Inhibition of human DNA alkylation damage repair enzyme ALKBH2 by HIV protease inhibitor ritonavir.

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA and is overexpressed in several cancers. However, there are no known inhibitors available for this crucial DNA repair enzyme. The aim of this study was to examine whether the first-generation HIV protease inhibitors having strong anti-cancer activity can be repurposed as inhibitors of ALKBH2. We selected four such inhibitors and performed in vitro binding analysis against ALKBH2 based on alterations of its intrinsic tryptophan fluorescence and differential scanning fluorimetry. The effect of these HIV protease inhibitors on the DNA repair activity of ALKBH2 was also evaluated. Interestingly, we observed that one of the inhibitors, ritonavir, could inhibit ALKBH2-mediated DNA repair significantly via competitive inhibition and sensitized cancer cells to alkylating agent methylmethane sulfonate (MMS). This work may provide new insights into the possibilities of utilizing HIV protease inhibitor ritonavir as a DNA repair antagonist.

Analysis of transcriptional response in haploid and diploid Schizosaccharomyces pombe under genotoxic stress.

Whole genome duplications are implicated in genome instability and tumorigenesis. Human and yeast polyploids exhibit increased replication stress and chromosomal instability, both hallmarks of cancer. In this study, we investigate the transcriptional response of Schizosaccharomyces pombe to increased ploidy generally, and in response to treatment with the genotoxin methyl methanesulfonate (MMS). We find that treatment of MMS induces upregulation of genes involved in general response to genotoxins, in addition to cell cycle regulatory genes. Downregulated genes are enriched in transport and sexual reproductive pathways. We find that the diploid response to MMS is muted compared to the haploid response, although the enriched pathways remain largely the same. Overall, our data suggests that the global S. pombe transcriptome doubles in response to increased ploidy but undergoes modest transcriptional changes in both unperturbed and genotoxic stress conditions.

REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster.

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

Liver-on-chip model and application in predictive genotoxicity and mutagenicity of drugs.

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.

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.

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, 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 (DPCMdG), 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 DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG 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, DPCMdG 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.

Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals similar roles for RTT109 and ASF1 in development and DNA damage response.

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.

Proteomic investigation reveals the role of bacterial laccase from Bacillus pumilus in oxidative stress defense.

The wide distribution of laccases in nature makes them involved in different biological processes. However, little information is known about how laccase participates in the defense machinery of bacteria against oxidative stress. The present study aimed to elucidate the oxidative stress response mechanism of Bacillus pumilus ZB1 and the functional role of bacterial laccase in stress defense. The oxidative stress caused by methyl methanesulfonate (MMS) significantly induced laccase activity and its transcript level. The morphological analysis revealed that the defense of B. pumilus ZB1 against oxidative stress was activated. Based on the proteomic study, 114 differentially expressed proteins (DEPs) were up-regulated and 79 DEPs were down-regulated. In COG analysis, 66.40% DEPs were classified into the category "Metabolism". We confirmed that laccase was up-regulated in response to MMS stress and its functional annotation was related to "Secondary metabolites biosynthesis, transport and catabolism". Based on protein-protein interaction prediction, two up-regulated DEPs (YcnJ and GabP) showed interaction with laccase and contributed to the formation of laccase stability and adaptability. The overexpressed laccase might improve the antioxidative property of B. pumilus ZB1. These findings provide an insight and the guidelines for better exploitation of bioremediation using bacterial laccase. SIGNIFICANCE: Bacillus pumilus is a gram-positive bacterium that has the potential for many applications, such as bioremediation. The expression of bacterial laccase is significantly influenced by oxidative stress, while the underlying mechanism of laccase overexpression in bacteria has not been fully studied. Elucidation of the biological process may benefit the bioremediation using bacteria in the future. In this study, the differentially expressed proteins were analyzed using a TMT-labeling proteomic approach when B. pumilus was treated with methyl methanesulfonate (MMS). Reactive oxygen species induced by MMS activated the secondary metabolites biosynthesis, transport, and catabolism in B. pumilus, including laccase overexpression. Moreover, the simultaneously up-regulated YcnJ and GabP may benefit the synthesis and the stability of laccase, then improve the antioxidative property of B. pumilus against environmental stress. Our findings advance the understanding of the adaptive mechanism of B. pumilus to environmental conditions.

Nestin Regulates Müller Glia Proliferation After Retinal Injury.

Purpose: The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the proliferative response of mammalian Müller glia, we comparatively analyzed the expression and function of nestin, an intermediate filament protein established as a neural stem cell marker, in the mouse and rat retinas after injury. Methods: Nestin expression in the retinas of C57BL/6 mice and Wistar rats after methyl methanesulfonate (MMS)-induced photoreceptor injury was examined by immunofluorescence and Western blotting. Adeno-associated virus (AAV)-delivered control and nestin short hairpin RNA (shRNA) were intravitreally injected to rats and Müller glia proliferation after MMS-induced injury was analyzed by BrdU incorporation and immunofluorescence. Photoreceptor removal and microglia/macrophage infiltration were also analyzed by immunofluorescence. Results: Rat Müller glia re-entered the cell cycle and robustly upregulated nestin after injury whereas Müller glia proliferation and nestin upregulation were not observed in mice. In vivo knockdown of nestin in the rat retinas inhibited Müller glia proliferation while transiently stimulating microglia/macrophage infiltration and phagocytic removal of dead photoreceptors. Conclusions: Our findings suggest a critical role for nestin in the regulation of Müller glia proliferation after retinal injury and highlight the importance of cross species analysis to identify the molecular mechanisms regulating the injury responses of the mammalian retina.

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.

The Comet Assay in Drosophila: A Tool to Study Interactions between DNA Repair Systems in DNA Damage Responses In Vivo and Ex Vivo.

The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze DNA repair. We have used this approach to study interactions between DNA repair pathways in vivo. Additionally, we have implemented an ex vivo comet assay, in which nucleoids from treated and untreated cells were incubated ex vivo with cell-free protein extracts from individuals with distinct repair capacities. Four strains were used: wild-type OregonK (OK), nucleotide excision repair mutant mus201, dmPolQ protein mutant mus308, and the double mutant mus201;mus308. Methyl methanesulfonate (MMS) was used as a genotoxic agent. Both approaches were performed with neuroblasts from third-instar larvae; they detected the effects of the NER and dmPolQ pathways on the DDR to MMS and that they act additively in this response. Additionally, the ex vivo approach quantified that mus201, mus308, and the double mutant mus201;mus308 strains presented, respectively, 21.5%, 52.9%, and 14.8% of OK strain activity over MMS-induced damage. Considering the homology between mammals and Drosophila in repair pathways, the detected additive effect might be extrapolated even to humans, demonstrating that Drosophila might be an excellent model to study interactions between repair pathways.

SUMOylation of yeast Pso2 enhances its translocation and accumulation in the mitochondria and suppresses methyl methanesulfonate-induced mitochondrial DNA damage.

Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While recent work has revealed that Pso2/Snm1 is dual-localized in the nucleus and mitochondria, it remains unclear whether cell-intrinsic and -extrinsic factors regulate its subcellular localization and function. Herein, we show that Pso2 undergoes ubiquitination and phosphorylation, but not SUMOylation, in unstressed cells. Unexpectedly, we found that methyl methanesulfonate (MMS), rather than ICL-forming agents, induced robust SUMOylation of Pso2 on two conserved residues, K97 and K575, and that SUMOylation markedly increased its abundance in the mitochondria. Reciprocally, SUMOylation had no discernible impact on Pso2 translocation to the nucleus, despite the presence of steady-state levels of SUMOylated Pso2 across the cell cycle. Furthermore, substitution of the invariant residues K97 and K575 by arginine in the Pso2 SUMO consensus motifs severely impaired SUMOylation and abolished its translocation to the mitochondria of MMS-treated wild type cells, but not in unstressed cells. We demonstrate that whilst Siz1 and Siz2 SUMO E3 ligases catalyze Pso2 SUMOylation, the former plays a dominant role. Notably, we found that the phenotypic characteristics of the SUMOylation-defective mutant Pso2K97R/K575R closely mirrored those observed in the Pso2Δ petite mutant. Additionally, leveraging next-generation sequencing analysis, we demonstrate that Pso2 mitigates MMS-induced damage to mitochondrial DNA (mtDNA). Viewed together, our work offers previously unknown insights into the link between genotoxic stress-induced SUMOylation of Pso2 and its preferential targeting to the mitochondria, as well as its role in attenuating MMS-induced mtDNA damage.

Mec1-independent activation of the Rad53 checkpoint kinase revealed by quantitative analysis of protein localization dynamics.

The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator Mec1, and in some scenarios independent of Tel1 or the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.

N-terminal acetyltransferase NatB regulates Rad51-dependent repair of double-strand breaks in Saccharomyces cerevisiae.

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.

Downregulation of MUS81 expression inhibits cell migration and maintains EBV latent infection through miR-BART9-5p in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) infection is associated with the occurrence and development of gastric cancer (GC). Methyl methanesulfonate and ultraviolet-sensitive gene 81 (MUS81) is the catalytic component of a structure-specific endonuclease and plays an important role in chromosomal stability. However, the link between EBV infection and MUS81 remains unclear. In the present study, we found that MUS81 expression was much lower in EBV-associated GC cells than in EBV-negative GC. MUS81 acts as an oncogene in GC by inducing the cell migration and proliferation. Western blot and luciferase reporter assays revealed that miR-BART9-5p directly targeted MUS81 and downregulated its expression. Additionally, overexpression of MUS81 in EBV-positive GC cells inhibited the expression of EBV nuclear antigen 1 (EBNA1). EBNA1 is critical for the pathogenesis of EBV-associated tumors and the maintenance of a stable copy number of the viral genomes. Altogether, these results indicated that the lowering MUS81 expression might be a mechanism by EBV to maintain its latent infection.

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.

Novel In Vivo CometChip Reveals NDMA-Induced DNA Damage and Repair in Multiple Mouse Tissues.

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 N-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.

Royal jelly reduce DNA damage induced by alkylating agent in mice.

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.

Suitability of salivary leucocytes to assess DNA repair ability in human biomonitoring studies by the challenge-comet assay.

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.

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.