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International Journal of Molecular... Oct 2022The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand...
The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand breaks, abasic sites and alkali-sensitive sites. Furthermore, the assay can also be modified to include purified DNA glycosylases so that alkylated and oxidized bases can be detected. The CometChip is a higher throughput version of the traditional comet assay and has been used to study cultured cells. Here, we have tested its utility for studies of DNA damage present in vivo. We show that the CometChip is effective in detecting DNA damage in multiple tissues of mice exposed to the direct-acting methylating agent methylmethane sulfonate (MMS) and to the metabolically activated methylating agent -nitrosodimethylamine (NDMA), which has been found to contaminate food, water, and drugs. Specifically, results from MMS-exposed mice demonstrate that DNA damage can be detected in cells from liver, lung, kidney, pancreas, brain and spleen. Results with NDMA show that DNA damage is detectable in metabolically competent tissues (liver, lung, and kidney), and that DNA repair in vivo can be monitored over time. Additionally, it was found that DNA damage persists for many days after exposure. Furthermore, glycosylases were successfully incorporated into the assay to reveal the presence of damaged bases. Overall, this work demonstrates the efficacy of the in vivo CometChip and reveals new insights into the formation and repair of DNA damage caused by MMS and NDMA.
Topics: Alkalies; Animals; Comet Assay; DNA; DNA Damage; DNA Glycosylases; DNA Repair; Dimethylnitrosamine; Methyl Methanesulfonate; Mice
PubMed: 36233095
DOI: 10.3390/ijms231911776 -
Toxics Aug 2022The safety evaluation of food contact materials requires excluding mutagenicity and genotoxicity in migrates. Testing the migrates using in vitro bioassays has been...
The safety evaluation of food contact materials requires excluding mutagenicity and genotoxicity in migrates. Testing the migrates using in vitro bioassays has been proposed to address this challenge. To be fit for that purpose, bioassays must be capable of detecting very low, safety relevant concentrations of DNA-damaging substances. There is currently no bioassay compatible with such qualifications. High-performance thin-layer chromatography (HPTLC), coupled with the planar SOS Umu-C (p-Umu-C) bioassay, was suggested as a promising rapid test (~6 h) to detect the presence of low levels of mutagens/genotoxins in complex mixtures. The current study aimed at incorporating metabolic activation in this assay and testing it with a set of standard mutagens (4-nitroquinoline--oxide, aflatoxin B1, mitomycin C, benzo(a)pyrene, -ethyl nitrourea, 2-nitrofluorene, 7,12-dimethylbenzanthracene, 2-aminoanthracene and methyl methanesulfonate). An effective bioactivation protocol was developed. All tested mutagens could be detected at low concentrations (0.016 to 230 ng/band, according to substances). The calculated limits of biological detection were found to be up to 1400-fold lower than those obtained with the Ames assay. These limits are lower than the values calculated to ensure a negligeable carcinogenic risk of 10. They are all compatible with the threshold of toxicological concern for chemicals with alerts for mutagenicity (150 ng/person). They cannot be achieved by any other currently available test procedures. The p-Umu-C bioassay may become instrumental in the genotoxicity testing of complex mixtures such as food packaging, foods, and environmental samples.
PubMed: 36136466
DOI: 10.3390/toxics10090501 -
International Journal of Radiation... 2023Many articles describe the effects of extremely low-frequency magnetic fields (MFs) on DNA damage induction. However, the mechanism of MF interaction with living matter...
PURPOSE
Many articles describe the effects of extremely low-frequency magnetic fields (MFs) on DNA damage induction. However, the mechanism of MF interaction with living matter is not yet known with certainty. Some works suggest that MF could induce an increase in the efficacy of reactive oxygen species (ROS) production. This work investigates whether pulsed MF exposure produces alterations in genomic DNA damage induced by co-exposure to DNA damaging agents (bleomycin and methyl methanesulfonate (MMS)).
MATERIALS AND METHODS
Genomic DNA, prepared from cultures, was exposed to pulsed MF (1.5 mT peak, 25 Hz) and MMS (0-1%) (15-60 min), and to MF and bleomycin (0-0.6 IU/mL) (24-72 h). The damage induced to DNA was evaluated by electrophoresis and image analysis.
RESULTS
Pulsed MF induced an increment in the level of DNA damage produced by MMS and bleomycin in all groups at the exposure conditions assayed.
CONCLUSIONS
Pulsed MF could modulate the cytotoxic action of MMS and bleomycin. The observed effect could be the result of a multifactorial process influenced by the type of agent that damages DNA, the dose, and the duration of the exposure to the pulsed MF.
Topics: Saccharomyces cerevisiae; Magnetic Fields; DNA Damage; Methyl Methanesulfonate; DNA; Genomics
PubMed: 36069754
DOI: 10.1080/09553002.2022.2121873 -
Photoacoustics Sep 2022How DNA damage and repair processes affect the biomechanical properties of the nucleus interior remains unknown. Here, an opto-acoustic microscope based on time-domain...
How DNA damage and repair processes affect the biomechanical properties of the nucleus interior remains unknown. Here, an opto-acoustic microscope based on time-domain Brillouin spectroscopy (TDBS) was used to investigate the induced regulation of intra-nuclear mechanics. With this ultrafast pump-probe technique, coherent acoustic phonons were tracked along their propagation in the intra-nucleus nanostructure and the complex stiffness moduli and thicknesses were measured with an optical resolution. Osteosarcoma cells were exposed to methyl methanesulfonate (MMS) and the presence of DNA damage was tested using immunodetection targeted against damage signaling proteins. TDBS revealed that the intra-nuclear storage modulus decreased significantly upon exposure to MMS, as a result of the chromatin decondensation and reorganization that favors molecular diffusion within the organelle. When the damaging agent was removed and cells incubated for 2 h in the buffer solution before fixation the intra-nuclear reorganization led to an inverse evolution of the storage modulus, the nucleus stiffened. The same tendency was measured when DNA double-strand breaks were caused by cell exposure to ionizing radiation. TDBS microscopy also revealed changes in acoustic dissipation, another mechanical probe of the intra-nucleus organization at the nano-scale, and changes in nucleus thickness during exposure to MMS and after recovery.
PubMed: 36068801
DOI: 10.1016/j.pacs.2022.100385 -
Chemosphere Nov 2022The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the...
The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the kinetics of repair process. Peripheral blood mononuclear cells (PBMC) are the cells most frequently employed in human biomonitoring studies using the challenge-comet assay, but having a validated alternative of non-invasive biomatrix would be highly convenient for certain population groups and circumstances. The objective of this study was to validate the use of salivary leucocytes in the challenge-comet assay. Leucocytes were isolated from saliva samples and challenged (either in fresh or after cryopreservation) with three genotoxic agents acting by different action mechanisms: bleomycin, methyl methanesulfonate, and ultraviolet radiation. Comet assay was performed just after treatment and at other three additional time points, in order to study repair kinetics. The results obtained demonstrated that saliva leucocytes were as suitable as PBMC for assessing DNA damage of different nature that was efficiently repaired over the evaluated time points, even after 5 months of cryopreservation (after a 24 h stimulation with PHA). Furthermore, a new parameter to determine the efficacy of the repair process, independent of the initial amount of damage induced, is proposed, and recommendations to perform the challenge-comet assay with salivary leucocytes depending on the type of DNA repair to be assessed are suggested. Validation studies are needed to verify whether the method is reproducible and results reliable and comparable among laboratories and studies.
Topics: Biological Monitoring; Bleomycin; Comet Assay; DNA Damage; DNA Repair; Humans; Leukocytes, Mononuclear; Methyl Methanesulfonate; Ultraviolet Rays
PubMed: 36007734
DOI: 10.1016/j.chemosphere.2022.136139 -
Mutation Research 2022Royal jelly (RJ) is a creamy white-yellow liquid that is secreted by the mandibular and hypopharyngeal glands of bees to nourish the larvae. RJ has gained increasing...
Royal jelly (RJ) is a creamy white-yellow liquid that is secreted by the mandibular and hypopharyngeal glands of bees to nourish the larvae. RJ has gained increasing interest in recent years owing to its antioxidant potential. However, little is known about adequate RJ dosing and its effects on genetic material. Thus, the aim of this study was to evaluate the in vivo effects of RJ on genotoxicity and mutagenicity induced by the alkylating agent methyl methanesulfonate (MMS). In this study, 3-month-old Swiss albino male mice (N = 66) were divided into 11 groups for experimentation. Experiments were performed by administering lyophilized RJ (150 mg/kg, 300 mg/kg, and 1000 mg/kg) or water via gavage as pre- and posttreatment processes with the alkylating agent MMS. After treatment, blood samples were collected from the mice via an incision at the end of the tail to conduct comet assays at times of 24 h and 48 h posttreatment. The mice were then euthanized to remove the bone marrow for a micronucleus test. Overall, regardless of dose, RJ did not exhibit genotoxic, mutagenic activity and the administration of high doses, mainly in the form of posttreatment, presented antigenotoxic and antimutagenic actions. Further, a dose-response correlation was observed in the RJ posttreatment groups. These results demonstrate that RJ administration was effective in reversing the damage caused by the alkylating agent MMS.
Topics: Mice; Bees; Animals; Alkylating Agents; DNA Damage; Fatty Acids; Comet Assay; Methyl Methanesulfonate; Mutagens
PubMed: 36007462
DOI: 10.1016/j.mrfmmm.2022.111796 -
International Journal of Molecular... Jul 2022is an important pathogen that causes a devastating disease in rice. It has been reported that the dual-specificity LAMMER kinase is conserved from yeast to animal...
is an important pathogen that causes a devastating disease in rice. It has been reported that the dual-specificity LAMMER kinase is conserved from yeast to animal species and has a variety of functions. However, the functions of the LAMMER kinase have not been reported in . In this study, we identified the unique LAMMER kinase MoKns1 and analyzed its function in . We found that in a deletion mutant, growth and conidiation were primarily decreased, and pathogenicity was almost completely lost. Furthermore, our results found that MoKns1 is involved in autophagy. The Δ mutant was sensitive to rapamycin, and MoKns1 interacted with the autophagy-related protein MoAtg18. Compared with the wild-type strain 70-15, autophagy was significantly enhanced in the Δ mutant. In addition, we also found that MoKns1 regulated DNA damage stress pathways, and the Δ mutant was more sensitive to hydroxyurea (HU) and methyl methanesulfonate (MMS) compared to the wild-type strain 70-15. The expression of genes related to DNA damage stress pathways in the Δ mutant was significantly different from that in the wild-type strain. Our results demonstrate that MoKns1 is an important pathogenic factor in involved in regulating autophagy and DNA damage response pathways, thus affecting virulence. This research on pathogenesis lays a foundation for the prevention and control of .
Topics: Ascomycota; Fungal Proteins; Gene Expression Regulation, Fungal; Magnaporthe; Oryza; Plant Diseases; Spores, Fungal; Virulence
PubMed: 35897680
DOI: 10.3390/ijms23158104 -
International Journal of Molecular... Jul 2022The infection of a mammalian host by the pathogenic fungus involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the...
The infection of a mammalian host by the pathogenic fungus involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage-inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in . Here, we explored the global transcription profile in response to MMS in and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as , , and , showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene and glutathione metabolism genes and showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited -related regulation. Intriguingly, the transcription profile in response to MMS in shared a limited similarity with the pattern in , including , , and . Overall, cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.
Topics: Actin Capping Proteins; Animals; Candida albicans; DNA Damage; DNA-Binding Proteins; Fungal Proteins; Gene Expression Regulation, Fungal; Mammals; Methyl Methanesulfonate; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35886903
DOI: 10.3390/ijms23147555 -
Cell Biology and Toxicology Aug 2023DDI2 and DDI3 (DDI2/3) are two identical genes in Saccharomyces cerevisiae encoding cyanamide (CY) hydratase. They are not only highly induced by CY, but also by a...
DDI2 and DDI3 (DDI2/3) are two identical genes in Saccharomyces cerevisiae encoding cyanamide (CY) hydratase. They are not only highly induced by CY, but also by a DNA-damaging agent methyl methanesulfonate (MMS), and the regulatory mechanism is unknown. In this study, we performed a modified genome-wide genetic synthetic array screen and identified Fzf1 as a zinc-finger transcriptional activator required for CY/MMS-induced DDI2/3 expression. Fzf1 binds to a DDI2/3 promoter consensus sequence CS2 in vivo and in vitro, and this interaction was enhanced in response to the CY treatment. Indeed, experimental over production of Fzf1 alone was sufficient to induce DDI2/3 expression; however, CY and MMS treatments did not cause the accumulation or apparent alteration in migration of cellular Fzf1. To test a hypothesis that Fzf1 is activated by covalent modification of CY and MMS, we performed mass spectrometry of CY/MMS-treated Fzf1 and detected a few modified lysine residues. Amino acid substitutions of these residues revealed that Fzf1-K70A completely abolished MMS-induced and reduced CY-induced DDI2/3 expression, indicating that the Fzf1-K70 methylation activates Fzf1. This study collectively reveals a novel regulatory mechanism by which Fzf1 is activated by chemical modifications and in turn induces the expression of its target genes for detoxification.
Topics: Transcriptional Activation; Saccharomyces cerevisiae; Transcription Factors
PubMed: 35809138
DOI: 10.1007/s10565-022-09745-x -
ACS Omega Jun 2022Ammonium salt derivatives with a neopentyl moiety are remarkably stable against Hofmann elimination, but the neopentyl moiety slows nucleophilic substitution,...
Ammonium salt derivatives with a neopentyl moiety are remarkably stable against Hofmann elimination, but the neopentyl moiety slows nucleophilic substitution, complicating their synthesis. To identify the best leaving group for the synthesis of the ammonium salts, we prepared six 1,1,1-tris(X-methyl)ethane derivatives, where X is chloride, bromide, iodide, methanesulfonate, -toluenesulfonate, and trifluoromethanesulfonate (triflate), and studied the kinetics of their reactions with sodium, cesium, or tetramethylammonium azide in deuterated dimethylsulfoxide (DMSO) at 100 °C by NMR spectroscopy. Iodide and bromide were found to be more reactive than -toluenesulfonate and methanesulfonate. As expected, the best leaving group for nucleophilic substitution was triflate. Despite the usual high reactivity and instability of primary alkyl triflates, neopentyl triflate can be used as a stable but sufficiently reactive reactant for nucleophilic substitution on neopentyl skeletons.
PubMed: 35721974
DOI: 10.1021/acsomega.2c01965