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Chemico-biological Interactions Mar 1986Quantitation of 7-methyldeoxyguanosine (m7dG) produced in the in vitro methyl methanesulfonate (MeMS) action on calf-thymus DNA is achieved by enzymatic degradation,...
Quantitation of 7-methyldeoxyguanosine (m7dG) produced in the in vitro methyl methanesulfonate (MeMS) action on calf-thymus DNA is achieved by enzymatic degradation, liquid chromatographic separation and chemical ionization mass spectrometry. The total degree of methylation, measured by uptake of [14C]MeMS was 0.35%. Mass spectral analysis shows that m7dG constitutes 84% of the total methylated product. It is also shown that tandem mass spectrometry allows detection of m7dG, as the protonated base, down to 1 pmol level, suggesting that MS/MS analysis can be the method of choice in quantitation of the adducts of in vivo DNA modifications.
Topics: Animals; Cattle; DNA; Deoxyguanosine; Mass Spectrometry; Methyl Methanesulfonate; Thymus Gland
PubMed: 3698118
DOI: 10.1016/0009-2797(86)90004-9 -
Mutation Research Dec 2007The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central...
Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate.
The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O(6)-methyl-2'-deoxyguanosine (O(6)-mdGuo) by LC-MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10(9) nucleotides while O(6)-mdGuo was almost 50-fold lower (32 adducts per 10(9) nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the "doubling dose", i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7microM MMS were deduced for 7-mG and O(6)-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66microM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose-response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose-response relationships and for the setting of tolerable exposure levels are discussed.
Topics: Animals; Cell Line, Tumor; Chromatography, Liquid; DNA Adducts; DNA Breaks; DNA Methylation; Deoxyguanosine; Dose-Response Relationship, Drug; Guanine; Leukemia L5178; Methyl Methanesulfonate; Mice; Models, Biological; Mutagens; Tandem Mass Spectrometry
PubMed: 17586535
DOI: 10.1016/j.mrfmmm.2007.05.007 -
Toxicology and Applied Pharmacology May 1997The cogenotoxicity of Cd has been recognized. This effect may stem from Cd inhibition of DNA repair. We studied the effects of Cd on DNA repair of methyl...
The cogenotoxicity of Cd has been recognized. This effect may stem from Cd inhibition of DNA repair. We studied the effects of Cd on DNA repair of methyl methanesulfonate (MMS)-damaged Chinese hamster ovary cells (CHO-K1) by single-cell alkaline electrophoresis. The results indicate that in the presence of Cd, DNA strand breaks accumulated in MMS-treated cells. Using hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) to block DNA polymerization, DNA strand breaks accumulated and Cd had little inhibitory effects on these accumulations. However, Cd inhibited the rejoining of these DNA strand breaks, which could be rejoined 6 hr after release from Hu plus AraC blockage. These results indicate that the potency of Cd inhibition of DNA repair replication and/or ligation may be greater than the inhibition of DNA adduct excision. To further elucidate this mechanism, we used an in vitro cell-free assay system to analyze the Cd effects on DNA repair synthesis, DNA polymerization, and DNA ligation. We have shown a dose-dependent inhibition of these three activities by Cd in CHO-K1 cell extract. The IC50s of Cd were 55, 26, and 10 microM, respectively. Moreover, Cd inhibition of DNA ligation in cell extract could be recovered partially by thiol compounds such as glutathione, beta-mercaptoethanol, dithiothreitol, and metallothionein. Since both in vivo and in vitro studies demonstrated that Cd was more effectively involved in interfering with the DNA ligation step and that thiol agents could partially remove Cd inhibition of DNA ligation, we speculate that part of the Cd inhibition of DNA repair may be through binding of Cd to the proteins participating in DNA ligation.
Topics: Animals; CHO Cells; Cadmium; Cell Extracts; Cricetinae; DNA; DNA Damage; DNA Repair; Drug Interactions; Methyl Methanesulfonate
PubMed: 9169081
DOI: 10.1006/taap.1997.8116 -
Mutation Research Oct 1997The suppressive effect of S-methyl methanethiosulfonate (MMTS) on aflatoxin B1 (AFB1)- or methyl methanesulfonate (MMS)-induced chromosome aberrations (CA) in rat bone...
The suppressive effect of S-methyl methanethiosulfonate (MMTS) on aflatoxin B1 (AFB1)- or methyl methanesulfonate (MMS)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. MMTS significantly suppressed CA induced by both AFB1 (an indirect-acting carcinogen) and MMS (a direct-acting carcinogen). Suppression was observed at all periods (6, 12, 18, 24 and 48 h) after AFB1 or MMS treatment and in all doses of AFB1 (5, 10 and 20 mg/kg) or MMS (50, 75 and 100 mg/kg) investigated. AFB1-induced CA was potently suppressed by MMTS given between 2 h before and 6 h after the AFB1 injection. The suppression of AFB1-induced CA by MMTS paralleled the dose of MMTS when MMTS was given in a dose range of 1-20 mg/kg body weight. MMS-induced CA was potently suppressed by MMTS given between 2 h before and 2 h after the MMS injection. The suppressive effect of MMTS on MMS-induced CA paralleled the dose of MMTS when MMTS was given in a dose range of 1-15 mg/kg body weight. Diphenyl disulfide, which modifies -SH groups in proteins like MMTS, also significantly suppressed both AFB1- and MMS-induced CA. Although other mechanisms are not excluded, the suppression of carcinogen-induced CA by MMTS may result from the ability of MMTS to modify -SH groups in proteins. The juices of cabbage and onion, which contain considerable amounts of MMTS and S-methyl-L-cysteinesulfoxide (the precursor of MMTS), also significantly suppressed AFB1- or MMS-induced CA. These results suggest that MMTS is a possible chemopreventive agent against cancer.
Topics: Aflatoxin B1; Animals; Antimutagenic Agents; Bone Marrow Cells; Brassica; Chromosome Aberrations; Disulfides; Male; Methyl Methanesulfonate; Mutagens; Onions; Plant Extracts; Rats; Rats, Wistar
PubMed: 9393623
DOI: 10.1016/s1383-5718(97)00116-2 -
Journal of Separation Science Sep 2016Methyl methanesulfonate and ethyl methanesulfonate are potential genotoxic impurities in imatinib mesylate. In this work, a simple, sensitive, reliable, and fast gas...
Methyl methanesulfonate and ethyl methanesulfonate are potential genotoxic impurities in imatinib mesylate. In this work, a simple, sensitive, reliable, and fast gas chromatography with mass spectrometry method for the simultaneous determination of methyl methanesulfonate and ethyl methanesulfonate was developed and validated. Total analysis time was only 7 min. An n-hexane/water solution was used to dissolve samples, and then extracted-ion-chromatogram mode was used to quantify methyl methanesulfonate and ethyl methanesulfonate. Calibration curves showed good linearity over the studied range for methyl methanesulfonate and ethyl methanesulfonate. The correlation coefficient of fit exceeded 0.999 for each impurity. The LOD and LOQ of methyl methanesulfonate and ethyl methanesulfonate were as low as 0.001 and 0.005 μg/mL, respectively, with RSDs of the peak area within 1.06-1.96%. Method accuracy was within 97.2-99.8% for methyl methanesulfonate and ethyl methanesulfonate. Therefore, this method can be used to quantify methyl methanesulfonate and ethyl methanesulfonate impurities at extremely low levels in imatinib mesylate.
Topics: Drug Contamination; Ethyl Methanesulfonate; Gas Chromatography-Mass Spectrometry; Imatinib Mesylate; Methyl Methanesulfonate; Mutagens
PubMed: 27461842
DOI: 10.1002/jssc.201600389 -
Molecular & General Genetics : MGG Sep 1979A mutant of Haemophilus influenzae which does not discriminate between low efficiency (LE) and high efficiency (HE) markers has been isolated. The mutant does not differ...
A mutant of Haemophilus influenzae which does not discriminate between low efficiency (LE) and high efficiency (HE) markers has been isolated. The mutant does not differ wild type in its sensitivity to ultraviolet radiation, methyl methanesulfonate (MMS) mitomycin C, and nitrous acid. Spontaneous mutation frequencies for three loci studied are 10- to 30-fold higher in the mutant than in the wild type strain. Low- and high-efficiency transforming markers are equally UV-resistant when assayed on this mutant. This mutant is thus similar to the hex mutant of Streptococcus pneumoniae.
Topics: DNA Repair; Genetic Markers; Haemophilus influenzae; Methyl Methanesulfonate; Mitomycins; Mutation; Nitrous Acid; Phenotype; Transformation, Bacterial; Ultraviolet Rays
PubMed: 316097
DOI: 10.1007/BF00425533 -
Mutation Research Oct 1977Lethality induced in larval populations of Drosophila melanogaster was recorded after treatment with (1) caffeine, (2) MMS or (3) caffeine plus MMS. The mixture of...
Lethality induced in larval populations of Drosophila melanogaster was recorded after treatment with (1) caffeine, (2) MMS or (3) caffeine plus MMS. The mixture of caffeine plus MMS was less toxic than expected from the effects observed after treatment with either substance individually. It is postulated that in the combined treatment the caffeine, by inhibiting semiconservative DNA replication, allows for some additional time for repair of alkylated DNA by a repair pathway which is not sensitive to caffeine, possibly excision repair.
Topics: Animals; Caffeine; DNA Repair; DNA Replication; Drosophila melanogaster; Genes, Lethal; Larva; Mesylates; Methyl Methanesulfonate; Mutagens; Mutation
PubMed: 199836
DOI: 10.1016/0027-5107(77)90041-0 -
Mutation Research Aug 2010A new, high-throughput version of the comet assay was developed using human fibroblasts (Stang and Witte, 2009). The present study examines the suitability of other...
A new, high-throughput version of the comet assay was developed using human fibroblasts (Stang and Witte, 2009). The present study examines the suitability of other adherent and non-adherent cell types in this high-throughput assay. We found that in addition to V79 human fibroblasts, HeLa cells, Hep-G2 cells, and lymphocytes can be used. The time intervals needed for attachment on the agarose-coated 96-well multi-chamber plate (MCP, specially developed for the high-throughput comet assay) differed for all adherent cell lines mentioned. V79 cells needed 6h for attachment, fibroblasts 2-4h, Hep-G2 required 18 h, and HeLa cells 16 h. After this period, chemical treatment could occur. Non-adherent lymphocytes could be treated with the chemicals directly after they had been pipetted into the wells of the MCP and centrifuged. We compared the sensitivities of these five cell types toward the directly DNA-damaging compounds methyl methanesulfonate (MMS), and hydrogen peroxide (H(2)O(2)), and toward the indirectly acting agent pentachlorophenol (PCP). Except for Hep-G2 cells, exposure to PCP was conducted in the presence of an S9 microsome fraction. DNA damage, measured as an increase in the percentage of DNA in the tail region of the comets, occurred in a concentration-dependent manner. Under the test conditions used in this study, human lymphocytes were the most sensitive cells toward the three chemicals tested, fibroblasts showed a similar sensitivity toward the directly acting MMS and H(2)O(2), but were less sensitive toward PCP. HeLa, V79, and Hep-G2 cells reacted with similar sensitivity.
Topics: Animals; Cell Line; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Fibroblasts; Humans; Hydrogen Peroxide; Lymphocytes; Methyl Methanesulfonate; Mutagens; Pentachlorophenol
PubMed: 20399888
DOI: 10.1016/j.mrgentox.2010.04.011 -
Mutation Research Sep 1981Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for...
Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine.
Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16h. DNA synthesis peaked 20 h after removal of nutrient stress and declined. Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.
Topics: Acetoxyacetylaminofluorene; Cell Cycle; Cell Line; Cytological Techniques; Dose-Response Relationship, Drug; Drug Resistance; Humans; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mutation; Skin; Thioguanine
PubMed: 7029258
DOI: 10.1016/0027-5107(81)90009-9 -
Mutation Research Mar 1983
Topics: Caffeine; DNA; DNA Repair; Drug Synergism; Male; Methyl Methanesulfonate; Methylation; Spermatids
PubMed: 6682172
DOI: 10.1016/0027-5107(83)90131-8