No Modifying Effect of NAT1, GSTM1, and GSTT1 on the Relation between Smoking and Colorectal Cancer Risk

Cigarette smoke contains potent carcinogenic compounds, including polycyclic aromatic hydrocarbons, heterocyclic amines, and nitrosamines. When entering the body, cigarette carcinogens have relatively weak mutagenic potential. After being metabolized, more potent mutagens are formed, which could result in DNA adduct formation. The genes involved in this metabolism include NAT1, NAT2, GSTM1, and GSTT1, and several polymorphisms could influence their enzyme activity. For NAT2, we found smoking to be a risk factor for colorectal cancer, especially in women with rapid NAT2 imputed phenotype (1). No consistent association has been found between NAT1 and colorectal cancer, as reported in a review (2). The glutathione S-transferases are a family of enzymes responsible for the detoxification of mutagenic electrophiles including polycyclic aromatic hydrocarbons. Homozygotes for the null alleles (deletion) of glutathione S-transferase μ (GSTM1) and glutathione S-transferase θ (GSTT1) lack activity of the respective enzymes. Because it seems likely that genetically determined variation in enzymatic (in)activation may modify colorectal cancer risk associated with smoking, the aim of the present study was to investigate the combined effect of smoking and genetic polymorphisms in each of the relevant metabolic genes (NAT1, GSTM1, and GSTT1).

A population-based screening program for early detection of breast cancer was started in 1974, in Utrecht, the Netherlands: the so-called “DOM” (Diagnostisch Onderzoek Mammacarcinoom) project. A total of 25,769 women, born between 1911 and 1945 attended the screening and provided a urine sample, which was stored at −20°C. The participation rate was 70% (1).

Follow-up from 1976 until 1987 revealed 56 deaths because of colon cancer and 8 deaths because of rectal cancer. From 1987 to January 1, 1996, 161 incident colon cancer cases and 73 incident rectal cancer cases were identified. For the present study, a subcohort of 1000 women was randomly selected from the source population. The institutional review board for human studies of the University Medical Center Utrecht approved the study.

Urine samples of 1298 study participants were retrieved and thawed, and 50 ml were removed for DNA isolation. Four NAT1 polymorphisms were detected in duplicate by radioactive allele-specific oligonucleotide hybridization. For genotype determination four X-ray films were read at the same time. The samples were classified in duplicate by two independent observers as NAT1*3, NAT1*4, NAT1*10, or NAT1*11 homozygote or heterozygote.

The presence or absence of the GSTM1 and GSTT1 gene was determined by multiplex PCR with an internal control. All laboratory analyses were performed blind with respect to the case or control status.

Smoking habits were assessed at baseline by self-administered questionnaire. For 10 women, the smoking status was not known; for 267 participants, DNA was insufficient or genotyping method failed (23% cases and 20% subcohort) for the NAT1 gene, and for 306 women (26% cases and 23% subcohort), we could not determine GSTM1 or GSTT1 genotype.

Women with at least one NAT1*10 allele were classified as rapid acetylators, and the remaining women were classified as slow acetylators. We used Poisson regression models to estimate incidence rate ratios and corresponding 95% CIs² for colorectal cancer. Because the 1000 women constitute a random sample of the total cohort, multiplication of the person-years in the reference group by 25.9 (the inverse of the subcohort non-cases sampling fraction) enabled us to analyze the data as a full cohort analysis, in which the person-years provide unbiased estimates of true person-years. Robust 95% CIs were calculated to account for additional variance introduced by sampling for the cohort.

There were no differences between the genotyped and nongenotyped women in any of the following baseline characteristics: age; height; and weight. About one-third of the women reported ever having smoked. None of the three putative at risk genotypes (NAT1*10 allele, GSTM1-null genotype, or GSTT1-null genotype) showed increased colorectal cancer risk. Women who had ever smoked were at increased risk for colorectal cancer (RR = 1.33; 95% CI, 1.00–1.79). This risk was equal for colon cancer (RR = 1.37; 95% CI, 0.99–1.90) and rectal cancer (RR = 1.26; 95% CI, 0.76–2.08), respectively. Compared with nonsmoking women with slow NAT1 genotype, women with rapid NAT1 genotype who smoked showed a nonsignificant increase of 26% (RR = 1.26; 95% CI, 0.75–2.11; Table 1).

The present study shows an increased colorectal cancer risk for women who reported ever having smoked; however, modification by NAT1, GSTM1, or GSTT1 genotype was not observed.

According to a recent review regarding smoking, a long latency period (30–40 years) is suggested to be necessary before colorectal cancer develops (3). Women started smoking in the Netherlands in the late 1950s, and sufficiently long follow-up time up to 1990 is necessary to observe carcinogenic effects of smoking on the colon and rectum in women.

Because the cohort started as a population-based screening program for early detection of breast cancer, colorectal cancer registry started only after 1987. However, a mortality registry was set up from the beginning and identified 64 deaths due to colorectal cancer. Restriction of the analyses to only the incident cases led to the same results. Recently, it was also reported that colorectal cancer survival was not associated with GSTM1- or GSTT1-null genotype (4). We therefore think that the absence of interaction is real. The power to detect a doubling of risk associated with smoking, NAT1 rapid genotype, GSTM1-null genotype, or GSTT1-null genotype is more than 95%, and the power to detect a 1.5× increased risk is 70%, 70%, 73%, and 61%, respectively.

We did not observe effects of GSTM1- or GSTT1-null genotype, which is in accordance with the hypothesis and a meta-analysis in which no association of either GSTM1 (10 studies) or GSTT1 (8 studies) deficiency with colorectal cancer risk was observed (5). The hypothesized higher colorectal cancer risk in women who smoked and who have a lower detoxification capacity, due to GSTM1- or GSTT1-null genotype, is not supported by our data. Because all of the enzymes examined in this study are involved in carcinogen metabolism, but with low substrate specificity, it is possible that inadequate activation or detoxification due to polymorphisms in one gene is compensated for by another gene. An interaction between NAT2 slow acetylator and GSTT1-null status was reported by Welfare et al. (Ref. 6; OR = 2.33; 95% CI, 1.1–5.0). We therefore classified women into categories with increasing number of putative risk genotype. However, no effect on colorectal cancer risk was observed, and numbers became too small to study a modifying effect on smoking.

In conclusion, this study supports smoking to be a risk factor for colorectal cancer, but no evidence that NAT1*10 genotype, GSTM1-null genotype, and GSTT1-null genotype increase this risk was found.