Introduction
There is growing evidence that oxidative stress induced by reactive oxygen species (ROS) is involved in colorectal carcinogenesis by affecting cellular processes critical in tumor development (1). Crucial genes in the oxidative stress pathway include catalase (CAT), manganese superoxide dismutase (MnSOD), myeloperoxidase (MPO), and endothelial nitric oxide synthase (eNOS), coding for enzymes that are related to oxidative stress mechanisms by either neutralizing or generating ROS. Genetic polymorphisms in oxidative stress genes could alter colorectal cancer risk, and this association could be modified by increased ROS exposure following smoking. Tobacco smoke contains highly reactive free radicals and has been associated with colorectal cancer risk in a dose-dependent fashion. In accordance with the biological hypothesis that smoking may act as an initiator of colorectal tumorigenesis and, thus, requires a long induction period (2, 3), we found that long duration of smoking at a high cumulative dose increases colorectal cancer risk (4). In the present study, we assessed potential effect modification by long-term and high-dose smoking on the association between functional genetic polymorphisms in oxidative stress genes [CAT C262T (rs1001179), MnSOD Val9Ala (rs4880), MPO G463A (rs2333227), and eNOS Glu298Asp (rs1799983)] and colorectal cancer risk in a German case-control study.
Materials and Methods
Study Population
As described previously, we conducted a population-based case-control study on incident colorectal cancer in the southwest of Germany (4-6). Cases were identified by treating clinicians. Controls were randomly selected from population registers and frequency matched to the cases by sex, 5-y age groups, and county of residence. Eligible participants were of ages ≥30 y, resident of the study region, German speaking, and physically and mentally able to participate in a personal interview. The study was approved by the ethics committee of the University of Heidelberg and the medical boards of Baden-Wuerttemberg and Rhineland-Palatinate. All participants provided written informed consent.
Comprehensive information on colorectal cancer risk factors including lifetime smoking habits was collected. Pathology records and discharge letters were collected for the cases. Between January 2003 and December 2004, 671 cases enrolled, with 643 (96%) providing a biological sample. Of the 1,391 eligible controls, 613 (44.1%) enrolled and provided a biological sample.
Genotyping
According to the manufacturer's instructions, genomic DNA was extracted from whole-blood samples using the FlexiGene DNA Kit (Qiagen) or from mouthwash samples using the QiAmp DNA Mini Kit (Qiagen). DNA isolation was successful for 632 (98.3%) cases and 606 (98.7%) controls. Single-nucleotide polymorphisms in CAT, MnSOD, MPO, and eNOS were analyzed using PCR followed by Pyrosequencing technology (Biotage, Uppsala, Sweden). Genotyping was done blinded to case-control status. A 10% random sample was genotyped twice for quality assurance, which yielded 100% concordance.
Statistical Analysis
Conditional logistic regression adjusted for potential confounders was used to assess the association between genetic polymorphisms and colorectal cancer risk and potential effect modification by smoking. Analyses were stratified by sex and 5-y age groups. Test for trend was done by using the Wald statistic.
Statistical interaction was assessed using a multiplicative interaction model. Interaction terms of the dichotomous genotype variable and the dummy-coded ordinal variables for pack-years of smoking (never smoking, 1 to 29 pack-years, ≥30 pack-years) were included in the model. Statistical significance of interaction was analyzed by comparing the model with and without the respective interaction terms by means of the likelihood-ratio test with corresponding degrees of freedom. To consider potential effect modification by high cumulative dose solely, we also assessed interaction including only the variable for ≥30 pack-years in the model.
Results
All single-nucleotide polymorphisms were in Hardy-Weinberg equilibrium and the observed allele frequencies were comparable to those reported for Caucasians in the dbSNP database. Consistent with previous reports, differences between cases and controls were observed with respect to body mass index, endoscopic screening examinations, regular use of nonsteroidal anti-inflammatory drugs, and education level. Smoking status was not associated with colorectal cancer risk in multivariate analyses. However, an increased risk of colorectal cancer was observed in smokers who had smoked for ≥30 pack-years [odds ratio (OR), 1.33; 95% confidence interval (95% CI), 0.87–2.04]. None of the genetic polymorphisms were associated with colorectal cancer risk (Table 1). No significant interaction was observed between pack-years of smoking and genetic polymorphisms for the two models that included two levels of cumulative dose (1–29 pack-years, ≥30 pack-years) or smoking ≥30 pack-years only (Table 2).
ORs for colorectal cancer according to polymorphisms in CAT, MnSOD, MPO, and eNOS
Genetic polymorphism . | n (cases/controls) . | Crude model* [OR (95% CI)] . | Adjusted model† [OR (95% CI)] . | Ptrend . |
---|---|---|---|---|
CAT | ||||
C/C | 374/348 | 1 | 1 | 0.64 |
C/T | 235/231 | 0.95 (0.75–1.20) | 0.95 (0.73–1.22) | |
T/T | 23/26 | 0.82 (0.46–1.47) | 0.92 (0.49–1.73) | |
T carriers | 0.94 (0.75–1.18) | 0.94 (0.74–1.21) | ||
MnSOD | ||||
T/T | 136/146 | 1 | 1 | 0.4 |
C/T | 321/294 | 1.18 (0.89–1.57) | 1.18 (0.86–1.60) | |
C/C | 166/163 | 1.1 (0.80–1.52) | 1.17 (0.83–1.65) | |
C carriers | 1.15 (0.88–1.51) | 1.17 (0.88–1.57) | ||
MPO | ||||
G/G | 396/373 | 1 | 1 | 0.92 |
G/A | 202/206 | 0.92 (0.72–1.17) | 0.94 (0.73–1.22) | |
A/A | 29/24 | 1.25 (0.71–2.20) | 1.24 (0.68–2.27) | |
A carriers | 0.95 (0.76–1.20) | 0.97 (0.76–1.25) | ||
eNOS | ||||
G/G | 289/271 | 1 | 1 | 0.98 |
G/T | 285/272 | 0.99 (0.78–1.25) | 1.02 (0.79–1.31) | |
T/T | 58/61 | 0.90 (0.60–1.33) | 0.99 (0.64–1.52) | |
T carriers | 0.97 (0.78–1.22) | 1.01 (0.79–1.29) |
Genetic polymorphism . | n (cases/controls) . | Crude model* [OR (95% CI)] . | Adjusted model† [OR (95% CI)] . | Ptrend . |
---|---|---|---|---|
CAT | ||||
C/C | 374/348 | 1 | 1 | 0.64 |
C/T | 235/231 | 0.95 (0.75–1.20) | 0.95 (0.73–1.22) | |
T/T | 23/26 | 0.82 (0.46–1.47) | 0.92 (0.49–1.73) | |
T carriers | 0.94 (0.75–1.18) | 0.94 (0.74–1.21) | ||
MnSOD | ||||
T/T | 136/146 | 1 | 1 | 0.4 |
C/T | 321/294 | 1.18 (0.89–1.57) | 1.18 (0.86–1.60) | |
C/C | 166/163 | 1.1 (0.80–1.52) | 1.17 (0.83–1.65) | |
C carriers | 1.15 (0.88–1.51) | 1.17 (0.88–1.57) | ||
MPO | ||||
G/G | 396/373 | 1 | 1 | 0.92 |
G/A | 202/206 | 0.92 (0.72–1.17) | 0.94 (0.73–1.22) | |
A/A | 29/24 | 1.25 (0.71–2.20) | 1.24 (0.68–2.27) | |
A carriers | 0.95 (0.76–1.20) | 0.97 (0.76–1.25) | ||
eNOS | ||||
G/G | 289/271 | 1 | 1 | 0.98 |
G/T | 285/272 | 0.99 (0.78–1.25) | 1.02 (0.79–1.31) | |
T/T | 58/61 | 0.90 (0.60–1.33) | 0.99 (0.64–1.52) | |
T carriers | 0.97 (0.78–1.22) | 1.01 (0.79–1.29) |
NOTE: MnSOD, T = Val, C = Ala; eNOS, G = Glu, T = Asp.
*Conditional logistic regression stratified by sex and age in 5-y age groups.
†Conditional logistic regression stratified by sex and age in 5-y age groups adjusted for education level, endoscopic screening, family history of colorectal cancer, body mass index, regular use of nonsteroidal anti-inflammatory drugs, frequency of red meat consumption, pack-years of smoking, and average lifetime daily alcohol consumption.
ORs for colorectal cancer according to polymorphisms in CAT, MnSOD, MPO, and eNOS by pack-years of smoking
Genetic polymorphism . | Never smokers . | 1–29 pack-years . | ≥30 pack-years . | P* . | P† . | |||
---|---|---|---|---|---|---|---|---|
Ca/Co . | OR (95% CI)‡ . | Ca/Co . | OR (95% CI)‡ . | Ca/Co . | OR (95% CI)‡ . | |||
CAT | ||||||||
C/C | 180/174 | 1 | 139/138 | 1 | 49/32 | 1 | ||
T carriers | 120/112 | 0.98 (0.69–1.41) | 103/121 | 0.76 (0.51–1.12) | 32/24 | 1.53 (0.52–4.57) | 0.41 | 0.61 |
MnSOD | ||||||||
T/T | 60/68 | 1 | 57/69 | 1 | 15/9 | 1 | ||
C carriers | 237/216 | 1.18 (0.77–1.81) | 180/190 | 1.28 (0.81–2.02) | 65/47 | 0.82 (0.23–2.90) | 0.86 | 0.61 |
MPO | ||||||||
G/G | 191/174 | 1 | 148/163 | 1 | 50/36 | 1 | ||
A carriers | 108/110 | 0.86 (0.60–1.23) | 90/96 | 0.95 (0.63–1.42) | 31/20 | 1.19 (0.43–3.24) | 0.76 | 0.52 |
eNOS | ||||||||
G/G | 141/125 | 1 | 102/113 | 1 | 40/29 | 1 | ||
T carriers | 159/160 | 1.01 (0.71–1.44) | 140/146 | 1.08 (0.73–1.61) | 41/27 | 1.35 (0.53–3.43) | 0.96 | 0.79 |
Genetic polymorphism . | Never smokers . | 1–29 pack-years . | ≥30 pack-years . | P* . | P† . | |||
---|---|---|---|---|---|---|---|---|
Ca/Co . | OR (95% CI)‡ . | Ca/Co . | OR (95% CI)‡ . | Ca/Co . | OR (95% CI)‡ . | |||
CAT | ||||||||
C/C | 180/174 | 1 | 139/138 | 1 | 49/32 | 1 | ||
T carriers | 120/112 | 0.98 (0.69–1.41) | 103/121 | 0.76 (0.51–1.12) | 32/24 | 1.53 (0.52–4.57) | 0.41 | 0.61 |
MnSOD | ||||||||
T/T | 60/68 | 1 | 57/69 | 1 | 15/9 | 1 | ||
C carriers | 237/216 | 1.18 (0.77–1.81) | 180/190 | 1.28 (0.81–2.02) | 65/47 | 0.82 (0.23–2.90) | 0.86 | 0.61 |
MPO | ||||||||
G/G | 191/174 | 1 | 148/163 | 1 | 50/36 | 1 | ||
A carriers | 108/110 | 0.86 (0.60–1.23) | 90/96 | 0.95 (0.63–1.42) | 31/20 | 1.19 (0.43–3.24) | 0.76 | 0.52 |
eNOS | ||||||||
G/G | 141/125 | 1 | 102/113 | 1 | 40/29 | 1 | ||
T carriers | 159/160 | 1.01 (0.71–1.44) | 140/146 | 1.08 (0.73–1.61) | 41/27 | 1.35 (0.53–3.43) | 0.96 | 0.79 |
NOTE: MnSOD, T = Val, C = Ala; eNOS, G = Glu, T = Asp.
Abbreviations: Ca, cases; Co, controls.
*P value for interaction of conditional logistic regression model between genotype and two categories of smoking (2 degrees of freedom).
†P value for interaction of conditional logistic regression model between genotype and ≥30 pack-years of smoking (1 degree of freedom).
‡Conditional logistic regression stratified by sex and age in 5-y age groups adjusted for education level, endoscopic screening, family history of colorectal cancer, body mass index, regular use of nonsteroidal anti-inflammatory drugs, frequency of red meat consumption, and average lifetime daily alcohol consumption.
Discussion
We observed no effect modification either by low- or high-level smoking on the association between genetic polymorphisms in CAT, MnSOD, MPO, or eNOS and colorectal cancer risk. Focusing particularly on smoking for a long duration and at high intensity, which has been shown to contribute to colorectal carcinogenesis, we observed also no significant effect modification. We had more than 80% power to detect an interaction OR of 3.0 between ≥30 pack-years of smoking and CAT, MnSOD, MPO, or eNOS genotype and nearly 70% to detect an interaction OR of 2.5. Because the effect of smoking on colorectal cancer risk was quite modest, only strong interaction with oxidative stress gene polymorphisms would have been detectable in this study.
As tobacco smoke has the potential to generate oxidative stress (7), smoking could modify the association between polymorphisms in oxidative stress genes. Variant alleles of CAT C262T or MnSOD Val9Ala have been associated with increased ROS levels and, thus, were suggested to increase cancer risk, whereas variant alleles of MPO G463A and eNOS Glu298Asp, both related to low activity of the corresponding enzymes, have been proposed to reduce cancer risk. Epidemiologic studies considering effect modification by smoking on the association between genetic polymorphisms in oxidative stress genes and cancer risk are scarce. Despite a biological plausible hypothesis, this has thus far not been assessed for colorectal cancer. With regard to breast cancer risk, both significant (8) and nonsignificant (9-11) interactions between MnSOD Val9Ala and smoking were observed. However, studies done in other cancer sites did not reveal modification of smoking effects by CAT C262T (12), MPO G463A (13-19), or eNOS Glu298Asp (19). Large studies will be required to investigate potential interaction between smoking and polymorphisms in oxidative stress genes with respect to colorectal cancer, which is likely to be modest.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
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