous chemicals present in industry, cooked food and as environmental contaminants. Examples include 4-aminobiphenyl and 2-naphthylamine, which are found in dye-manufacturing and cigarette smoke, heterocyclic arylamines formed during heat processing of meat and polycyclic nitroaromatic hydrocarbons in diesel exhaust (3). Acetylation polymorphism in humans is regulated at the NAT2 gene locus on the short arm of chromosome 8 (4). Point mutations in coding and non-coding regions of the gene account for differences in enzyme activity and hence influence individual susceptibility to arylamine-induced cancers. Of these, mutations at positions 481 (giving rise to the NAT2*5A allele), 590 (NAT2*6A) and 857 (NAT2*7A) appear to be most important in determining acetylator phenotype (5). NAT2 is known to be expressed in the liver and in colonic and bladder mucosa in humans, and its role in the aetiology of bladder cancer has been confirmed in many studies (3,6,7). Slow acetylator activity confers a higher risk of bladder cancer, due to reduced activity in the hepatic N-acetylation pathway which detoxifies compounds such as 4-aminobiphenyl and competes with N-oxidation of these compounds by cytochrome P4501A2 to reactive hydroxylamines (3,8). The role of NAT2 in colon cancer is more controversial, but it has been suggested that rapid acetylators have a higher risk of the disease, an observation explained by the role of NAT2 in the O-acetylation and subsequent activation of N-hydroxy arylamines in the colonic mucosa to potentially DNA-binding forms (3,9,10). The lung is exposed to a variety of xenobiotics, including polycyclic aromatic hydrocarbons and aromatic amines, from both inhalational and non-inhalational routes (11). However, results from studies of NAT2 among lung cancer subjects have been conflicting, some reporting no association with acetylator status (1214) and others reporting an increased risk among rapid acetylators (15) or slow acetylators (16,17).

The role of cigarette smoking in the aetiology of the disease is widely accepted and, in the West, trends in lung cancer have closely paralleled trends in smoking and tobacco sales (18,19). Epidemiological data suggest that there are important ethnic differences in the relationship between smoking and lung cancer. Chinese female populations are distinctive in having a high incidence of lung cancer despite a low prevalence of smoking (20,21). Singapore Chinese women, for example, have had an overall smoking prevalence of only 3% since the 1980s (22), yet the incidence of lung cancer in the 1980s was comparable with that among women in Canada and the UK, where smoking prevalence was much higher (23,24). Studies among female lung cancer patients have reported smoking rates of between 16 and 52% for Chinese in Hawaii, China, San Francisco and Singapore, compared with 77–90% among women in North America and the UK, and 81–99% among men worldwide (21). In a combined analysis of eight case-control studies in Chinese, the odds ratio (OR) for smoking was only 2.3 (25), which is much lower than in Western populations. All these data taken together indicate that risk factors apart from cigarette smoking play an important role in the aetiology of lung cancer in this population.

These observed ethnic differences in risk suggest that genetic factors influence individual susceptibility to the disease. The objective of the present study was to determine if NAT2 acetylator genotype modifies risk of lung cancer among female Chinese living in Singapore, which is a small island republic with a resident population of 3.1 million. Of these, 78% are ethnic Chinese, many of whom, particularly the elderly, were immigrants from mainland China, Hong Kong or Taiwan. The annual incidence rate of lung cancer for the years 1988–1992 in this population was 19.6 per 100 000 (26).

A total of 153 patients with pathologically confirmed primary lung cancer and 141 age-matched controls were included in this study. Only Chinese females who were Singapore citizens or permanent residents were eligible for participation. Cases were incident cancers diagnosed at three of the major hospitals in Singapore between January 1997 and December 1998. Controls were patients admitted to the same hospital as the cases, frequency-matched for age (within the same 10 year age group) with no history of cancer or any chronic respiratory condition. They included patients drawn from internal medicine (22.5%), orthopaedic (42.6%), surgical/trauma (24.8%) and eye (8.5%) wards. A blood sample (6 ml) was obtained from each subject by venepuncture. Demographic information and data on smoking were obtained by standardized questionnaire administered in person by a research nurse. Subjects were classified as smokers if they had ever smoked at least one cigarette a day for 1 year or more. Ex-smokers were smokers who had stopped smoking for 1 year or more. The pathology specimens of all cases were reviewed and classified independently by two study pathologists; only pathologically confirmed cases where both agreed on a diagnosis of squamous cell carcinoma, small cell carcinoma, adenocarcinoma or large cell carcinoma were included.

Isolation of genomic DNA was carried out using a standard proteinase K–phenol–chloroform extraction procedure (27). An aliquot of 5 µl of genomic DNA obtained by this method was then subjected to PCR according to the method described by Blum et al. (5). Specific primers for the wild-type allele NAT2*4 and the mutant alleles NAT2*5A, NAT2*6A and NAT2*7A were used in separate PCRs (Figure 1Go). Primer 2 is complementary to the NAT2 gene at position 481–494, primer 3 recognizes the mutation C481->T of NAT2*5A. Primer pairs 5,6 and 7,8 test for the presence of NAT2*6A (G590->A) and NAT2*7A (G857->A), respectively. The common primer used for the reactions with primers 2, 3, 7 and 8 is primer 1, and for the reactions with primers 5 and 6, is primer 4. All PCRs were carried out in a total volume of 50 µl in the presence of 10 mM Tris–HCl pH 8.3, 50 mM KCl, 0.01% gelatin, each dNTP at 0.2 mM, each primer at 0.2 µM, 1 U Taq polymerase (Perkin Elmer, Branchburg, NJ), 300–600 ng of genomic DNA and 1.5 mM MgCl2. For each reaction, denaturing (30 cycles at 94°C for 1 min each) was carried out, followed by annealing at 48°C (NAT2*5A), 55°C (NAT2*6A) and 35°C (NAT2*7A) for 1.5 min. The extension period was 7 min at 72°C. The PCR products were then electrophoresed on a 1.2% agarose gel containing ethidium bromide. All stages of the analysis were carried out blind to the patient's disease status.



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Fig. 1. Identification of nucleotides at positions 481 (C or T), 590 (G or A) and 857 (G or A) of the NAT2 gene using allele-specific PCR. (A) 481 C only, 590 G and A, 857 G and A (number 125). (B) 481 C only, 590 G only, 857 G only (number 186). (C) 481 C and T, 590 G and A, 857 G only (number 261). Lane M is the 100 bp DNA ladder. Primers used in the amplification, as described in the text, are as follows: Primer 1 (5'-AATTAGTCACACGAGGA-3', position –74 to –58); Primer 2 (5'-CTGATTTGGTCCAG-3', position 481–494); Primer 3 (5'-CTGATTTGGTCCAA-3', position 481–494); Primer 4 (5'-TCTAGCATGAATCACTCTGC-3', position 1119–1138); Primer 5 (5'-TTTACGCTTGAACCTCG-3', position 574–590); Primer 6 (5'-TTTACGCTTGAACCTCA-3', position 574–590); Primer 7 (5'-AATAGTAAGGGATC-3', position 857–870); and Primer 8 (5'-AATAGTAAGGGATT-3', position 857–870). Bold represents the base that is different between the two primer pairs.

 
Subjects were designated as slow or rapid acetylators based on the combination of NAT2 alleles detected; those homozygous or heterozygous for the NAT2*4 (`wild-type') allele being classified as rapid acetylators and those homozygous for any of the three remaining (`mutant') alleles as slow acetylators.

Differences in NAT2 acetylator activity between cases and controls were expressed as ORs and 95% confidence intervals (CI). Logistic regression analyses were used to obtain age- and birthplace-adjusted ORs. All calculations were performed using the SPSSWIN v.8.0 statistical package (SPSS, Chicago, IL).

Of the 294 individuals in the study population, 217 (73.8%) reported never having smoked, and 76 (25.9%) were either current or ex-smokers. Information on smoking history was not available for one control subject. The proportion of current and ex-smokers among the cases was 39.9% (61 women), and that among the controls was 10.6% (15 women). Data on the mean age, dialect group and place of birth of our subjects by smoking status are given in Table IGo. In general, cases tended to be marginally older and there was a slight over-representation of Cantonese women among cases compared with controls. Among non-smokers, cases were more likely to be foreign-born, particularly migrants from China.


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Table I. Characteristics of study populationa
 
The large majority of cancers diagnosed among non-smokers were adenocarcinomas (72.8%), while the distribution of histological types among smokers included a larger proportion of squamous and small cell carcinomas (55.7% combined). The proportion of large cell undifferentiated carcinomas, which were a subgroup of non-small cell carcinomas which could not reliably be assigned to specific types, was similar between smokers and non-smokers (Table IIGo).


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Table II. Distribution of histological types and NAT2 genotype by smoking status
 
The distribution of NAT2* genotypes is given in Table IIGo; the allelic frequencies of NAT2*4, NAT2*5A, NAT2*6A and NAT2*7A among controls in this study were 0.53, 0.05, 0.23 and 0.19, respectively. These were similar to that reported previously among healthy Chinese in Singapore and Hong Kong (28,29), and consistent with the higher frequency of the wild-type (NAT2*4) allele observed in Asian, compared with Western populations (30). Among these three alleles, the frequency of the NAT2*7A allele, in particular, was higher among cases than controls (26.5 and 19.1%, respectively; data not shown). Based on the system described above, 39.2% of our cases and 25.5% of controls were classified as slow acetylators (homozygous mutant alleles). The OR for lung cancer among non-smoking women was 1.9 (95% CI 1.1–3.7) for slow acetylators compared with rapid acetylators, and this elevation in risk was independent of age and place of birth (Table IIIGo). There was no association between acetylator status and lung cancer among current or ex-smokers (adjusted OR 0.8).


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Table III. Odds ratios for NAT2 acetylator genotype and lung cancer by smoking status and by histological type
 
As smoking was an obvious effect modifier, we chose to restrict further analysis to non-smokers, the subgroup of particular interest to us. Among these women, there was an elevation in risk for all histological types, but this was statistically significant only for adenocarcinomas. Our results show a 2-fold increase in risk of lung adenocarcinoma among slow acetylators, an effect which was again independent of age and birthplace. The small number of cases with other histological types accounts for the wide CIs, and the ORs should be interpreted with caution.

Our results demonstrate, for the first time, an association between slow NAT2 acetylator genotype and increased risk of lung cancer, particularly adenocarcinomas, in female non-smokers. While NAT2 has been one of the most intensely investigated metabolic polymorphisms, its effect on lung cancer has not been studied in this particular group. The homogenous nature of our study population in terms of gender and ethnicity allow control for possible confounding by hormonal and other related effects. As the prevalence of smoking in Chinese female populations is among the lowest worldwide (21), the fairly large number (n = 217) of non-smokers in our study population enables estimates of the OR to be reasonably precise. It was not possible to validate self-reported smoking status in hospital, but a comparison of our data with the smoking history in the medical records of 301 lung cancer cases indicated a high degree of reproducibility (concordance rate 96%, k = 0.93). Any misclassification of a smoker as a non-smoker is most likely unrelated to NAT2 status and the effect would be to bias the results toward the null.

There are a few studies in the literature which have examined the role of NAT2 in lung cancer, and of these, two (13,14), which included mainly smokers (95.4 and 100%, respectively), found no significant association with NAT2 acetylator genotype. Another study (~70% smokers in the study population) (15) found an over-representation of a rapid (NAT2*4/*4) genotype among cases compared with controls, but there was no difference in the phenotypic distribution in this and another similar study (92.3% smokers) (12). The study populations in all these were Caucasian and included both males and females. In a Japanese study (16), there was an increased risk of adenocarcinoma among slow acetylators (OR 2.01, 95% CI 0.99–4.05). Interestingly, non-smoking bus maintenance workers in Scandinavia exposed to diesel exhaust, and who had a combination of NAT2 slow acetylator and GSTM1-negative genotype, demonstrated higher aromatic DNA adduct levels in peripheral blood (17).

The observed differences in frequency of acetylator status among non-smoking lung cancer cases and controls suggest that this polymorphism may be one of the factors involved in susceptibility to the disease in this population. Our finding that slow acetylators are at increased risk also suggests that the mechanism by which this occurs involves deactivation pathways, or pathways that compete with the metabolic activation of arylamine carcinogens, as has been described for bladder carcinogenesis (1,3). The presence of 4-ABP–DNA adducts in peripheral lung tissue of both smokers and non-smokers (11) demonstrates that uptake of reactive forms of this chemical carcinogen can take place at this site. It is also worth noting that in animal studies, the feeding of the heterocyclic arylamines 2-amino-3-methylimidazo(4,5-f)quinoline and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline to mice was associated with the development of lung tumours in addition to tumours of the liver and forestomach (31,32).

Occupations found to increase risk among Chinese women in China, such as metal smelting, coal dust, burning fuel and automobile manufacturing (33), are almost exclusively undertaken by males in Singapore. Exposure to arylamines in our study population is therefore likely to be domestic in origin, either through inhalation or ingestion. Diet is a known source of heterocyclic amines, and the Chinese diet is no exception, but exposure to inhalants during cooking is of particular significance in this population since domestic exposure to cooking fumes has been implicated in several studies.

Several epidemiologic studies have pointed to the role of domestic exposure to cooking fumes in the aetiology of lung cancer among Chinese women (34,35). Apart from other mutagens, several workers have demonstrated that heterocyclic amines are volatilized during frying (36,37), particularly when high temperatures and oil are used to cook meat and meat products (38). Older Chinese women in Singapore, especially those originating in the south of China, have continued to practice traditional Chinese wok cooking, characterized by `bao', a form of rapid stir-frying in oil over high heat (39).

It has been estimated from population-based studies that 52% of non-smoking women in Singapore have been exposed to environmental tobacco smoke (40), which has been shown to increase risk of lung cancer (41). The concentration of 4-aminobiphenyl (4-ABP) in sidestream smoke is calculated to be 31 times that in mainstream smoke (23), and the amount inhaled through passive smoking is not insignificant. The lack of effect with regard to NAT2 acetylator activity among smokers in our study, and the others described above, is consistent with the observation that inter-individual variability in susceptibility to carcinogens is more important at low degrees of environmental exposure. Since we propose that NAT2 affects arylamine-induced lung carcinogenesis through deactivation pathways, it is very reasonable that the activity of these pathways does not influence risk in persons exposed to high concentrations of carcinogens. This is supported by the observations of Vineis et al. (42) who showed that 4-ABP–Hb adduct levels in peripheral blood were higher in slow acetylators, but the difference was less evident with increasing nicotine-cotinine levels.

In conclusion, our finding that NAT2 acetylator polymorphism affects susceptibility to lung cancer in this population supports the hypothesis that domestic and other sources of arylamines are important contributors to risk of the disease among non-smoking Chinese women. Further studies will be needed to confirm this and ascertain the scope for prevention.


    Acknowledgments
 
This project was supported by a research grant from the Singapore National Medical Research Council.


    Notes
 
7 To whom correspondence should be addressed Email: cofseowa{at}nus.edu.sg Back


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Received March 19, 1999; revised June 8, 1999; accepted June 9, 1999.