Reduced DNA repair of benzo[a]pyrene diol epoxide-induced adducts and common XPD polymorphisms in breast cancer patients

Qiuling Shi1, Li-E. Wang1, Melissa L. Bondy1, Abenaa Brewster2, S. Eva Singletary3 and Qingyi Wei1,4

1 Department of Epidemiology, 2 Department of Clinical Cancer Prevention and 3 Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

4 To whom correspondence should be addressed Email: qwei{at}mdanderson.org


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Environmental chemicals are thought to play a role in the etiology of breast cancer, because polycyclic acromatic hydrocarbon (PAH)–DNA adducts are detectable in normal and malignant breast tissues. Peripheral blood lymphocytes (PBLs) from female breast cancer patients were more sensitive to in vitro exposure to benzo[a]pyrene diol epoxide (BPDE) than those from healthy controls. Therefore, we hypothesized that reduced DNA repair is associated with risk of breast cancer in women and the risk may be modulated by polymorphisms of DNA repair genes. In a case-control pilot study, we included 69 previously untreated female breast cancer patients and 79 controls frequency matched to the cases on age and ethnicity. The PBLs were used to measure DNA repair capacity (DRC) by using the host-cell reactivation (HCR) assay with a reporter gene damaged by exposure to 60 µM BPDE prior to transfection. We also genotyped for two common XPD polymorphisms Lys751Gln and Asp312Asn. We found that the mean DRC level was significantly lower in breast cancer patients (10.1%) than in controls (11.1%) (P = 0.008). Subjects with DRC lower than the median level of controls (11.0%) had >3-fold increased risk (OR = 3.36, 95% CI = 1.15–9.80) for breast cancer than did those with higher DRC after adjustment for age, smoking status and assay-related variables. None of the genotypes was statistically significantly associated with an increased risk of breast cancer, which may be due to the small number of observations in each subgroup. The XPD variant genotypes in general predicted the DRC better in the controls than in the cases, suggesting genetic variants of other DNA repair genes may be involved in these breast cancer patients. These findings suggest that women with reduced DRC may be at an increased risk of developing breast cancer. Large studies are warranted to confirm these preliminary findings.

Abbreviations: BPDE, benzo[a]pyrene diol epoxide; DRC, DNA repair capacity; HCR, host-cell reactivation; NER, nucleotide excision repair; PAH, polycyclic acromatic hydrocarbon; PBLs, peripheral blood lymphocytes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is the most frequently diagnosed cancer in women for all ethnic groups, and it is estimated that in 2004 there will be 215 990 new invasive breast cancer cases and 40 110 breast cancer deaths among women in the US (1). Although the contribution of environmental exposure (including tobacco smoke) to breast cancer risk is under vigorous investigation, few environmental factors, except for radiation, have been identified as risk factors for breast cancer (2). Epidemiological studies have been inconclusive for the roles of some of the most suspected risk factors such as cigarette smoking, organo chlorine compounds and well-done meat intake (3).

Tobacco smoke contains hundreds of chemicals, many of which, such as polycyclic aromatic hydrocarbons (PAHs), are known mammary carcinogens in laboratory animals (4). Studies also demonstrated that PAHs are involved in the carcinogenesis of the human breast (5). Although the role of tobacco smoke in the etiology of breast cancer remains controversial (6), PAH-like bulky adducts have been consistently found in both breast normal and tumor tissues (7,8), which was later found to be related to PAH exposure (9). Furthermore, the level of these PAH-like adducts can be modulated by genetic and environmental factors such as GST null genotype and alcohol use (10,11), and genetic polymorphisms in DNA repair genes have been shown to be associated with breast cancer risk in smokers (12).

One of the well-studied tobacco PAHs, benzo[a]pyrene, can be bioactivated to form benzo[a]pyrene diol epoxide (BPDE), an ultimate carcinogen that can induce BPDE–DNA adducts. These BPDE-induced DNA adducts are mainly repaired by the nucleotide excision repair (NER) pathway (13), and unrepaired BPDE–DNA adducts may block the transcription of essential genes (14). Increasing evidence suggests that the rate of removing DNA damage and mutation fixation may be influenced by genetically determined DNA repair capacity (15,16). In a recent study, we demonstrated that peripheral blood lymphocytes (PBLs) from breast cancer patients were more sensitive to in vitro exposure to BPDE compared with normal controls (17), suggesting suboptimal repair for BPDE may exist in breast cancer patients.

XPD is one of the seven genetic complementation groups encoding for proteins involved in the NER pathway. Although some rare germ line mutations in XPD result in defective NER phenotypes (18,19), the functional relevance of some common polymorphisms in XPD has not been determined. Understanding the correlation between DNA genotypes and phenotypes is an important step towards determining how polymorphic genotypes are associated with cancer in the general population. In previous studies, we found that two XPD polymorphisms modulated DNA repair capacity (DRC), measured by the host-cell reactivation (HCR) assay, using the ultimate tobacco carcinogen BPDE as a DNA damaging agent in lung cancer patients (20).

Therefore, we hypothesize that suboptimal DNA repair capacity is associated with risk of developing environmentally induced breast cancer and this risk may be modulated by XPD polymorphisms. To test this hypothesis, we conducted a pilot molecular epidemiological study with 69 cases and 79 cancer-free controls and measured DRC by the HCR assay of BPDE-damaged plasmids harboring a reporter gene. In addition, we typed two common XPD polymorphisms Lys751Gln and Asp312Asn in both case and control subjects.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
All the subjects were females and non-Hispanic whites. The 69 cases were patients with newly diagnosed breast cancer and consecutively recruited between 2002 and 2003 at the Breast Cancer Center of The University of Texas M. D. Anderson Cancer Center. The 79 cancer-free controls frequently matched to the cases by age (±5 years) were randomly recruited from hospital visitors who were genetically unrelated to the cases over a similar period of time. The subjects completed short questionnaires that elicited information about demographic variables and smoking history. The exclusion criteria included prior chemotherapy or radiotherapy for cancer, infectious diseases and blood transfusion within the last 6 months to control their confounding effects on lymphocyte stimulation. After the subjects provided their informed consent, each subject donated 30 ml of blood collected into heparinized tubes. The research protocol was approved by the M. D. Anderson Cancer Center institutional review board.

Blood processing and lymphoblastoid cell lines
Each subject donated a 30-ml blood sample that was drawn into heparinized Vacutainers (BD Biosciences, Franklin Lakes, NJ). The lymphocytes were isolated from each sample within 8 h of blood collection by Ficoll-gradient centrifugation (21), re-suspended and frozen in freezing medium (50% fetal bovine serum, 40% RPMI-1640, 10% dimethyl sulfoxide), and stored at –80°C. All samples were stored within 20 months before being thawed, short-term cultured and assayed. Four Epstein–Barr virus immortalized human lymphoblastoid cell lines from the human genetic Mutant Cell Repositories (Camden, NJ) were used as experimental controls. Two of the cell lines, GM00892 and GM00131, have apparently normal DRCs of ~20 and 30%, respectively, as determined by the HCR assay using plasmids damaged by 60 µM BPDE (Figure 1). The other two cell lines were derived from xeroderma pigmentosum (XP) patients and are deficient in nucleotide excision repair: GM02345 is an XP group A line with a DRC of <1%, and GM02246 is an XP group C line with a DRC of ~5% (Figure 1).



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Fig. 1. The DRC curve in normal (repair-proficient) and repair-deficient lymphoblastoid cell lines. This is a representative of two separate experiments. The data show that XP cells had much lower DRC than the apparently normal cells as measured by the HCR assay.

 
Plasmids and BPDE-treatment
The plasmid expression vector pCMVcat (5.0 kb, a gift from Dr Lawrence Grossman, The Johns Hopkins University, Baltimore, MD) was used for the CAT assays. BPDE (NCI L0137, 99% purity) was purchased from Midwest Research Institute (Kansas City, MO) as a white powder and was completely dissolved in tetrahydrofuran (Sigma Chemical, St Louis, MO). The stock concentration (1 mM, or 0.3 mg/ml) was further diluted for the working solution, which was prepared once in a dark room, aliquoted into Eppendorf tubes, and kept at –20°C without exposure to air or light. For plasmid treatment, purified plasmid was dissolved in Tris–EDTA buffer (pH 7.9) at a concentration of 500 µg/ml, and 1-ml aliquots of the solution were placed in Eppendorf tubes. The HCR assay uses the BPDE-damaged plasmids, and the conformation change in the plasmids occurs when the dose of BPDE used is >60 µM (data not shown), which reduces transfection efficiency. Therefore, we selected BPDE doses of 60 µM and smaller that effectively induced BPDE–DNA adducts without detectable changes in plasmid conformation (22,23). To establish a repair curve, the BPDE working solution was added to the tubes containing the plasmids to reach final concentrations of 0, 30, 45 and 60 µM BPDE and the mixtures were incubated for 3 h in a dark room. The BPDE treatments were performed in one batch for all HCR assays. After treatment, the plasmids were precipitated with 100% ethanol and washed three times with 70% ethanol, then dissolved in Tris–EDTA buffer at a final concentration of 50 µg/ml, and assessed for conformation changes by using 0.8% agarose gel electrophoresis.

HCR assay
The frozen lymphocytes in each vial (~1.5 ml) were thawed in batches later for the HCR. The thawing medium consisted of 50% fetal bovine serum, 40% RPMI 1640 and 10% dextrose (Sigma Chemical), which ensured a viability of 90% after thawing. After washing with the thawing medium, the cells were incubated in RPMI 1640 supplemented with 20% fetal bovine serum and 112.5 µg/ml phytohemagglutinin (Murex Diagnostics, Norcross, GA) for stimulation at 37°C for 72 h. For transfection, 2 x 106 cells were transfected using the diethylaminoethyl-dextran (Phamacia Biotech, Piscataway, NJ) method with 0.25 µg of untreated plasmid or plasmid treated by 60 µM BPDE. The transfections were performed in duplicate for each dose. Forty hours after transfection, the expression of the repaired CAT gene was measured by scintillation counting of tritiated [3H]monoacetylated and diacetylated chloramphenicols formed by the reaction of chloramphenicol and tritiated-acetyl coenzyme A catalyzed by the CAT protein in the cell extract.

DRC was defined as the CAT activity of cells transfected with plasmids treated with 60 µM BPDE divided by that of cells transfected with untreated plasmids x100%. The CAT activities of cells transfected with the undamaged plasmids provides an experimental internal control, because it is derived under the same experimental conditions as the CAT activities of cells transfected with damaged plasmids and from the same number of cells from the same individual.

Genotyping methods
The XPD genotypes were determined by PCR–RFLP analysis of DNA samples extracted from the samples used for DNA repair assays. The PCR primers for the Lys751Gln were as described previously (20): forward, 5'-GCCCGCTCTGGATTATACG-3'; and reverse, 5'-CTATCATCTCCTGGCCCCC-3'. PCR was performed in 10-µl containing 2 mM MgCl2, 0.04 mM deoxynucleotide triphosphates, 1.0 U of Taq polymerase, and the manufacturer's buffer [20 mM Tris–HCl (pH 8.4) and 50 mM KCl]. After an initial denaturation at 94°C for 3 min, there were 38 cycles of 45 s at 94°C, 45 s at 60°C, and 60 s at 72°C, and then a final extension step of 7 min at 72°C. After overnight digestion of the PCR product with PstI, 5 µl of the digested products were resolved on a 3% agarose gel (5 V/cm) containing ethidium bromide. The homozygous wild-type allele (Lys 751) produced two DNA bands (290 and 146 bp), whereas the variant allele (Gln 751) produced three DNA bands (227, 146 and 63 bp). Heterozygotes displayed all four bands (290, 227, 146 and 63 bp). For amplification of the exon 10 region of XPD, which contains the polymorphic StyI restriction site, we used the oligonucleotide primers 5'-CTGTTGGTGGGTGCCCGTATCTGTTGGTCT-3' and 5'-TAATATCGGGGCTCACCCTGCAGCACTTCCT-3' (20). PCR was performed in 10 µl reaction mixtures containing 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 3% DMSO, 0.2 µM primers, 1 µg of template DNA and 1.5 U of Taq polymerase in PCR buffer [10 mM Tris–HCl (pH 9.0 at 25°C), 50 mM KCl and 0.1% Triton X-100 (Promega)]. After an initial denaturation at 94°C for 4 min, the DNA was amplified by 30 cycles of 30 s at 94°C, 30 s at 60°C and 60 s at 72°C, and then by a final extension step of 5 min at 72°C. Five microliters of the PCR product was digested with StyI for 8 h at 37°C. The digestion products were then resolved on a 3% agarose gel (5 V/cm) containing ethidium bromide. The homozygous wild-type (Asp/Asp) was identified by two DNA bands (507 and 244 bp), the homozygous mutant type (Asn/Asn) produced three bands (474, 244 and 33 bp); and heterozygotes (Asp/Asn) displayed all four bands (507, 474, 244 and 33 bp).

Statistical analysis
The differences between the cases and controls in the distribution of demographic variables and known risk factors were examined by using the {chi}2 test. The Hardy–Weinberg equilibrium was tested by a goodness-of-fit {chi}2 test to compare the observed genotype frequencies with expected genotype frequencies among the cases and controls. DRC was first analyzed as a continuous variable and the Student's t test was used to compare differences in DRC between case and control subjects and XPD genotype groups. We used the median DRC of control subjects (11.0%) as the cut-off value to calculate crude odds ratios (ORs) and 95% confidence intervals (CIs). Values greater than the median value (11.0%) were considered to reflect proficient repair capacity, and values less than or equal to the median value (≤11.0%) were considered to reflect deficient (i.e. suboptimal) repair capacity. For logistic regression analysis, dummy variables of risk factor classes and the median values for DRC and genotypes were created to calculate the ORs and 95% CIs. Adjusted ORs were calculated by fitting unconditional logistic regression models with adjustment for age, smoking status and for DRC; assay-related variables (i.e. blastogenic rate, cell storage time and baseline level of CAT activity) were also included in the model. All statistical analyses were performed with the use of Statistical Analysis System software (version 8.0; SAS Institute, Cary, NC). All statistical tests were two-sided.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The four repair-proficient and -deficient lymphoblastoid cell lines were first assayed to determine the appropriate dose of BPDE. Figure 1 shows the does–response curve of DRCs in the four established cell lines. To maximize the damage and saturate host-cell DRC, a dose of 60 µM BPDE was chosen for the assays of PBLs. This dose reduced the DRC to 10–30% of the normal cells' DRC, consistent with previous reports (21).

As shown in Table I, the frequency matching on age was effective, because no difference was observed in the distribution of age between the cases and controls (P = 0.274). Although there were more ever smokers among the cases (52.2%) than among the controls (40.5%), this difference was not significant statistically (P = 0.160). For the histological types, there were 72.5% invasive breast cancer and 27.5% in situ breast cancer. When DRC was analyzed as a continuous variable (Figure 2), the mean DRC was 10.1% in the cases and 11.1% in the controls, representing an average reduction of 10% in DRC in the cases relative to that of the controls (P = 0.008) (Table I).


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Table I. Distribution of selected characteristics and DRC in breast cancer cases and cancer-free controls

 


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Fig. 2. Box-plot for distributions of DRC in the cases and controls. Horizontal lines within the boxes are medians, lines above the medians are at the 75th percentile, lines below the medians are at 25th percentile, the vertical bars above and below the boxes are the upper and lower quartiles, respectively, and the open circle is an outlier value. The data show a lower median of DRC in the cases than in the controls.

 
Further stratification of DRC revealed that patients who were younger at diagnosis of breast cancer (<45 years old) had significantly lower mean DRC than the subjects of the same age group (10.3 versus 11.8%, P = 0.013), but in subjects older than 45 years the difference between the cases and controls did not reach statistical significance (P = 0.082). In both never smokers and ever smokers, the cases exhibited lower mean DRC levels than the controls, but both the differences were borderline significant (P = 0.054 for non-smokers and P = 0.100 for ever smokers), which may be due to smaller sample sizes in the subgroups. For histological types, significant lower mean DRC level was lower only in patients with invasive breast cancer compared with the controls (10.0 versus 11.1%, P = 0.006), but not in patients with in situ breast cancer (Table I); the number of cases (n = 19) with in situ breast cancer was too small for a meaningful comparison.

The effect of DRC on risk for breast cancer was further evaluated by logistic regression analysis. Because the DRC assays were performed at different times, the assay-related variables (such as blastogenic rate after stimulation of the thawed cells, cell storage time and baseline CAT expression level of cells bearing the undamaged plamids) may have an effect on the DRCs. Therefore, we adjusted for these variables in addition to age and smoking status in the same logistic regression model. Using the median of the DRCs of controls (11.0%) as the cut-off value, the crude and adjusted ORs for breast cancer risk associated with DRC of ≤11.0% were significantly statistically elevated (OR = 2.02, 95% CI = 1.04 – 3.93; ORadj = 3.36, 95% CI = 1.15 – 9.80) (Table II).


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Table II. Association between DRC and XPD variant alleles and genotypes and risk for breast cancer

 
The frequencies for 751Gln and 312Asn alleles were 0.341 and 0.348, respectively, for the cases (Table II), and 0.297 and 0.247, respectively, for the controls, which are consistent with the results of published studies with larger sample sizes of different study populations (20,24). The distributions of the polymorphic genotypes in the controls did not depart from the values predicted from the Hardy–Weinberg equilibrium model (P = 0.596 for Lys751Gln and P = 0.473 for Asy312Asn). Compared with the homozygous 751Lsy variant genotype, the risk for breast cancer was non-significantly elevated for 751Lys/Gln heterozygotes (adjusted OR = 1.14, 95% CI = 0.57–2.28) and 751Gln/Gln homozygotes (adjusted OR = 1.49, 95% CI = 0.46–4.86). Compared with the homozygous 312Asp variant genotype, the risk for breast cancer was also non-significantly elevated for 312Asp/Asn heterozygotes (adjusted OR = 2.00, 95% CI = 0.98–4.08) and 312Asn/Asn homozygotes (adjusted OR = 2.06, 95% CI = 0.63–6.69) (Table II). Because of the small sample size of variant homozygotes, we combined the variant homozygotes and heterozygotes as one group for both Lys751Gln and Asp312Asn polymorphisms in the final analysis. Subjects with the variant 312Asn genotype (Asp/Asn + Asn/Asn) showed a significantly higher risk for breast cancer (adjusted OR = 2.01, 95% CI = 1.03–3.94) compared with Asp/Asp homozygotes. However, this significantly increased risk was not observed for the Lys751Gln polymorphism (Table II).

We then determined genotype–phenotype correlations. Among the controls, those with the 751Lys/Lys homozygous genotype exhibited higher mean DRC (11.7%) than the 751Gln variant genotypes DRC (10.5%) (P = 0.019) and those with the 312Asp/Asp homozygous genotype exhibited higher mean DRC (11.5%) than the 312Asn genotypes (10.4%) (P = 0.018) (Table III). Among the cases, these patterns were less evident and no significant differences in DRC were exhibited amongst both Lys751Gln and Asp312Asn polymorphisms (Table III).


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Table III. Difference in DRC (%)a between the breast cancer cases and cancer-free controls by XPD genotypes

 
We also estimated risk for suboptimal DRC associated with the XPD polymorphisms. As shown in Table IV, the odds ratio (95% CI) for exhibiting suboptimal DRC was 2.66 (1.03–6.82) for subjects with the 751Gln variant (Lys/Gln + Gln/Gln) genotype and 3.12 (1.20–8.13) for those with the 312Asn variant (Asp/Asn + Asn/Asn) genotype in the controls, but these variant genotypes were not associated with suboptimal DRC in the cases.


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Table IV. Association between XPD genotypes and risk for suboptimal DRC

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this pilot study, we demonstrated that reduced repair of BPDE-induced DNA damage was associated with an increased risk of breast cancer in women, especially in the subgroups younger than 45 years, suggesting that suboptimal DRC may play a role in susceptibility to breast cancer. In addition, our results also suggested that the DRC of removing BPDE-induced adducts can be modulated by genetic polymorphisms in XPD. Although the study size was relatively small and the results are preliminary, several lines of evidence support these findings.

BPDE is a well-studied tobacco carcinogen and can induce DNA adducts that are repaired mainly by the NER pathway, and XP cells, which are deficient in NER, cannot efficiently repair damage induced by BPDE (21). Although smoking as a risk factor for breast cancer is not yet established, it has been demonstrated that mononuclear cells, including lymphocytes, are a valid surrogate tissue for estimating the burden of DNA adducts in the breast (25,26); therefore, the DRC measured by using the HCR assay in PBLs should be a valid estimation of repair in the target tissue. Our previous studies on lung cancer (with BPDE-damaged plasmids) and cutaneous malignant melanoma (with UV-induced photoproducts) have adapted this method of using PBLs as a surrogate tissue and demonstrated that reduced DRCs are associated with risk of those cancers (23,27). Furthermore, the carcinogenic effect of PAHs on the mammary gland of laboratory animals has been well established. Extrapolation from rodent models for chemical-induced mammary cancer suggests the possibility that human mammary epithelial cells in situ might contain DNA adducts due to exposure to environmental chemicals (4). Experimental studies also showed that a number of chemicals found in cigarette smoke, ambient air, workplace environments, drinking water and food can bind to DNA in breast epithelial cells (28).

Direct population data also demonstrated that the mean aromatic/hydrophobic–DNA adduct level in tissues from breast cancer women was higher than in tissues from non-cancer patients; furthermore, smokers with breast cancer displayed a pattern of adducts associated with exposure to tobacco smoking, while tissue from non-smokers did not show this pattern (29). A recent case-control study among women from Long Island has shown that a modest 50% increase in the risk of breast cancer was associated with the highest quartile of PAH–DNA adducts levels compared with the lowest quartile; and the two main sources of the adduct formation identified were active or passive cigarette smoking (30). In the present study, our data further suggest that breast cancer patients may have suboptimal DRC that could result in less efficient removal of these PAH adducts in the breast tissues, making these women at a higher risk if they had smoked. To compare mechanistically to previously published data (23), our new findings from the present study suggest that increased BPDE mutagen sensitivity in these patients may be due partly to the suboptimal DRC of the host cells, in which inefficient NER as measured by the HCR assay may have left more nicks on either strand of the DNA templates, increasing the probability of forming double-strand breaks that eventually lead to chromosomal breaks. However, this hypothesis needs to be tested in future studies.

Our previously published data on BPDE-induced mutagen sensitivity have demonstrated that sensitivity to BPDE-induced chromosomal aberrations may be modulated by both genetic (GSTT1) and environmental factors (smoking) (17). These findings suggest a possible role of gene–environment interaction in breast cancer development. This is supported by the Carolina Breast Cancer study of XRCC1 and breast cancer, in which smoking duration was positively associated with breast cancer risk among women with the XRCC1 Arg/Arg genotype, but not the Arg/Glu or Glu/Glu genotypes, in both white and African-American populations (12). Additionally, the XPD Lys751Gln polymorphism was shown to be associated with an elevated risk of developing breast cancer in women (31).

For the relationship between DRC and XPD genotypes, we reported previously that the Lys751Gln and Asp312Asn polymorphisms have modulating effects on DRC for both UV damage in a normal population and BPDE-induced DNA adducts in lung cancer patients (20,32,33). Interestingly, one study found that the XPD 751Lys/Lys, but not 312Asn/Asn, homozygous genotype increased the risk of sub-optimal DNA repair of X-ray-induced DNA damage that is repaired by the base-excision, but not NER, pathway (31). A more recent study found that smoking is associated with increased levels of 4-aminobiphenyl–DNA adducts in human breast tissue, but the XPD 751Gln variant did not have a significant effect on the formation of DNA adducts in breast cancer cases (34). In the present study, our finding of the association between the XPD variant genotypes and DRC in controls, but not in the cases, suggest that the XPD variant genotypes may affect the DRC phenotype in the general population. As we demonstrated in larger studies (20,32,33), the DRC may be homogeneously low or other XPD polymorphisms or polymorphisms of genes other than XPD may play a role in the low DRC phenotype in these breast cancer patients. However, these hypotheses need to be tested in larger studies.

Although tobacco smoking is a main source of PAHs, we did not find any significant difference of mean DRC level of the cases and controls in either ever smokers or never smokers. This may be due to the possibility that the DRC phenotype is genetically determined and that smoking does not have an effect on women's DRC phenotype, which is consistent with our previous finding that the apparent smoking-related enhancement in DRC was limited to men (23), who smoke more heavily than women. It is also possible that this study size was too small to detect such a smoking effect. Because of the small study size, our findings may also be due to chance, and because this was a retrospective study, it cannot be ruled out that the disease status may also have an effect on the DRC phenotype in the cases. In spite of the relatively small sample size and the sizable variability (~5-fold) of the HCR assays, our findings provide a clue that DRC may be a potential biomarker for breast cancer susceptibility. However, unless significant improvement can be made to reduce the variability in the measurements, the HCR assay will remain as a research tool rather than a screening or diagnostic tool. Large studies preferably with repeated samples over time are warranted to confirm our preliminary findings. Because the role of tobacco smoking in breast cancer is still unclear, other environmental exposures should also be considered in further studies.


    Acknowledgments
 
The authors thank Dr Lawrence Grossman (Johns Hopkins University) for providing pCMVcat and scientific advice, Ms Margaret Lung for recruiting the subjects, Ms Yawei Qiao and Mr Zhaozheng Guo for laboratory assistance, Dr Maureen Goode for scientific editing and Ms Joanne Sider for manuscript preparation. This study was supported in part by National Institutes of Health grants CA 89608 (to M.B.) and ES 11740 (to Q.W.), and ES07784 (a center grant from National Institute of Environmental Health Sciences).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 22, 2004; revised April 2, 2004; accepted April 6, 2004.