DNA damage and breast cancer risk
Tasha R. Smith1,
Mark S. Miller1,2,
Kurt K. Lohman3,
L.Douglas Case2,3 and
Jennifer J. Hu1,4
1 Department of Cancer Biology, Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, NC 27157, USA
2 Comprehensive Cancer Center, Wake Forest University Health Sciences, Winston-Salem, NC, USA
3 Department of Public Health Sciences, Wake Forest University Health Sciences, Winston-Salem, NC, USA
4 To whom correspondence should be addressed Email: jenhu{at}wfubmc.edu
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Abstract
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To evaluate whether deficient DNA repair contributes to elevated DNA damage and breast carcinogenesis, we used the comet assay (single-cell alkaline gel electrophoresis) to measure the levels of DNA damage in peripheral lymphocytes from 70 breast cancer cases and 70 controls. DNA damage, measured as the comet tail moment, was not influenced by age, family history (FH), age at menarche, age at first birth or parity. The results showed that cancer cases had significantly higher DNA damage compared with controls; the comet tail moments (mean ± SD) for cases and controls were: 10.78 ± 3.63 and 6.86 ± 2.76 (P < 0.001) for DNA damage at baseline (DB), 21.24 ± 4.88 and 14.97 ± 4.18 (P < 0.001) for DNA damage after exposure to 6 Gy of ionizing radiation (DIR), and 14.76 ± 5.35 and 9.75 ± 3.35 (P < 0.001) for DNA damage remaining after 10 min repair following exposure to 6 Gy of IR (DRP), respectively. Body mass index (BMI) affected DNA damage differently for cases and controls. Damage decreased with increasing BMI for controls, while damage increased with increasing BMI for cases. Above-median DNA damage was significantly associated with breast cancer risk; the age-adjusted odds ratio (OR) = 13.44 [95% confidence interval (CI) = 5.9730.24] for DB, 13.65 (6.0730.71) for DIR and 6.54 (3.1113.79) for DRP, respectively. This association was stronger in women with above-median BMI. Our results, although based on a relatively small group of subjects, indicate that elevated DNA damage is significantly associated with breast cancer risk and warrant larger studies to further define the molecular mechanisms of DNA damage/repair in breast cancer susceptibility.
Abbreviations: BMI, body mass index; DB, DNA damage at baseline; DIR, DNA damage after exposure to ionizing radiation; DRP, DNA damage remaining after repair; FH, family history; IR, ionizing radiation; OR, odds ratio; ROS, reactive oxygen species
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Introduction
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In the US, breast cancer is the second leading cause of cancer deaths in women. In 2002,
205,000 American women will be diagnosed with breast cancer, and 40,000 will die from the disease (1). Age-adjusted US breast cancer incidence rates have remained relatively unchanged during the last decade, while death rates have declined, due, in part to improved therapies and earlier detection (2). Three well-established risk factors, later age at first birth and nulliparity, higher income and family history (FH), account for
40% of US breast cancer cases. Therefore,
60% of cases are without defined risk factors (3). Many studies suggest that environmental factors and genetic susceptibility contribute to human breast cancer risk.
Mammalian cells are constantly exposed to exogenous and endogenous DNA-damaging agents. Specific DNA repair mechanisms have evolved to repair different types of DNA damage and to maintain genomic integrity. Ionizing radiation (IR), an established etiologic agent for breast cancer, and other suspected risk factors, such as chemical carcinogens, alcohol, estrogen and diet, result in reactive oxygen species (ROS), oxidized bases, bulky DNA adducts and DNA strand breaks (4,5). Under these circumstances, women may develop cytogenetic alterations, such as deletions, amplifications and/or mutations in critical oncogenes and tumor suppressor genes, leading to cellular transformation and neoplasm (68). Higher levels of DNA damage and deficient DNA repair may predispose individuals to breast cancer (916). Previous studies have shown that breast cancer patients and healthy women with a positive FH of breast cancer may have deficient DNA repair and are hypersensitive to IR, compared with healthy women without a FH (1016). Thus, a combination of genetic susceptibility and elevated exposure to genotoxic agents may contribute to breast cancer.
Persistent basal DNA damage may reflect higher exposure to carcinogens and deficient DNA repair (1719). Higher levels of DNA adducts and oxidative base lesions have been reported in normal adjacent and tumor tissues of breast cancer patients compared with controls (2025). These findings suggest that the accumulation of DNA damage may contribute to breast carcinogenesis. Various biomarkers have been used to determine cellular DNA damage; cytogenetic measurements include chromosomal aberrations, micronuclei and sister chromatid exchanges. These genotoxic endpoints have been utilized extensively in biomonitoring studies (26).
The single-cell gel electrophoresis, or the Comet assay, is a novel approach for the assessment of DNA strand breakage in a single cell. It is based on the alkaline lysis of labile DNA at sites of damage. The unwound, relaxed DNA is able to migrate out of the cell during electrophoresis and can be visualized by SYBR Green staining. Cells that have accumulated DNA damage appear as fluorescent comets with tails of DNA fragments, whereas normal, undamaged DNA does not migrate far from the cell origin. The assay is relatively easy to perform and well-suited for population-based studies (2731). Previous studies using this technique have shown that high levels of cellular DNA damage in non-neoplastic urothelial cells may predispose smokers to urinary bladder cancer (32). Patients with cervical dysplasia exhibited longer comet tails in their cervical epithelial cells and peripheral blood lymphocytes (19). To the best of our knowledge, only one group has used the comet assay in a published breast cancer risk study (18). They reported elevated levels of DNA damage in breast cancer cases and in women with a FH of breast cancer at baseline after treatment with a mutagen, N-methyl-N-nitro N-nitrosoguanidine, and following DNA repair.
In this study, we used samples collected from a case-control study to evaluate whether deficient DNA repair may contribute to elevated DNA damage and breast cancer risk. The comet assay was used to measure the levels of DNA damage in lymphocytes.
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Materials and methods
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Study population
The details of the study population have been described previously (10). In brief, breast cancer cases and controls were recruited at Georgetown University Medical Center from August 1995 to November 1996 as part of the Breast Cancer Biomarker Resource of the Lombardi Cancer Center. Study subjects received a detailed description of the study protocol and signed their informed consent as approved by the medical centre's Institutional Review Board. Breast cancer cases were recruited from the Breast Cancer Section of the Division of Hematology/Oncology. Study controls were recruited from the Comprehensive Breast Center and the Cancer Assessment and Risk Evaluation program. Each woman was asked to complete a self-administered questionnaire establishing demographic information, breast cancer risk factors, medical conditions and FH of breast cancer. A woman with at least one first-degree relative with breast cancer is considered to have a positive FH. All the laboratory and questionnaire data were coded, entered and verified; neither the laboratory nor the data entry personnel had knowledge of the subjects' case-control status.
Comet assay
The comet assay protocol developed by Singh et al. (33) was employed with some modifications. A human lymphoblastoid cell line, GM00131B (Human Cell Repository, Camden, NJ), was used as an internal control for each batch, which consisted of samples from both cancer cases and controls. Cryopreserved lymphocytes were thawed, suspended in thawing medium (50% FBS, 40% RPMI 1640 and 10% dextrose), and centrifuged at 1100 r.p.m. at 4°C for 10 min. Cell viability was >90% for all samples, as determined by Trypan Blue exclusion. Lymphocytes were resuspended at a final concentration of 3 x 105 cells/ml in RPMI 1640 without phenol red. Fifty microliters of each subject's cell suspension was mixed with 500 µl of low melting point agarose (0.5% Metaphor agarose in 1x PBS, Ca2+, Mg2+ free), and from this mixture, 50 µl was applied onto three comet slide wells (Trevigen, Gaithersburg, MD).
Slides were allowed to solidify at 4°C for 15 min. For DNA damage at baseline (DB), slides were assessed without any further treatment. For DNA damage after exposure to ionizing radiation (DIR) and DNA damage remaining after repair (DRP), slides were exposed to 6-Gy
-irradiation. DRP slides were further incubated in RPMI 1640 without phenol red at 37°C for 10 min of repair. All slides were then placed in pre-chilled lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris base, 1% sodium lauryl sarcosinate and 0.01% Triton X-100) for 30 min. After lysis, slides were treated with an alkali solution (300 mM NaOH and 1 mM EDTA) at room temperature for 1 h and electrophoresed in 1x TBE at
0.8 V/cm for 20 min. Slides were fixed in 100% ethanol for 5 min and stored at 4°C until image analysis. Cellular DNA was stained with 1:10 000 SYBR Green in TE buffer, followed by the addition of antifade solution (0.37 mM p-phenylenediamine dihydrochloride/90% glycerol in 1x PBS, Ca2+, Mg2+ free) and a coverslip. The slides were analyzed using the LAI Comet Assay Analysis System (Loats Associates, Westminster, MD). The results were expressed as the mean comet tail moment of 50 cells. The comet tail moment is defined as the product of the percentage of cellular DNA in the comet tail and the length of DNA tail migration. The higher the comet tail moment value, the greater the amount of cellular DNA strand breaks.
Statistical analyses
Characteristics of breast cancer cases and controls were compared using Student's t-test,
2 test, or Fisher's exact test. Analysis of covariance was used to assess group case/control differences in mean comet tail moments after adjustment for participant characteristics and to assess the two-way interactions involving case/control status. Logistic regression was used to calculate crude and adjusted odds ratios (OR) and 95% confidence intervals (CI).
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Results
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The general characteristics of cases and controls are summarized in Table I. Age, race, age at menarche, age at first birth, parity and body mass index (BMI) did not significantly differ between cases and controls. However, FH differed significantly between cases and controls (P = 0.002), because we recruited a greater proportion of controls with positive FH in order to evaluate whether FH is associated with deficient DNA repair. To evaluate batch-to-batch variation of the comet assay, a lymphoblastoid cell line, GM00131B, was run simultaneously with each batch of samples. With 16 batches over a 14-week period, the coefficient of variation was 22, 22 and 23% for DB, DIR and DRP, respectively.
In the original study,
30% of the study subjects (36% cases and 25% controls) did not return the risk questionnaire (10). According to the Institutional Review Board guidelines, we could not contact them. Two groups of cases were recruited: (i) cases diagnosed with breast cancer within 5 years and free of cancer/treatments for at least 6 months prior to study entry (n = 49), and (ii) newly diagnosed breast cancer cases before any treatment (n = 21). The comet tail moments (mean ± SD) for cases from groups 1 and 2 were: 11.27 ± 3.57 and 9.63 ± 3.60 (P = 0.09) for DB; 21.83 ± 4.93 and 19.87 ± 4.58 (P = 0.12) for DIR, and 15.51 ± 5.37 and 13.02 ± 4.99 (P = 0.07) for DRP, respectively. The three measurements of DNA damage were not significantly different between the two groups. Therefore, combined data were used for all subsequent statistical analyses.
Table II summarizes the mean comet tail moments in breast cancer cases and controls by age or FH. The levels of DNA damage were not influenced by these factors. However, comet tail moments differed significantly between cases and controls; the mean ± SD comet tail moment for cases and controls were (A) 10.78 ± 3.63 and 6.86 ± 2.76 (P < 0.001) for DB, (B) 21.24 ± 4.88 and 14.97 ± 4.18 (P < 0.001) for DIR and (C) 14.76 ± 5.35 and 9.75 ± 3.35 (P < 0.001) for DRP, respectively. To demonstrate the variations in DNA damage for each group, Figure 1 shows the box plot of DNA damage in cases and controls: (A) DB, (B) DIR and (C) DRP, respectively. The solid line indicates the median for each group. The box edges mark the 25th and 75th percentiles of the observed values, and the T-bars indicate the 10th and 90th percentiles.

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Fig. 1. Box plots of comet tail moment in breast cancer cases and controls. (A) DB, (B) DIR and (C) DRP. The solid line indicates the median for each group. The box edges mark the 25th and 75th percentiles of the observed values, and the T-bars indicate the 10th and 90th percentiles.
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Interestingly, we observed that the association between BMI and DNA damage differed between cases and controls. The levels of DB, DIR and DRP increased with increasing BMI for cases, whereas they decreased with increasing BMI in controls (P = 0.05, 0.04 and 0.04, respectively) as shown in Figure 2. The estimated regression lines are: (A) DB = 6.59 + 0.15 BMI for cases and DB = 12.39 0.23 BMI for controls, (B) DIR = 13.43 + 0.31 BMI for cases and DIR = 21.84 0.29 BMI for controls and (C) DRP = 5.52 + 0.38 BMI for cases and DRP = 14.47 0.20 BMI for controls, respectively.

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Fig. 2. Correlation between BMI and comet tail moment by case/control status. (A) DB, (B) DIR and (C) DRP.
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The comet tail moment was dichotomized to high or low by the median of all study subjects. Multiple logistic regression models were used to assess the association between high DNA damage and breast cancer risk. The data presented in Table III show that above-median comet tail moments for all DNA damages were significantly associated with breast cancer risk; OR = 13.44 (95% CI = 5.9730.24) for DB, OR = 13.65 (95% CI = 6.0730.71) for DIR, and OR = 6.54 (95% CI = 3.1113.79) for DRP, after adjustment for age. Given the results we found earlier regarding BMI, we also conducted a subgroup analysis (BMI data are available for 45 controls and 35 cases) to determine the association between elevated DNA damage and breast cancer risk, stratified by BMI. As shown in Table III, a stronger association between elevated DNA damage and breast cancer risk was observed in women with above-median BMI. Age-adjusted ORs and 95% CIs for below- versus above-median BMI are as follows: 3.92 (0.9216.64) versus 40.69 (5.93279.05) for DB, 9.32 (1.9345.12) versus 17.15 (3.4485.50) for DIR and 2.77 (0.6711.44) versus 26.24 (3.95174.44) for DRP, respectively.
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Discussion
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Breast cancer etiology encompasses multiple, diverse risk factors, such as environmental agents, hormonal exposures and genetics. Previous studies have suggested that elevated DNA damage levels and suboptimal DNA repair may be associated with breast cancer risk (916). DNA damage inflicted by carcinogenic agents, such as IR, ROS and estrogen metabolites, may contribute to genetic alterations that are critical in breast carcinogenesis. Therefore, we hypothesized that DNA damage due to exposure and/or genetic susceptibility, may serve as a breast cancer risk biomarker.
Methodologies for measuring DNA damage differ between laboratories and depend upon the DNA-damaging agent used, DNA repair kinetics, the endpoint measured and ways to measure the endpoint (quantitatively or qualitatively). The comet assay protocol used in this study is adequate to detect significant differences in single strand breaks between breast cancer cases and controls. Other studies have also reported higher levels of baseline DNA damage in blood and lymphocytes of cancer cases than in benign controls (9,18,19,34). In this study, DNA damage was
1.51.75-fold higher in breast cancer cases than in controls, which is in agreement with other studies using chromosomal aberrations as the study endpoint (35,36). However, the magnitude of difference in our study is much lower than that reported by Rajeswari et al. (18), who observed a 7-fold difference in comet tail length between breast cancer cases and controls in India. It is not clear what exposure or genetic factors may contribute to the different results between the two studies. However, a strength of our study is that we used the LAI Automated Comet Assay Analysis System for digital image collection and quantitative analysis of comet images to eliminate subjective comet assessments. Therefore, our approach provides a completely objective, fully automated delineation and analysis of head and tail regions within damaged cells, which are critical for larger population-based studies.
When determining cancer susceptibility, it would be ideal to evaluate DNA damage and sensitivity in the target tissue, such as mammary epithelial cells. However, peripheral lymphocytes are most often utilized in risk assessment studies, because it is easy to acquire them from a blood draw. However, we must ask whether measurements in lymphocytes can serve as surrogates for target tissue. Using the comet assay, Udumudi et al. (19) reported that comet tail lengths significantly differed between cancer patients and controls in cervical epithelial cells, as well as in peripheral blood leukocytes. In addition, higher levels of DNA adducts have been detected in normal adjacent breast tissue and altered DNA repair has been observed in cultured skin fibroblasts of breast cancer patients (21,37). These studies suggest that genetic defects in DNA repair may contribute to higher levels of DNA damage in lymphocytes and target tissue in cancer patients.
Previous studies reported that age did not have a significant effect on comet tail moments/lengths (30,31,38). In this study, potential confounding factors of breast cancer, such as age and FH did not influence DNA damage. However, we observed a differential relationship between BMI and DNA damage in cases versus controls. DNA damage increased with increasing BMI in cases, while it decreased with increasing BMI in controls. We propose two possible explanations for our findings.
First, for cancer cases, a higher BMI may be associated with higher levels of lipophilic aromatic compounds, such as polychlorinated biphenyls, aromatic and heterocyclic amines, and polycyclic aromatic hydrocarbons, stored in breast adipose tissue, leading to a continuous exposure to DNA-damaging agents (3941). Deficient DNA repair may contribute to the accumulation of unrepaired damage in both lymphocytes and target tissue. This concept is supported by the evidence that higher levels of oxidative adducts were detected in breast cancer cases than in controls (2025,41). Our findings are supported by a larger study showing an increased breast cancer risk with the intake of well-done red meat (a potential carcinogen source) that was more pronounced in women with a high BMI (42). Secondly, for controls, low BMI may be associated with more lean body mass, higher metabolic rate and elevated ROS generation (43,44). Therefore, BMI is inversely correlated with DNA damage in our study controls. We are currently conducting a larger study to further evaluate this interesting relationship between BMI and DNA damage in breast cancer cases and controls.
ROS, produced by IR, environmental carcinogens and endogenous activities such as cellular respiration and lipid peroxidation may contribute to breast carcinogenesis (16,27,45). Mutagenic products of ROS have been detected at elevated levels in breast cancer patients and in women with an increased risk for breast cancer (34,46). BRCA1, a breast cancer susceptibility gene required for transcription-coupled repair of oxidative damage, has been found to have reduced expression in sporadic breast tumor cases (47,48). Thus, considering the extensive daily base oxidation of DNA,
104 bases/cell/day, even a slight decrease in DNA repair capacity would lead to accumulation of genetic lesions (49). The higher levels of DNA damage measured in breast cancer cases may be related to a combination of exposure to ROS and genetic deficiencies in DNA repair (5052).
We consider two limitations of this study. First,
30% of the study subjects did not return their risk questionnaires in the original study. Although age and FH did not influence DNA damage levels, we still cannot rule out the possibility that other potential confounders or risk modifiers may influence the study results. Secondly, although newly diagnosed cases before treatment are generally preferred in case-control studies, biomarker measurements in surrogates, such as lymphocytes, may be influenced by tumor-associated factors (i.e. breast cancer associated antigens and cytokines). Therefore, we believe it is important to also evaluate DNA damage levels in samples collected from cancer-free women who were diagnosed previously with breast cancer. To minimize potential survival bias, we limited our recruitment to women who were diagnosed with breast cancer within 5 years and free of cancer/treatments for at least 6 months prior to study entry. Our study results suggest that DNA damage measured by the comet assay may serve as a susceptibility marker for breast cancer, because the levels were not influenced by disease status. However, it is obvious that the findings from this study need to be validated in prospective studies before we can draw the conclusion that elevated DNA damage is a predisposition marker for breast cancer.
A large percentage of breast malignancies cannot be explained by known risk factors, such as age at first birth, nullparity and FH (3). To assist with early detection/prevention of breast cancer, reliable risk biomarkers are urgently needed. The comet assay is a simple, quantitative way to determine the level of basal DNA damage and cellular response to DNA damage. With a limited sample size, this pilot study supports the hypothesis that persistent DNA damage and IR sensitivity may contribute to breast carcinogenesis. Larger studies are warranted to provide more definitive data on the role of DNA damage/repair in breast cancer susceptibility.
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Acknowledgments
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The authors acknowledge the contributions of Andrea Golden, Bruce Trock, Caryn Lerman, Susan Honig and Linus Truong. This study was supported by NIH/NCI grants CA-73629 (to J.J.H.), CA-91221 (to J.J.H.), CA81330 (to M.S.M.); ACS grant #RPG-97-115-01 (to J.J.H.); and a grant from the Friends you can count on foundation (to M.S.M.).
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References
|
---|
- Jemal,A., Thomas,A., Murray,T. and Thun,M. (2002) Cancer Statistics, 2002. CA Cancer J. Clin., 52, 2347.[Abstract/Free Full Text]
- Ries,L.A.G., Eisner,M.P., Kosary,C.L., Hankey,B.F., Miller,B.A., Clegg,L. and Edwards,B.K. (eds) (2001) SEER Cancer Statistics Review, 19731998. National Cancer Institute, Bethesda, MD.
- Madigan,M.P., Ziegler,R.G., Benichou,J., Byrne,C. and Hoover,R.N. (1995) Proportion of breast cancer cases in the United States explained by well-established risk factors. J. Natl Cancer Inst., 87, 16811685.[Abstract]
- Ron,E. (1998) Ionizing radiation and cancer risk: evidence from epidemiology. Radiat. Res., 150, S3041.[ISI][Medline]
- Johnson-Thompson,M.C. and Guthrie,J. (2000) Ongoing research to identify environmental risk factors in breast carcinoma. Cancer, 88, 12241229.[CrossRef][ISI][Medline]
- Gonzalez,R., Silva,J.M., Dominguez,G., Garcia,J.M., Martinez,G., Vargas,J., Provencio,M., Espana,P. and Bonilla,F. (1999) Detection of loss of heterozygosity at RAD51, RAD52, RAD54, and BRCA1 and BRCA2 loci in breast cancer: pathological correlations. Br. J. Cancer, 81, 503509.[CrossRef][ISI][Medline]
- Lininger,R.A., Zhuang,Z.P., Man,Y.G., Park,W.S., Emmert-Buck,M. and Tavassoli,F.A. (1999) Loss of heterozygosity is detected at chromosomes 1p35-36 (NB), 3p25 (VHL), 16p13 (TSC2/PKD1), and 17p13 (TP53) in microdissected apocrine carcinomas of the breast. Mod. Path., 12, 10831089.[ISI][Medline]
- Katsama,A., Sourvinos,G., Zachos,G. and Spandidos,D.A. (2000) Allelic loss at the BRCA1, BRCA2, and TP53 loci in human sporadic breast carcinoma. Cancer Lett., 150, 165170.[CrossRef][ISI][Medline]
- Djuric,Z., Heilbrun,L.K., Lababidi,S., Berzinkas,E., Simon,M.S. and Kosir,M.A. (2001) Levels of 5-hydroxymethyl-2'-deoxyuridine in DNA from blood of women scheduled for breast biopsy. Cancer Epidemiol. Biomarkers Prev., 10, 147149.[Abstract/Free Full Text]
- Hu,J.J., Smith,T.R., Miller,M.S., Lohman,K. and Case,L.D. (2002) Genetic regulation of ionizing radiation sensitivity and breast cancer risk. Environ. Mol. Mutagen., 39, 208215.[CrossRef][ISI][Medline]
- Helzlsouer,K.J., Harris,E.L., Parshad,R., Perry,H.R., Price,F.M. and Sanford,K.K. (1996) DNA repair proficiency: potential susceptibility factor for breast cancer. J. Natl Cancer Inst., 88, 754755.[Free Full Text]
- Parshad,R., Price,F.M., Bohr,V.A., Cowans,K.H., Zujewski,J.A. and Sanford,K.K. (1996) Deficient DNA repair capacity, a predisposing factor in breast cancer. Br. J. Cancer, 74, 15.[ISI][Medline]
- Hu,J.J., Roush,G.C., Dubin,N., Berwick,M., Roses,D.F. and Harris,M.N. (1997) Poly(ADP-ribose) polymerase in human breast cancer: a case-control analysis. Pharmacogenetics, 7, 309316.[ISI][Medline]
- Chakraborty,R., Little,M.P. and Sankaranarayanan,K. (1997) Cancer predisposition, radiosensitivity and the risk of radiation-induced cancers. III. Effects of incomplete penetrance and dose-dependent radiosensitivity on cancer risks in populations. Radiat. Res., 147, 309320.[ISI][Medline]
- Patel,R.K., Trivedi,A.H., Arora,D.C., Bhatavdekar,J.M. and Patel,D.D. (1997) DNA repair proficiency in breast cancer patients and their first-degree relatives. Int. J. Cancer, 73, 2024.[CrossRef][ISI][Medline]
- Scott,D., Barber,J.B., Spreadborough,A.R., Burrill,W. and Roberts,S.A. (1999) Increased chromosomal radiosensitivity in breast cancer patients: a comparison of two assays. Int. J. Radiat. Biol., 75, 110.[CrossRef][ISI][Medline]
- Grossman,L., Matanoski,G., Farmer,E., Hedayati,M., Ray,S., Trock,B., Hanfelt,J., Roush,G., Berwick,M. and Hu,J.J. (1999) DNA repair as a susceptibility factor in chronic diseases in human populations. In Dizdaroglu,M. and Karakaya,A.E. (eds) Advances in DNA Damage and Repair. Kluwer Academic/Plenum Publishers, New York, pp. 149167.
- Rajeswari,N., Ahuja,Y.R., Malini,U., Chandrashekar,S., Balakrishna,N., Rao,K.V. and Khar,A. (2000) Risk assessment in first degree female relatives of breast cancer patients using the alkaline Comet assay. Carcinogenesis, 21, 557561.[Abstract/Free Full Text]
- Udumudi,A., Jaiswal,M., Rajeswari,N., Desai,N., Jain,S., Balakrishna,N., Rao,K.V. and Ahuja,Y.R. (1998) Risk assessment in cervical dysplasia patients by single cell gel electrophoresis: a study of DNA damage and repair. Mutat. Res., 412, 195205.[ISI][Medline]
- Li,D., Wang,M., Dhingra,K. and Hittelman,W.N. (1996) Aromatic DNA adducts in adjacent tissues of breast cancer patients: clues to breast cancer etiology. Cancer Res., 56, 287293.[Abstract]
- Li,D., Zhang,W., Sahin,A.A. and Hittelman,W.N. (1999) DNA adducts in normal tissue adjacent to breast cancer: a review. Cancer Detect. Prev., 23, 454462.[CrossRef][ISI][Medline]
- Li,D., Zhang,W., Zhu,J., Chang,P., Sahin,A., Singletary,E., Bondy,M., Hazra,T., Mitra,S., Lau,S.S., Shen,J. and DiGiovanni,J. (2001) Oxidative DNA damage and 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase in human breast cancer. Mol. Carcinogen., 31, 214223.[CrossRef][ISI][Medline]
- Musarrat,J., Arezina-Wilson,J. and Wani,A.A. (1996) Prognostic and aetiological relevance of 8-hydroxyguanosine in human breast carcinogenesis. Eur. J. Cancer, 32A, 12091214.[CrossRef]
- Malins,D.C. and Haimanot,R. (1991) Major alterations in the nucleotide structure of DNA in cancer of the female breast. Cancer Res., 51, 54305432.[Abstract]
- Rundle,A., Tang,D., Hibshoosh,H., Estabrook,A., Schnabel,F., Cao,W., Grumet,S. and Perera,F.P. (2000) The relationship between genetic damage from polycyclic aromatic hydrocarbons in breast tissue and breast cancer. Carcinogenesis, 21, 12811289.[Abstract/Free Full Text]
- Kassie,F., Parzefall,W. and Knasmüller,S. (2000) Single cell gel electrophoresis assay: a new technique for human biomonitoring studies. Mutat. Res., 463, 1331.[ISI][Medline]
- Moller,P., Knudsen,L.E., Loft,S. and Wallin,H. (2000) The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidemiol. Biomarkers Prev., 9, 10051015.[Abstract/Free Full Text]
- Calderon-Garciduenas,L., Osnaya,N., Rodriguez-Alcaraz,A. and Villareal-Calderon,A. (1997) DNA damage in nasal respiratory epithelium from children exposed to urban pollution. Environ. Mol. Mutagen., 30, 1120.[CrossRef][ISI][Medline]
- Vodicka,P., Tvrdik,T., Osterman-Golkar,S. et al. (1999) An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers. Mutat. Res., 445, 205224.[ISI][Medline]
- Andreoli,C., Leopardi,P. and Crebelli,R. (1997) Detection of DNA damage in human lymphocytes by alkaline single cell gel electrophoresis after exposure to benzene or benzene metabolites. Mutat. Res., 377, 95104.[ISI][Medline]
- Zhu,C.Q., Lam,T.H., Jiang,C.Q., Wei,B.X., Lou,X., Liu,W.W., Lao,X.Q. and Chen,Y.H. (1999) Lymphocyte DNA damage in cigarette factory workers measured by the Comet Assay. Mutat. Res., 444, 16.[ISI][Medline]
- Gontijo,A.M., Elias,F.N., Salvadori,D.M., de Oliveira,M.L., Correa,L.A., Goldberg,J., Trindade,J.C. and de Camargo,J.L. (2001) Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers. Cancer Epidemiol. Biomarkers Prev., 10, 987993.[Abstract/Free Full Text]
- Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184191.[ISI][Medline]
- Frenkel,K., Karkoszka,J., Glassman,T., Dubin,N., Toniolo,P., Taioli,E., Mooney,L.A. and Kato,I. (1998) Serum autoantibodies recognizing 5-hydroxymethyl-2'-deoxyuridine, an oxidized DNA base, as biomarkers of cancer risk in women. Cancer Epidemiol. Biomarkers Prev., 7, 4957.[Abstract]
- Roy,S.K., Trivedi,A.H., Bakshi,S.R., Patel,R.K., Shukla,P.H., Patel,S.J., Bhatavdekar,J.M., Patel,D.D. and Shah,P.M. (2000) Spontaneous chromosomal instability in breast cancer families. Cancer Genet. Cytogenet., 118, 5256.[CrossRef][ISI][Medline]
- Trivedi,A.H., Roy,S.K., Bhachech,S.H., Patel,R.K., Dalal,A.A., Bhatavdekar,J.M. and Patel,D.D. (1998) Cytogenetic evaluation of 20 sporadic breast cancer patients and their first degree relatives. Breast Cancer Res. Treat., 48, 187190.[CrossRef][ISI][Medline]
- Hannan,M.A., Siddiqui,Y., Rostom,A., Al-Ahdal,M.N., Chaudhary,M.A. and Kunhi,M. (2001) Evidence of DNA repair/processing defects in cultured skin fibroblasts from breast cancer patients. Cancer Res., 61, 36273631.[Abstract/Free Full Text]
- Jaloszynski,P., Kujawski,M., Czub-Swierczek,M., Markowska,J. and Szyfter,K. (1997) Bleomycin-induced DNA damage and its removal in lymphocytes of breast cancer patients studied by comet assay. Mutat. Res., 385, 223233.[ISI][Medline]
- McTiernan,A. (2000) Associations between energy balance and body mass index and risk of breast carcinoma in women from diverse racial and ethnic backgrounds in the U.S. Cancer, 88, 12481255.[CrossRef][ISI][Medline]
- Gorlewska-Roberts,K., Green,B., Fares,M., Ambrosone,C.B. and Kadlubar,F.F. (2002) Carcinogen-DNA adducts in human breast epithelial cells. Environ. Mol. Mutagen., 39, 184192.[CrossRef][ISI][Medline]
- Lucena,R.A., Allam,M.F., Costabeber,I.H., Villarejo,M.L.J. and Navajas,R.F-C. (2001) Breast cancer risk factors: PCB congeners. Eur. J. Cancer Prev., 10, 117119.[CrossRef][ISI][Medline]
- Dai,Q., Shu,X-o., Jin,F., Gao,Y-T., Ruan,Z-X. and Zheng,W. (2002) Consumption of animal foods, cooking methods, and risk of breast cancer. Cancer Epidemiol. Biomarkers Prev., 11, 801808.[Abstract/Free Full Text]
- Shah,M., Miller,D.S. and Geissler,C.A. (1988) Lower metabolic rates of post-obese versus lean women: thermogenesis, basal metabolic rate and genetics. Eur. J. Clin. Nutr., 42, 741752.[ISI][Medline]
- Loft,S., Vistisen,K., Ewertz,M., Tjonneland,A., Overvad,K. and Poulsen,H.E. (1992) Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Carcinogenesis, 13, 22412247.[Abstract]
- Frenkel,K. (1992) Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol. Ther., 53, 757773.
- Shibutani,S., Takeshita,M. and Grollman,A.P. (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature, 349, 431434.[CrossRef][ISI][Medline]
- Gowan,L.C., Avrutskaya,A.V., Latour,A.M., Koller,B.H. and Leadon,S.A. (1998) BRCA1 required for transcription-coupled repair of oxidative damage. Science, 281, 10091013.[Abstract/Free Full Text]
- Sourvinos,G. and Spandidos,D.A. (1998) Decreased BRCA1 expression levels may arrest the cell cycle through activation of p53 checkpoint in human sporadic breast tumors. Biochem. Biophys. Res. Commun., 245, 7580.[CrossRef][ISI][Medline]
- Shigenaga,M.K., Gimeno,C.J. and Ames,B.N. (1989) Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative damage. Proc. Natl Acad. Sci. USA, 86, 96979701.[Abstract]
- Hu,J.J., Smith,T.R., Miller,M.S., Mohrenweiser,H.W., Golden,A. and Case,L.D. (2001) Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis, 22, 917922.[Abstract/Free Full Text]
- Hu,J.J., Mohrenweiser,H.W., Bell,D.A., Leadon,S.A. and Miller,M.S. (2002) Symposium overview: genetic polymorphisms in DNA repair and cancer risk. Toxicol. Appl. Pharmacol., 185, 6473.[CrossRef][ISI][Medline]
- Smith,T.R., Miller,M.S., Lohman,K., Lange,E.M., Case,L.D., Mohrenweiser,H.W. and Hu,J.J. (2003) Polymorphisms of XRCC1 and XRCC3 genes and susceptibility to breast cancer. Cancer Lett., 190, 183190.[CrossRef][ISI][Medline]
Received November 4, 2002;
revised February 21, 2003;
accepted February 24, 2003.