Mutation Research/Genetic Toxicology最新文献

Among the main DNA-reactive metabolites of 1,3-butadiene (BD), both 1,2:3,4-butadiene diepoxide (BDE) and 1,2-epoxy-3-butene (BME) have been reported in mice and rats exposed to BD, but blood and tissue levels of these metabolites are much higher in mice than in rats under similar exposure conditions. BDE, being more reactive and genotoxic than BME, is thought to be responsible for the greater susceptibility of mice to BD carcinogenicity. While BDE is a DNA-alkylating agent and some BDE adducts have been characterized, no sufficiently sensitive methods has been reported for studying BDE-DNA binding in vivo. In the present investigation, a modified dinucleotide/monophosphate version of the ³²P-postlabeling assay was applied to detect BDE-DNA adducts, which were prepared by reacting BDE with calf thymus DNA or deoxyribooligonucleotides [(AC)₁₀, (AG)₁₀, (CCT)₇ and (GGT)₇] in vitro or with skin DNA of mice in vivo upon topical treatment. Optimal resolution by 2-D PEI-cellulose TLC of the highly polar 5′-monophosphate adducts was achieved at +4°C using 0.3 M LiCl (D1) and 0.4 M NaCl, 0.04 M H₃BO₃, pH 7.6 (D2). The profiles of the ³²P-postlabeled adducts were similar for calf thymus and skin DNA, with 3 major spots being detected. Adducts obtained in in vitro and in vivo experiments were compared by re- and cochromatography in 4 or 5 different solvents, and these experiments provided evidence that corresponding BDE adducts, for the most part, were identical and represented adenine derivatives. Guanine adducts were not detected by this method although literature data indicate their formation. Quantitatively, the assay responded linearly to adduct concentration, as shown in an experiment where BDE-modified skin DNA was serially diluted up to 81-fold with control DNA. The limit of detection was approximately 1 adduct in 10⁸ normal nucleotides. Further, in an in vivo dosimetry study, skin DNA from groups of 8 individual mice treated with different doses of BDE (1.9, 5.7, 17, 51 and 153 μmol/mouse) for 3 days exhibited a linear relationship (r ≥ 0.992) between adduct levels and dose. The results suggest that the ³²P-postlabeling assay described herein will have utility in mechanistic studies and biomonitoring of DNA adduct formation from BDE and possibly other polar epoxides.

Dinitrochlorobenzene (DNCB) is clinically efficacious in the therapy of alopecia areata, but its use was limited when it was found to be mutagenic in the Ames test. However, there has been renewed interest in the immunomodulatory benefits of topically applied dinitrochlorobenzene in patients with human immunodeficiency virus and systemic lupus erythematosus. The current study examines the genotoxicity of dinitrochlorobenzene in human skin fibroblasts using sister chromatid exchange. Dinitrochlorobenzene caused a significant increase in sister chromatid exchange at concentrations ranging from 2.5 to 10 μM. Thus, dinitrochlorobenzene is genotoxic in human skin fibroblasts at concentrations well below those used clinically. The potential for long-term toxicity from dinitrochlorobenzene will have to be weighed against the severity and prognosis of the diseases for which it is used.

Workers in the open pit uranium mine in Namibia appear to suffer from health problems including malignant diseases at a much higher prevalence when compared with the general population. The objective of the present study was to determine whether long-term exposure to low-dose uranium increases the risk of biological radiation damage which could lead to malignant diseases. In order to investigate this risk, we measured the relative frequency of chromosome alterations using Fluorescence in situ hybridization (FISH). A representative cohort of 11 non-smoking miners, were compared to a control group of 9 individuals with no occupational history in mining. We determined a significant increase in chromosome aberrations in the circulating lymphocytes of miners versus the non-smoking controls (p = 0.0000096). Therefore, we concluded that these uranium exposed miners are at an increased risk to acquire genetic damage, which may be associated with an increased risk for malignant transformation.

Highly mutagenic water of the Katsura River, Kyoto, and moderately mutagenic of the Asahi River, Okayama, were used to evaluate the efficacy of three concentration techniques, the blue-chitin column, the blue-rayon hanging, and the XAD-2 column. These two river waters have been shown to exhibit high mutagenicity in the assay with Salmonella typhimurium TA98 with metabolic activation. With this assay as a measure, two water samples from the Katsura, collected on different dates, and a sample from the Asahi were submitted to the column concentration techniques, blue-chitin and XAD-2. Blue-chitin was more efficient than XAD-2 for all of these samples: e.g., for one Katsura sample, the mutagenicity found was 913 ± 53 (mean ± SD, n = 3) revertants/500 ml with blue-chitin, and 419 ± 129 (n = 3)/500 ml with XAD-2. Blue rayon (0.5 g) hung in the Asahi for 24 h gave 563 ± 74 (n = 3) revertants, while the water spot-sampled at the start of the hanging showed 253 ± 10 (n = 3) revertants per 5 liter with the blue-chitin column technique. We conclude that for quantitative measurement of the ‘Salmonella TA98 + S9’ mutagens in these rivers, the blue-chitin column is more efficient and accurate than the XAD-2 column and that for judging the presence of mutagens, the blue-rayon hanging is the most sensitive and convenient among the three methods examined.

DNA damage results from a wide variety of external agents such as chemicals and radiation. The consequences of exposure to agents that damage DNA have been traditionally studied from the perspective of cell survival and mutagenesis. Mutations are late endpoints of DNA damage. Cells respond to the earlier stages of DNA damage by inducing the expression of several genes, including those specific to the nature of the lesion. These early transcriptional responses are likely to predetermine the later fate of the damaged cell. Genes activated during this early response include those involved in DNA repair, replication, and growth control. We are interested in the transcriptional mechanisms by which cells respond to DNA damaging agents. To facilitate the measurement of gene induction, we used seven different reporter constructs integrated stably into the RKO cell line derived from a human colon carcinoma. These constructs were derived from promoters and/or response elements isolated from genes associated with DNA damage responses in human cells, and were fused to the bacterial reporter gene, choramphenicol acetyl transferase (CAT). The cell lines generated in this manner contain the promoters and/or response elements representing DNA polymerase β, p53, gadd (growth arrest and DNA damage) 45 and 153, c-fos, TPA response element, and tissue-type plasminogen activator. These recombinant cell lines were assembled in a 96-well microtiter plate permitting their simultaneous exposure to compounds and subsequent CAT protein measurement. This assembly has been designated the CAT-Tox (D) assay. These cell lines were exposed to different classes of DNA damaging agents including those which covalently join bases to form dimers (e.g., UVC irradiation), generate DNA adducts by alkylation (e.g., methylmethane sulfonate [MMS], ethylmethane sulfonate [EMS], N-methyl-N-nitro-N-nitrosoguanine [MNNG], dimethylnitrosamine [DMN]), cross-link DNA (e.g., mitomycin C), and inhibit DNA replication by intercalative (e.g., actinomycin D) and noninterlalative (e.g., hydroxyurea) mechanisms. The transcriptional responses were measured as a function of the accumulation of CAT protein using antibodies against CAT protein in a standard ELISA. Endogenous cellular responses were evaluated for a number of the genes represented in the assay at both the mRNA and protein levels by Northern and Western blot analysis, respectively. These data corroborate the stress-induced responses measured by CAT ELISA in the CAT-Tox (D) assay, demonstrating the usefulness of this assay as a rapid and sensitive method for detection of DNA damaging agents in human cells.

When metabolically activated, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine isolated from cooked food, is clastogenic in cultured Chinese hamster and human cells. Secondary metabolites of PhIP are formed via acetyltransferase (AT) and sulfotransferase (ST) activity; however, which is responsible for its clastogenic effect is unknown. We addressed this question. We used a parental Chinese hamster lung cell line and three sublines transfected with different AT genes to test the clastogenic (i.e., micronucleus-inducing) effects of metabolically activated PhIP and 7,12-dimethylbenz[a]anthracene (DMBA) in the presence and absence of pentachlorophenol (PCP), a ST inhibitor. PhIP was significantly more clastogenic in the three AT-enriched sublines than in the parental line (p < 0.001). DMBA (a ST-activated mutagen), on the other hand, equally induced MNs in all the cell lines. When PCP was added to the test system, the MN-induction ability of DMBA, but not of PhIP, decreased significantly (p < 0.001). These findings strongly suggest that PhIP clastogenicity is due to AT activity and not to ST activity.

Using the Salmonella/microsome assay, the antimutagenic effects of specific components of the extracts from eggplant fruits were investigated. The eggplant fruit juice exhibited an antimutagenic activity against 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) induced mutagenicity. In some of the fractions extracted with several organic solvents (acetone, petroleum ether, ethyl acetate, and methanol), the activity was recognized. No mutagenicity or toxicity for Salmonella typhimurium TA98 in the presence of S9 mixture was observed with any of the extracts. It is suggested that there are multiple components of the activities that exist in the eggplant fruit. We isolated lutein from the 84% methanol (methanol/water, v/v) layer, pheophorbide or chlorophyllide from the 70% methanol layer and tannins containing sugar-moieties from the water layer. Pheophytin a and b, Mg-free derivatives of chlorophyll a and b, were isolated from the petroleum ether layer as possible antimutagens. The pheophytin a with S9 mix inhibited by 30–40% the mutagenicity of Trp-P-2.

The herbicide trifluralin was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and micronuclei (MN) were scored as genetic endpoints. To detect eventual metabolic modification in the genotoxicity of this herbicide, the cultures for SCE and MN demonstration were also treated with S9 fraction. From our results we can conclude that trifluralin was able to exert a weak cytotoxic effect, reducing both the proliferative rate index (PRI) and the cytokinesis block proliferation index (CBPI), and also to induce a slight but statistically significant increase in the frequency of SCE. Under our conditions of testing, no genotoxic effects of trifluralin were observed in the CA and MN assays.

The mutagenic activity of bile was compared between Chilean and Japanese female patients having cholelithiasis by the Ames assay using Salmonella typhimurium tester strain TA98 in the presence of S9 mix with blue rayon adsorption technique. A reason for conducting the present investigation is that Chile and Japan have the highest mortality rates for the gallbladder cancer (GBC) in the world. Of 24 bile samples collected in Chile, 20 (83.3%) samples showed mutagenicity. In the case of Japanese bile, 21 (80.8%) of 26 and 5 (19.2%) of 26 cases were mutagenic in samples from high- and low-risk areas for GBC, respectively. Therefore, both the Chilean and the Japanese samples collected in high-risk areas showed higher mutagenic rates than the Japanese ones in a low-risk area, with atatistical significance (p < 0.001, chi-square test). The average number of revertant colonies were 128 ± 92 (mean ± SD), 62 ± 14 and 66 ± 13, respectively, when the blue rayon extracts of 200 μl bile were applied to the Ames test. Thus, Chilean bile had a tendency to show a higher mutagenic activity than Japanese.