Investigation of the abasic sites induced by hydrogen peroxide and methyl methanesulfonate in calf thymus DNA and BEAS-2B cells.

The primary goals of this study were to investigate the formation of abasic sites (AP sites) induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2), and to characterize specific types of these pro-mutagenic DNA lesions in calf thymus DNA (CT-DNA), and BEAS-2B human lung normal cell line. Furthermore, these profiles were compared with those observed in leukocytes derived from healthy controls (HC), breast cancer patients (BCP) before treatment, and 5-year survivors. Results indicated that both H2O2 and MMS induced the concentration- and time-dependent formation of AP sites in CT-DNA. To characterize the specific types of AP sites induced by H2O2 or MMS, we performed AP site cleavage assay using putrescine, T7 exonuclease (T7 Exo), and exonuclease III (Exo III). Results showed that the AP sites induced by H2O2 in CT-DNA were predominantly 5'-and 3'-nicked AP sites and no intact AP sites were detected. By contrast, the majority of AP sites generated by MMS in CT-DNA are not excisable and are classified as residual and intact AP sites. Similar approaches were performed in human BEAS-2B cells and comparable observations were confirmed in the cell-based model. Further investigation indicated that the profile of the AP sites observed in Taiwanese HC is identical to that of BEAS-2B cells treated with H2O2 whereas the pattern of AP sites detected in BCP is similar to that of CT-DNA exposed to H2O2, suggesting that these AP sites were produced primarily through reactive oxygen species (ROS) generation. More than 70 % of the AP sites in leukocytes derived from BCP were 5'-nicked and residual AP sites. Furthermore, the characteristics of the AP sites detected in 5-year survivors are comparable with the ones in HC by using putrescine cleavage assay. Overall, we speculate that deficiency in the DNA repair cascade may play a role in mediating the formation of specific types of AP sites detected in BCP.

The Assessment of Methyl Methanesulfonate Absorption by Amphipods from the Environment Using Lux-Biosensors.

The ability of aquatic mesofauna representatives involved in trophic chains to sorb and accumulate toxicants is important for understanding the functioning of aquatic ecosystems and for fishing industry. This study investigated the capacity of marine amphipod Gammarus oceanicus and freshwater amphipods Eulimnogammarus vittatus and Gammarus lacustris to absorb the DNA-alkylating agent methyl methanesulfonate (MMS). The presence of alkylating agents in the environment and in the tissues of the amphipods was determined using whole-cell lux-biosensor Escherichia coli MG1655 pAlkA-lux, in which the luxCDABE genes from Photorhabdus luminescens, enabling the luminescence of the cell culture, are controlled by the PalkA promoter of DNA glycosylase. It was shown that within one day of incubation in water containing MMS at a concentration above 10 µM, the amphipods absorbed the toxicant and their tissues produce more alkylation damage to biosensor cells than the surrounding water. Concentrations of MMS above 1 mM in the environment caused the death of the amphipods before the toxicant could be significantly concentrated in their tissues. The sensitivity and the capacity to absorb MMS were found to be approximately the same for the marine amphipod G. oceanicus and the freshwater amphipods E. vittatus and G. lacustris.

DNA Damage Checkpoints Govern Global Gene Transcription and Exhibit Species-Specific Regulation on HOF1 in Candida albicans.

DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in Saccharomyces cerevisiae and other fungi has expanded our comprehension of the downstream pathways involved in DNA damage response. While the function of checkpoint kinases, specifically Rad53, is well documented in the fungal pathogen Candida albicans, their targets remain poorly understood. In this study, we explored the impact of deleting RAD53 on the global transcription profiles and observed alterations in genes associated with ribosome biogenesis, DNA replication, and cell cycle. However, the deletion of RAD53 only affected a limited number of known DNA damage-responsive genes, including MRV6 and HMX1. Unlike S. cerevisiae, the downregulation of HOF1 transcription in C. albicans under the influence of Methyl Methanesulfonate (MMS) did not depend on Dun1 but still relied on Rad53 and Rad9. In addition, the transcription factor Mcm1 was identified as a regulator of HOF1 transcription, with evidence of dynamic binding to its promoter region; however, this dynamic binding was interrupted following the deletion of RAD53. Furthermore, Rad53 was observed to directly interact with the promoter region of HOF1, thus suggesting a potential role in governing its transcription. Overall, checkpoints regulate global gene transcription in C. albicans and show species-specific regulation on HOF1; these discoveries improve our understanding of the signaling pathway related to checkpoints in this pathogen.

Characterization of structural, genotoxic, and immunological effects of methyl methanesulfonate (MMS) induced DNA modifications: Implications for inflammation-driven carcinogenesis.

Genotoxic DNA damaging agents are the choice of chemicals for studying DNA repair pathways and the associated genome instability. One such preferred laboratory chemical is methyl methanesulfonate (MMS). MMS, an SN2-type alkylating agent known for its ability to alkylate adenine and guanine bases, causes strand breakage. Exploring the outcomes of MMS interaction with DNA and the associated cytotoxicity will pave the way to decipher how the cell confronts methylation-associated stress. This study focuses on an in-depth understanding of the structural instability, induced antigenicity on the DNA molecule, cross-reactive anti-DNA antibodies, and cytotoxic potential of MMS in peripheral lymphocytes and cancer cell lines. The findings are decisive in identifying the hazardous nature of MMS to alter the intricacies of DNA and morphology of the cell. Structural alterations were assessed through UV-Vis, fluorescence, liquid chromatography, and mass spectroscopy (LCMS). The thermal instability of DNA was analyzed using duplex melting temperature profiles. Scanning and transmission electron microscopy revealed gross topographical and morphological changes. MMS-modified DNA exhibited increased antigenicity in animal subjects. MMS was quite toxic for the cancer cell lines (HCT116, A549, and HeLa). This research will offer insights into the potential role of MMS in inflammatory carcinogenesis and its progression.

Spot Assay and Colony Forming Unit (CFU) Analyses-based sensitivity test for Candida albicans and Saccharomyces cerevisiae.

Cellular sensitivity is an approach to inhibit the growth of certain cells in response to any non-permissible conditions, as the presence of a cytotoxic agent or due to changes in growth parameters such as temperature, salt, or media components. Sensitivity tests are easy and informative assays to get insight into essential gene functions in various cellular processes. For example, cells having any functionally defective genes involved in DNA replication exhibit sensitivity to non-permissive temperatures and to chemical agents that block DNA replication fork movement. Here, we describe a sensitivity test for multiple strains of Saccharomyces cerevisiae and Candida albicans of diverged genetic backgrounds subjected to several genotoxic chemicals simultaneously. We demonstrate it by testing the sensitivity of DNA polymerase defective yeast mutants by using spot analysis combined with colony forming unit (CFU) efficiency estimation. The method is very simple and inexpensive, does not require any sophisticated equipment, can be completed in 2-3 days, and provides both qualitative and quantitative data. We also recommend the use of this reliable methodology for assaying the sensitivity of these and other fungal species to antifungal drugs and xenobiotic factors.

In silico interactions and deep neural network modeling for toxicity profile of methyl methanesulfonate.

In this study, the toxicity induced by the alkylating agent methyl methanesulfonate (MMS) in Allium cepa L. was investigated. For this aim, bulbs were divided into 4 groups as control and application (100, 500 and 4000 µM MMS) and germinated for 72 h at 22-24 °C. At the end of the germination period root tips were collected and made ready for analysis by applying traditional preparation methods. Germination, root elongation, weight, mitotic index (MI) values, micronucleus (MN) and chromosomal abnormality (CAs) numbers, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities and anatomical structures of bulbs were used as indicators to determine toxicity. Moreover the extent of DNA fragmentation induced by MMS was determined by comet assay. To confirm the DNA fragmentation induced by MMS, the DNA-MMS interaction was examined with molecular docking. Correlation and principal component analyses (PCA) were performed to examine the relationship between all parameters and understand the underlying structure and relationships among these parameters. In the present study, a deep neural network (DNN) with two hidden layers implemented in Matlab has been developed for the comparison of the estimated data with the real data. The effect of MDA levels, SOD and CAT activities at 4 different endpoints resulting from administration of various concentrations of MMS, including MN, MI, CAs and DNA damage, was attempted to be estimated by DNN model. It is assumed that the predicted results are in close agreement with the actual data. The effectiveness of the model was evaluated using 4 different metrics, MAE, MAPE, RMSE and R2, which together show that the model performs commendably. As a result, the highest germination, root elongation, weight gain and MI were measured in the control group. MMS application caused a decrease in all physiological parameters and an increase in cytogenetic (except MI) and biochemical parameters. MMS application caused an increase in antioxidant enzyme levels (SOD and CAT) up to a concentration of 500 µM and a decrease at 4000 µM. MMS application induced different types of CAs and anatomical damages in root meristem cells. The results of the comet assay showed that the severity of DNA fragmentation increased with increasing MMS concentration. Molecular docking analysis showed a strong DNA-MMS interaction. The results of correlation and PCA revealed significant positive and negative interactions between the studied parameters and confirmed the interactions of these parameters with MMS. It has been shown that the DNN model developed in this study is a valuable resource for predicting genotoxicity due to oxidative stress and lipid peroxidation. In addition, this model has the potential to help evaluate the genotoxicity status of various chemical compounds. At the end of the study, it was concluded that MMS strongly supports a versatile toxicity in plant cells and the selected parameters are suitable indicators for determining this toxicity.

Opi1-mediated transcriptional modulation orchestrates genotoxic stress response in budding yeast.

In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.

Olive juice dry extract containing hydroxytyrosol, as a nontoxic and safe substance: Results from pre-clinical studies and review of toxicological studies.

Products derived from olives, such as the raw fruit and oils, are widely consumed due to their taste, and purported nutritional/health benefits. Phenolic compounds, especially hydroxytyrosol (HT), have been proposed as one of the key substances involved in these effects. An olive juice extract, standardized to contain 20% HT ("OE20HT"), was produced to investigate its health benefits. The aim of this study was to demonstrate the genotoxic safety of this ingredient based on in vitro Ames assay and in vitro micronucleus assay. Results indicated that OE20HT was not mutagenic at concentrations of up to 5000 µg/plate, with or without metabolic activation, and was neither aneugenic nor clastogenic after 3-hour exposure at concentrations of up to 60 µg/mL with or without metabolic activation, or after 24-hour exposure at concentrations of up to 40 µg/mL. To further substantiate the safety of OE20HT following ingestion without conducting additional animal studies, a comprehensive literature review was conducted. No safety concerns were identified based on acute or sub-chronic studies in animals, including reproductive and developmental studies. These results were supported by clinical studies demonstrating the absence of adverse effects after oral supplementation with olive extracts or HT. Based on in vitro data and the literature review, the OE20HT extract is therefore considered as safe for human consumption at doses up to 2.5 mg/kg body weight/day.

Genomic DNA damage induced by co-exposure to DNA damaging agents and pulsed magnetic field.

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 S. cerevisiae 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.

Fate of micronuclei and micronucleated cells after treatment of HeLa cells with different genotoxic agents.

Although micronuclei are well-known biomarkers of genotoxic damage, the biological consequences of micronucleus induction are only poorly understood. To further elucidate these consequences, HeLa cells stably expressing histone 2B coupled with green fluorescent protein were used for long-term live cell imaging to investigate the fate of micronuclei and micronucleated cells after treatment of cells with various genotoxic agents (doxorubicin (20, 30 and nM), tert-butyl hydroperoxide (tBHP, 50, 100 and 150 µM), radiation (0.5, 1 and 2 Gy), methyl methanesulfonate (MMS, 20, 25 and 30 µg/ml) and vinblastine (1, 2 and 3 nM)). Most micronuclei persist for multiple cell cycles or reincorporate while micronucleated cells were more prone to cell death, senescence and fatal mitotic errors compared to non-micronucleated cells, which is consistent with previous studies using etoposide. No clear substance-related effects on the fate of micronuclei and micronucleated cells were observed. To further investigate the fate of micronuclei, extrusion of micronuclei was studied with treatments reported as inducing the extrusion of micronuclei. Since extrusion was not observed in HeLa cells, the relevance of extrusion of micronuclei remains unclear. In addition, degradation of micronuclei was analysed via immunostaining of γH2AX, which demonstrated a high level of DNA damage in micronuclei compared to the main nuclei. Furthermore, transduction with two reporter genes (LC3B-dsRed and LaminB1-dsRed) was conducted followed by long-term live cell imaging. While autophagy marker LC3B was not associated with micronuclei, Lamin B1 was found in approximately 50% of all micronuclei. While degradation of micronuclei was not observed to be a frequent fate of micronuclei, the results show impaired stability of DNA and micronuclear envelope indicating rupture of micronuclei as a pre-step to chromothripsis.

Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate.

The infection of a mammalian host by the pathogenic fungus Candida albicans 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 C. albicans 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 C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, 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 CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited RAD53-related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.

Decitabine improves MMS-induced retinal photoreceptor cell damage by targeting DNMT3A and DNMT3B.

Introduction: Retinitis pigmentosa (RP) is a group of neurodegenerative retinopathies causing blindness due to progressive and irreversible photoreceptor cell death. The alkylating agent methyl methanesulfonate (MMS) can induce selective photoreceptor cell death, which is used to establish RP animal models. MMS induces DNA base damage by adding alkyl groups to DNA, and epigenetic modifications influence DNA damage response. Here, we aimed to explore the relationship between DNA methylation and DNA damage response in dying photoreceptors of RP. Methods: The mouse RP model was established by a single intraperitoneal injection of MMS. The retinal structure and function were assessed by H&E, OCT, TUNEL, and ERG at several time points. The expression of DNA methylation regulators was assessed by qPCR and Western blot. DNMT inhibitor 5-aza-dC was applied to inhibit the activity of DNA methyltransferases and improve the retinal photoreceptor damage. Results: The outer nuclear layer (ONL) and IS/OS layer were significantly thinner and the retinal function was impaired after MMS treatment. The cell death was mainly located in the ONL. The retinal damage induced by MMS was accompanied by hyperexpression of DNMT3A/3B. The application of DNMT inhibitor 5-aza-dC could suppress the expression level of DNMT3A/3B, resulting in the remission of MMS-induced photoreceptor cell damage. The ONL and IS/OS layers were thicker than that of the control group, and the retinal function was partially restored. This protective effect of 5-aza-dC was associated with the down-regulated expression of DNMT3A/3B. Conclusion: These findings identified a functional role of DNMT3A/3B in MMS-induced photoreceptor cell damage and provided novel evidence to support DNMTs as potential therapeutic targets in retinal degenerative diseases.Graphical Abstract.

Antiproliferative, apoptotic, and antimutagenic properties of stem bark and seed fractions of Polyalthia cerasoides (Roxb.).

AIMS: The aim was to compare the anticancer and antimutagenic potency of Polyalthia cerasoides seeds and stem bark. AIM OF THE STUDY: The aim of this study was to investigate the antiproliferative, apoptotic, antioxidation to DNA, and antimutagenic activity of alcoholic (PS-1 and PS-3) and petroleum ether (PS-2 and PS-4) stem bark and seed fractions of P. cerasoides. METHODS: P. cerasoides stem bark and seeds were extracted with ethanol: water mixture (9:1 ratio v: v) and fractionated with petroleum ether. Fractions were investigated for antiproliferative effect using cell by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole assay (cell line used liver [HepG2] and cervical [HeLa] cancer cell lines), DNA damage protection using hydroxyl radical and antimutagenic effect using chromosome aberration test. RESULTS: PS-1 (IC50 10 µg/ml) and PS-3 (IC50 11 µg/ml) showed maximum antiproliferative activity against HepG2 cell lines, whereas, PS-1 (IC50 10 µg/ml), PS-2 (IC50 24 µg/ml), and PS-3 (IC50 11 µg/ml) showed better antiproliferative activity against HeLa cell lines. PS-3 and PS-4 were protective against oxidation to the supercoiled DNA molecule. Further, petroleum ether extract of both seed (PS-2) and stem bark (PS-4) showed good antimutagenicity as revealed by the less chromosomal aberrations compared to PS-1 and PS-3 fractions. CONCLUSIONS: This study demonstrated the beneficial effect of fractions against oxidation of DNA, antiproliferative, apoptotic, and antimutagenic activity. Probably, this property would be attributable by their phenolic and steroid constituents. Therefore, this plant could be used as a potential source of nutraceutical agents.

The Alkylating Agent Methyl Methanesulfonate Triggers Lipid Alterations at the Inner Nuclear Membrane That Are Independent from Its DNA-Damaging Ability.

In order to tackle the study of DNA repair pathways, the physical and chemical agents creating DNA damage, the genotoxins, are frequently employed. Despite their utility, their effects are rarely restricted to DNA, and therefore simultaneously harm other cell biomolecules. Methyl methanesulfonate (MMS) is an alkylating agent that acts on DNA by preferentially methylating guanine and adenine bases. It is broadly used both in basic genome stability research and as a model for mechanistic studies to understand how alkylating agents work, such as those used in chemotherapy. Nevertheless, MMS exerts additional actions, such as oxidation and acetylation of proteins. In this work, we introduce the important notion that MMS also triggers a lipid stress that stems from and affects the inner nuclear membrane. The inner nuclear membrane plays an essential role in virtually all genome stability maintenance pathways. Thus, we want to raise awareness that the relative contribution of lipid and genotoxic stresses when using MMS may be difficult to dissect and will matter in the conclusions drawn from those studies.

Homolog of Saccharomyces cerevisiae SLX4 is required for cell recovery from MMS-induced DNA damage in Candida albicans.

SLX4 is a scaffold to coordinate the action of structure-specific endonucleases that are required for homologous recombination and DNA repair. In view of ScSLX4 functions in the maintenance and stability of the genome in Saccharomyces cerevisiae, we have explored the roles of CaSLX4 in Candida albicans. Here, we constructed slx4Δ/Δ mutant and found that it exhibited increased sensitivity to the DNA damaging agent, methyl methanesulfonate (MMS) but not the DNA replication inhibitor, hydroxyurea (HU). Accordingly, RT-qPCR and western blotting analysis revealed the activation of SLX4 expression in response to MMS. The deletion of SLX4 resulted in a defect in the recovery from MMS-induced filamentation to yeast form and re-entry into the cell cycle. Like many other DNA repair genes, SLX4 expression was activated by the checkpoint kinase Rad53 under MMS-induced DNA damage. In addition, SLX4 was not required for the inactivation of the DNA damage checkpoint, as indicated by normal phosphorylation of Rad53 in slx4Δ/Δ cells. Therefore, our results demonstrate SLX4 plays an important role in cell recovery from MMS-induced DNA damage in C. albicans.

Influence of Kv11.1 (hERG1) K+ channel expression on DNA damage induced by the genotoxic agent methyl methanesulfonate.

Besides their crucial role in cell electrogenesis and maintenance of basal membrane potential, the voltage-dependent K+ channel Kv11.1/hERG1 shows an essential impact in cell proliferation and other processes linked to the maintenance of tumour phenotype. To check the possible influence of channel expression on DNA damage responses, HEK293 cells, treated with the genotoxic agent methyl methanesulfonate (MMS), were compared with those of a HEK-derived cell line (H36), permanently transfected with the Kv11.1-encoding gene, and with a third cell line (T2) obtained under identical conditions as H36, by permanent transfection of another unrelated plasma membrane protein encoding gene. In addition, to gain some insights about the canonical/conduction-dependent channel mechanisms that might be involved, the specific erg channel inhibitor E4031 was used as a tool. Our results indicate that the expression of Kv11.1 does not influence MMS-induced changes in cell cycle progression, because no differences were found between H36 and T2 cells. However, the canonical ion conduction function of the channel appeared to be associated with decreased cell viability at low/medium MMS concentrations. Moreover, direct DNA damage measurements, using the comet assay, demonstrated for the first time that Kv11.1 conduction activity was able to modify MMS-induced DNA damage, decreasing it particularly at high MMS concentration, in a way related to PARP1 gene expression. Finally, our data suggest that the canonical Kv11.1 effects may be relevant for tumour cell responses to anti-tumour therapies.

Evaluation of the genotoxic and antigenotoxic potential of lignin-derivative BP-C2 in the comet assay in vivo.

The genotoxic and antigenotoxic potential of BP-C2, a novel lignin-derived polyphenolic composition with ammonium molybdate, was investigated as a radioprotector/radiomitigator for civil applications and as a medical countermeasure for radiation emergencies. Using the alkaline comet assay and methyl methanesulfonate (MMS, 40 mg/kg) as the DNA-damaging agent, these effects of BP-C2 on liver, bone marrow cells and blood leukocytes in rats were studied. The DNA damage was estimated by the DNA content in the comet tail (TDNA, %) 1, 6 and 18 h post exposure to MMS. BP-C2 at doses of 20, 200 and 2000 mg/kg did not exert genotoxic activity in the tested tissues in rats. BP-C2 administered at doses of 20, 100 and 200 mg/kg 1 h before MMS significantly (p < 0.01) mitigated MMS-induced DNA damage, showing a strong genoprotective effect in the liver. In blood leukocytes and bone marrow samples of animals treated with BP-C2, the TDNA % was slightly higher than in the negative control (vehicle) but significantly lower than in the positive control (MMS). Thus, BP-C2 exerted a genoprotective effect against MMS-induced DNA damage to a greater extent towards liver cells, requiring further evaluation of this substance as a genoprotective agent.

Genome protection: histone H4 and beyond.

Histone proteins regulate cellular factors' accessibility to DNA, and histone dosage has previously been linked with DNA damage susceptibility and efficiency of DNA repair pathways. Surplus histones are known to impede the DNA repair process by interfering with the homologous recombination-mediated DNA repair in Saccharomyces cerevisiae. Here, we discuss the recent finding of association of methyl methanesulfonate (MMS) resistance with the reduced histone H4 gene dosage in the pathogenic yeast Candida glabrata. We have earlier shown that while the low histone H3 gene dosage led to MMS susceptibility, the lack of two H4-encoding ORFs, CgHHF1 and CgHHF2, led to resistance to MMS-induced DNA damage. This resistance was linked with a higher rate of homologous recombination (HR). Taking these findings further, we review the interactome analysis of histones H3 and H4 in C. glabrata. We also report that the arginine residue present at the 95th position in the C-terminal tail of histone H4 protein is required for complementation of the MMS resistance in the Cghhf1Δhhf2Δ mutant, thereby pointing out a probable role of this residue in association with HR factors. Additionally, we present evidence that reduction in H4 protein levels may constitute an important part of varied stress responses in C. glabrata. Altogether, we present an overview of histone H4 dosage, HR-mediated repair of damaged DNA and stress resistance in this opportunistic human fungal pathogen.

Comet assay on one- and two-cell mouse embryos.

DNA damage quantified as the comet tail length was assessed using in vitro and in vivo comet assay on one- and two-cell mouse embryos obtained by natural mating. The use of a protocol with three layers of agarose reduces the embryo loss and makes it possible to study a small number of embryos. A significantly lower level of basal, but not induced DNA damage was found in embryos with cleaved zona pellucida compared to embryos with intact zona pellucida. There were no significant differences in the length of the comet's tail between embryos lysed in different lysis solutions, both in cases of basal and induced DNA damage. A significant increase in the comet tail length was detected in one-cell embryos of mice treated with methyl methanesulfonate and etoposide compared to the control. The data show that DNA damage induced in maternal germ cells persists, which can be detected in embryos using the comet assay.

Analyzing Somatic DNA Repair in Arabidopsis Meiotic Mutants.

Meiotic and somatic recombination share a common set of factors. Thus, the analysis of somatic DNA repair in meiotic mutant lines should be of special interest. Growth defects of mutant plants induced by specific genotoxins can thereby hint to DNA repair functions of the affected proteins. Here, we describe two kinds of approaches to characterize deficiencies in DNA repair in mutant lines of Arabidopsis thaliana, after genotoxin treatment.