Carcinogenesis vol.19 no.10 pp.1803–1807, 1998
A pilot study testing the association between N-acetyltransferases 1 and 2 and risk of oral squamous cell carcinoma in Japanese people
Takahiko Katoh1,2, Shigeru Kaneko3, Robert Boissy1, Mary Watson1, Kunio Ikemura3 and Douglas A.Bell1,4 1National
Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA, 2Occupational Health Science Course, School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu and 3Department of Oral Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
4To
whom correspondence should be addressed Email:
[email protected]
Risk of oral cancer has been associated with exposure to tobacco smoke, alcohol and with genetic predisposition. The aromatic amines and their metabolites, a class of carcinogens present in tobacco smoke, undergo metabolism (activation or detoxification) through an N- or O-acetylation pathway by the polymorphic enzymes, N-acetyltransferases (NAT)1 or NAT2. The genes that encode these enzymes, NAT1 and NAT2, have a variety of high and low activity alleles and we analyzed these genetic polymorphisms in 62 oral squamous cell carcinoma cases, and 122 healthy control subjects from Japan. NAT1 alleles tested were NAT1*3 (C1095A), NAT1*4 (functional reference allele), NAT1*10 (T1088A,C1095A), NAT1*11(9 bp deletion), NAT1*14 (G560A), NAT1*15 (C559T) and NAT1*17 (C190T). No low activity alleles (NAT1*14, NAT1*15 and NAT1*17) were observed in these Japanese subjects. We observed significantly increased risk [odds ratio 3.72; 95% confidence interval (CI) 1.56–8.90; P < 0.01] associated with the NAT1*10 allele, an allele that contains a variant polyadenylation signal. Stratifying by smoking status we found odds ratios of 5.9 (95% CI 1.13–30.6; P < 0.05) for non-smokers with the NAT1*10 allele and 3.1 (95% CI 1.09–9.07; P , 0.05) for smokers, but these risks were not significantly different from each other. Thus, we did not observe that NAT1*10 alleles confer differential risk among smokers and non-smokers. NAT2 rapid acetylation genotype was not a significant risk factor for oral cancer in this Japanese study population. This is the first study to test for oral cancer risk associated with polymorphism in the NAT1 and NAT2 genes, and these positive findings in our pilot study, while based on small numbers, suggest that the NAT1*10 allele may be a genetic determinant of oral squamous cell carcinoma among Japanese people. Introduction Humans are routinely exposed to highly mutagenic and carcinogenic aromatic amines (including arylamines and heterocyclic amines) via tobacco smoking, cooked foods and other sources (1–3). Acetylation is an important route of biotransformation Abbreviations: AS-PCR, allele-specific PCR; CI, confidence interval; GST, glutathione S-transferase; NAT1, N-acetyltransferase 1 gene; NAT2, N-acetyltransferase 2 gene; OLA, oligonucleotide ligation assay; OR, odds ratio; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism. © Oxford University Press
for these chemicals. In humans, two N-acetyltransferases, designated NAT1 and NAT2, catalyze N- and O-acetylation of various arylamines. NAT1 and NAT2 have been shown to be polymorphic enzymes that segregate independently into a large number of polymorphic genotypes that correspond to rapid and slow acetylator phenotypes (4,5). Genetic polymorphisms in these genes influence the metabolism of environmental arylamines and modulate the risk of certain human cancers, such as those of the colon and bladder (6–13). Of note, recent studies have indicated that a specific NAT1 polymorphism (NAT1*10 allele) is associated with higher enzyme activity in colon, bladder and liver tissues (14–16). However, the molecular basis for these differences has not been demonstrated. Cancers of the oral cavity have been strongly associated with exposure to tobacco and alcohol (17–19). Oral tissue is relatively unique from the standpoint of carcinogen exposure, because direct exposures to tobacco smoke, food carcinogens and alcohol are likely to be more important than pathways involving the liver and other organs (13). Metabolism of carcinogenic aromatic amines is complex and many potential pathways exist, but N-hydroxylation by cytochrome P450 oxidases, followed by O-acetylation by NAT1 or NAT2 would be a possible route for activation in oral tissues. In this pilot study, we analyzed a group of 62 oral squamous cell carcinoma patients, and 122 control subjects for genetic polymorphisms in both the NAT1 and NAT2 genes. We found a significantly increased risk for oral squamous cell carcinoma associated with a specific NAT1 polyadenylation signal sequence polymorphism (NAT1*10), but no risk associated with NAT2 slow acetylator polymorphism. Materials and methods Subjects The case group was comprised of 62 patients with oral squamous cell carcinoma (40 men, 22 women, mean age 61.7 6 11.0) from Kitakyushu, Japan. The patients were consecutive cases at the University of Occupational and Environmental Health Hospital who had been referred to there through dental health clinics or general health clinics, and had been histologically diagnosed with oral squamous cell carcinoma during the period September 1992 to June 1995. None of the patients refused participation. The control group was comprised of 122 subjects (72 men, 50 women, mean age 5 62.4 6 16.5 years) who were recruited from three general health clinics in Kitakyushu City (Tochiku Hospital, Mihagino Hospital, Yahatanishi Hospital) between September 1993 and April 1995. They were consecutive patients seen for general health check-ups. Data on glutathione S-transferase (GST)M1 and GSTT1 genotype for control subjects were reported in Katoh et al. (20). Case and control subjects did not originate from the same clinics, but all clinics involved in the study drew patients from the general population of Kitakyushu City. Case and control subjects were not specifically matched, although both mean ages and proportion of males/females were similar. Control subjects had no current or previous diagnosis of cancer. All participants were given an explanation of the nature of the study, and informed consent was obtained. All study subjects completed a questionnaire administered by the clinical staff, covering medical, residential, occupational and smoking history. For stratified analysis, smokers were defined as being sometimes or non-smokers (Table V). In logistic regression analysis, smokers were categorized (categorical variable) as to their smoking index (cigarettes
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Table I. Oligonucleotides used for NAT1 oligonucleotide ligation assays Type of oligo
Name
Starta
Enda
Length (nt)
Modifications
Sequence (59–39)
PCR primers
NT1.365-U NT1.1566-L NT1-LL.p630 NT1-LR.630.bio.*G NT1-LR.630.bio.*A NT1.UR.p999 NT1.UL.Bio999*C NT1.UL.Bio999*T NT1.UR.p1000 NT1.UL.Bio1000*G NT1.UL.Bio1000*A
365 1566 609 630 630 1000 977 977 1001 977 977
394 1595 629 647 647 1021 999 999 1022 1000 1000
30 30 21 18 18 22 23 23 22 24 24
none none 59-phosphate 59-biotin 59-biotin 59-Phosphate 59-Biotin 59-Biotin 59-Phosphate 59-Biotin 59-Biotin
CAT AAT TAG CCT ACT CAA ATC CAA GTG TAA ATT AAA AGC TTT CTA GCA TAA ATC ACC AAT ATT TCT TCT CAC AAC TTG ATC GAG ACA CCA TCC ACC CCG GAG ACA CCA TCC ACC CCA GAA AAA TCT ACT CCT TTA CTC T TCT CCT AGA AGA CAG CAA ATA CC TCT CCT AGA AGA CAG CAA ATA CT AAA AAT CTA CTC CTT TAC TCT T TCT CCT AGA AGA CAG CAA ATA CCG TCT CCT AGA AGA CAG CAA ATA CCA
NAT1*17 OLA (C190T) NAT1*15 OLA (C559T) NAT1*14 OLA (G560A) aThese
values for nucleotide numbering are from GenBank sequence accession no. X17059.
per day3years of smoking): non-smoker; smoking index 1–400, smoking index .400. Alcohol consumption information, an important risk factor in oral cancer, was collected for oral cancer cases but, unfortunately, not for control subjects. Data for other oral squamous cell carcinoma risk factors, such as dietary habits, oral hygiene, dental health and infection with human papillomavirus were not available. The relative associations between cases and controls were assessed by calculating crude odds ratios from contingency tables. The corresponding chisquare tests on the oral squamous cell carcinoma patients and controls were carried out and 95% confidence intervals (95% CI) were determined. Statistical analysis was carried out using Stat view 4.0J (Hulinks) or Epistat programs (Finnish Institute of Occupational Health). Adjusted analyses (age, gender, smoking) and tests of interactions were carried out using logistic regression with the SAS statistical program (SAS, Cary, NC). Genotype analysis Genomic DNA was isolated from peripheral leukocytes by proteinase K digestion and phenol–chloroform extraction. We used a polymerase chain reaction (PCR)–RFLP (MboII; New England Biolabs, Beverly, MA) and an allele-specific PCR method to detect the four most common alleles of the NAT1 gene [NAT1*3 (C1095A), NAT1*4 (functional reference allele), NAT1*10 (T1088A,C1095A) and NAT1*11] (8,14). The presence of rare NAT1 alleles that have lower activity [NAT1*14 (G560A), NAT1*15 (C559T), NAT1*17 (C190T)] (21–23) was determined using an oligonucleotide ligation assay (OLA) (24) that was modified for implementation on a Hamilton Microlab 2200 liquid handling robot (Boissy et al., manuscript in preparation). Briefly, PCR primers were selected to amplify a 1231 bp fragment containing all three polymorphic sites (Table I). PCR reactions (16 µl) contained 50 ng of genomic DNA, 1 µl Tfl polymerase reaction buffer (Promega, Madison, WI), 1.0 M betaine monohydrate (Fluka) (25), 0.2 mM dNTPs, 4 mM Mg(OAc)2, 800 nM of each primer, and 0.02 U/µl of Tfl polymerase (Promega). Each 96-well PCR plate contained two positive (heterozygote) controls for each polymorphic site, and two no-template controls. PCR cycling conditions were 7 min at 94°C; 40 cycles of 0.5 min at 94°C then 2 min at 68°C; 6 min at 72°C; hold at 4°C. The resulting PCR product was diluted 9-fold to allow for aliquoting into ligation plates (15 µl diluted PCR product per well). NAT2 genotypes were determined using a PCR–RFLP method described previously (9). This method discriminates the five most common functional (NAT2*4) and low activity alleles (NAT2*5, NAT2*6, NAT2*7, NAT2*14), which correspond to the trivial names WT, M1, M2, M3 and M4, respectively. Individuals with any two low activity NAT2 alleles were considered slow acetylators, while those with either one or two NAT2 functional alleles were considered rapid acetylators.
Results and discussion Table II displays the calculated NAT1 allele frequencies for the groups in this study. In our Japanese study population, we found no individuals with the NAT1*11, NAT1*14 (G560A), NAT1*15 (C559T) or NAT1*17 (C190T) alleles. This is consistent with data from other Asian populations (23; H.Lin, personal communication; D.A.Bell, unpublished data). The allele frequency distribution among oral squamous cell carcinoma patients differed from the control group. The 1804
NAT1*4 allele was less frequent among cases, whereas the NAT1*10 allele was more frequent among cases. Pooling the rare NAT1*3 alleles with the NAT1*4 alleles (these alleles share the consensus polyadenylation signal), the overall frequency distributions among cases and controls were significantly different (P 5 0.008). The NAT1*10 allele frequency among the Japanese control population (0.42) was significantly higher than English (0.16; P , 0.001; data from ref. 8) and European-American (0.19; P , 0.001; D.A.Bell, unpublished data) control populations we have previously genotyped. The low activity alleles (NAT1*14, NAT1*15 and NAT1*17), which are absent among Japanese would hypothetically be protective. The non-occurrence of low activity alleles and the much higher frequency of NAT1*10 alleles among Japanese could potentially impact the relative incidence of NAT1 mediated diseases compared with European populations. The frequencies (%) of NAT1 genotypes among oral squamous cell carcinoma patients and controls are shown in Table III. Both heterozygous and homozygous genotypes containing the NAT1*10 allele were more frequent among oral squamous cell carcinoma cases (58.1 and 27.4%, respectively) compared with the control group (41 and 21.3%, respectively). There was a 3.6-fold risk associated with inheriting either homozygous and heterozygous NAT1*10 genotypes, but no significant gene dosage effect was observed for NAT1*10 alleles (OR*4/*10 5 3.6, 95% CI 16–8.5; OR *10/*10 5 3.3, 95% CI 1.3–8.6). This result was surprising given that the frequency of homozygous NAT1*10 genotypes should have been high enough in this study population to detect such an effect. Previous results from this laboratory suggested that the NAT1*10 allele was associated with increased risk of advanced stage colorectal cancer (8) and smoking-associated bladder cancer (26). Furthermore, earlier studies have shown the NAT1*10 alleles are associated with higher tissue levels of NAT1 enzyme activity in human bladder and colon tissue (14,15), and higher levels of DNA adducts (15). However, recent unpublished work from other laboratories has not confirmed the relationship between NAT1*10 genotype and NAT1 activity levels in tissues (27; D.M.Grant, personal communication). Studies focusing on the molecular and biochemical properties of different NAT1 alleles are currently in progress. Despite the uncertainty regarding the mechanistic relationship between the NAT1*10 polyadenylation polymorphism and NAT1 activity, in the present study we have observed a statistically significant association between NAT1*10 genotype and oral cancer risk.
NAT1 and oral squamous cell carcinoma
Table II. NAT1 allele frequencies among control subjects and oral squamous cell carcinoma patients
Controls Oral squamous cell carcinoma patients aNo
NAT1*4 (n)
NAT1*3 (n)
NAT1*10 (n)
NAT1*11, NAT1*14, NAT1*15, NAT1*17 (n)
Total chromosomes tested
0.56 (136) 0.43 (53)
0.025 (6) 0.008 (1)
0.42 (102) 0.56 (70)b
0.00 (0)a 0.00 (0)
244 124
NAT1*11, NAT1*14, NAT1*15 or NAT1*17 alleles found in these populations. allele comparison between groups, χ2 5 7.66; P 5 0.02; 2 df.
bThree
Table III. NAT1 genotype frequencies and estimated risks (crude ORs) among cases and controls % frequency (n)
NAT1*4 (or NAT1*3) Homozygote NAT1*4/NAT1*10 NAT1*10/NAT1*10 Risk for any NAT1*10 allele versus NAT1*3 genotypes aP bP cP
Case
Control
Risk (OR, 95% CI)
14.5 (9) 58.1 (36) 27.4 (17)
37.7 (46) 41.0 (50) 21.3 (26)
1 3.7 (1.60–8.46)b 3.3 (1.31–8.56)a 3.6 (1.61–7.90)c
5 0.012, risk relative to NAT1*4/NAT1*4 (or NAT1*3). Adjusted for age, gender and smoking category OR 5 3.72 (95% CI 1.41–9.75). 5 0.002, risk relative to NAT1*4/NAT1*4 (or NAT1*3). Adjusted for age, gender and smoking category OR 5 4.02 (95% CI 1.70–9.48). 5 0.001, risk relative to NAT1*4/NAT1*4 (or NAT1*3). Adjusted for age, gender and smoking category OR 5 3.92 (95% CI 1.72–8.89).
Table IV. NAT2 genotype frequencies and risks (crude ORs) among the oral squamous cell carcinoma patients and controls % frequency (n)
NAT2 rapid acetylator NAT2*4/NAT2*4 NAT2 intermediate (1 low activity allele) NAT2 slow acetylator (2 low activity alleles) Risk for NAT2 slow genotypes versus combined rapid/intermediate genotypes aP
Case
Control
Risk (OR, 95% CI)
41.9 (26) 46.8 (29) 11.3 (7)
50.0 (61) 44.3 (54) 5.7 (7)
1 1.3 (0.66–2.4)a 2.3 (0.8–7.2)b 2.1 (0.7–6.1)c
5 0.48; bP50.14; cP 5 0. 18.
Table IV shows NAT2 genotype frequencies among oral squamous cell carcinoma patients and controls. Individuals with two low activity alleles were classified as slow acetylators, whereas individuals with one low activity allele were intermediate acetylators and two low activity alleles were classified as rapid acetylators. Consistent with other studies (15), NAT2 slow acetylation genotypes were present at much lower frequency among Japanese (5.7% in controls) relative to European (55%) and African-American populations [41% (9)]. The NAT2 slow acetylator genotype was more common among cases (11.3%) than among controls (5.7%), but this difference was not statistically significant. The calculated odds ratio of 2.1 (95% CI 0.7–6.1; P 5 0.18) for slow versus rapid genotypes suggests a possible risk but the very small number of NAT2 slow acetylator genotypes among the cases (seven cases, 11%) makes it difficult to assess any role for this gene. NAT1 codes for a protein with a wide tissue distribution. In contrast, NAT2 codes for a protein expressed predominantly in liver (10–12). Degawa et al. observed that NAT1 enzyme activity was present in laryngeal mucosa but no NAT2 enzyme activity could be detected (13). No data are available regarding the expression of the NAT1 enzyme in oral tissue. However, the similarity of oral and laryngeal tissue (both squamous cell) would suggest that the NAT1 enzyme, but not NAT2, would
be expressed in oral tissues. Our finding that the NAT1*10 polymorphism, but not NAT2 genotype, was significantly associated with the risk of oral squamous cell carcinoma is consistent with this hypothesis. A link between tobacco smoking and oral squamous cell carcinoma has been shown by numerous epidemiological studies (reviewed in refs 17–19). Cigarette smoke is a complex mixture of compounds that includes carcinogenic polycyclic aromatic hydrocarbons, nitrosamines and aromatic amines. In our study, the smoking frequency was somewhat higher in the oral squamous cell carcinoma patient group (64.5%) than in the healthy control group (58.2%, P 5 0.6; among males it was 92.5 versus 77.5%) but neither of these differences were statistically significant. Analysis of the mean smoking index (cigarettes per day3years of smoking) showed that cases had a higher cumulative exposure to cigarettes compared with the control subjects (452 versus 375, P 5 0.009). While these results are compatible with previous epidemiological data that show a strong association between oral squamous cell carcinoma and smoking (17–19), the high rate of smoking in the present Japanese control group limits the statistical power for analysis of smoking effects in this case-control study. Data on alcohol use were available for oral cancer subjects but not controls. Among cases, alcohol use was correlated with tobacco 1805
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Table V. Smoking and NAT1 genotype: stratified analysis % frequency (n)
NAT1*4 (or NAT1*3) homozygote NAT1*10 genotypes Non-smokers NAT1*4 (or NAT1*3) homozygote NAT1*10 genotypes Smokers NAT1*4 (or NAT1*3) homozygote NAT1*10 allele
Case
Control
Risk (OR, 95% CI)
14.5 (9) 85.5 (53)
37.7 (46) 62.3 (76)
1 3.6 (1.61–7.90)a
9.1 (2) 90.9 (20)
35.3 (18) 64.7 (33)
1 5.5 (1.14–26.0)b
17.5 (7) 82.5 (33)
39.4 (28) 60.6 (43)
1 3.1 (1.61–7.90)c
aP 5 0.012, risk relative to NAT1*4/NAT1*4 (or NAT1*3). Adjusted for age, gender and smoking category OR 5 3.72 (95% CI 1.41–9.75). bP 5 0.022. cP 5 0.017; estimated risks for NAT1*10 among non-smokers (OR 5 5.5) and smokers (OR 5 3.1) were not significantly different (P . 0.05).
consumption (P , 0.05) but not NAT genotype. It is likely that alcohol use is an important risk factor for oral cancer in this study; however, it was not possible to directly assess this because alcohol use information was lacking for control subjects. Based on a hypothesized role for NAT1 in modulating the effects of carcinogens present in tobacco smoke, we investigated a combined role for smoking and NAT1 genotype. These data are shown in Table V. Following stratification by smoking, the frequency of the NAT1*10 allele was higher among both smoking and non-smoking cases relative to controls, with ORs of 5.88 (P , 0.05) for non-smoker cases and 3.14 (P , 0.05) for smokers. The higher value for non-smokers was not significantly different than that for smokers. This suggests that individuals with NAT1*10 alleles are at higher risk for oral squamous cell carcinoma, but that smoking history (as measured in this study) may not play a role in this genetic relationship. Smoking behavior in cases or controls (either smoker or non-smoker index) was not associated with any NAT genotypes. It should be noted that the small number of non-smokers in the study limited the power to detect combined gene by smoking effects, and this is reflected in the wide confidence intervals around the point estimates of risk. When we calculated the OR for individuals with NAT1*10 genotypes and adjusted for gender, age and smoking, we found that oral cancer cases were 3.9-fold more likely to have NAT1*10 genotypes (Table III, footnote). The observation that NAT2 slow acetylator genotype frequencies were higher (although not significantly higher) among cases suggested the possibility that the two NAT genes might have some combined effect on risk. This phenomenon has been observed in previous studies (8,15,26). We carried out statistical tests of gene–gene interaction between NAT1 and NAT2 genotypes using a series of logistic regression models and found no evidence for interaction (P 5 0.8 for test of interaction). This study is the first test of association between NAT1 alleles and oral cancer and the first analysis of NAT1 genotype in a Japanese population. In this study we tested for recently discovered rare, low activity NAT1 alleles (21,23) and find that these were absent in this Japanese study population. We observed that the NAT1*10 polymorphism was associated with the occurrence of oral squamous cell carcinoma in a Japanese population. The role for NAT1 appears to be independent of 1806
smoking behavior; we presume other exposure factors, such as carcinogens in the diet, are modulated in some way. Unfortunately, only partial information about risk factors in subjects was available in this small pilot study and this limits the interpretation of the results. While the mechanism for the risk associated with the NAT1*10 allele is unclear, the present finding suggests that carcinogens metabolized through a NAT1 pathway may be important in oral squamous cell carcinoma. Acknowledgements We thank Richard Morris and Renee Jarmillo, Analytical Sciences Inc., Durham, NC, for performing logistic regression analyses and Gary Pittman, NIEHS, for helpful comments on the manuscript.
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