Well-done meat consumption, NAT1 and NAT2 acetylator genotypes and prostate cancer risk: The Multiethnic Cohort study

Abstract

Background

Prostate cancer (PC) is the most common male malignancy in the U.S. and disparities in risk exist among ethnic/racial groups. A high intake of well-done meat and the presence of the rapid NAT1 and slow NAT2 acetylator genotypes, as modifiers of the carcinogenic effect of heterocyclic amines, were hypothesized to increase PC risk and possibly explain these ethnic differences in risk.

Methods

This study examined the associations between well-done (red) meat consumption, NAT1 and NAT2 acetylator genotypes and PC risk among five ethnicities (African American, Native Hawaiian, Japanese American, Latino and Caucasian) in a case-control study of PC nested within the Multiethnic Cohort study. Cases (n=2,106) and controls (n=2,063) were genotyped for eight single nucleotide polymorphisms (SNPs) in NAT1 and seven SNPs in NAT2 that characterize all common alleles for these genes. Well-done meat intake was computed based on responses to a detailed food frequency questionnaire including a question on meat preference. Conditional logistic regression was used in the analysis.

Results

There was no evidence of an increased risk associated with preference for well-done meat, intake of well-done meat and NAT1 or NAT2 genotypes (jointly or separately).

Conclusions

These results do not support the hypothesis that exposure to heterocyclic amines is associated with risk of PC. However, additional studies with more precise exposure measures are needed.

Introduction

Prostate cancer (PC) is the most common male malignancy in the U.S. and risk varies by ethnicity which could partially be due to differential exposure to heterocyclic aromatic amines (HAAs), a class of carcinogens formed when meat is cooked at high temperature (1-8). The rapid NAT1 and the slow NAT2 genotypes are suspected to increase PC risk due to their effect on HAA activation by O-acetylation in the prostate and decreased detoxification of HAAs in the liver, respectively (9-11). We examined associations between well-done meat and PC risk, and the modifying effects of NAT1 and NAT2 acetylator genotypes, among five ethnic/racial groups.

Materials and Methods

This case-control study nested in the Multiethnic Cohort (MEC) was approved by the Institutional Review Boards at the University of Hawaii and the University of Southern California. Participants (N>215,000) were recruited from Hawaii and Los Angeles in 1993-1996, were aged 45-75 years at entry and were primarily comprised of African American, Native Hawaiian, Japanese American, Latino and Caucasian men and women (12,13). Incident PC cases since January 1995 were identified through Surveillance Epidemiology and End Results cancer registries (14). Blood samples were generally obtained after diagnosis (15). Controls were frequency-matched by ethnicity and age.

NAT1 and NAT2 were determined using TaqMan allele discrimination assays (Applied Biosystems) (16,17) with a successful genotyping rate of ≥98.7% and genotype concordance (among 5% blind QC duplicates) of ≥98.5%. The genotype distributions among controls were in Hardy-Weinberg equilibrium (p>0.05) for each ethnic group. Through genotyping of seven single nucleotide polymorphisms (SNPs) occurring with >1% frequency in at least one ethnicity [G191A (R64Q), C282T, T341C (I114T), C481T, G590A (R197Q), A803G (K268R), G857A (G286T)], 26 of the common NAT2 allelic variants can be detected (NAT2*4; NAT2*5A,B,C,D,E,G,J; NAT2*6A,B,C,E; NAT2*7A,B; NAT2*11A; NAT2*12A,B,C; NAT2*13; NAT2*14A,B,C,D,E,F,G) (18). Similarly, all common NAT1 allelic variants (NAT1*3; NAT1*4; NAT1*10; NAT1*11A,B,C; NAT1*14A,B; NAT1*15; NAT1*17; NAT1*19; NAT1*22) can be characterized by genotyping eight SNPs [C97T (R33Stop), C1095A (3′-UTR), C190T (R64W), G445A (V149I), C559T (R187Stop), G560A (R187Q), A752T (D251V), T1088A (3′-UTR)] (16,17). Individuals with two “rapid” alleles (NAT2*4, NAT2*11A, NAT2*12A,B,C and NAT2*13), two “slow” phenotypes and with one “rapid” and one “slow” allele were assigned to the “rapid”, “slow” and “intermediate” NAT2 genotype, respectively. The NAT1*10 allele was designated as the “at risk” phenotype. NAT1 was categorized as “NAT1*10”, “NAT1*10/other NAT1 allele” and “any combination of other NAT1 alleles”, represented as “2”, “1” and “0 copies”, respectively. Missing SNP results were imputed when certainty was ≥95% using PHASE (version 2.1) (18,19).

The validated food frequency questionnaire (FFQ) included questions on preference for well-done meat and the amount and frequency of consumption of different types of meat over the past year (12, 13). The meat groups were computed as the sum of all corresponding food items and the relevant proportion from mixed dishes.

Conditional logistic regression stratified by 5-year age groups and ethnicity and adjusted for energy, BMI, education, family history and smoking was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) using SAS, version 9.1 (SAS, Cary, NC, USA). Adjustment for fat was not included because fat intake was not found to have any effect on PC risk in the MEC. Interactions between ethnicity, well-done red meat, NAT1 and NAT2 were examined by a Wald test of cross-product terms. Results for Native Hawaiians are not presented separately because of the small sample size, although they were included in the combined group.

Results

Among cases and controls, more African Americans and Latinos consumed well-done meat than other ethnicities (Table 1). African Americans had a higher prevalence than Caucasians for the high risk NAT1*10 allele but not for the NAT2 slow genotype.

Table 1.

Odds ratios (ORs) and 95% confidence intervals (CIs) for risk of prostate cancer associated with meat preference, NAT1 and NAT2 genotype

	Total			African American		Japanese American		Latino		Caucasian
	Cases/ controls	OR (95% CI), adjusted for age and ethnicity*	OR (95% CI), multivariate adjusted†	Cases/controls	OR (95% CI)‡	Cases/controls	OR (95% CI)‡	Cases/controls	OR (95% CI)‡	Cases/controls	OR (95% CI)‡
Meat preference
Rare / No Meat	184 / 160	1.00	1.00	22/15	1.00	38/40	1.00	39/29	1.00	75/69	1.00
Medium	902 / 892	0.86 (0.68-1.09)	0.93 (0.76-1.14)	173/169	0.68 (0.34-1.36)	269/252	1.01 (0.62-1.65)	191/209	0.67 (0.40-1.13)	230/229	0.94 (0.64-1.37)
Well-done	1020 / 1011	0.85 (0.67-1.08)	0.84 (0.66-1.07)	404/387	0.73 (0.37-1.43)	117/128	0.87 (0.51-1.47)	366/350	0.72 (0.44-1.21)	116/123	0.84 (0.55-1.27)
p for trend §		0.30	0.23		0.94		0.41		0.69		0.38
Genotypes
NAT1*10
0 copy	864 / 841	1.00	1.00	200/187	1.00	145/118	1.00	229/245	1.00	282/281	1.00
1 copy	878 / 873	0.98 (0.86-1.13)	0.98 (0.86-1.13)	278/265	1.00 (0.77-1.30)	179/204	0.72 (0.53-1.00)	262/257	1.09 (0.85-1.39)	125/121	1.04 (0.77-1.40)
2 copies	364 / 349	1.01 (0.84-1.21)	1.01 (0.84-1.22)	121/119	0.96 (0.70-1.33)	100/98	0.84 (0.58-1.21)	105/86	1.30 (0.92-1.82)	14/19	0.73 (0.36-1.49)
P for trend §		0.99	0.96		0.83		0.27		0.14		0.73
NAT2
Rapid	379 / 355	1.00	1.00	65/48	1.00	202/204	1.00	68/65	1.00	27/21	1.00
Intermediate	909 / 894	0.93 (0.78-1.12)	0.92 (0.77-1.11)	254/275	0.68 (0.45-1.02)	169/175	0.97 (0.73-1.29)	284/268	1.02 (0.70-1.48)	167/147	0.88 90.48-1.62)
Slow	818 / 814	0.91 (0.75-1.11)	0.91 (0.75-1.11)	280/248	0.82 (0.55-1.24)	53/41	1.28 (0.81-2.01)	244/255	0.92 (0.63-1.35)	227/253	0.70 (0.38-1.27)
P for trend §		0.42	0.42		0.87		0.49		0.50		0.07
Intermediate/	1288 / 1249	1.00	1.00	319/323	1.00	371/379	1.00	352/333	1.00	194/168	1.00
Slow	818 / 814	0.97 (0.85-1.10)	0.97 (0.95-1.11)	280/248	1.14 (0.90-1.43)	53/41	1.29 (0.84-1.99)	244/255	0.91 (0.72-1.15)	227/253	0.78 (0.60-1.03)

^*Adjusted for age groups and ethnicity as strata in a conditional logistic regression model;

^†Adjusted for age groups and ethnicity as strata in a conditional logistic regression model, and for energy, body mass index, years of education, family history of prostate cancer and smoking status (never/former/current) as covariates;

^‡Adjusted for age groups as strata in a conditional logistic regression model;

^§Wald statistic for trend variables assigned the number of variant alleles for NAT1 (0, 1 and 2 copies, respectively) and NAT2 (slow, intermediate and rapid, respectively).

The age- and ethnicity-adjusted and multivariate-adjusted ORs were similar in all models. No statistically significant association was observed between meat preference (p_{heterogeneity}=0.72) (Table 1) or types of meat by level of doneness and PC risk. There was no association with PC risk for 1 copy or 2 copies of NAT1*10 compared to 0 copies, the intermediate or slow NAT2 compared to the rapid genotype (p_{heterogeneity}=0.37 for NAT1 and 0.25 for NAT2) (Table 1) or NAT1 and NAT2 jointly (data not shown). The OR for men with 2 copies of NAT1*10 and the slow NAT2 genotype was 0.81 (0.54-1.21) compared to those with 0 copies and the rapid genotype (p_{heterogeneity}=0.22). The two-way (Table 2) and three-way interactions of NAT1*10, NAT2 and preference for well-done meat were not significant. All results were also null in an analysis of advanced PC.

Table 2.

Odds ratios (ORs) and 95% confidence intervals (CIs) for risk of prostate cancer associated with the two-way interaction between NAT1/NAT2 and preference for well-done meat

NAT	Preference for well-done meat	Cases / Controls	OR, adjusted for age and ethnicity *	95% CI	OR, multivariate adjusted †	95% CI
NAT1*10 (copies)
0	No	469 / 446	1.00		1.00
0	Yes	395 / 395	0.94	0.77-1.14	0.92	0.76-1.12
1 or 2	No	617 / 606	0.97	0.81-1.26	0.96	0.80-1.15
1 or 2	Yes	625 / 616	0.95	0.79-1.14	0.93	0.78-1.13
p for interaction (1 df)‡				0.72		0.67
NAT2
Intermediate/Rapid	No	693 / 638	1.00		1.00
Intermediate/Rapid	Yes	595 / 611	0.88	0.75-1.04	0.88	0.74-1.03
Slow	No	393 / 414	0.86	0.72-1.04	0.87	0.72-1.05
Slow	Yes	425/ 400	0.95	0.79-1.15	0.94	0.78-1.14
p for interaction (1 df) ‡				0.08		0.10

^*Adjusted for age groups and ethnicity as strata in a conditional logistic regression model.

^†Adjusted for age groups and ethnicity as strata in a conditional logistic regression model and for energy, body mass index, years of education, family history of prostate cancer and smoking status (never/former/current) as covariates.

^‡The p for interaction is based on a Wald test of cross-product terms.

Discussion

This study did not find significant associations for well-done meat, NAT1 and NAT2 with PC risk overall, by ethnicity or among advanced PC cases. Our null findings for meat and PC risk agree with a previous cohort study (20). In another study, high consumption of red meat doubled the PC risk for African Americans (21), while in two largely Caucasian cohorts a direct association was observed for high intake of red meat and well-done meat with PC risk (4,22). The slow NAT2 genotype has been associated with a lowered PC risk while the rapid NAT2 genotype has been associated with a non-significantly elevated PC risk (23,24). Among Japanese, the NAT1*10 was related to a higher PC risk (25) and the slow NAT2 genotype was more common in PC cases than controls (26). In agreement with our results, other studies also found no relationship between NAT2 and PC (27,28). The combination of the NAT1*10 and the slow NAT2 genotype has been associated with a five-fold higher PC risk and the very slow NAT2 genotype with a seven-fold elevated PC risk (11). In one small case-control study, the associations of meat and NAT1/NAT2 with PC were also not significant (29).

This study is the first large nested case-control study to investigate the ethnic-specific effect of well-done meat, NAT1 and NAT2 on PC risk. A FFQ developed specifically for this population was used to ensure standardized data collection, and a comprehensive number of NAT1 and NAT2 SNPs were genotyped. Since exposure to dietary HAAs is difficult to measure, as it depends on the type of meat, as well as the duration and temperature of cooking, additional studies with more direct measurement of HAAs would be useful.

In conclusion, these data do not support the hypothesis that consumption of well-done meat, NAT1, NAT2 or their interactions are associated with PC risk.