Affiliation of authors: M. B. Schabath, M. R. Spitz, K. Zhang, P.-J. Zheng, X. Wu (Department of Epidemiology), H. B. Grossman, C. P. Dinney (Department of Urology), The University of Texas M. D. Anderson Cancer Center, Houston, TX.
Correspondence to: Xifeng Wu, M.D., Ph.D., The University of Texas M. D. Anderson Cancer Center, Department of Epidemiology, Box 189, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: xwu{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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A variety of assays can assess genetic instability and DNA repair capacity in population-based studies, including those that assess metaphase chromosomal aberrations, micronuclei, sister chromatid exchanges, and host cell reactivation. However, these methods are typically laborious and time-consuming (e.g., host cell reactivation) or require highly trained technicians to accurately read and interpret slides (e.g., micronuclei and chromosomal aberrations). Over the past decade, the comet assay, or single-cell gel electrophoresis, has been used to study processes ranging from DNA repair to genotoxicity, a measure of the adverse effects of a toxin on DNA (5). Automated imaging technology facilitates use of this assay to determine DNA damage, which has been shown to be associated with an individuals susceptibility to cancer. The comet assay is relatively high-throughput, requires small numbers of cells, detects primary DNA damage in individual cells (5), provides results within a few hours of sampling, and is cost-effective (6). It is also a simple, sensitive, and reliable laboratory method that is suited for assessing in vitro genetic instability in molecular epidemiologic studies.
The challenge mutagen induces a specific type of DNA damage and provides information on possible deficiencies in DNA repair pathways. -Radiation induces single- and double-strand breaks that are repaired by base excision and/or double-strand break repair pathway(s). Benzo[a]pyrene diol epoxide (BPDE), a metabolite of the tobacco smoke procarcinogen benzo[a]pyrene, induces DNA adducts that are repaired by the nucleotide excision repair pathway. Two DNA repair genes, XPD (belonging to the nucleotide excision repair pathway) and XRCC1 (belonging to the base excision repair pathway), have been shown to be associated with an increased cancer risk in several casecontrol studies. Previous reports (714) indicate that variant genotypes in XPD (exons 10 and 23) and in XRCC1 (exon 6 wild-type and exon 10) are putative adverse genotypes that appear to be associated with increased levels of DNA damage.
In an ongoing hospital-based casecontrol study, we used the comet assay to quantify the baseline and BPDE- and -radiation-induced DNA damage in the peripheral blood lymphocytes of 114 patients with incident bladder cancer and 145 healthy control subjects and assessed whether individuals with latent genetic instability, as reflected in higher levels of DNA damage, have an increased estimated relative risk of bladder cancer.
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PATIENTS AND METHODS |
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Patients with incident urinary bladder cancer were accrued from August 10, 1999, through August 17, 2001, from the patient population at The University of Texas M. D. Anderson Cancer Center in Houston, Texas. All case patients had been diagnosed with bladder cancer within 1 year of accrual, their bladder cancer was histologically confirmed, and they had not been previously treated with chemotherapy or radiotherapy. There were no age, sex, ethnic, or cancer-stage restrictions. Case patients were identified by M. D. Anderson staff interviewers from daily review of computerized appointment schedules for clinics in which patients with bladder cancer were treated (Departments of Urology and Genitourinary Medical Oncology). Each new patient was initially screened by using a brief eligibility questionnaire that assessed prior cancer therapy and willingness to participate in the epidemiologic study. If the patient was willing to participate, the interviewer escorted the study participant to a private room or examination room to conduct the interview. We accrued a total of 114 case patients with bladder cancer and 145 healthy matched controls for this study.
The control subjects without a history of cancer (except nonmelanoma skin cancer) were recruited from a large pool of potential volunteers from the Kelsey-Seybold clinics, Houstons largest multispecialty physician group. As of May 1, 2001, there were more than 79 000 potential control subjects in the database, with 10001200 subjects added monthly. A control subject was selected to match to a case patient by age (±5 years), sex, and ethnicity. Control subjects were then contacted by telephone to confirm their willingness to participate, and an appointment was scheduled at a Kelsey-Seybold clinic convenient to the volunteer.
Epidemiologic Data
After written informed consent was provided, all study participants completed a 45-minute personal interview that was administered by trained M. D. Anderson staff interviewers. The interview elicited information regarding demographics, smoking history, alcohol consumption, family history of cancer, medical history, and occupational history. Additionally, a 60-minute food-frequency questionnaire was administered to assess diet during the year before diagnosis for case patients and diet during the year before the interview for control subjects. Both questionnaires consisted of a fixed script and included introductory and transitional statements. All interviewers were trained in the appropriate use of probes. A Spanish language version of both questionnaires was available. At the conclusion of the interview, a 40-mL blood sample was drawn into coded heparinized tubes. All study participants were compensated with a gift certificate to a local retail grocery store. Approval for research on human subjects was obtained from the M. D. Anderson and the Kelsey-Seybold Institutional Review Boards.
An individual who had smoked at least 100 cigarettes in his or her lifetime was defined as an ever smoker. Ever smokers include former smokers, current smokers, and recent quitters (who quit smoking within the previous year). For the case patients, a former smoker was a person who had quit smoking at least 1 year before diagnosis. For the control subjects, a former smoker was a person who had quit smoking at least 1 year before the interview.
BPDE Preparation
Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±) (i.e., BPDE) was obtained from the National Cancer Institute (NCI) Chemical Carcinogen Reference Standard Repository (NCI product L0137; Midwest Research Institute, Kansas City, MO) and was dissolved in tetrahydrofuran (Sigma Chemical Co., St. Louis, MO). Stock solutions (10 mM) prepared in tetrahydrofuran were stored at 20 °C in the dark to prevent photo-oxidation, and working solutions (0.4 mM) diluted with dimethyl sulfoxide (Sigma Chemical Co.) were freshly prepared for each experiment.
Tissue Cultures
Blood cultures were established immediately upon receipt of peripheral blood samples. Three blood cell cultures were prepared for each study subject to measure comets at baseline (untreated), and in -radiation-treated, and BPDE-treated cells. Phytohemagglutinin (PHA)-stimulated cultures were established in 60-mm x 15-mm Petri dishes (Falcon product 351007; Becton-Dickinson Labware, Franklin Lakes, NJ) with 0.4 mL of the fresh peripheral blood sample and 1.6 mL of RPMI-1640 medium (Life Technologies, Rockville, MD) supplemented with 15% fetal bovine serum (Life Technologies) and 1.25% PHA (Murex Diagnostics, Norcross, GA). After a 72-hour incubation, the comet assay was performed. From previous doseresponse experiments (data not shown), we determined that 10 µL of 0.4 mM BPDE (final BPDE concentration = 2 µM) added 24 hours before the comet assay was sufficient to induce nuclear DNA damage but was not cytotoxic. From previous experiments (15), we also determined that 1.5 Gy from a 137Cs source at room temperature was the optimal dose of
-radiation to induce nuclear DNA damage. After
-irradiation, blood cultures were placed on ice to slow DNA repair. Cells were embedded in agarose, which was attached to slides approximately 15 minutes after
-irradiation. Thus, the comet cells reflected the net result of DNA damage and repair because the fast component of DNA repair was unavoidable with this approach.
Comet Assay
To minimize extraneous DNA damage from ambient ultraviolet radiation, all steps were performed with reduced illumination. We slightly modified the comet assay under alkaline conditions as originally described by Singh et al. (16). Briefly, fully frosted slides were precoated on each end with 50 µL of 1% agarose in phosphate-buffered saline (PBS, pH 7.4), covered with a 22-mm x 22-mm glass coverslip, and allowed to stand at room temperature for 2030 minutes. For both baseline and mutagen-exposed cells, 50 µL of the blood culture was gently mixed with 150 µL of 0.5% low-melting-point agarose (Life Technologies) in PBS and maintained at 42 °C on a dry-bath incubator (Fisher Scientific, Houston, TX). A 50-µL aliquot of the mixture was immediately spread onto each end of a precoated slide and covered with a fresh glass coverslip. The agarose on the slides was allowed to solidify on a metal plate at 4 °C for approximately 10 minutes. A final layer of 0.5% low-melting-point agarose in PBS was placed on top of the cell suspension/low-melting-point agarose layer and covered with a new glass coverslip, and the slide was placed on a metal plate at 4 °C for an additional 10 minutes. Cells were lysed by submersing the slides in freshly prepared 1x lysis buffer (2.5 M NaCl, 100 mM EDTA, 1% N-lauroylsarcosine sodium salt, 10 mM Tris, adjusted to pH 10 with NaOH; 10% dimethyl sulfoxide and 1% Triton X-100 were added before use) for approximately 1 hour at 4 °C. To allow for DNA denaturation and unwinding and the exposure of the alkali-labile sites, slides were kept for 30 minutes in a horizontal electrophoresis box without power that was filled with freshly prepared alkali buffer (300 mM NaOH and 1 mM EDTA at pH >13) at 4 °C. To separate the damaged DNA from the intact nuclei, a constant electric current of 295300 mA was then applied for 23 minutes at 4 °C. After electrophoresis, the slides were neutralized with three 5-minute washes in 0.4 M TrisHCl (pH 7.4). Finally, the slides were fixed in 100% methanol for 510 minutes and stored in the dark at room temperature until analysis.
Quantification of DNA Damage
Immediately before imaging analysis, slides were hydrated in fresh 0.4 M TrisHCl (pH 7.4) for approximately 30 minutes and then stained for 1 minute with a fresh solution of 0.4 M TrisHCl containing 300 µL of ethidium bromide at 10 mg/mL. Fifty consecutive cells (25 cells from each end of the slide) were manually selected and were automatically quantified with Komet version 4.0.2 (Kinetic Imaging Ltd., Bromborough, Wirral, U.K.) imaging software attached to a fluorescent microscope (Nikon, Melville, NY), which also determined the Olive tail moment parameter [(tail mean head mean) x (% tail DNA/100)] used to quantify DNA damage (17). There are no expressible units for DNA damage measured by the comet assay. The head of the comet is the nucleus of the cell, and the tail of the comet is the damaged DNA that has been liberated from the nucleus by electrophoresis. The tail mean is the tail DNA intensity subtracted from background intensity, the head mean is the head DNA intensity subtracted from background intensity, and the percentage of tail DNA is the fraction of DNA that has migrated from the head. The difference between the tail mean and the head mean represents the difference in the distance between the center of gravity of the DNA distribution in the comet head and the center of gravity of the DNA distribution in the comet tail. For each subject, the averages of the Olive tail moment were calculated for the baseline comets, -radiation-induced comets, and BPDE-induced comets.
Polymerase Chain ReactionRestriction Fragment Length Polymorphism Assays for XPD and XRCC1
Previously described polymerase chain reactionrestriction fragment length polymorphism assays were used to amplify the polymorphic regions for XPD exons 10 and 23 (13) and XRCC1 exons 6 and 10 (14). For the XPD exon 10 polymorphism (Asp312Asn), the wild-type (Asp/Asp) genotype produced two DNA bands (507 and 244 base pairs [bp]), the variant genotype (Asn/Asn) produced three bands (474, 244, and 33 bp), and the heterozygous genotype (Asp/Asn) produced all four bands (507, 474, 244, and 33 bp). For the XPD exon 23 polymorphism (Lys751Gln), the homozygous wild-type genotype (Lys/Lys) produced two DNA bands (290 and 146 bp), the variant genotype (Gln/Gln) produced three DNA bands (227, 146, and 63 bp), and the heterozygous genotype (Lys/Gln) produced all four bands (290, 227, 146, and 63 bp). For the XRCC1 exon 6 polymorphism (Arg194Trp), the wild-type genotype (Arg/Arg) produced one band (490 bp), the heterozygous genotype (Arg/Trp) produced three bands (490, 294, and 196 bp), and the variant genotype (Trp/Trp) produced two bands (294 and 196 bp). For the XRCC1 exon 10 polymorphism (Arg399Gln), the homozygote genotype (Arg/Arg) produced two bands (269 and 133 bp), the heterozygous genotype (Arg/Gln) produced three bands (402, 269, and 133 bp), and the variant genotype (Gln/Gln) produced one band (402 bp). Positive and negative controls were used in each experiment, and 10% of the total samples were randomly selected and reanalyzed with 100% concordance.
Statistical Analysis
All statistical analyses were performed with the Intercooled Stata 7.0 statistical software package (Stata Corporation, College Station, TX). The 2 test was used to test for differences between the case patients and the control subjects in the distribution of sex, ethnicity, and smoking status, and the Students t test was used to test for differences between the case patients and control subjects for age, pack-years (among ever smokers), and Olive tail moments. Odds ratios (ORs) were calculated as estimates of relative risk by dichotomizing the tail moment at the median (50th percentile) value in control subjects. Unconditional multivariable logistic regression was performed to control for possible confounding by age, sex, ethnicity, and smoking status, where appropriate. To detect patterns of association between the degree of risk and higher levels of DNA damage for baseline and mutagen-induced comets, the tail moment was categorized by the tertile values in the control subjects, and the two new variables were analyzed simultaneously in one model. All statistical tests were two-sided.
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RESULTS |
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DISCUSSION |
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When the data were dichotomized at the median value for the control subjects, there was a statistically significantly increased estimated relative risk of cancer associated with higher levels of baseline (spontaneous or uninduced) DNA damage (OR = 1.84, 95% CI = 1.07 to 3.15). Thus, categorizing individuals into discrete groups may be useful for identifying subsets of individuals who are at higher risk for cancer. Other studies using the comet assay have also shown greater baseline DNA damage in patients with cancer, including those with oral squamous cell carcinoma (18), cancer of the uterine cervix (19), and breast cancer (20,21). McKelvey-Martin et al. (22) noted statistically significantly greater DNA damage in cells from bladder washings of patients with bladder cancer than in cells from bladder washings of control subjects. We also observed this association between genetic instability and the estimated relative risk of bladder cancer when data on DNA damage were separated into tertiles. Specifically, individuals in the highest tertile of DNA damage at baseline were at a nearly twofold greater estimated relative risk than those in the first tertile (OR = 1.95, 95% CI = 1.02 to 3.74); individuals in the second tertile of DNA damage at baseline were at a 1.3-fold greater estimated relative risk (OR = 1.31, 95% CI = 0.65 to 2.62). Therefore, our data support the hypothesis that individuals with genetic instability are at an increased estimated relative risk for bladder cancer. Indeed, categorizing the level of DNA damage by this approach may be particularly useful for identifying individuals with susceptible genetic profiles. Results of the comet assay could be combined with data on cigarette smoking, family history of cancer, occupation, and dietary intake to construct comprehensive risk profiles.
Latent genetic instability can be identified by a mutagen challenge. Therefore, a mutagen challenge with both BPDE and -radiation, which induce different types of DNA damage, can assess overall host susceptibility to bladder cancer because it yields data on the net result of DNA damage and repair for two distinct repair pathways. BPDE and
-radiation have both been used previously to induce DNA damage that can be detected with the comet assay (23,24). We observed an increased estimated relative risk of bladder cancer associated with
-radiation- and BPDE-induced DNA damage and a 2.2-fold increased estimated relative risk associated with elevated levels of both (Table 3
). The OR for the intermediate category containing individuals classified as above the median value for either mutagen challenge was not statistically significant (OR = 0.75, 95% CI = 0.35 to 1.63). This result may reflect the small sample size or may represent biologic effects in that the individuals segregated readily to both high levels of BPDE- and
-radiation-induced DNA damage or low levels of BPDE- and
-radiation-induced DNA damage. When the data from control subjects were separated into tertiles, we observed an increasing estimated relative risk with higher levels of
-radiation- and BPDE-induced damage (Table 4
). Previous studies using the comet assay have demonstrated similar casecontrol differences in induced DNA damage with a variety of test mutagens for other cancers. Colleu-Durel et al. (25) noted that lymphocyte DNA from patients with various cancers had higher levels of damage than that of control subjects with or without irradiation. Rajaee-Behbahani et al. (26) reported that the number of comet cells in bleomycin-treated peripheral blood lymphocytes, measured before DNA repair could occur, was statistically significantly higher in lung cancer patients than in tumor-free hospital control subjects. Kleinsasser et al. (27) used the comet assay to demonstrate that N'-nitrosodiethylamine induced statistically significantly more DNA migration in lymphocytes from patients with nasopharyngeal cancer (tail moment = 9.8 ± 3.1) than in lymphocytes from nontumor patients (tail moment = 8.2 ± 2.3) (P = .03).
A useful feature of the comet assay is that it can be modified to measure various types of DNA damage and to measure the repair of that damage. We propose that the mutagen-induced comet cells in our study reflect the net result of DNA damage and repair. In the BPDE experiments, the peripheral blood lymphocytes were treated once with BPDE and incubated for an additional 24 hours before the comet assay was performed. During this 24-hour period, cells were continuously removing and repairing BPDE-induced DNA damage. Breaks would be only transiently present as cells repaired lesions by nucleotide excision (28), so that a high level of breaks in our study after a 24-hour incubation should reflect poor nucleotide excision repair. A similar argument could be made for -radiation-induced DNA damage; the observed differences should be the net results of DNA damage and repair. Approximately 50% of
-radiation-induced DNA damage occurs within 15 minutes (29), and the rapid repair of DNA, but not the slow component, occurs within 5 minutes. Additionally, cells from patients with ataxia-telangiectasia have a reduced fast-repair component in both G1- and G2-phase cells that operates on DNA double-strand breaks and chromosomal breaks. However, these differences are not observed in the slow component of DNA repair (29). In our approach,
-irradiation was performed at room temperature. Cells were then placed on ice blocks to slow DNA repair, embedded in agarose, and attached to the slides approximately 15 minutes after
-radiation. Consequently,
-radiation-induced comet cells should reflect the net result of DNA damage and repair because the fast component of DNA repair was not inhibited. Our data therefore indicate that the latent genetic instability, identified by using a mutagen challenge followed by the comet assay, is associated with an increased estimated relative risk of bladder cancer.
The data were also analyzed by smoking status to ensure that smoking behavior did not contribute to the differences observed between the case patients and the control subjects (Table 5). There were no differences in DNA damage when former smokers and ever smokers were compared with never smokers. Thus, smoking status apparently does not explain the substantial differences in DNA damage between case patients and control subjects. Further analysis of the DNA damage by casecontrol status revealed a normal distribution, which supports the idea that the DNA damage in case patients was apparently not influenced by a subset of case patients with higher values, which would have contributed to an artificially increased estimated relative risk (data not shown).
We also report preliminary data showing a non-statistically significant association between DNA damage as measured by the comet assay and the polymorphic DNA repair genes XRCC1 (exons 6 and 10) and XPD (exons 10 and 23). XRCC1 is an important gene in the base excision repair pathway and is involved in the removal of -radiation-induced DNA damage. The deleterious XRCC1 genotypes (exon 6 wild-type genotype and exon 10 variant genotypes) were associated with greater
-radiation-induced DNA damage than genotypes thought to be associated with more proficient DNA repair (exon 6 variants and exon 10 wild-type). Additional support for our findings comes from previous studies showing that the XRCC1 exon 10 variant genotypes are associated with greater mean sister chromatid exchange frequencies (7) and higher levels of aflatoxin B1DNA adducts and erythrocyte glycophorin A somatic mutations (variant NN) (8) than the wild-type genotype. XRCC1 exon 10 variant genotypes have also been associated with increased risk for head and neck cancer (9) and gastric cancer, and XRCC1 exon 6 genotypes modulate the risks for bladder cancer (11), oral and pharyngeal cancers (9), and gastric cardia cancer (10).
XPD is an essential gene in the nucleotide excision repair pathway and is involved in the removal of BPDE-induced DNA adducts. In our study, a higher level of BPDE-induced DNA damage among control subjects was associated with the variant genotypes of XPD (exons 10 and 23) than with the XPD wild-type genotypes (exons 10 and 23). These results are consistent with previous findings of more efficient DNA repair with XPD wild-type genotypes than with XPD variant genotypes at both of these exons (12,13). The small sample size in the present study limited extensive analyses of the XPD and XRCC1 genotypes. Nonetheless, we did note that the putative adverse genotypes of XPD and XRCC1 were observed more frequently among case patients than among control subjects, but these differences did not reach statistical significance. The small sample size could also explain why we did not observe a statistically significant association between the DNA repair genotypes and DNA damage in case patients. Because cancer is a multigenic disease and each gene involved has only a small independent effect on the disease phenotype (13), a much larger sample size is required to determine whether each polymorphism was associated with risk for bladder cancer.
In conclusion, genetic instability (both spontaneous and mutagen-induced) appears to be associated with the estimated relative risk of bladder cancer. Measuring mutagen sensitivity with the comet assay is a promising approach for determining genetic instability and thus the susceptibility of an individual to cancer. Patients with bladder cancer and control subjects appear to have subtle differences in the degree of genetic instability, as shown by the mean differences in spontaneous and mutagen-induced DNA damage that were detected by tail moments of comet cells. However, by separating the data into categories, we may identify individuals at higher estimated relative risk for developing bladder cancer. Although this study may not be large enough (sufficiently powered) to detect the complex associations between DNA repair genotypes and DNA damage, this study is an initial step to demonstrate that the comet assay can be used as a tool to assess genetic instability for cancer risk. Large population-based cohort studies are required to further validate these findings and determine whether the comet assay will have clinical utility and applications in large-scale molecular epidemiologic studies.
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NOTES |
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We gratefully acknowledge Beth Notzon for editorial assistance.
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Manuscript received May 3, 2002; revised January 23, 2003; accepted February 6, 2003.
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