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Toxicology Jan 2024DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1-2...
Further development of CometChip technology to measure DNA damage in vitro and in vivo: Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay.
DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1-2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer's protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer's protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO, 0-4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer's protocol), using TK6 cells with MMS (100-800 µM, 1 h) and hydrogen peroxide (HO 7.7-122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.
Topics: Male; Animals; Rats; Comet Assay; Rats, Wistar; DNA Damage; DNA Repair Enzymes; Gels
PubMed: 38040084
DOI: 10.1016/j.tox.2023.153690 -
Journal of Proteomics Feb 2024The wide distribution of laccases in nature makes them involved in different biological processes. However, little information is known about how laccase participates in...
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.
Topics: Bacillus pumilus; Laccase; Proteomics; Methyl Methanesulfonate; Bacterial Proteins; Oxidative Stress
PubMed: 37981008
DOI: 10.1016/j.jprot.2023.105047 -
Bio-protocol Nov 2023Cellular 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...
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 and 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.
PubMed: 37969749
DOI: 10.21769/BioProtoc.4872 -
Investigative Ophthalmology & Visual... Nov 2023The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the...
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.
Topics: Animals; Mice; Rats; Cell Proliferation; Eye Injuries; Methyl Methanesulfonate; Mice, Inbred C57BL; Nestin; Rats, Wistar; Neuroglia
PubMed: 37934159
DOI: 10.1167/iovs.64.14.8 -
Biochemical and Biophysical Research... Dec 2023During cell cycle progression in Saccharomyces cerevisiae, spindle pole bodies (SPBs) are duplicated during the G1/S-phase transition. SPBs are crucial for the...
During cell cycle progression in Saccharomyces cerevisiae, spindle pole bodies (SPBs) are duplicated during the G1/S-phase transition. SPBs are crucial for the organization of both the spindle and astral microtubules, and their orientation defines the direction of nuclear division. In this process, an old SPB, which serves as the template SPB during the duplication process, is oriented toward the bud side. The patterning microtubule plus-end tracking protein, Kar9, plays an important role in the orientation of SPBs by asymmetrically localizing to the old SPB. Here, methylglyoxal (MG), a metabolite derived from glycolysis, was found to perturb asymmetric Kar9 localization and influence proper positioning of the old SPB. MG activated the DNA damage checkpoint pathway, and MG-induced perturbation of asymmetric Kar9 localization was abolished by the deletion of MEC1, a sensor for the DNA damage checkpoint pathway. Methyl methanesulfonate, a DNA-alkylating agent, also perturbed asymmetric Kar9 localization. Our results suggest that activation of the DNA damage checkpoint pathway perturbs the asymmetric Kar9 localization required for proper positioning of SPBs.
Topics: DNA Damage; Microtubules; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus; Spindle Pole Bodies
PubMed: 37918324
DOI: 10.1016/j.bbrc.2023.149157 -
Data in Brief Dec 2023Toxicological analysis of the effects of natural compounds is frequently mandated to assess their safety. In addition to more simple cellular systems, more complex...
Toxicological analysis of the effects of natural compounds is frequently mandated to assess their safety. In addition to more simple cellular systems, more complex biological systems can be used to evaluate toxicity. This dataset is comprised of bright-field microscopy images of chicken-embryo blood cells, a complex biological model that recapitulates several features found in human organisms, including circulation in blood stream and biodistribution to different organs. In the presented collection of blood smear images, cells were exposed to the flavonoid quercetin, and the two mutagens methyl methanesulfonate (MMS) and cadmium chloride (CdCl). models offer a unique opportunity to investigate the effects of various substances, pathogens, or cancer treatments on developing embryos, providing valuable insights into potential risks and therapeutic strategies. In toxicology, models allow for early detection of harmful compounds and their impact on embryonic development, aiding in the assessment of environmental hazards. In immunology, these models offer a controlled system to explore the developing immune responses and the interaction between pathogens and host defenses. Additionally, models are instrumental in oncology research as they enable the study of tumor development and response to therapies in a dynamic, rapidly developing environment. Thus, these versatile models play a crucial role in advancing our understanding of complex biological processes and guiding the development of safer therapeutics and interventions. The data presented here can aid in understanding the potential toxic effects of these substances on hematopoiesis and the overall health of the developing organism. Moreover, the large dataset of blood smear images can serve as a resource for training machine learning algorithms to automatically detect and classify blood cells, provided that specific optimized conditions such as image magnification and background light are maintained for comparison. This can lead to the development of automated tools for blood cell analysis, which can be useful in research. Moreover, the data is amenable to the use as teaching and learning resource for histology and developmental biology.
PubMed: 37876742
DOI: 10.1016/j.dib.2023.109673 -
Environmental Science and Pollution... Nov 2023In 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...
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.
Topics: Methyl Methanesulfonate; Molecular Docking Simulation; Antioxidants; Plant Roots; Meristem; Superoxide Dismutase; Chromosome Aberrations; Onions; DNA; DNA Damage
PubMed: 37874518
DOI: 10.1007/s11356-023-30465-0 -
The EMBO Journal Dec 2023R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we...
R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.
Topics: Humans; DNA Replication; RNA; Ribonucleases; DNA; Hydroxyurea; Ribonuclease H
PubMed: 37855233
DOI: 10.15252/embj.2022113104 -
Scientific Reports Sep 2023Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages....
Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages. It has been shown that the high-efficiency DNA-repair capacity of hMSCs is decreased during their differentiation. However, the underlying its mechanism during adipogenesis and osteogenesis is unknown. Herein, we investigated how alkyl-damage repair is modulated during adipogenic and osteogenic differentiation, especially focusing on the base excision repair (BER) pathway. Response to an alkylation agent was assessed via quantification of the double-strand break (DSB) foci and activities of BER-related enzymes during differentiation in hMSCs. Adipocytes showed high resistance against methyl methanesulfonate (MMS)-induced alkyl damage, whereas osteoblasts were more sensitive than hMSCs. During the differentiation, activities, and protein levels of uracil-DNA glycosylase were found to be regulated. In addition, ligation-related proteins, such as X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase β, were upregulated in adipocytes, whereas their levels and recruitment declined during osteogenesis. These modulations of BER enzyme activity during differentiation influenced DNA repair efficiency and the accumulation of DSBs as repair intermediates in the nucleus. Taken together, we suggest that BER enzymatic activity is regulated in adipogenic and osteogenic differentiation and these alterations in the BER pathway led to different responses to alkyl damage from those in hMSCs.
Topics: Humans; Adipogenesis; Osteogenesis; Bone Marrow; Cells, Cultured; Cell Differentiation; DNA Repair; Mesenchymal Stem Cells; X-ray Repair Cross Complementing Protein 1
PubMed: 37773206
DOI: 10.1038/s41598-023-43737-z -
Research in Microbiology 2023DNA integrity in bacteria is regulated by various factors that act on the DNA. trans-translation has previously been shown to be important for the survival of...
DNA integrity in bacteria is regulated by various factors that act on the DNA. trans-translation has previously been shown to be important for the survival of Escherichia coli cells exposed to certain DNA-damaging agents. However, the mechanisms underlying this sensitivity are poorly understood. In this study, we explored the involvement of the trans-translation system in the maintenance of genome integrity using various DNA-damaging agents and mutant backgrounds. Relative viability assays showed that SsrA-defective cells were sensitive to DNA-damaging agents, such as nalidixic acid (NA), ultraviolet radiation (UV), and methyl methanesulfonate (MMS). The viability of SsrA-defective cells was rescued by deleting sulA, although the expression of SulA was not more pronounced in SsrA-defective cells than in wild-type cells. Live cell imaging using a Gam-GFP fluorescent reporter showed increased double-strand breaks (DSBs) in SsrA-defective cells during DNA damage. We also showed that the ribosome rescue function of SsrA was sufficient for DNA damage tolerance. DNA damage sensitivity can be alleviated by partial uncoupling of transcription and translation by using sub-lethal concentrations of ribosome inhibiting antibiotic (tetracycline) or by mutating the gene coding for RNase H (rnhA). Taken together, our results highlight the importance of trans-translation system in maintaining genome integrity and bacterial survival during DNA damage.
Topics: Ultraviolet Rays; Protein Biosynthesis; Escherichia coli; DNA Damage; Anti-Bacterial Agents; DNA
PubMed: 37690591
DOI: 10.1016/j.resmic.2023.104136