Genetic variants of myeloperoxidase and lung cancer risk

Matthew B. Schabath1,2, Margaret R. Spitz1, Xinmei Zhang1, George L. Delclos2 and Xifeng Wu1,2,3

1 The University of Texas M.D. Anderson Cancer Center, Department of Epidemiology, Box 189, 1515 Holcombe Boulevard, Houston and
2 University of Texas, The School of Public Health, The University of Texas Health Science Center at Houston, 1200 Herman Pressler, Houston, TX 77030, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cytochrome P450 family of enzymes is responsible for many of the initial metabolic conversions of procarcinogenic compounds in tobacco smoke to reactive metabolites. However, other enzyme-based systems such as myeloperoxidase (MPO) may also be involved in this metabolic process. MPO is a phase I metabolic enzyme that has a polymorphic region upstream of the gene that appears to reduce transcriptional activity. The polymorphic G->A nucleotide base shift negates the binding region for the general transcription factor SP1. Thus, individuals with the variant allele may be provided with a protective effect due to decreased metabolic conversion of carcinogenic compounds in tobacco smoke. This study has investigated the hypothesis that individuals with the variant allele may be at a reduced risk for lung cancer. Our results demonstrate that the protective effects of the MPO variant allele reduced overall lung cancer risk in Caucasians by 48% (OR = 0.52, 95% CI 0.30–0.90, P = 0.02). There was a 72% protective effect (OR = 0.28, 95% CI 0.12–0.61, P < 0.05) evident in men but not in women. Additionally, in younger individuals (<61 years) there was a statistically significant 72% reduction in risk (OR = 0.28, 95% CI 0.11–0.69, P < 0.05) but not in older individuals. A protective effect was observed for current smokers (OR = 0.24, 95% CI 0.10–0.58, P < 0.05) but not in former smokers and those who had never smoked. These data demonstrate that there is a reduction in lung cancer risk associated with a variant allele of MPO that is evident in men, younger individuals and current smokers.

Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxide; HMO, health maintenance organization; MPO, myeloperoxidase; MS, multiple sclerosis; RFLP, restriction fragment length polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cytochrome P450 family of enzymes is responsible for many of the initial metabolic conversions of procarcinogenic compounds in tobacco smoke to reactive metabolites. However, other enzyme-based systems can also be involved, including lipoxygenases, cyclooxygenases, epoxide hydrolase, dihydrodiol dehydrogenase, UDP-glucuronosyl transferase and myeloperoxidase (MPO) (1,2). MPO is a phase I metabolic enzyme found in monocytes/macrophages and primary granules of neutrophils (3) and is involved in the oxidation of procarcinogens (4). Acute inflammation of lung tissue results from procarcinogen exposures in environmental pollutants, occupational exposures and tobacco smoke. Trush et al. (5) suggested that an immune response resulting in the metabolic activation of chemical carcinogens to genotoxic derivatives could contribute to carcinogenesis. As part of a mediated immune response, neutrophils will be recruited and accumulate at the sites of pulmonary inflammation (6). Subsequently, MPO is released into the local environment (7,8) for the metabolic biotransformation of specific carcinogen metabolites. This enzymatic biotransformation process is necessary to convert lipophilic carcinogens to hydrophilic forms that are more readily excreted. However, during metabolic biotransformation, highly carcinogenic metabolites can be formed which are capable of deleterious effects on genomic integrity.

MPO has been shown to convert metabolites of the tobacco smoke procarcinogen benzo[a]pyrene (B[a]P) to the highly reactive and carcinogenic (+)-anti-diol epoxide (benzo[a] pyrene diol epoxide, BPDE) (4,9). BPDE is carcinogenic and mutagenic since it is capable of forming DNA adducts as well as causing sister chromatid exchanges. In the presence of neutrophils, activation of the B[a]P intermediate B[a]P-7,8-diol to the carcinogenic BPDE occurs via an MPO-dependent system (4). However, there is a polymorphic site near the MPO gene that may modify the level of carcinogen metabolism. The polymorphism is located in a hormone response element region 463 bp upstream of the MPO gene (10,11). The polymorphic G->A nucleotide base shift negates the binding region for the general transcription factor SP1. Piedrafita et al. (11) demonstrated that individuals with the A allele had an overall weaker transcriptional activity of the MPO gene due to reduced binding of SP1. Since overall transcriptional activity is decreased in individuals with the variant allele (A allele), less enzyme would ultimately be available for conversion of the B[a]P intermediate to the highly carcinogenic BPDE.

We hypothesized that individuals with the variant allele may be at a reduced risk for lung cancer. Polymorphisms of MPO have already been linked with numerous diseases, including acute promyelocytic leukemia (12), cystic fibrosis (13) and multiple sclerosis (MS) (14). MPO enzyme activity has been reported as being involved in carcinogen metabolism (4,9), inhibition of DNA repair (15) and DNA damage (16). However, there are limited data regarding a possible link between MPO and carcinogenesis. London et al. (17) were the first to report an association between the variant allele of MPO and lung cancer risk (OR = 0.30, 95% CI 0.10–0.93, P = 0.04, n = 182 cases). The purpose of this study was to confirm this previous report and further examine the association between genetic polymorphisms of the MPO gene and lung cancer risk by selected smoking characteristics.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
Lung cancer cases were accrued from The University of Texas M.D. Anderson Cancer Center. All cases were newly diagnosed, previously untreated (chemotherapy or radiotherapy) and histologically confirmed. There were no age, gender or stage restrictions. Since there were inadequate numbers of ethnic minorities, this analysis focused only on the Caucasian subset of cases. Healthy controls were recruited from a large multi-specialty health maintenance organization (HMO) in the Houston metropolitan area. The controls did not have a previous diagnosis of any cancer (except non-melanoma of the skin) and were matched to the cases on age (±5 years), gender, ethnicity and smoking status. Identification and recruitment of the control study subjects is thoroughly detailed in Hudmon et al. (18). Briefly, during patient registration the HMO personnel distributed a one page questionnaire that elicits relevant criteria for control selection and participant interest. The potential control participants were instructed to complete the questionnaire and deposit it in a conspicuously placed collection box in the waiting area. Subjects who indicated willingness to answer more questions were contacted by phone at a later date for confirmation of participation and to set an appointment for the interview. The appointment was scheduled at a time and HMO clinic location convenient to the participant.

Data collection
All study subjects completed a 60 min personal interview that was administered by highly trained staff personnel. The interview elicited information regarding sociodemographics and smoking history. At the conclusion of the interview, a 30 ml peripheral blood sample was drawn into coded heparinized tubes for immediate DNA isolation and subsequent molecular analysis.

MPO genotyping
Genomic DNA was isolated from peripheral blood lymphocytes, coded and subjected to restriction fragment length polymorphism (RFLP)–PCR. PCR was used to amplify a 350 bp region upstream of the MPO gene containing the polymorphic site. The primers 5'-CGG TAT AGG CAC ATG CTG AG -3' and 5'-GCA ATG GTT CAA GCG ATT CTT C-3' (GenoSys) were used in the MPO assay. Briefly, the RFLP–PCR reaction was performed in a 50 µl reaction containing 1x PCR buffer (500 mM KCl, 100 mM Tris–HCl, 1.0% Triton X-100; Promega), 1.25 mM MgCl2, 0.25 mM dNTPs, 0.3 µM each primer, 200 µg of template DNA and 2.5 U of Taq polymerase in storage buffer B (20 mM Tris–HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% Nonidet-P40 and 0.5% Tween 20; Promega).

The PCR cycling conditions for the MPO assay were 94°C for 5 min, followed by 38 cycles at 94°C for 60 s, 56°C for 60 s and 72°C for 60 s, with a final extension step at 72°C for 7 min. After PCR amplification, a 20 µl aliquot of PCR product was digested overnight at 37°C in a 30 µl reaction containing 12.5 U of AciI (New England BioLabs), 1.0 µl 10x NEBuffer 3 (100 mM NaCl, 50 mM Tris–HCl, 10 mM MgCl2, 1 mM dithiothreitol) and 6.5 µl dH2O. After overnight digestion, the products were resolved and separated for 30 min at 220 V on a 2.5% agarose gel stained with ethidium bromide. The predominant wild-type genotype (G/G) will resolve bands at 169, 120 and 61 bp. The heterozygote genotype (G/A) will resolve bands at 289, 169, 120 and 61 bp and the infrequent mutant genotype (A/A) will resolve bands at 289 and 61 bp. The 61 bp band is used as an internal control to ensure proper digestion for all three genotypes.

Statistical analysis
Smoking status and pack-years were defined in a previous report (19). Briefly, an ever smoker is defined as an individual who has smoked at least 100 cigarettes in his or her lifetime. A former smoker is defined as an individual who had smoked cigarettes in the past but stopped at least 1 year prior to diagnosis. For the controls, a former smoker is defined as an individual who had stopped smoking 1 year before entering the study. All statistical analyses were performed using STATA statistical software (20). Pearson's {chi}2 was used to test the differences in the distributions between cases and controls. Hardy–Weinberg equilibrium was tested by a goodness-of-fit {chi}2 test to compare the observed frequencies with the expected frequencies among the cases and controls. All odds ratios were calculated as estimates of the relative risk. The crude odds ratios were calculated by the method of Woolf (21). The adjusted odds ratios were calculated by logistic regression to control for age, gender and smoking status where appropriate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IGo displays the selected characteristics of our Caucasian population of 93 cases and 121 controls. There were no statistically significant differences between the cases and controls in age, gender, smoking status or any of the smoking variables, indicating successful matching.


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Table I. Distribution of select characteristics in cases and controls
 
Table IIGo displays the MPO genotype distributions for the cases and controls. A statistically significant difference was noted in the genotype distribution between the cases and controls (Pearson {chi}2 = 8.62). However, the cases and controls were in Hardy–Weinberg equilibrium by the goodness-of-fit {chi}2 test. The controls exhibited a higher frequency of the rare A allele (0.30) than the cases (0.18). The normal theoretical test for the equality of proportions demonstrated that the A allele frequencies between the cases and controls were significantly different (P = 0.04). Only one case was observed with the A/A variant genotype. The cases had a higher percentage of the wild-type genotype (64.5%) compared with the controls (48.8%). The normal theoretical test for the equality of proportions also demonstrated that the wild-type genotype frequencies between the cases and controls were significantly different (P = 0.02).


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Table II. Distribution of the MPO genotypes and A allele frequency by case/control status
 
The risk estimates for the MPO variant allele are displayed in Table IIIGo. The overall reduction in estimated risk was 48% (OR = 0.52, 95% CI 0.30–0.90, P = 0.02). When stratified by gender, we observed a significant reduced estimate of risk only in men (OR = 0.28, 95% CI 0.12–0.61, P < 0.05) while there was no significant association in women (OR = 1.02, 95% CI 0.44–2.34, P = 0.97). When stratified by the median age (61 years), younger individuals (<61 years) were observed to have a significantly reduced estimate of risk (OR = 0.28, 95% CI 0.11–0.69, P < 0.05) compared with older individuals (>=61 years) (OR = 0.78, 95% CI 0.37–1.64, P = 0.52). An interesting trend was observed when the data were analyzed by smoking status. There was a significant protective effect (OR = 0.24, 95% CI 0.10–0.58, P < 0.05) for current smokers with the variant allele but not for former smokers nor those who had never smoked. Significant results were also observed when we stratified current smokers by the median age. In younger current smokers there was an 89% reduction in risk (OR = 0.11, 95% CI 0.03–0.40, P < 0.05) of lung cancer, while the protective effect was less evident in older current smokers (OR = 0.65, 95% CI 0.18–2.36, P = 0.44). A significant 62% reduction in risk (OR = 0.38, 95% CI 0.17–0.86, P = 0.02) was observed in individuals who smoked >30 cigarettes/day, compared with individuals who smoked <30 cigarettes/day (OR = 0.69, 95% CI 0.30–1.57, P = 0.38). Similar results regarding tobacco smoke exposure were observed when the data were stratified on the median years smoked. Individuals above the median were at a 64% reduced risk (OR = 0.36, 95% CI 0.17–0.77, P < 0.05), while in individuals below the median there was a near null odds ratio (OR = 0.92, 95% CI 0.39–2.17, P = 0.85).


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Table III. Risk estimates for the variant MPO genotypes among Caucasians
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genetic polymorphisms and enzyme activity of MPO have previously been implicated in numerous diseases and biological processes (1216,22,23), including carcinogenesis (4,9,17). Reynolds et al. (23) reported that individuals with the wild-type genotype (G/G) are at a greater risk for two types of acute promyelocytic leukemias, suggesting that higher levels of MPO are associated with an increased risk for these leukemias. The wild-type genotype was over-represented in cases with acute promyelocytic leukemias M3 and M4 (80%) as compared with the general population (60%). Additionally, the wild-type genotype was over-represented in patients with early onset MS in females (14), suggesting that higher levels of MPO in macrophages accelerate neural damage. The wild-type genotype was observed in 74% of females with MS, while only 61% of the general population carried the wild-type genotype. Lastly, the variant allele was observed to decrease lung cancer risk in Caucasians by 70% (17), with case and control A allele frequencies of 0.18 and 0.23, respectively.

During the MPO-dependent biotransformation process, MPO converts the tobacco smoke intermediate B[a]P-7,8-diol to the highly reactive and carcinogenic product BPDE (4). Since the variant allele may be associated with weaker transcriptional activity, less enzyme would be available for activation of B[a]P-7,8-diol. Thus, the variant allele of MPO may be associated with reduced metabolic activity resulting in an overall decrease in lung cancer risk. Our data support this hypothesis, however, the protective effects were evident only in men, younger individuals and current smokers.

When the data were stratified by gender we observed a significant 72% protective effect in men but not women. A recent report demonstrated that the wild-type genotype was associated with an increased incidence of Alzheimer's disease in females but a decreased incidence in males (23). These results suggest a gender-specific effect of the MPO variants. Additionally, Zang and Wynder (24) concluded that gender differences in overall lung cancer risk cannot be completely explained by baseline exposure, smoking history, body size or occupational exposure. However, variations in physiological mechanisms such as the metabolic activation of carcinogens could account for gender differences in overall lung cancer risk. In support of this theory, Ryberg et al. (25) reported that DNA adduct levels were higher in females than in male lung cancer patients and concluded that women may be at a greater risk of tobacco-induced lung cancer. Previous studies utilizing animal models have also shown gender differences in the bioactivation of specific compounds by P450 metabolic enzymes (2628).

We observed that a 76% protective effect was evident only for current smokers, but not in former smokers and those who had never smoked. Since MPO is involved in the metabolism of specific tobacco smoke intermediates, current smokers with the variant allele should benefit most from reduced carcinogen activation. Specifically, the metabolic conversion of specific procarcinogenic compounds found in tobacco smoke may only occur when there is a biologically available dose. Therefore, the MPO-dependent pathway that produces reactive intermediates from the tobacco smoke carcinogen B[a]P would not be initiated unless there is a bioavailable dose. Carcinogenic metabolites formed by MPO would ultimately be found in the highest concentrations among current smokers. Furthermore, among cases, the average duration of cessation for former smokers prior to enrollment was 14.13 ± 10.04 years. In our study, enrollment is an approximation of diagnosis date, therefore, it is plausible that the protective effect of the variant allele is limited to only current smokers, as observed in our data. We similarly noted a significant reduction in risk for heavier smokers when we categorized the data by the median number of cigarettes smoked per day and median years smoked.

Lastly, we analyzed the data to determine if there was an age effect. Since MPO is associated with a mediated immune response, it is certainly plausible that older individuals may have a weaker immune system as compared with younger individuals, resulting in less of a protective effect. Additionally, susceptibility factors may play a role in early age onset patients, which is reflected in the protective effects in younger individuals as compared with older ones. When the data were stratified by the median age (61 years), we observed a 72% reduced risk in younger individuals (<61 years) compared with a non-significant 22% reduced risk in older individuals. These findings are similar to data reported for the susceptible phenotypes involving mutagen sensitivity and DNA repair capacity (29,30). However, a much larger study is required to substantiate the theory that this genetic factor may play a major role in younger lung cancer patients. Additionally, the protective effects of the MPO variant allele may not provide a beneficial effect in older individuals due to the protective effects being overwhelmed by cumulative environmental exposures and poorer DNA repair capacity.

In summary, our results support a previous report of an overall reduction in lung cancer risk associated with the variant allele of MPO. However, this protective effect was confined to men, younger individuals and heavy smokers. Nonetheless, these data provide epidemiological support that MPO is involved in the metabolic activation of tobacco smoke carcinogens as related to lung cancer risk. We plan to confirm these findings in African-Americans and Mexican-Americans.


    Notes
 
3 To whom correspondence should be addressed at: The University of Texas M.D. Anderson Cancer Center, Department of Epidemiology, Box 189, 1515 Holcombe Boulevard, Houston, TX 77030, USA Email: xwu{at}mdanderson.org Back


    Acknowledgments
 
We thank Susan Honn and Qiong Dong for their excellent contributions in data quality management. This study was supported by grants CA 55769, CA 68437 and CA 74880 from the National Cancer Institute.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 14, 1999; revised January 28, 2000; accepted February 17, 2000.