Mutagen sensitivity in patients with familial and non-familial urothelial cell carcinoma

Due to variation in individual susceptibility, only a fraction of all individuals exposed to environmental carcinogens will develop cancer. Our aim was to assess whether mutagen sensitivity plays a role in developing urothelial cell carcinoma (UCC) and whether this sensitivity is different in familial and non-familial cases. Intrinsic susceptibility was quantified by a mutagen sensitivity assay (mean number of chromatid breaks per cell after damage induction with bleomycin in the late S-G2 phase of the cell cycle). Patients were classified as sporadic (n = 25), familial (2 patients in 1 nuclear family, n = 23) or hereditary (2 patients <60 years or 3 patients in 1 nuclear family, n = 13) and compared with control subjects without a history of cancer. Information on demographic factors, smoking history and family history of UCC was collected by postal questionnaires. Differences in mutagen sensitivity were assessed by ANOVA and logistic regression analysis. Overall, UCC patients showed a higher mutagen sensitivity score compared with control subjects [mean number of chromatid breaks per cell 0.91, 95% confidence interval (CI) 0.84-0.97, and 0.74, 95% CI 0.69-0.79, respectively; p = 0.001). Sporadic and familial patients exhibited the highest susceptibility (0.94, 95% CI 0.82-1.06, and 0.93, 95% CI 0.83-1.03, respectively). Hereditary patients (0.79, 95% CI 0.72-0.86) showed a susceptibility similar to controls. Mutagen sensitivity increases the risk of non-hereditary UCC. The relatively low mutagen sensitivity score among hereditary patients points to a different carcinogenic pathway.     

Sensitivity to benzo(a)pyrene diol-epoxide associated with risk of breast cancer in young women and modulation by glutathione S-transferase polymorphisms: a case-control study.

Mounting epidemiological evidence suggests that smoking may play a role in the etiology of breast cancer. Because smoking-related DNA adducts are detectable in both normal and malignant breast tissues, we hypothesized that breast cancer patients may be sensitive to tobacco-induced carcinogenesis, and this sensitivity could be modulated by variants of metabolic genes. To test this hypothesis, we evaluated benzo(a)pyrene diol-epoxide (BPDE)-induced mutagen sensitivity and polymorphisms of GSTM1 and GSTT1 in a pilot case-control study of breast cancer. Short-term cell cultures were established from blood samples of 100 female breast cancer patients and 105 healthy controls. After 5 h of in vitro exposure to 4 microM of BPDE, we harvested the lymphocytes for cytogenetic evaluation and recorded and compared the frequency of BPDE-induced chromatid breaks between cases and controls. We used a multiplex PCR-based assay to simultaneously detect polymorphisms of GSTM1 and GSTT1 from genomic DNA. We performed univariate and multivariate logistic regression analyses and calculated odds ratios (OR) and 95% confidence intervals (CIs). Cases had a significantly higher frequency of chromatid breaks than did controls (P < 0.0001). The level of chromatid breaks greater than the median value of controls was associated with a >3-fold increased risk of breast cancer [adjusted odds ratio (ORadj) = 3.11; 95% CI = 1.72-5.64]. The risk was more pronounced in those who were < 45 years (ORadj = 4.79; 95% CI = 1.87-12.3), ever-smokers (ORadj = 5.55; 95% CI = 1.85-16.6), alcohol drinkers (ORadj = 4.64; 95% CI = 1.70-12.7), and those who had the GSTT1 null variant (ORadj = 8.01; 95% CI = 1.16-55.3). These data suggest that sensitivity to BPDE-induced chromosomal aberrations may contribute to the risk of developing breast cancer, and such sensitivity may be modulated by both genetic and environmental factors. Larger studies are needed to confirm our findings.     

Radiation-induced chromatid breaks as a predictor of breast cancer risk

PURPOSE: In in vivo models, radiation-induced genomic instability correlates with the risk of breast cancer development. In addition, homozygous mutations in tumor suppressor genes associated with breast cancer development adversely affects the processing and repair of radiation-induced DNA damage. We performed a case-control study to determine whether an assay measuring radiation-induced chromatid breaks correlated with the risk of having bilateral breast cancer. METHODS AND MATERIALS: Patients were prospectively studied on an institutional review board-approved protocol. We included only women with bilateral breast cancer as cases to obtain patients with a presumed genetic susceptibility for breast cancer. Controls were healthy women without a previous cancer history. A mutagen sensitivity assay using gamma-radiation was performed on lymphocytes obtained from 26 cases and 18 controls. One milliliter of whole blood was cultured with 9 mL of blood medium for 91 h and then treated with 125 cGy using a Cs-137 irradiator. Following an additional 4 h in culture, cells were treated with Colcemid for 1 h to arrest cells in metaphase. The number of chromatid breaks per cell was counted using a minimum of 50 metaphase spreads for each sample. RESULTS: Cases had a statistically higher number of gamma-radiation-induced chromatid breaks per cell than controls, with mean values of 0.61 +/- 0.24 vs. 0.45 +/- 0.14, respectively (p = 0.034, Wilcoxon rank sum test). Using the 75th percentile value in the control group as a definition of radiation sensitivity, the radiation-sensitive individuals had a 2.83-fold increased odds ratio for breast cancer development compared with individuals who were not radiation sensitive (95% confidence intervals of 0.83 and 9.67). CONCLUSIONS: These preliminary data suggest that sensitivity to radiation-induced chromatid breaks in lymphocytes correlates with the risk of bilateral breast cancer. Although the differences between cases and controls were statistically significant, the small sample size necessitates that this finding be validated in a larger study. More data are also needed to determine whether this sensitivity is limited to breast cancer patients with a genetic susceptibility for the disease or also applies to the general breast cancer population.     

Joint effect of mutagen sensitivity and insulin-like growth factors in predicting the risk of developing secondary primary tumors and tumor recurrence in patients with head and neck cancer.

PURPOSE: Early-stage head and neck cancer patients are at high risks for tumor recurrence and secondary primary tumor (SPT) development. We hypothesized that latent genetic instability and proliferation potential may be associated with elevated risks of SPT and recurrence. EXPERIMENTAL DESIGN: We conducted a nested case-control study within a randomized, placebo-controlled chemoprevention trial in patients with early-stage head and neck cancer. We compared prediagnostic bleomycin-induced chromatid breaks in peripheral blood lymphocyte cultures (as an indicator of latent genetic instability) between 298 cases (patients with SPT/recurrence) and 693 controls (patients without SPT/recurrence). We also determined the joint effects of latent genetic instability and elevated proliferation potential [indicated by serum insulin-like growth factor (IGF) level] in modulating the risk of SPT and recurrence. RESULTS: In the Cox proportional hazards model, patients with higher mutagen sensitivity (using a cutoff of > or =0.50 breaks per cell) exhibited a significantly increased risk of developing SPT/recurrence [hazard ratio, 1.38; 95% confidence interval (95% CI), 1.02-1.86]. Cases also exhibited significantly higher levels of IGF-I and IGF-binding protein-3 than controls (P = 0.022 and 0.042, respectively). Moreover, there were joint effects between mutagen sensitivity and IGFs in modulating SPT/recurrence risk. Using patients with low IGF-I level and low mutagen sensitivity profile as the reference group, the odds ratios of developing SPT/recurrence for patients with high IGF-I level alone, high mutagen sensitivity alone, and both high IGF-I level and high mutagen sensitivity were 2.85 (95% CI, 0.92-8.82), 3.92 (95% CI, 1.28-11.97), and 6.16 (95% CI, 2.03-18.71), respectively. A similar joint effect was observed for mutagen sensitivity and IGF-binding protein-3 level. CONCLUSIONS: This is the largest prospective study to evaluate mutagen sensitivity as a prognosis marker in head and neck cancer because mutagen sensitivity data were derived from baseline samples drawn before the development of SPT or tumor recurrence. The results also show for the first time that latent genetic instability and elevated proliferation potential jointly elevate the risk of second tumors in early-stage head and neck cancers.     

Joint effect of insulin-like growth factors and mutagen sensitivity in lung cancer risk

BACKGROUND: We hypothesize that accumulation of genetic damage is dependent on an individual's intrinsic carcinogen sensitivity and on various humoral factors (e.g., insulin-like growth factors [IGFs]) that enhance proliferation, resistance to apoptotic cell death, and clonal outgrowth of genetically damaged cells. We tested this hypothesis by determining whether proliferation potential and genetic instability are associated with the risk of lung cancer. METHODS: In a study of 183 lung cancer patients and 227 matched control subjects, we examined the joint effects of latent genetic instability (measured as mutagen sensitivity) and elevated proliferation potential (assessed by measuring IGFs) in lung cancer risk. Levels of IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3) in plasma were measured by use of immunoassay kits. Mutagen sensitivity was assessed by quantitating bleomycin- and benzo[a]pyrene diol epoxide (BPDE)-induced chromatid breaks in peripheral blood lymphocyte cultures. RESULTS: Although not statistically significant, the mean levels of IGF-I and the molar ratio of IGF-I/IGFBP-3 were higher in patients with advanced or poorly differentiated disease than in patients with early or well-differentiated disease. Variation in IGFs was not associated with any specific histologic type or tumor stage. High levels of IGF-I and enhanced mutagen sensitivity were individually associated with increased risk of lung cancer: odds ratio (OR) of 2.13 (95% confidence interval [CI] = 1.20-3.78) for IGF-I, 2.50 (95% CI = 1. 49-4.20) for bleomycin sensitivity, and 2.95 (95% CI = 1.72-5.06) for BPDE sensitivity. The OR was statistically significantly elevated to 8.88 for both higher IGF-I and bleomycin sensitivity (95% CI = 3.67-21.50) and to 13.53 for higher IGF-I and BPDE sensitivity combined (95% CI = 4.48-40.89). With all three risk factors considered together, the OR was 17.09 (95% CI = 4.16-70.27). High levels of IGFBP-3 alone were associated with reduced lung cancer risk: OR = 0.59 (95% CI = 0.33-1.05). CONCLUSIONS: Our data suggest that individuals with genetic instability and higher proliferation potential are at enhanced risk for lung cancer.     

Chromosome instability and risk of squamous cell carcinomas of head and neck

In 895 subjects with squamous cell carcinoma of the head and neck (SCCHN) and 898 cancer-free controls matched by age, sex, and ethnicity, we validated our previous finding that mutagen sensitivity as measured by the frequency of chromatid breaks in vitro induced by benzo[a]pyrene diol epoxide (BPDE) is an independent risk factor for SCCHN. Using a previously established concentration of 4 micromol/L BPDE to treat short-term cultured primary lymphocytes for 5 hours, we evaluated chromatid breaks in 50 well-spread metaphases for each blood sample. The mean frequency of BPDE-induced chromatid breaks was significantly higher in cases than in controls in non-Hispanic Whites (P = 0.0003) but not in other ethnic groups (P = 0.549 for Hispanic Americans and 0.257 for African Americans). The odds ratio associated with risk of SCCHN for the frequency of chromatid breaks greater than median value of controls was 1.56 (95% confidence interval, 1.27-1.91) in non-Hispanic Whites (767 cases and 763 controls) after adjustment for age, sex, smoking status, and drinking status. When the quartiles of the controls were used as the cutoff values, there was a dose response between the degree of mutagen sensitivity and risk of SCCHN in non-Hispanic Whites (P(trend) = 0.0001). However, none of these associations in non-Hispanic Whites was identified in Hispanic Americans (69 cases and 70 controls) or African Americans (59 cases and 65 controls), possibly because of the small samples of these ethnic groups or ethnic difference in genetic variation, which needs to be confirmed in future studies.     

Increased mutagen sensitivity in head-and-neck squamous-cell carcinoma patients,particularly those with multiple primary tumors

Mutagen sensitivity is a constitutional factor which may be used to identify head-and-neck squamous-cell carcinoma (HNSCC) patients at high risk for the development of multiple primary tumors (MPT). In this retrospective study, mutagen sensitivity was measured in HNSCC patients with a single primary tumor (SPT), HNSCC patients who have already developed MPT and control subjects with no tumor history. In vitro, lymphocytes were challenged with bleomycin and chromosomal damage was quantified by scoring chromatid breaks of 100 cells. A significant difference in the mean number of breaks per cell (b/c) was found between SPT patients and controls. Patients with MPT showed a significantly higher mean b/c value than SPT patients. This increase in mutagen sensitivity in HNSCC patients was not related to well-known cancer risk factors such as age, or life-style factors such as smoking and alcohol drinking habits. In addition, tumor site but not tumor stage was found to be related to mutagen sensitivity. On the basis of our findings, we propose that mutagen sensitivity is not an independent risk factor but a constitutional factor which reflects the way in which genotoxic compounds are dealt with and is thereby directly related to cancer risk.     

Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies

There is an increasing need for viable lymphocytes in performing phenotypic assays for biomarker studies. Both fresh and cryopreserved lymphocytes have been used for cell culture-based functional assays. However, fresh lymphocytes do not allow assays to be done in batches and cryopreservation of isolated lymphocytes results in a considerable loss of viable cells. To investigate the feasibility of using cryopreserved whole blood as a source of viable lymphocytes in molecular epidemiology studies, two well-established biomarkers, the host-cell reactivation (HCR) and mutagen sensitivity assays, were used to compare the method of cryopreserving whole blood with the traditional methods. In 25 paired blood samples assayed for DNA repair capacity (DRC) by the HCR assay, the DRC values of frozen whole blood (mean +/- SD, 11.59 +/- 3.07) were similar to those of frozen isolated lymphocytes (11.08 +/- 3.50). The correlation between the paired DRC values was 0.77 (P < 0.001). In 31 paired blood samples assayed for the gamma-radiation-induced chromatid breaks by the mutagen sensitivity assay, there was no significant difference between the baseline level of chromatid breaks in lymphocytes from frozen blood (0.05 +/- 0.03) and fresh blood (0.06 +/- 0.03). The blastogenic rate and mitotic index of the cells used for the two assays were compared between the different processing methods. The lymphocytes from frozen whole blood were more sensitive to gamma-radiation, with a higher mean level of chromatid breaks (0.68 +/- 0.21) than that in fresh blood (0.42 +/- 0.12, P < 0.01), and the correlation between the numbers of chromatid breaks in the paired samples was statistically significant (r = 0.61, P < 0.001). These data suggest that within the limits of the parameters investigated here, cryopreserved whole blood is a good source of viable lymphocytes for biomarker assays in molecular epidemiological studies.     

Evaluation of glutathione S-transferase polymorphisms and mutagen sensitivity as risk factors for the development of second primary tumors in patients previously diagnosed with early-stage head and neck cancer

BACKGROUND: The objective of this study was to evaluate the effects of polymorphisms in 2 genes in the glutathione S-transferase (GST) family and the mutagen-sensitivity phenotype on the risk of second primary tumors (SPTs) in patients with previously diagnosed early-stage head and neck squamous cell carcinoma. Data were available for 303 patients who were enrolled in a placebo-controlled chemoprevention trial of low-dose 13-cis-retinoic acid to reduce the occurrence of SPTs. METHODS: A Cox proportional hazards model and survival tree analysis were used to evaluate the association between specified genetic variations and the development of SPTs. The average number of bleomycin-induced chromatid breaks per cell was used to quantify mutagen sensitivity as an individual patient's degree of sensitivity to genotoxicity. RESULTS: The GST-M1 null genotype was associated with an increased risk for any SPTs (hazard ratio [HR], 1.99; 95% confidence interval [95% CI], 1.11-3.56) and for tobacco-related SPTs (HR, 2.16; 95% CI, 1.01-4.62) after adjusting for covariates. The GST-T1 null genotype and bleomycin-induced chromatid breaks were not associated with a statistically significant increased risk for SPTs or tobacco-related SPTs after similar adjustment. Simultaneous nonnull status for both GST genotypes was associated with a decreased risk for any SPTs (HR, 0.52; 95% CI, 0.28-0.96) and tobacco-related SPTs (HR, 0.50; 95% CI, 0.22-1.11) compared with null status for GST-M1 accompanied by nonnull status for GST-T1. CONCLUSIONS: An association was observed between the development of SPTs and the GST-M1 null genotype after successful treatment for early-stage head and neck squamous cell carcinoma. The GST-T1 null genotype and bleomycin-induced chromatid breaks were not associated with an increased risk, and no significant interactions were identified.     

Family history of cancer, mutagen sensitivity, and increased risk of head and neck cancer

To evaluate individual cancer susceptibility, 170 previously untreated patients with pathologically-confirmed squamous cell carcinoma of the oral cavity, pharynx, and larynx, and 175 age- and sex-matched health controls were investigated for the occurrence of cancer in first-degree relatives along with other established risk factors for head and neck cancer. More than 54% of these subjects were assayed for mutagen sensitivity by quantifying in-vitro bleomycin-induced chromosomal breaks within peripheral blood lymphocytes. After adjusting for age, gender, education, family income, tobacco and alcohol consumption, the odds ratio associated with three or more first-degree relatives with cancer at any site was 3.79 (95% CI 0.9-15.9) with a linearly-increased trend in risk (P = 0.040). Significantly elevated risk was found to be associated with a history of cancer within siblings (OR = 2.61, 1.2-5.6, P = 0.014). Patients with a family cancer history and mutagen sensitivity were at greatest risk (OR = 7.88, 2.5-25.3, P = 0.005), indicating an additive interactive effect. The findings suggested that genetic familial influence is important in the causation of head and neck cancer.     

Prospective analysis of DNA damage and repair markers of lung cancer risk from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial

Mutagen challenge and DNA repair assays have been used in case-control studies for nearly three decades to assess human cancer risk. The findings still engender controversy because blood was drawn after cancer diagnosis so the results may be biased, a type called 'reverse causation'. We therefore used Epstein-Barr virus-transformed lymphoblastoid cell lines established from prospectively collected peripheral blood samples to evaluate lung cancer risk in relation to three DNA repair assays: alkaline Comet assay, host cell reactivation (HCR) assay with the mutagen benzo[a]pyrene diol epoxide and the bleomycin mutagen sensitivity assay. Cases (n = 117) were diagnosed with lung cancer between 0.3 and 6 years after blood collection and controls (n = 117) were frequency matched on calendar year and age at blood collection, gender and smoking history; all races were included. Case and control status was unknown to laboratory investigators. In unconditional logistic regression analyses, statistically significantly increased lung cancer odds ratios (OR(adjusted)) were observed for bleomycin mutagen sensitivity as quartiles of chromatid breaks/cell [relative to the lowest quartile, OR = 1.2, 95% confidence interval (CI): 0.5-2.5; OR = 1.4, 95% CI: 0.7-3.1; OR = 2.1, 95% CI: 1.0-4.4, respectively, P(trend) = 0.04]. The magnitude of the association between the bleomycin assay and lung cancer risk was modest compared with those reported in previous lung cancer studies but was strengthened when we included only incident cases diagnosed more than a year after blood collection (P(trend) = 0.02), supporting the notion the assay may be a measure of cancer susceptibility. The Comet and HCR assays were unrelated to lung cancer risk.     

Gamma radiation sensitivity and risk of malignant and benign salivary gland tumors: a pilot case-control analysis

BACKGROUND: The salivary gland is a highly radiosensitive organ. Exposure to gamma radiation is a risk factor for both malignant (MSTs) and benign salivary gland tumors (BSTs), but the exact mechanisms remain unknown. The objectives of the current study were to determine whether gamma radiation-induced chromatid breaks increase the risk of MSTs and BSTs and whether there is any difference in risk between these two diseases. METHODS: The authors performed a pilot case-control study of 57 patients with salivary gland diseases (45 patients with MSTs and 12 patients with BSTs) and 105 cancer-free controls. Peripheral blood lymphocytes from these participants were cultured and exposed to gamma radiation (1.5 grays). Five hours later, metaphase spread slides were evaluated. The chromatid breaks in 50 well-spread metaphase slides were counted to determine the average number of chromatid breaks per cell (b/c). RESULTS: Multivariate logistic regression analyses revealed that gamma radiation-induced b/c values greater than the median of the controls were a significant risk factor for salivary gland tumors (adjusted odds ratio [OR], 17.25; 95% confidence interval [CI], 4.92-60.49). The risk remained significant for MSTs (adjusted OR, 40.45; 95% CI, 5.27-310.17) but was of borderline significance for BSTs (adjusted OR, 4.73; 95% CI, 0.94-23.87) when these tumors were analyzed separately. CONCLUSIONS: In the current study, high levels of chromatid breaks in lymphocytes induced by gamma irradiation were associated with an independent risk for MSTs and were likely to increase the risk of BSTs. However, larger studies are needed to verify these findings.     

Bleomycin-induced chromosome breaks as a risk marker for lung cancer: a case-control study with population and hospital controls

Environmental exposure to carcinogens and individual susceptibility play significant roles in cancer risk. Suboptimal DNA repair capability, measured by quantifying mutagen-induced chromosome breaks, might explain variable host susceptibility to environmental carcinogens. In an ongoing lung cancer case-control study, we compared individual sensitivity to bleomycin-induced chromosome breaks in 152 non-small cell lung cancer patients with 94 population controls and 85 hospital controls with no history of cancer. Mutagen sensitivity was measured by mean number of chromatid breaks per cell in cultured peripheral blood lymphocytes treated with bleomycin. Non-parametric tests and chi(2) tests were used to determine the statistical significance of the crude case-control comparisons, followed by logistic regression to adjust for important covariates. The mean number of bleomycin-induced breaks per cell was 1.01 for the cases compared with 0.86 for hospital controls (P < 0.01) and 0.89 for population controls (P < 0.01). The mean number of breaks per cell was 1.01 for those >65 years old and 0.81 for those < or = 65 years old (P < 0.01) among population controls. Defining bleomycin sensitive as >0.84 break/cell (the median level in population controls), 67% of the cases were bleomycin sensitive compared with 49% of the hospital controls [adjusted odds ratio (OR) = 2.69, 95% confidence interval (CI) = 1.44, 5.04], and 51% of the population controls (adjusted OR = 2.18, 95% CI = 1.13, 4.21). Our data indicate that the increased number of bleomycin-induced chromosome breaks was significantly associated with an increased risk of lung cancer in the first 331 subjects.     

Chromosome instability in lymphocytes: a potential indicator of predisposition to oral premalignant lesions

Oral premalignant lesions (OPLs) are related to tobacco use and mark individuals at high risk for oral cancer development. Increased mutagen sensitivity as measured by an in vitro mutagen challenge assay has been shown to be a risk factor for upper aerodigestive tract cancers. In this case control study, we used two assays with mutagens relevant to tobacco exposure (benzo[a]pyrene diol epoxide (BPDE) and bleomycin) to see whether sensitivity to these mutagens could be used as biomarkers for assessing risk of premalignant lesions. Furthermore, we evaluated whether 3p21.3 is a molecular target of BPDE damage in lymphocytes of patients with OPLs. There were 82 patients with OPLs and 89 healthy controls frequency matched to the cases on age, sex, ethnicity, and smoking status. These subjects' lymphocytes were treated in two separate experiments with either 2 microM BPDE for 24 h or 0.03 units/ml bleomycin for 5 h, and the frequency of induced chromatid breakage in Giemsa-stained preparations was determined. BPDE-induced 3p21.3 aberrations were scored by fluorescent in situ hybridization technique in 1000 interphases/sample. We found that the mean BPDE-induced chromatid breaks per cell were higher in cases than controls (1.05 +/- 0.40 and 0.55 +/- 0.27, respectively; P < 0.01). Similar results were evident with bleomycin-induced chromatid breaks per cell (0.78 +/- 0.37 and 0.57 +/- 0.31, respectively; P < 0.01). After adjusting for age, sex, ethnicity, and smoking status, significantly elevated odds ratios (95% confidence interval) for OPL risk were noted for BPDE sensitivity [12.96 (5.51, 30.46)] and bleomycin sensitivity [3.33 (1.64, 6.77)]. When subjects were categorized into quartiles of the number of breaks per cell, a dose response was observed for both assays. The adjusted odds ratios for subjects with increasing numbers of breaks per cell in quartiles were 2.34, 9.14, and 54.04 for BPDE sensitivity and 1.92, 3.33, and 7.15 for bleomycin sensitivity, respectively. Subjects sensitive to both mutagens had a 50-fold increased risk for OPLs. In addition, there were significantly more BPDE-induced chromosome aberrations at the 3p21.3 locus in cases (51.13/1000) than in controls (40.93/1000; P < 0.0001). However, no such difference was observed for 3q13, a control locus. BPDE-induced 3p21.3 aberrations were associated with an elevated risk for OPLs of 6.08 (2.57, 14.4). The degree of BPDE sensitivity at 3p21.3 and risk for OPLs increased in a dose-dependent manner. In summary, BDPE sensitivity and bleomycin sensitivity appear to be individually and jointly associated with elevated risk of OPLs. Furthermore, 3p21.3 may be a molecular target of BPDE in OPLs. This is the first study to examine mutagen sensitivity in a premalignant condition. The next step is to correlate these findings in surrogate (lymphocyte) tissue with molecular events in the target tissue.     

Deletion in poly(ADP-ribose)polymerase pseudogene and lung cancer risk

The poly(ADP-ribose)polymerase (PADPRP) gene has been implicated in carcinogenesis through its role in DNA repair, replication and recombination. A two-allele polymorphism in the chromosome 13 PADPRP pseudogene has been studied in several racial groups. It has been suggested that the B allele, which results from a 193-bp deletion in the gene, predisposes to myeloma in Blacks. We assessed the association between chromosome 13 PADPRP pseudogene genotype, mutagen sensitivity (a marker reflecting host DNA repair capability), cigarette smoking, and lung cancer risk in a minority lung cancer case-control study. The chromosome 13 PADPRP pseudogene polymorphism was detected by polymerase chain reaction-based analysis. Mutagen sensitivity was measured by an in vitro assay that quantified bleomycin-induced chromatid breaks in peripheral blood lymphocyte cultures. We examined 121 cases (80 African- Americans and 41 Mexican-Americans) with previously untreated lung cancer and 171 matched controls. Our results suggested that the distribution of the PADPRP pseudogene genotype frequencies was significantly different among African-American and Mexican-American controls (P < 0.001). The susceptibility genotype (i.e. at least one B allele) was found in 82.5% of African-American cases, 79.4% of African- American controls, 53.7% of Mexican-American cases, and 32.4% of Mexican-American controls. The odds ratios (OR) and 95% confidence intervals for the PADPRP susceptibility genotypes were 2.3 (95% CI = 0.7-8.0) and 3.2 (95% CI = 1.0-10.3) for African-Americans and Mexican- Americans respectively, after adjustment by age, sex, pack-years and mutagen sensitivity. Patients with the susceptibility genotype appeared to have more mutagen-induced breaks than did patients with the other genotype. Only adenocarcinoma was significantly associated with the PADPRP susceptibility genotype (OR = 3.8). Mutagen sensitivity (> or = 1 break/cell) was significantly associated with lung cancer risk for both ethnic groups with increased ORs of above three-fold. On stratified analysis, synergistic interactions were noted for the PADPRP susceptibility genotype, mutagen sensitivity and smoking status. In Mexican-Americans, the ORs for PADPRP susceptibility genotype, mutagen sensitivity and both risk factors combined were 1.3, 2.7 and 17.1 respectively. The combined OR for the PADPRP susceptibility genotype and smoking status was 15.6. Therefore, this polymorphism appears to be associated with lung cancer risk. However, it is likely that no single genotype is sufficiently predictive of risk and that a panel of susceptibility markers is needed to define the high-risk subgroup.      

172G>T variant in the 5' untranslated region of DNA repair gene RAD51 reduces risk of squamous cell carcinoma of the head and neck and interacts with a P53 codon 72 variant

RAD51 participates in homologous recombination (HR) repair of double-stranded DNA breaks (DSBs) that may cause genomic instability and cancer. Two single-nucleotide polymorphisms (SNPs) and three P53 binding sites have been found in the RAD51 promoter and 5' untranslated region. We hypothesized that RAD51 and P53 SNPs may interact and alter risk of squamous cell carcinoma of the head and neck (SCCHN) and we genotyped for RAD51 135G>C and 172G>T and P53 Arg72Pro SNPs in 716 SCCHN patients and 719 matched controls (all non-Hispanic whites) and evaluated their effects on gamma radiation-induced mutagen sensitivity. We found that RAD51 172TT homozygotes had a significantly decreased risk [adjusted odds ratio (OR) = 0.66, 95% confidence interval (CI) = 0.50-0.87] of SCCHN, compared with carriers of other genotypes, particularly in P53 Arg72Arg homozygotes (adjusted OR = 0.60, 95% CI = 0.41-0.89) (homogeneity test P = 0.047), although no alterations in the risk were associated with the RAD51 135G>C and P53 Arg72Pro SNPs. Consistent with a protective effect of the 172TT genotype, significantly fewer gamma radiation-induced chromatid breaks per cell were present in 172TT homozygotes (mean +/- SD = 0.36 +/- 0.13) than in subjects with other genotypes (mean +/- SD = 0.46 +/- 0.13, P < 0.001) among 148 control subjects we tested. The finding that the functional RAD51 172G>T SNP, particularly in the presence of the P53 Arg72Arg genotype, may be a marker of susceptibility to SCCHN needs to be validated by larger studies of different ethnic populations.     

Inherited susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes.

BACKGROUND: Susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes may reflect the way a person deals with carcinogenic challenges. This susceptibility (also referred to as mutagen sensitivity) has been found to be increased in patients with environmentally related cancers, including cancers of the head and neck, lung, and colon, and, in combination with carcinogenic exposure, this susceptibility can greatly influence cancer risk. The purpose of this study was to assess the heritability of mutagen sensitivity. METHODS: Heritability was determined by use of a maximum likelihood method that employed the FISHER package of pedigree analysis. Bleomycin-induced breaks per cell values for 135 healthy volunteers without cancer were determined. These individuals were from 53 different pedigrees and included 25 monozygotic twin pairs (n = 50), 14 pairs of dizygotes (twin pairs and siblings, n = 28), and 14 families selected on the basis of a first-degree relative who was successfully treated for head and neck cancer and who had no sign of recurrence for at least 1 year. All data were analyzed simultaneously, and different models of familial resemblance were fitted to the data. All P values are two-sided. RESULTS: Our results showed no evidence for the influence of a shared family environment on bleomycin-induced chromatid breaks. Genetic influences, however, were statistically significant (P =. 036) and accounted for 75% of the total variance. CONCLUSIONS: The high heritability estimate of the susceptibility to bleomycin-induced chromatid breaks indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage--and thereby a susceptibility to cancer--may exist in the general population.     

Gamma-ray-induced mutagen sensitivity and risk of sporadic breast cancer in young women: a case–control study

Hypersensitivity to radiation exposure has been suggested to be a risk factor for the development of breast cancer. In this case–control study of 515 young women (≤55 years) with newly diagnosed sporadic breast cancer and 402 cancer-free controls, we examined the radiosensitivity as measured by the frequency of chromatid breaks induced by gamma-radiation exposure in the G2 phase of phytohemagglutinin-stimulated and short-term cultured fresh lymphocytes. We found that the average chromatid breaks per cell from 50 well-spread metaphases were statistically significantly higher in 403 non-Hispanic White breast cancer patients (0.52 ± 0.22) than that in 281 non-Hispanic White controls (0.44 ± 0.16) (P value < 0.001), and in 60 Mexican American breast cancer patients (0.52 ± 0.19) than that in 65 Mexican American controls (0.44 ± 0.16) (P value = 0.021), but the difference was not significant in African Americans (52 cases [0.45 ± 0.16] versus 56 controls [0.47 ± 0.16], P = 0.651). The frequency of chromatid breaks per cell above the median of control subjects was associated with two-fold increased risk for breast cancer in non-Hispanic Whites and Mexican Americans. A dose–response relationship was evident between radiosensitivity and risk for breast cancer (P _trend < 0.001) in these two ethnic groups. We concluded that gamma-ray-induced mutagen sensitivity may play a role in susceptibility to breast cancer in young non-Hispanic White and Mexican American women.     

Bleomycin-induced chromosome damage in lymphocytes indicates inefficient DNA repair capacity in breast cancer families

In vitro mutagen susceptibility has been observed as a predictor of cancer risk. To evaluate susceptibility to mutagen, we have studied the response to in vitro bleomycin (BLM) treatment in cultured peripheral blood lymphocytes (PBL) of 9 breast cancer families (BCFs). Eleven breast cancer patients (BCPs) and 36 healthy blood relatives (HBRs) from BCFs were included in the study. Data were compared with 22 healthy control women. The frequencies of chromosomal aberrations were evaluated after exposure to BLM in the last five hours. Mean frequency of BLM-induced chromosomal aberrations per cell (CA) observed among BCPs was significantly higher as compared to their HBRs as well as control subjects. Moreover, mean BLM-induced CA/cell value observed for HBRs was also significantly higher than that of control subjects. In comparison to controls, it was observed that there was four times more cancer risk in BCPs (OR=4.148, 95% CI=5.83-687.46) and 2.5 times more cancer risk in HBRs (OR=2.67, 95% CI=5.31-39.25). Lymphocytes from 90% of BCPs and 69% of HBRs were found to be sensitive to BLM (using a cutoff value = controls group mean + 1 SD). Thus, lymphocytes of BCPs and their HBRs were more sensitive to BLM exposure as compared to controls. Our finding indicated inefficient DNA repair capacity in BCFs. The HBRs in BCFs, having increased BLM-sensitivity, may be at higher risk to develop a similar cancer.     

Mutagen sensitivity and genetic variants in nucleotide excision repair pathway: genotype-phenotype correlation.

The rationale behind gene-disease association studies is that genetic variants (polymorphisms) result in alterations in intermediate phenotypes. However, genotype-phenotype correlations have not been established for most polymorphisms. In this study, we correlated genotype data of genes involved in the nucleotide excision repair pathway with mutagen sensitivity phenotype, quantified by benzo(a)pyrene diol epoxide (BPDE)-induced chromatid breaks in peripheral blood lymphocytes in 422 healthy subjects recruited into a twin study that included 138 pairs of monozygotic twins, 51 pairs of dizygotic twins, and 44 siblings. Among a panel of single nucleotide polymorphisms examined, we found that BPDE sensitivity was modified by individual polymorphisms in XPC, RAD23B, and XPA genes. Specific haplotypes and diplotypes of XPC also modified BPDE sensitivity profiles. In addition, a more consistent and stronger correlation was observed between mutagen sensitivity phenotype and the combination of multiple polymorphisms in the nucleotide excision repair pathway. Specifically, when XPC-PAT, XPC Lys939Gln, XPA A23G, and RAD23B Val249Ala were analyzed together, we observed a significant dose-response relationship between increasing mutagen sensitivity with increasing number of adverse alleles: mutagen sensitivity for those carrying zero to two, three to five, and six or more adverse alleles were 0.64, 0.68, and 1.06, respectively (P for trend = 0.008), and the results remained significant after adjusting for multiple comparisons. Using individuals carrying zero to two adverse alleles as the reference group, the risks of being mutagen sensitive (mutagen sensitivity values greater than the median) were 1.05 (95% confidence interval, 0.68-1.64) and 4.48 (95% confidence interval, 1.21-16.61) for those carrying three to five and six or more adverse alleles, respectively. Analyses of the effects of genotype combinations yielded similar results. These findings underscore the importance of assessing the collective effects of a panel of polymorphisms in the same pathway in modulating mutagen sensitivity. As risk assessment for cancer risk is moving toward a multigenic pathway-based approach, future genotype-phenotype correlation studies should also investigate the combined effects of multiple genetic variants.