DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

The COVID-19 pandemic has led to the emergence of acute and chronic post-COVID syndromes, which present diverse clinical manifestations. The underlying pathophysiology of these conditions is not yet fully understood, but genetic instability has been proposed as a potential contributing factor. This study aimed to explore the differential impact of physical and psychological health factors on genetic instability in individuals with acute and chronic post-COVID syndromes. In this study, three groups of subjects were analyzed: a control group, an acute post-COVID group, and a chronic post-COVID group, with a total of 231 participants. The participants were assessed using a questionnaire for long-COVID-19COVID, and female participants reported more symptoms than male participants in areas related to fatigue, memory, mental health, and well-being during the chronic phase. Genetic instability was assessed using the comet assay, and participants\' physical and psychological profiles were evaluated. The overall results showed no significant differences in DNA damage, as measured by the comet assay, among the three groups, suggesting that genetic instability, as assessed by this method, may not be a primary driver of the distinct clinical presentations observed in post-COVID syndromes. However, when gender was considered, male participants in the acute long COVID group exhibited higher levels of genetic instability compared to females. Multiple linear regression analysis revealed that gender, age, and waist circumference were significant predictors of DNA damage. Among females in the acute group, sexual health, and eye-related symptoms significantly influenced the increase in DNA damage. These findings indicate the need for further investigation on the gender-specific differences in genetic instability and their potential implications for the pathophysiology of post-COVID syndromes. Exploring alternative markers of genetic instability and the interplay between genetic, inflammatory, and cellular processes could provide valuable insights for the management of these debilitating post-viral sequelae.

In this study, the protective role of Urtica dioica extract (Udex) against Li2CO3 toxicity in Allium cepa L. was investigated using various parameters such as germination rates, root growth, weight gain, mitotic index (MI), malondialdehyde (MDA), micronucleus (MN), antioxidant enzyme activity, chromosomal abnormalities (CAs) and anatomical changes. As the biological activity of Udex is related to its active content, the profile of phenolic compounds was determined by LC-MS/MS analysis. Li2CO3 caused abnormalities in the tested parameters and serious regressions in germination parameters. Application of 100 mg/L Li2CO3 reduced the chlorophyll a and b contents by 73.04% and 65.7%, respectively. Li2CO3 application exhibited a cytotoxic effect by inducing significant decreases in MI and increases in the frequency of MN, and also showed a genotoxic effect by causing CAs. After 100 mg/L Li2CO3 treatment, MDA, proline, superoxide dismutase, and catalase levels increased by 54.9%, 58.5%, 47.8%, and 52.3%, respectively. Li2CO3 and Udex co-administration resulted in a regression in increased biochemical parameters and genotoxicity parameters, and an improvement in germination parameters. Furthermore, Udex demonstrated efficacy in mitigating the detrimental effects of Li2CO3 on the root tip, particularly in the 200 µg/mL Udex-treated group. The thickening of the cortex cell wall and conduction tissue, which is commonly induced by Li₂CO₃, was not observed in the Udex-treated group. The protective effect of Udex can be explained by the phenolic compounds it contains. Rutin was detected as the major component in Udex and other phenolics were listed according to their presence rate as protecatechuic acid > caffeic acid > p-coumaric acid > syringic acid > rosemarinic acid > epicatechin. Li ions, which increase in the environment after industrialization, are an important environmental pollutant and exhibit toxicity that affects many pathways in organisms. Scientific research should not only detect these toxic effects but also develop solutions to such problems. In this study, it was determined that the Udex application had a toxicity-reducing role against Li2CO3 toxicity. Also, it has been demonstrated that A. cepa is an important indicator in determining this toxicity and toxicity-reducing applications.

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1\'s nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6-14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology.

Olive mill wastewater (OMWW), with its high level of phenolic compounds, simultaneously represents a serious environmental challenge and a great resource with potential nutraceutical activities. To increase the knowledge of OMWW\'s biological effects, with an aim to developing a food supplement, we performed a chemical characterisation of the extract using the Liquid Chromatography-Quadrupole Time-of-flight spectrometry (LC-QTOF) and an in vitro genotoxicity/antigenotoxicity assessment on HepaRG ™ cells. Chemical analysis revealed that the most abundant phenolic compound was hydroxytyrosol. Biological tests showed that the extract was not cytotoxic at the lowest tested concentrations (from 0.25 to 2.5 mg/mL), unlike the highest concentrations (from 5 to 20 mg/mL). Regarding genotoxic activity, when tested at non-cytotoxic concentrations, the extract did not display any effect. Additionally, the lowest tested OMWW concentrations showed antigenotoxic activity (J-shaped dose-response effect) against a known mutagenic substance, reducing the extent of DNA damage in the co-exposure treatment. The antigenotoxic effect was also obtained in the post-exposure procedure, although only at the extract concentrations of 0.015625 and 0.03125 mg/mL. This behaviour was not confirmed in the pre-exposure protocol. In conclusion, the present study established a maximum non-toxic OMWW extract dose for the HepaRG cell model, smoothing the path for future research.

Microplastics (MP) are now present in all ecosystems and undergo weathering processes, including physical or chemical degradation. Although most studies have been carried out on MP toxicity in the marine ecosystem, interest is growing for the terrestrial and entire aquatic compartments. However, the interface between both environments, also known as the soil/water continuum, is given little consideration in MP toxicity studies. Only a few studies considered the toxicity of artificially aged or soil field-collected MP on species living at this interface. The present study evaluates the impact of artificial and field aging polyethylene (PE) MP on the bivalve Scrobicularia plana, a key organism of the estuarine compartment, living at the soil/water interface. Clams were exposed for 21 days to environmental concentrations (0.008, 10 and 100 μg L-1) of unaged as well as artificially and field aged PE MP. Toxicity was assessed from individual to molecular levels including condition index, clearance rate, burrowing behavior, energy reserves, enzyme activities and DNA damage. Results showed differential effects at all biological levels depending on the type and the concentration of the MP tested. Indeed, a decrease in burrowing behavior was observed in S. plana exposed to aged and field PE at low concentration (0.008 μg L-1). In the gills of clams, exposures to aged PE (0.008 and 100 μg L-1), virgin PE (10 μg L-1) and field PE (all tested concentrations) decreased CAT activity while DNA damage increased after exposure to virgin PE (0.008 μg L-1 and 10 μg L-1) and field PE (0.008 μg L-1). Our findings suggest that aging modifies the toxicity profile of PE polymer on S. plana and considering plastic from field at environmental concentrations is important when performing ecotoxicological studies.

UNASSIGNED: Moringa oleifera and Tinospora cordifolia is extensively used as an ingredient of food and in traditional medicine for the management of a variety of diseases.
UNASSIGNED: The extracts of leaf of Moringa oleifera and stem of Tinospora cordifolia were assessed to examine their ability to inhibit the oxidative DNA damage (by DNA protection assay), cytoprotective and genoprotective potential (by Comet assay) in V79 cells individually and in combinations.
UNASSIGNED: It was found that these extracts could significantly inhibit the OH-dependent damage of pUC18 plasmid DNA. M. oleifera extract (160 and 320 μg/mL) and Tinospora cordifolia extract (640, 1,280 and 2,560 μg/mL) individually showed higher DNA protection activity. M. oleifera (1,280 μg/mL) combined with Tinospora cordifolia (640 μg/mL) showed best cytoprotective and genoprotective activities among different concentration combinations and various concentrations of individual plants in V79 cell line against hydrogen peroxide induced cytotoxicity and genotoxicity.
UNASSIGNED: This study demonstrates the cytoprotective and genoprotective activity of M. oleifera and Tinospora cordifolia individually or in combination.

Fipronil (FP) is an insecticide used in the treatment and control of pests, but it also adversely affects bees. Currently, there is no data on the genotoxic effects of FP in the brain of bumblebees. Thus, through the comet assay and routine morphological analysis, we analyzed the morphological effects and potential genotoxicity of environmentally relevant concentrations of FP on the brain of Bombus atratus. Bumblebees were exposed at concentrations of 2.5 μg/g and 3.5 μg/g for 96 hours. After the exposure, the brains were removed for morphological and morphometric analysis, and the comet assay procedure - used to detect DNA damage in individual cells using electrophoresis. Our data showed that both concentrations (2.5 μg/g and 3.5 μg/g) caused DNA damage in brain cells. These results corroborate the morphological data. We observed signs of synapse loss in the calyx structure, intercellular spaces between compact inner and non-compact inner cells, and cell swelling. This study provides unprecedented evidence of the effects of FP on DNA and cellular structures in the brain of B. atratus and reinforces the need to elucidate its toxic effects on other species to allow future risk assessments and conservation projects.

Brazil is one of the world\'s largest consumers of pesticides. This intense use impacts the environment and exposes a wide range of individuals to pesticides, including rural workers who are occupationally exposed and rural residents who are environmentally exposed. We aimed to evaluate the effects of occupational exposure to pesticides on the health of rural workers and rural residents. We conducted an epidemiological study with 104 farmers and 23 rural residents of Casimiro de Abreu (Rio de Janeiro, Brazil). A comparison group (urban residents) comprised 103 residents of the urban area of the same city. We determined the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using a modified version of Ellman\'s method to evaluate exposure. In addition, we performed genotoxic and mutagenic analyses with the comet assay and the cytokinesis-block micronucleus (CBMN) assay. There was a reduction in cholinesterase activity, mainly BChE, in rural workers and rural residents compared with urban residents (p = 0.002). There was an increase in genotoxic effects in rural workers compared with urban residents (comet assay, p < 0.001; CBMN assay, p < 0.001). In addition, there was a greater chance of genotoxic changes in rural workers exposed to pesticides based on the comet assay (odds ratio [OR] 7.6, 95 % confidence interval [CI] 6.6-15.9) and the CBMN assay (OR 22.7, 95 % CI 10.3-49.9). We found that individuals occupationally exposed to pesticides are more likely to have genotoxic effects. These findings are useful for the development of programs to monitor populations exposed to genotoxic substances and allow the development of strategies for the prevention, control, and surveillance of effects that result from occupational and environmental exposures to pesticides.

Coal is a mixture of several chemicals, many of which have mutagenic and carcinogenic effects and are a key contributor to the global burden of mortality and disease. Previous studies suggest that coal is related to telomeric shortening in individuals occupationally exposed, however little is known about the effects of mining and burning coal on the telomeres of individuals living nearby. Therefore, the primary objective of this investigation was to assess the impact of proximity to coal power plants and coal mines on the genomic instability of individuals environmentally exposed, while also exploring potential associations with individual characteristics, oxidative stress, inflammatory responses, and the presence of inorganic elements. This study involved 80 men participants from three cities around a thermoelectric power plant and one city unexposed to coal and byproducts. DNA was extracted from peripheral blood samples obtained from each participant, and the telomeres length (TL) was assessed using quantitative real-time polymerase chain reaction (qPCR) methodology. No significant difference was observed between exposed individuals (6227 ± 2884 bp) when compared to the unexposed group (5638 ± 2452 bp). Nevertheless, TL decrease was associated with age and risk for cardiovascular disease; and longer TL was found to be linked with increased concentrations of silicon and phosphorus in blood samples. No correlations were observed between TL with comet assay (visual score), micronucleus test, oxidative stress, and inflammatory results. Additional research is required to ascertain the potential correlation between these changes and the onset of diseases and premature mortality.