Mutagen Sensitivity Research Articles

DNA Repair Defects and Other Mustakes in Drosophila melanogaster

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.

Three Measures of Mutagen Sensitivity in a Cancer-Free Population

Different individuals appear to respond differently to the same carcinogen, and different mutagens act differently on cells. We conducted mutagen sensitivity assays by using three mutagens (bleomycin, a radiomimetic agent; 4-nitroquinoline-1-oxide [4-NQO], an ultraviolet light mimetic agent; and benzo[a]pyrene diol epoxide [BPDE], a tobacco mutagen) in parallel in healthy human subjects to determine the relationships among these assays. Our results showed that the mean breaks per cell values (b /c) ( ±SD) for bleomycin, 4-NQO, and BPDE sensitivity were 0.49 ( ±0.26), 0.53 ( ±0.30), and 0.66 ( ±0.41), respectively. Age, sex, smoking status, and family history of cancer were not correlated with any of these mutagen sensitivities. Also, there was no correlation between bleomycin and 4-NQO or 4-NQO and BPDE sensitivity, but a weak correlation between bleomycin and BPDE was observed (correlation coefficient = 0.289; P = 0.001). When the 75th percentile of b /c was used as a cutoff point in each assay, only one individual (1.8%) was sensitive to all three mutagens. Ten individuals (17.9%) were sensitive to two mutagens, 20 (35.7%) to one mutagen, and 25 (44.6%) to none of three mutagens. Our study suggests that these three mutagens may involve different DNA damage and repair pathways. The lack of correlation between the assay results may indicate the necessity of using a battery of mutagen sensitivity tests to refine our ability to identify a subpopulation at high cancer risk.

DNA repair in lymphoblastoid cell lines from patients with head and neck cancer.

To analyze and compare components of the 3 primary DNA repair pathways of Epstein-Barr virus-transformed lymphocyte (lymphoblastoid) cell lines derived from 9 patients with squamous cell carcinoma of the head and neck and 11 cancer-free controls. These cell lines were previously characterized by using an established cytogenetic marker of cancer susceptibility (mutagen sensitivity assay). To evaluate nucleotide excision repair (NER), we measured the reactivation level of a tobacco carcinogen-damaged plasmid containing a bacterial reporter gene transfected into these cells. To assess mismatch repair (MMR) and recombinational repair, selected gene transcript levels were quantified by using a multiplex reverse transcriptase-polymerase chain reaction assay. The results of these DNA repair assays were correlated with the previously measured mutagen sensitivity values. The NER capacities of the 2 groups were similar: 25.1% (range, 14.3%-33.3%) for the patient cell lines and 26.0% (range, 9.4%-47.7%) for the control lines. Transcriptase levels for 6 MMR genes (hMSH3, hMSH2, hPMS2, GTBP, hMLH1 and hPMS1) did not differ in the 2 groups. Transcript levels for 4 of 6 recombinational repair genes (XRCC7, XRCC6, XRCC1, and RAD51) were higher in the patient cell lines, though this difference was significant only for XRCC7 (P = .003). The mutagen sensitivity values correlated with the NER capacity (P = .05) and the expression of XRCC4 (P = .01) and RAD51 (P = .06) genes. As revealed by the above-named assays, these lymphoblastoid cell lines derived from patients with head and neck cancer had minor alterations in DNA repair function. However, these differences in DNA repair do appear to affect the cytogenetic marker of cancer susceptibility, mutagen sensitivity.

A comparison of bleomycin-induced damage in lymphocytes and primary oral fibroblasts and keratinocytes in 30 subjects

The number of chromatid breaks in peripheral blood lymphocytes (PBL) after exposure to bleomycin in the S/G2 phase of the cell cycle (in the literature referred to as 'mutagen sensitivity') is associated with an increased risk of environmentally related cancers, including oral cancer. The aim of this study was to elucidate whether mutagen sensitivity measured in lymphocytes actually reflects chromosomal instability of normal cells in the areas in which tumors develop. Therefore, bleomycin-induced chromosomal damage in and growth inhibition of cultured oral fibroblasts and oral keratinocytes from 30 persons were compared with the standard mutagen sensitivity score in PBL. A correlation was found for the percentage of aberrant metaphases between PBL and oral fibroblasts but not for the number of breaks per cell. These data do not allow a conclusion to be drawn on the use of fibroblasts to study cancer risk. Within the fibroblasts it was found that a high number of breaks per cell was associated with less growth inhibition, indicative of damage-resistant growth. Oral keratinocytes were extremely sensitive to bleomycin, as indicated by a strong cell cycle block which resulted in a mitotic index too low to determine chromosomal breaks. Moreover, in the cell proliferation assay keratinocytes were found to be 100 times more sensitive as compared with fibroblasts. There was no correlation between bleomycin sensitivity of keratinocytes compared with fibroblasts from a single patient as measured by growth inhibition. This may be due to the strong influence of alcohol consumption by the subjects, which was found to increase the sensitivity of keratinocytes but not of PBL and fibroblasts. In conclusion, oral fibroblasts but not keratinocytes can be used to measure sensitivity for chromatid breaks. The apparent influence of environmental factors on keratinocytes makes them a useful source to study exposure characteristics but limits their application for the determination of genetic factors.

Mitomycin C induced chromosomal aberrations in young cancer patients

Mitomycin-C (MMC) induced Chromosomal aberration (CA) frequencies were studied in 48 h peripheral blood lymphocyte (PBL) cultures of untreated cancer patients of young age (maximum age 12 years, n=77). Control population ( n=71) consisted of age-matched group (maximum age 12 years, n=21); elder controls (minimum age 60 years, n=19) and healthy first degree relatives, i.e., parents or siblings of the pediatric cancer patients (mean age 24.3 years, n=31) as they share their genome and environment. Induced CA levels were found to be significantly higher among pediatric cancer patients as compared to control groups. The age-matched and elder control groups showed comparable CA levels. The first degree relatives controls showed higher induced CA levels as compared to pediatric and elder control groups. The present results indicate that there are different degrees of mutagen sensitivity prevailing in normal population. This may be responsible for differential cancer proneness. High degree of mutagen sensitivity in cancer patients may also be playing a major role in cancer onset at an early age.

Increased mutagen sensitivity in patients with head and neck cancer is less pronounced in patients with nasopharyngeal carcinoma.

Mutagen sensitivity tested with bleomycin sulfate can determine a susceptible phenotype, which is relevant only in organs and tissues that have direct contact with the external environment. Patients with head and neck cancers have more mutagen sensitivity than control subjects without cancer, and the hypersensitive phenotype has a risk for the development of a second primary cancer. Head and neck cancers, however, represent a heterogeneous group of neoplasm. The biological behavior of nasopharyngeal carcinoma (NPC) and other head and neck cancers differs. To evaluate the difference in mutagen sensitivity among patients without cancer, patients with NPC, patients with oral or oropharyngeal cancer (ORC), and patients with laryngeal or hypopharyngeal cancer (LHC). Peripheral blood was cultured at 37 degrees C, using 5% carbon dioxide, for 72 hours. After 67 hours of incubation, bleomycin in a concentration of 30 IU/L was added to induce chromatid breaks. The number of chromatid breaks per cell was scored in 50 metaphases of cultured lymphocytes and compared in the 4 groups. Patients with histologically proven squamous cell carcinoma of the mucosa of the upper digestive tract, which included 3 groups: patients with NPC, patients with ORC, and those with LHC. Control subjects were hospital inpatients with no tumor history. There were 35 patients in each group. The mean (+/-SD) number of breaks per cell in the control group and in the groups with NPC, ORC, and LHC were 0.80 (+/-0.32), 1.03 (+/-0.45), 1.30 (+/-0.44), and 1.35 (+/-0.46), respectively. All the cancer groups had significantly higher mean breaks per cell and a higher prevalence of hypersensitivity than the control group. Patients with NPC had a significantly lower mean number of breaks per cell than the group with ORC or that with LHC. Patients with NPC had less mutagen sensitivity than those with ORC or LHC. Our results support the clinical and epidemiological findings of a difference between NPC and other head and neck cancers. Environmental factors might play a less pronounced role in the carcinogenesis of NPC.

XRCC2 and XRCC3, New Human Rad51-Family Members, Promote Chromosome Stability and Protect against DNA Cross-Links and Other Damages

The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells.

Mutagen sensitivity as a predictor of tumor recurrence in patients with cancer of the upper aerodigestive tract.

Mutagen sensitivity as a predictor of tumor recurrence in patients with cancer of the upper aerodigestive tract.

Involvement of Brca2 in DNA Repair

Abnormalities precipitated by a targeted truncation in the murine gene Brca2 define its involvement in DNA repair. In culture, cells harboring truncated Brca2 exhibit a proliferative impediment that worsens with successive passages. Arrest in the G1 and G2/M phases is accompanied by elevated p53 and p21 expression. Increased sensitivity to genotoxic agents, particularly ultraviolet light and methylmethanesulfonate, shows that Brca2 function is essential for the ability to survive DNA damage. But checkpoint activation and apoptotic mechanisms are largely unaffected, thereby implicating Brca2 in repair. This is substantiated by the spontaneous accumulation of chromosomal abnormalities, including breaks and aberrant chromatid exchanges. These findings define a function of Brca2 in DNA repair, whose loss precipitates replicative failure, mutagen sensitivity, and genetic instability reminiscent of Bloom syndrome and Fanconi anemia.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells.

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Genetic susceptibility to head and neck cancer: interaction between nutrition and mutagen sensitivity.

The development of head and neck cancer may depend not only on exposure to environmental carcinogens but also on a genetically based susceptibility to carcinogen-induced damage. This thesis presents a case-control study that demonstrates the significance of mutagen sensitivity, a measure of an individual's intrinsic DNA repair capacity against free radical damage, as a risk factor for the disease. As part of the case-control analysis, 167 previously untreated patients and 177 age- and sex-matched healthy controls were assessed for various lifestyle factors including tobacco and alcohol habits, occupational exposures, and diet. Mutagen sensitivity expressed by each individual was determined by quantifying bleomycin-induced chromosomal breaks within peripheral blood lymphocytes in vitro. Consistent with our initial observations and those of others, mutagen hypersensitivity was strongly associated with increased risk of head and neck cancer (odds ratio, 4.95; 95% confidence interval, 2.67 to 9.17) after adjusting for age, sex, and race. Low intake of vitamins C and E was also associated with an increased risk of disease and was interactive with mutagen sensitivity in risk estimates. Individuals with both a low intake of various antioxidants and increased chromosomal sensitivity to oxidant-induced DNA damage were at greatest risk. This study supports the concept that the risk of head and neck cancer is determined by a balance of factors that either enhance or protect against free radical oxygen damage, including innate capacities for DNA repair.

Associations between cytochrome P4502E1 genotype, mutagen sensitivity, cigarette smoking and susceptibility to lung cancer.

Cytochrome P4502E1 (CYP2E1) is involved in the metabolic activation of carcinogenic N-nitrosoamines. We therefore assessed the genotype frequencies of PstI or RsaI CYP2E1 restriction fragment length polymorphisms and another susceptibility marker, mutagen sensitivity, in 137 lung cancer cases (92 African American and 45 Mexican American) and 206 controls (114 African American and 92 Mexican American) identified in a molecular epidemiological study of lung cancer. The CYP2E1 c1/c1 genotype was found in 86.7% of Mexican American cases, 70.6% of Mexican American controls, 89.1% of African American cases and 86.8% of African American controls. By multivariate analysis, this genotype was found to be associated with a 14.0-fold increased risk of lung cancer in Mexican Americans but not in African Americans; a 9.9-fold increased risk of lung cancer in Mexican American former smokers, but not in non-smokers or current smokers; a 15-fold increased risk of lung cancer in Mexican American males, but not in females. Patients with the susceptible genotype appeared to have developed cancer at an earlier age and with lower cigarette pack-year of exposure than did patients with the c1/c2 or c2/c2 genotypes. Stratified analysis suggested a greater than multiplicative interaction between cigarette smoking and CYP2E1 c1/c1 genotype, although not statistically significant. The odds ratios (ORs) for the CYP2E1 c1/c1 genotype, cigarette smoking and both risk factors combined were 1.3, 6.7 and 16.3, respectively. The association between CYP2E1 c1/c1 genotype and pack-years of smoking followed the same pattern. The interaction between mutagen sensitivity and CYP2E1 c1/c1 genotype was especially strong in former smokers (the ORs for the CYP2E1 c1/c1 genotype, mutagen sensitivity and both risk factors combined were 3.9, 5.4 and 23.0, respectively). Therefore, the data suggest that individuals who lack a c2 allele might be at higher risk for developing lung cancer.

Chromosome instability syndromes: lessons for carcinogenesis.

The ability to maintain genomic integrity in the face of DNA damage is critical for survival. Biological organisms are not merely passive targets of DNA-damaging agents but actively respond to DNA damage in a variety of ways, e.g., the SOS system in Escherichia coli (Battista et al. 1990). The means by which cells achieve this goal are complex and involve DNA repair, genetic recombination, alterations in the cell cycle, and programmed cell death. This article discusses the chromosome instability syndromes, human diseases in which these homeostatic processes have broken down, resulting in cancer, immunodeficiency, growth failure, neurologic abnormalities, and mutagen sensitivity. Genetic instability in these syndromes may result from changes in DNA topology, loss of cell cycle checkpoint control, or dysregulation of lymphokine-mediated signal-transduction pathways, illustrating the diverse nature of the cellular processes that can affect the integrity of the genome.

Survival, mutagenesis, and host cell reactivation in a Chinese hamster ovary cell ERCC1 knock-out mutant.

Positive selection-negative selection gene targeting was used to disrupt the nucleotide excision repair gene ERCC1 in a Chinese hamster ovary cell line, CHO-K1. Southern and Northern analysis showed that a cell clone isolated by this targeting approach, CHO-7-27, had an ERCC1 gene structure consistent with targeted disruption of ERCC1 exon V, and did not express ERCC1 mRNA. CHO-7-27 was further characterized with respect to UV and mitomycin C sensitivities, and was shown to exhibit severe mutagen sensitivity phenotypes consistent with those of other CHO cell ERCC1 mutants. Mutation frequency experiments showed that CHO-7-27 was UV-hypermutable at the hypoxanthine-guanine phosphoribosyltransferase locus. Experiments assessing host cell reactivation of viral DNA synthesis for UV-irradiated adenovirus showed that CHO7-27 exhibited a severely deficient HCR phenotype similar to that of UV20 cells. Our results demonstrate that CHOK1 cells are hemizygous for the ERCC1 gene, and show that the comparatively mild mutagen sensitivities and lack of severely deficient HCR phenotypes of conventionally derived CHO-K1 ERCC1 mutants, in contrast to the severe phenotypes of CHO-AA8-derived mutants, are not due to any intrinsic genetic differences between CHO-K1 and CHO-AA8 parental cell lines.

Plant-activation of the bicyclic aromatic amines benzidine and 4-aminobiphenyl

Benzidine and 4-aminobiphenyl (4-ABP) are promutagenic bicyclic aromatic amines that are activated into frameshift and base pair substitution mutagens by plant systems. Using the plant cell/microbe coincubation assay, plant-activated benzidine from 0 to 50 microM induced a concentration-response in Salmonella typhimurium. At concentrations above 5 microM, plant-activated benzidine induced frameshift and base pair substitution mutations in the N- or O-acetyltransferase over-expressing strains, DJ460, YG1024, and YG1029. With plant-activated 4-ABP, concentrations above 250 microM induced a significant mutagenic response in strains YG1024 and YG1029. A tobacco cell-free mixture, TX1MX, activated benzidine and 4-ABP into mutagenic metabolites in S. typhimurium strains YG1024, YG1029, and DJ460. The mutagenic sensitivities of plant-activated benzidine and 4-ABP were the same with two different types of plant activation systems, TX1 suspension cells and TX1MX cell-free medium. The plant activation of these aromatic amines is mediated by tobacco cell peroxidase. Plant-activated benzidine and 4-ABP are converted into intermediates that serve as substrates for bacterial or humanacetylCoA: N-hydroxyarylamine N-acetyltransferase to generate the ultimate mutagenic products.

Positional cloning of the Drosophila melanogaster mei-9 gene, the putative homolog of the Saccharomyces cerevisiae RAD1 gene

Mutation of the Drosophila melanogaster mei-9 gene causes enhanced mutagen sensitivity, DNA excision repair incision deficiency, germ line hypermutability, mitotic chromosome breakage and instability, and, in females, increased levels of meiotic non-disjunction and decreased levels of meiotic recombination. This paper reports on the utilization of a positional mapping strategy combined with a differential expression assay to clone the mei-9 gene. We report that mei-9 is homologous to the yeast repair gene, RAD1, and demonstrate that the mei-9 gene transcript is absent in the mei-9AT3 mutant.

The polymorphism of GSTM1, mutagen sensitivity in colon cancer and healthy control

The aim of this article is to study the relationship between GSTM1 polymorphism and colon cancer and to compare the chromosomal breakage induced by mutagen in a colon cancer group and healthy controls. Using PCR to identify the GSTM1 genotype, we found the frequency of GSTM1 −1 in colon cancer ( n = 19) and control group ( n = 23) was 36.8% and 26.1%, respectively ( p > 0.05, X 2-test). The bleomycin-induced chromosomal breakage (break/cell) in the patient group was 0.75 ±0.29, and in the control group 0.42 ±(0.24) ( p < 0.05, t-test). The percentage of mutagen sensitivity (b/c > 0.8) in the patient group (68%) was 4 times as high as that in the control group (20%). The mutagen hypersensitivity (b/c > 1.0) in the patient group (47%) was 5 times as high as that in the control group (12%). The odds radio was 6.6.

Mutagen sensitivity: Enhanced risk assessment of squamous cell carcinoma

Mutagen sensitivity: Enhanced risk assessment of squamous cell carcinoma

Mutagen sensitivity as a predisposing factor in familial oral cancer

Pedigree analysis of the oral cancer (OC) patients registered at our Centre had disclosed familial aggregation of oral cancer which hitherto has not been largely reported. There is a paucity of information on the genetic determinism for familial oral cancer predisposition. Therefore, we investigated constitutional chromosome abnormalities and bleomycin-induced chromosome sensitivity of 7 familial and 10 sporadic oral cancer patients and 14 unaffected family members (first-degree relatives) to determine whether these factors could give any clues regarding cancer-predisposing factors. Neither the oral cancer patients nor the unaffected family members showed any constitutional chromosomal abnormalities. However, with regard to bleomycin sensitivity, there was significant difference between the oral-cancer patients and unaffected relatives. The mean b/c value was 1.68 ± 0.48 for familial OC patients, 1.12 ± 0.36 for sporadic OC patients and 0.52 ± 0.18 for the unaffected family members (p < 0.001). A noteworthy observation was that one unaffected family member also showed bleomycin hypersensitivity and expressed a mean b/c value of 1.32, at the initiation of the study. That patient later developed oral carcinoma. This clearly demonstrates that mutagen hypersensitivity among unaffected relatives in OC families may be related to cancer predisposition. The mutagen sensitivity study is being continued in a larger series of subjects, for the development of a cytogenetic marker for prediction of cancer susceptibility. © 1996 Wiley-Liss, Inc.

Mutagen sensitivity as a predisposing factor in familial oral cancer.

Pedigree analysis of the oral cancer (OC) patients registered at our Centre had disclosed familial aggregation of oral cancer which hitherto has not been largely reported. There is a paucity of information on the genetic determinism for familial oral cancer predisposition. Therefore, we investigated constitutional chromosome abnormalities and bleomycin-induced chromosome sensitivity of 7 familial and 10 sporadic oral cancer patients and 14 unaffected family members (first-degree relatives) to determine whether these factors could give any clues regarding cancer-predisposing factors. Neither the oral cancer patients nor the unaffected family members showed any constitutional chromosomal abnormalities. However, with regard to bleomycin sensitivity, there was significant difference between the oral-cancer patients and unaffected relatives. The mean b/c value was 1.68 +/- 0.48 for familial OC patients, 1.12 +/- 0.36 for sporadic OC patients and 0.52 +/- 0.18 for the unaffected family members (p < 0.001). A noteworthy observation was that one unaffected family member also showed bleomycin hypersensitivity and expressed a mean b/c value of 1.32, at the initiation of the study. That patient later developed oral carcinoma. This clearly demonstrates that mutagen hypersensitivity among unaffected relatives in OC families may be related to cancer predisposition. The mutagen sensitivity study is being continued in a larger series of subjects, for the development of a cytogenetic marker for prediction of cancer susceptibility.