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Stem Cell Research Mar 2017Pluripotent cells have been reported to exhibit lower frequencies of point mutations and higher levels of DNA repair than differentiated cells. This predicts that...
Pluripotent cells have been reported to exhibit lower frequencies of point mutations and higher levels of DNA repair than differentiated cells. This predicts that pluripotent cells are less susceptible to mutagenic exposures than differentiated cells. To test this prediction, we used a lacI mutation-reporter transgene system to assess the frequency of point mutations in multiple lines of mouse pluripotent embryonic stem cells and induced pluripotent cells, as well as in multiple lines of differentiated fibroblast cells, before and after exposure to a moderate dose of the mutagen, methyl methanesulfonate. We also measured levels of key enzymes in the base excision repair (BER) pathway in each cell line before and after exposure to the mutagen. Our results confirm that pluripotent cells normally maintain lower frequencies of point mutations than differentiated cells, and show that differentiated cells exhibit a large increase in mutation frequency following a moderate mutagenic exposure, whereas pluripotent cells subjected to the same exposure show no increase in mutations. This result likely reflects the higher levels of BER proteins detectable in pluripotent cells prior to exposure and supports our thesis that maintenance of enhanced genetic integrity is a fundamental characteristic of pluripotent cells.
Topics: Animals; Cell Differentiation; Cell Line; DNA Damage; DNA Repair; Epigenomics; Methyl Methanesulfonate; Mice; Mice, Inbred C57BL; Mutagenesis; Stem Cells
PubMed: 28129601
DOI: 10.1016/j.scr.2016.12.029 -
Proceedings of the National Academy of... Dec 2002We performed a systematic screen of the set of approximately 5,000 viable Saccharomyces cerevisiae haploid gene deletion mutants and have identified 103 genes whose...
We performed a systematic screen of the set of approximately 5,000 viable Saccharomyces cerevisiae haploid gene deletion mutants and have identified 103 genes whose deletion causes sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). In total, 40 previously uncharacterized alkylation damage response genes were identified. Comparison with the set of genes known to be transcriptionally induced in response to MMS revealed surprisingly little overlap with those required for MMS resistance, indicating that transcriptional regulation plays little, if any, role in the response to MMS damage. Clustering of the MMS response genes on the basis of their cross-sensitivities to hydroxyurea, UV radiation, and ionizing radiation revealed a DNA damage core of genes required for responses to a broad range of DNA-damaging agents. Of particular significance, we identified a subset of genes that show a specific MMS response, displaying defects in S phase progression only in the presence of MMS. These genes may promote replication fork stability or processivity during encounters between replication forks and DNA damage.
Topics: DNA Damage; Gene Expression Profiling; Genome, Fungal; Hydroxyurea; Methyl Methanesulfonate; Mutation; S Phase; Saccharomyces cerevisiae
PubMed: 12482937
DOI: 10.1073/pnas.262669299 -
Carcinogenesis Dec 1997The effects of 2-butoxyethanol (2-BE) on poly(ADP-ribosyl)ation were studied in Syrian hamster embryo (SHE) cells by measuring the cellular concentrations of the polymer...
The effects of 2-butoxyethanol (2-BE) on poly(ADP-ribosyl)ation were studied in Syrian hamster embryo (SHE) cells by measuring the cellular concentrations of the polymer poly(ADP-ribose) (pADPr) and of NAD+, the substrate of poly(ADP-ribose) polymerase (PARP). As biotransformation pathways of ethylene glycol ethers involve NAD+-dehydrogenases, it was hypothesized that 2-BE could reduce poly(ADP-ribosyl)ation by consuming NAD+. As a result DNA repair could be altered, which would explain that 2-BE had been shown to potentiate the effects of clastogenic substances such as methyl-methanesulfonate (MMS). In this study, the effects of 2-BE on MMS-induced pADPr metabolism were analyzed. The results indicated that: (i) 2-BE (5 mM) by itself did not influence significantly pADPr or NAD+ levels. (ii) 2-BE inhibited pADPr synthesis in MMS (0.2 mM)-pretreated cells, without any change in NAD+ concentrations. (iii) MMS treatment, which rapidly increased pADPr levels, also affected the poly(ADP-ribosyl)ation system as a secondary effect by damaging cell structures. Membrane permeabilization, which occurred at concentrations >1 mM MMS, led to a dramatic leakage of cellular NAD+ resulting in a strong reduction in pADPr levels. (iv) A bleomycin pulse (100 microM) applied after MMS and/or 2-BE treatment confirmed that 2-BE reduced poly(ADP-ribosyl)ation capacities of MMS-treated cells, though the glycol ether had no effect alone. This study confirmed that the inhibition of pADPr synthesis could be responsible for the synergistic effects of 2-BE with genotoxic substances. The mechanism of this inhibition cannot be explained by a lack of NAD+ at the concentrations of 2-BE tested.
Topics: Animals; Cells, Cultured; Cricetinae; DNA Repair; Dose-Response Relationship, Drug; Ethylene Glycols; Mesocricetus; Methyl Methanesulfonate; NAD; Poly Adenosine Diphosphate Ribose; Time Factors
PubMed: 9450478
DOI: 10.1093/carcin/18.12.2333 -
Chemical Research in Toxicology May 2024The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion , but it is not believed to have a large direct physiological...
The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion , but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPC), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPC in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPC is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPC contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.
Topics: Humans; HeLa Cells; DNA; Deoxyguanosine; Methyl Methanesulfonate; Tandem Mass Spectrometry; Cell Survival; DNA Damage; Cross-Linking Reagents; DNA-Binding Proteins
PubMed: 38652696
DOI: 10.1021/acs.chemrestox.4c00076 -
Chemico-biological Interactions Jan 1981A study has been made of the unscheduled DNA synthesis (UDS) induced in early spermatid stages of the mouse by methyl nitrosourea (MNU), a methylating agent that reacts... (Comparative Study)
Comparative Study
A comparison of the molecular action of an SN1-type methylating agent, methyl nitrosourea and an SN2-type methylating agent, methyl methanesulfonate, in the germ cells of male mice.
A study has been made of the unscheduled DNA synthesis (UDS) induced in early spermatid stages of the mouse by methyl nitrosourea (MNU), a methylating agent that reacts predominantly by an SN1 type mechanism. In comparison with methyl methanesulfonate (MMS), a methylating agent that reacts predominantly by an SN2 mechanism, MNU induced more UDS by a factor of about 1.4. This result was in line with chemical dosimetry studies carrie out with both chemicals, which showed that 4 h after treatment with MNU, testicular DNA was methylated about 1.5 times more than it was 4 h after treatment with MMS. The UDS response in the spermatids fell off rapidly in the first half-day after treatment with either MNU or MMS. However, from 0.5 to 3 days after treatment the UDS response decreased with a t1/2 of 2.4 days after MNU treatment, but 1.2 days after MMS treatment. Chemical dosimetry studies with 3H-labeled MNU and MMS showed that the pattern of methylation produced in the developing sperm was different for each chemical and was generally correlated with the corresponding pattern of induced dominant-lethal mutations. However, on the basis of equal sperm-head methylation, MNU is as much as 17 times more effective than MMS in producing dominant lethals. It is suggested that more methylation by MNU at the O-6 position of guanine or phosphate groups in DNA in the developing germ cells may account for MNU's greater effectiveness in inducing dominant lethals. Greater methylation of these sites by MNU than by MMS might also account for the differences observed in the UDS response of the spermatids to these chemicals.
Topics: Alkylating Agents; Animals; DNA; Male; Methyl Methanesulfonate; Methylation; Methylnitrosourea; Mice; Nitrosourea Compounds; Sperm Head; Spermatozoa; Time Factors
PubMed: 7460068
DOI: 10.1016/0009-2797(81)90045-4 -
Mutation Research Aug 1989The effects of 2 routes of administration, intraperitoneal injection (i.p.) and oral gavage (p.o.), in the micronucleus test were evaluated using methyl methanesulfonate... (Comparative Study)
Comparative Study
The effects of 2 routes of administration, intraperitoneal injection (i.p.) and oral gavage (p.o.), in the micronucleus test were evaluated using methyl methanesulfonate (MMS) and 2 strains of mice (MS/Ae and CD-1). A small-scale acute toxicity study and a pilot micronucleus experiment were carried out first. On the basis of the results obtained, a final micronucleus test was performed at doses of 20, 40, 80, and 160 mg/kg (i.p.) and 40, 80, 160, and 320 mg/kg (p.o.), with a 24-h sampling time. MMS induced micronucleated polychromatic erythrocytes (MNPCEs) in both routes in both mouse strains under the conditions used. At 40 and 80 mg/kg, MMS induced a higher number of MNPCEs by the i.p. route in both strains. A 160 mg/kg MMS dose induced higher numbers of MNPCEs by the p.o. route in MS/Ae mice. The route-related difference with MMS on the basis of mg/kg disappeared when the difference was determined on the basis of a ratio of the LD50. In practice, both i.p. and p.o. routes are acceptable as routes of administration in the micronucleus test using this chemical.
Topics: Administration, Oral; Animals; Injections, Intraperitoneal; Lethal Dose 50; Male; Methyl Methanesulfonate; Mice; Micronucleus Tests; Mutagens; Pilot Projects
PubMed: 2747723
DOI: 10.1016/0165-1218(89)90091-8 -
Bulletin of Experimental Biology and... Jan 2018The effect of usnic acid enantiomers on the genotoxic effects of dioxidine and methyl methanesulfonate was studied in vitro in human peripheral blood lymphocytes by the...
The effect of usnic acid enantiomers on the genotoxic effects of dioxidine and methyl methanesulfonate was studied in vitro in human peripheral blood lymphocytes by the DNA comet method. We found that usnic acid enantiomers in a concentration range of 0.01-1.00 μM demonstrated pronounced antigenotoxic activity and reduced DNA damage induced by genotoxicants by 37-70%. In the same concentration range, the test enantiomers reduced the level of atypical DNA comets (hedgehogs) induced by genotoxicants by 23-61%. The test compounds did not modulate the effects of genotoxicants in a concentration of 10 μM and potentiated them in a concentration of 100 μM. The modifying activity of usnic acid did not depend on spatial configuration and on the used model genotoxicant.
Topics: Benzofurans; Comet Assay; DNA Fragmentation; Hormesis; Humans; Leukocytes, Mononuclear; Lichens; Methyl Methanesulfonate; Mutagens; Protective Agents; Quinoxalines; Stereoisomerism
PubMed: 29308568
DOI: 10.1007/s10517-018-3979-z -
Planta Medica Dec 2012Styrax camporum Pohl is a tall shrub or a tree with small white flowers, which grows in the states of São Paulo and Minas Gerais and is popularly used for the treatment...
In vivo protective activity of Styrax camporum hydroalcoholic extract against genotoxicity induced by doxorubicin and methyl methanesulfonate in the micronucleus and comet assays.
Styrax camporum Pohl is a tall shrub or a tree with small white flowers, which grows in the states of São Paulo and Minas Gerais and is popularly used for the treatment of gastroduodenal diseases. Considering this last fact, the aim of this study was to evaluate the genotoxic potential of S. camporum hydroalcoholic extract and its influence on genotoxicity induced by doxorubicin and methyl methanesulfonate in Swiss mice using the micronucleus and comet assays, respectively. The animals were treated by gavage with different doses of the extract (250, 500, and 1000 mg/kg body weight). For antigenotoxicity assessment, different doses of the S. camporum extract were administered simultaneously with doxorubicin (micronucleus test; 15 mg/kg) and methanesulfonate (comet assay; 40 mg/kg). The results showed that the S. camporum extract itself was not genotoxic in the mouse micronucleus or comet assay. The number of micronucleated polychromatic erythrocytes was significantly lower in animals treated with the S. camporum extract and doxorubicin when compared to animals treated only with doxorubicin. In the comet assay, the S. camporum extract, at the doses tested, significantly reduced the extent of DNA damage in liver cells induced by methanesulfonate. The putative activity of the active compounds of S. camporum extract may explain the effect of this plant on genotoxicity induced by doxorubicin and methanesulfonate.
Topics: Animals; Antimutagenic Agents; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Doxorubicin; Liposomes; Male; Methyl Methanesulfonate; Mice; Micronuclei, Chromosome-Defective; Micronucleus Tests; Plant Extracts; Plant Stems; Styrax
PubMed: 23254694
DOI: 10.1055/s-0032-1327901 -
Environmental and Molecular Mutagenesis 1991In order to determine the relationships among the reduction in relative cloning efficiency (RCE), sister-chromatid exchange (SCE) formation, and interference with...
Flow cytometric evaluation of cell-cycle progression in ethyl methanesulfonate and methyl methanesulfonate-exposed P3 cells: relationship to the induction of sister-chromatid exchanges and cellular toxicity.
In order to determine the relationships among the reduction in relative cloning efficiency (RCE), sister-chromatid exchange (SCE) formation, and interference with progression through the cell-cycle, human teratocarcinoma-derived (P3) cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate. The relationship between SCE and toxicity was quantified, the progression through the cell-cycle was evaluated with flow cytometric methods, and the effects of these chemicals on cell growth and average generation time (AGT) were determined. A strong correlation existed between RCE and SCE (r = -0.978, p less than .001) which was accompanied by an inhibition of growth as evidenced by a significant (p less than .0001) negative linear effect of concentration on the relative cell count from 24 to 72 hours after exposure and by a concentration-dependent increase (p less than .0001) in the AGT. Delays in the transit through S-phase were evident 4 hours after exposure to toxic concentrations of either carcinogen and by 8 to 12 hours post-exposure at the lower concentrations. Increases in the percentage of nuclei in G2 + M, indicative of G2 arrest, occurred from 12 to 24 hours after exposure. One interpretation of these results is that those effects of EMS and MMS exposure which result in S-phase delay and G2 arrest may be those elements common to the induction of SCE and cellular toxicity.
Topics: Cell Cycle; Dose-Response Relationship, Drug; Ethyl Methanesulfonate; Flow Cytometry; Humans; In Vitro Techniques; Methyl Methanesulfonate; Sister Chromatid Exchange; Teratoma; Time Factors; Tumor Cells, Cultured
PubMed: 1879406
DOI: 10.1002/em.2850180210 -
Mutation Research Oct 1988The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this...
The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK-/- mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK-/- mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 micrograms/ml MMS and either 261, 392, 560 or 712 micrograms/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12, 20, 35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased -7, -6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 micrograms/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 micrograms/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration.
Topics: Animals; Drug Synergism; Ethyl Methanesulfonate; Lymphoma; Methyl Methanesulfonate; Mice; Mutagenicity Tests; Mutagens; Mutation; Thymidine Kinase
PubMed: 3173391
DOI: 10.1016/0165-1218(88)90167-x