-
The Journal of Toxicological Sciences Dec 2008The single cell gel electrophoresis (comet) assay is a simple and effective method for detecting DNA damage in cells with or without the capability of cell division....
An in vivo comet assay of multiple organs (liver, kidney and bone marrow) in mice treated with methyl methanesulfonate and acetaminophen accompanied by hematology and/or blood chemistry.
The single cell gel electrophoresis (comet) assay is a simple and effective method for detecting DNA damage in cells with or without the capability of cell division. Methyl methanesulfonate (MMS), as a genotoxic compound that reacts with DNA directly, was confirmed for its DNA damage potential by in vivo comet assay in multiple organs such as liver, kidneys and bone marrow in mice and acetaminophen (APAP), a widely used analgesic drug, was evaluated for whether it possesses DNA damage potential or not. Furthermore, cytotoxicity was verified by hematology and /or blood chemistry simultaneously. Male Crj:CD1(ICR) mice were intraperitoneally once treated with MMS at 50, 100, and 150 mg/kg, and APAP at 12, 60, and 300 mg/kg. These organs were collected at 4 and 24 hr after treatment, and the comet assay was performed concomitantly with hematology and/or blood chemistry. The results showed that MMS induced a significant concentration-dependent increase in the frequency of tailed nuclei (DNA damage), tail moment, % DNA in the tail, and tail length in the liver, kidneys and bone marrow at both time points. With regard to hematology and blood chemistry results, nephrotoxic markers were not changed, but aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased in the 150 mg/kg-treated group, and bone marrow counts (BMC) decreased in all of the treatment groups 24 hr after treatment. These results suggested that DNA damage observed in the kidneys was due to genotoxicity, not nephrotoxicity. The DNA damage was more severe at 4 hr than 24 hr after treatment. This might indicate that the decrease in DNA damage was due to detoxification, repair of the lesions induced by the treatment, or cell turnover, all of which would reduce cellular damage. On the other hand, APAP induced increases in plasma AST and ALT levels in the highest dose group only, and the DNA damage in the liver increased at the same dose. These results suggest that the in vivo comet assay might be used to detect the DNA damage induced by MMS and the subsequent DNA repair in mouse liver, kidneys and bone marrow. APAP at the highest dose induces DNA damage in liver. Blood chemical results may indicate that the DNA damage by APAP treatment was attributable to hepato-cytotoxicity, because DNA damage and hepato-cytotoxicity were detected at the same doses.
Topics: Acetaminophen; Animals; Biomarkers; Blood Cell Count; Blood Chemical Analysis; Bone Marrow; Comet Assay; DNA Damage; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Kidney; Liver; Male; Methyl Methanesulfonate; Mice; Mice, Inbred Strains; Mutagens
PubMed: 19043273
DOI: 10.2131/jts.33.515 -
BMC Microbiology Sep 2009The Ada-dependent adaptive response system in Escherichia coli is important for increasing resistance to alkylation damage. However, the global transcriptional and...
BACKGROUND
The Ada-dependent adaptive response system in Escherichia coli is important for increasing resistance to alkylation damage. However, the global transcriptional and translational changes during this response have not been reported. Here we present time-dependent global gene and protein expression profiles following treatment with methyl methanesulfonate (MMS) in E. coli W3110 and its ada mutant strains.
RESULTS
Transcriptome profiling showed that 1138 and 2177 genes were differentially expressed in response to MMS treatment in the wild-type and mutant strains, respectively. A total of 81 protein spots representing 76 nonredundant proteins differentially expressed were identified using 2-DE and LC-MS/MS. In the wild-type strain, many genes were differentially expressed upon long-exposure to MMS, due to both adaptive responses and stationary phase responses. In the ada mutant strain, the genes involved in DNA replication, recombination, modification and repair were up-regulated 0.5 h after MMS treatment, indicating its connection to the SOS and other DNA repair systems. Interestingly, expression of the genes involved in flagellar biosynthesis, chemotaxis, and two-component regulatory systems related to drug or antibiotic resistance, was found to be controlled by Ada.
CONCLUSION
These results show in detail the regulatory components and pathways controlling adaptive response and how the related genes including the Ada regulon are expressed with this response.
Topics: DNA Repair; DNA, Bacterial; Electrophoresis, Gel, Two-Dimensional; Escherichia coli; Escherichia coli Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Methyl Methanesulfonate; Oligonucleotide Array Sequence Analysis; Proteome; Regulon; Tandem Mass Spectrometry
PubMed: 19728878
DOI: 10.1186/1471-2180-9-186 -
BMC Complementary and Alternative... Aug 2012Ayurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Rasayana is one of the eight branches of Ayurveda which refers...
BACKGROUND
Ayurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Rasayana is one of the eight branches of Ayurveda which refers to rejuvenant therapy. It has been reported that rasayanas have immuno-modulatory, antioxidant and antitumor functions, however, the genotoxic potential and modulation of DNA repair of many rasayanas have not been evaluated.
METHODS
The present study assessed the role of Brahmarasayana (BR) on Ethyl methanesulfonate (EMS)-and Methyl methanesulfonate (MMS)-induced genotoxicity and DNA repair in in vivo mouse test system. The mice were orally fed with BR (5 g or 8 mg / day) for two months and 24 h later EMS or MMS was given intraperitoneally. The genotoxicity was analyzed by chromosomal aberrations, sperm count, and sperm abnormalities.
RESULTS
The results have revealed that BR did not induce significant chromosomal aberrations when compared to that of the control animals (p >0.05). On the other hand, the frequencies of chromosomal aberrations induced by EMS (240 mg / kg body weight) or MMS (125 mg / kg body weight) were significantly higher (p<0.05) to that of the control group. The treatment of BR for 60 days and single dose of EMS or MMS on day 61, resulted in significant (p <0.05) reduction in the frequency of chromosomal aberrations in comparison to EMS or MMS treatment alone, indicating a protective effect of BR. Constitutive base excision repair capacity was also increased in BR treated animals.
CONCLUSION
The effect of BR, as it relates to antioxidant activity was not evident in liver tissue however rasayana treatment was observed to increase constitutive DNA base excision repair and reduce clastogenicity. Whilst, the molecular mechanisms of such repair need further exploration, this is the first report to demonstrate these effects and provides further evidence for the role of brahmarasayana in the possible improvement of quality of life.
Topics: Animals; Bone Marrow Cells; Cells, Cultured; Chromosome Aberrations; DNA Damage; DNA Repair; Ethyl Methanesulfonate; Humans; Male; Methyl Methanesulfonate; Mice; Plant Preparations; Plants, Medicinal
PubMed: 22853637
DOI: 10.1186/1472-6882-12-113 -
Environmental and Molecular Mutagenesis Mar 2017Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal...
Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n-ethyl-n-nitrosourea (ENU), 6-mercaptopurine (6-MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration-dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration-related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6-MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real-time PCR results showed that the mRNA level expression of p53 increased and bcl-2 decreased significantly with the increasing ENU, 6-MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6-MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6-MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99-107, 2017. © 2017 Wiley Periodicals, Inc.
Topics: Animals; Apoptosis; Cell Culture Techniques; Cell Survival; Cells, Cultured; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Ethylnitrosourea; Gene Expression; Male; Mercaptopurine; Methyl Methanesulfonate; Mice, Inbred Strains; Mutagens; Spermatozoa
PubMed: 28205273
DOI: 10.1002/em.22075 -
European Journal of Biochemistry Aug 1975Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens...
Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.
Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds.
Topics: Carcinogens; DNA Nucleotidyltransferases; DNA Repair; DNA Replication; Escherichia coli; Ethylnitrosourea; Mesylates; Methyl Methanesulfonate; Methylnitrosourea; Mitomycins; Nitrosourea Compounds; Nucleotides; Permeability; Radiation Effects; Ultraviolet Rays
PubMed: 170107
DOI: 10.1111/j.1432-1033.1975.tb02250.x -
Journal of Bacteriology Aug 2018In , high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated...
In , high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase () was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. binding assays and heterologous host expression studies show that VpsT acts directly at the promoter in response to c-di-GMP to induce expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade. is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in between a sessile biofilm-forming state and a motile lifestyle, both of which are important during environmental persistence and human infections. Here, we report that in c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in .
Topics: Bacterial Proteins; Cyclic GMP; DNA Repair; DNA, Bacterial; Gene Expression Regulation, Bacterial; Methyl Methanesulfonate; Vibrio cholerae
PubMed: 29610212
DOI: 10.1128/JB.00005-18 -
Nucleic Acids Research Jan 2015Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of...
Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.
Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Cell Survival; Cells, Cultured; Checkpoint Kinase 1; DNA Damage; DNA Replication; DNA-Directed DNA Polymerase; Methyl Methanesulfonate; Mice, Knockout; Mutation; Proliferating Cell Nuclear Antigen; Protein Kinases; S Phase; Ubiquitination
PubMed: 25505145
DOI: 10.1093/nar/gku1301 -
Archives of Toxicology Dec 2021The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a...
The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (HO) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than HO or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.
Topics: Antineoplastic Agents, Alkylating; Apoptosis; Cell Line; Cell Proliferation; Cell Survival; Comet Assay; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Etoposide; Humans; Hydrogen Peroxide; Methyl Methanesulfonate; Oxidants; Time Factors; Topoisomerase II Inhibitors
PubMed: 34609522
DOI: 10.1007/s00204-021-03164-3 -
Cells Oct 2021The DNA damage response revolves around transmission of information via post-translational modifications, including reversible protein ADP-ribosylation. Here, we applied...
The DNA damage response revolves around transmission of information via post-translational modifications, including reversible protein ADP-ribosylation. Here, we applied a mass-spectrometry-based Af1521 enrichment technology for the identification and quantification of ADP-ribosylation sites as a function of various DNA damage stimuli and time. In total, we detected 1681 ADP-ribosylation sites residing on 716 proteins in U2OS cells and determined their temporal dynamics after exposure to the genotoxins HO and MMS. Intriguingly, we observed a widespread but low-abundance serine ADP-ribosylation response at the earliest time point, with later time points centered on increased modification of the same sites. This suggests that early serine ADP-ribosylation events may serve as a platform for an integrated signal response. While treatment with HO and MMS induced homogenous ADP-ribosylation responses, we observed temporal differences in the ADP-ribosylation site abundances. Exposure to MMS-induced alkylating stress induced the strongest ADP-ribosylome response after 30 min, prominently modifying proteins involved in RNA processing, whereas in response to HO-induced oxidative stress ADP-ribosylation peaked after 60 min, mainly modifying proteins involved in DNA damage pathways. Collectively, the dynamic ADP-ribosylome presented here provides a valuable insight into the temporal cellular regulation of ADP-ribosylation in response to DNA damage.
Topics: ADP-Ribosylation; Cell Line, Tumor; DNA Damage; Humans; Hydrogen Peroxide; Methyl Methanesulfonate; Signal Transduction; Time Factors
PubMed: 34831150
DOI: 10.3390/cells10112927 -
Genes & Genetic Systems Sep 2023Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked...
Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked replication forks. Defects in HR and unscheduled HR can interfere with other cellular processes such as DNA replication and chromosome segregation, leading to genome instability and cell death. Therefore, the HR process has to be tightly controlled. Protein N-terminal acetylation is one of the most common modifications in eukaryotic organisms. Studies in budding yeast implicate a role for NatB acetyltransferase in HR repair, but precisely how this modification regulates HR repair and genome integrity is unknown. In this study, we show that cells lacking NatB, a dimeric complex composed of Nat3 and Mdm2, are sensitive to the DNA alkylating agent methyl methanesulfonate (MMS), and that overexpression of Rad51 suppresses the MMS sensitivity of nat3Δ cells. Nat3-deficient cells have increased levels of Rad52-yellow fluorescent protein foci and fail to repair DSBs after release from MMS exposure. We also found that Nat3 is required for HR-dependent gene conversion and gene targeting. Importantly, we observed that nat3Δ mutation partially suppressed MMS sensitivity in srs2Δ cells and the synthetic sickness of srs2Δ sgs1Δ cells. Altogether, our results indicate that NatB functions upstream of Srs2 to activate the Rad51-dependent HR pathway for DSB repair.
Topics: Acetyltransferases; DNA Repair; DNA-Binding Proteins; Homologous Recombination; Methyl Methanesulfonate; N-Terminal Acetyltransferase B; N-Terminal Acetyltransferases; Rad51 Recombinase; Rad52 DNA Repair and Recombination Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37331807
DOI: 10.1266/ggs.23-00013