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The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80 °C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The purposes of this review were to investigate the application of the comet assay in Allium cepa root cells to assess the genotoxicity of environmental samples and to analyse the experimental procedures employed. A literature search was performed selecting articles published between January 2000 and October 2023 from online databases using the combined search terms 'comet assay' and 'A. cepa'. Only 18 papers met the inclusion criteria. None of these were published in the first eight years (2000-2007), highlighting the increasing interest in using the comet assay on A. cepa to analyse environmental samples over the last decade. The majority of the selected studies (15/18, 83%) were performed on samples belonging to the water compartment on onion bulbs. Half of the selected studies (9/18) were conducted to demonstrate the DNA damaging effect of the sample, while the other half of the studies not only recognized the presence of genotoxic agents but also addressed possible remediation measures. Detailed analysis of the experimental procedures revealed heterogeneity in many key steps, such as exposure time, test controls, nuclei isolation solutions, duration of electrophoresis, and number of nuclei scored. This literature review has shown that the comet assay on A. cepa, although recognized as an appropriate tool, is underutilized in environmental toxicology. Greater standardization could lead to its more widespread use, providing valuable information on the genotoxicity of environmental samples and the ability of different processes to mitigate their negative effects on plants., (© The Author(s) 2024. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In male C57BL/6 mice (8-12 weeks) and male Wistar rats (12 weeks), the effect of dibornol (2,6-diisobornyl-4-methylphenol) on the level of spontaneous DNA damage in cells of the bone marrow, liver, kidneys, and rectum of mice (series I) and genotoxic effects in rat testicular cells after administration of cytostatic drugs with different mechanisms of action (series II) were studied using the DNA comet assay. In series I, dibornol was intragastrically administered to mice once at doses of 200, 400, and 2000 mg/kg; in series II, dibornol was intragastrically administered to rats at a dose of 10 mg/kg for 5 days before and 5 days after the cytostatic treatment (methotrexate, doxorubicin). It was found that dibornol in all studied doses did not produce the genotoxic (carcinogenic) effect and reduced the level of spontaneous DNA damage in the bone marrow. After combined administration of cytostatic drugs (doxorubicin, methotrexate) and dibornol, the level of DNA breaks was reduced to 38.5 and 49% of the control, respectively., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Rodents are considered good models for investigating genotoxic damage and mutagenic alterations caused by xenobiotic agents, due to their occupation of a wide variety of habitats. However, relatively few in situ studies have focused on DNA damage in wild rodents associated with environmental exposure. In this review, we investigate trends in the application of the micronucleus test and comet assay in in situ studies of wild rodents. A total of 33 papers were identified, distributed across 14 different countries. Brazil and Spain had the most published studies (six each), followed by Bulgaria (n = 5), Mexico (n = 4) and Italy (n = 3). Only 24 of the 2,652 recognized rodent species have been the subject of in situ studies, which have most frequently focus on species of the genus Mus. The protocols used for the micronucleus test and comet assay varied widely, although blood and bone marrow were the primary types of tissue used. Given the paucity of studies on wild rodents, we recommend further research, particularly focusing on the use of this group as bioindicators of environmental quality and the standardization of protocols., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Given the widespread applications in industrial and agricultural production, the health effects of rare earth elements (REEs) have garnered public attention, and the genotoxicity of REEs remains unclear. In this study, we evaluated the genetic effects of lanthanum nitrate, a typical representative of REEs, with guideline-compliant in vivo and in vitro methods. Genotoxicity assays, including the Ames test, comet assay, mice bone marrow erythrocyte micronucleus test, spermatogonial chromosomal aberration test, and sperm malformation assay were conducted to assess mutagenicity, chromosomal damage, DNA damage, and sperm malformation. In the Ames test, no statistically significant increase in bacterial reverse mutation frequencies was found as compared with the negative control. Mice exposed to lanthanum nitrate did not exhibit a statistically significant increase in bone marrow erythrocyte micronucleus frequencies, spermatogonial chromosomal aberration frequencies, or sperm malformation frequencies compared to the negative control (P > 0.05). Additionally, after a 24-h treatment with lanthanum nitrate at concentrations of 1.25, 5, and 20 μg/ml, no cytotoxicity was observed in CHL cells. Furthermore, the comet assay results indicate no significant DNA damage was observed even after exposure to high doses of lanthanum nitrate (20 μg/ml). In conclusion, our findings suggest that lanthanum nitrate does not exhibit genotoxicity., Competing Interests: Declaration of Competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objectives: To compare the genotoxic effects of desflurane and propofol using comet assay in patients undergoing elective discectomy surgery., Methods: This was a randomized controlled study. Patients who underwent elective lumbar discectomy under general anesthesia with propofol or desflurane were included in the study. Venous blood samples were obtained at 4 different time points: 5 minutes before anesthesia induction (T1), 2 hours after the start of anesthesia (T2), the first day after surgery (T3), and the fifth day following surgery (T4). Deoxyribonucleic acid damage in lymphocytes was assessed via the comet assay., Results: A total of 30 patients, 15 in each group, were included in the analysis. The groups were similar in terms of age and gender distribution. There were no significant differences in demographics, duration of surgery, total remifentanil consumption, and total rocuronium bromide consumption. The comet assay revealed that head length, head intensity, tail intensity, tail moment at T1 were similar in the desflurane and propofol groups. Head length, tail length and tail moment measured in the desflurane group at T4 were significantly higher compared to the propofol group. Tail lengths of the desflurane group at T1, T2 and T3 were significantly higher than the corresponding values in the propofol group., Conclusion: Propofol and desflurane do not appear to induce DNA damage in lymphocytes. However, when the quantitative data were compared, it was determined that propofol had relatively lower genotoxic potential than desflurane. ClinicalTrials.gov Reg. No.: NCT05185167 ., (Copyright: © Saudi Medical Journal.)

Endometrial cancer is one of the most common invasive gynecologic malignancies in developed countries. The aim of this study was to evaluate chromosomal instability and level of DNA damage in peripheral blood mononuclear cells (PBMCs) of newly diagnosed endometrial cancer patients in relation to health status (diagnosis), age, histological grade of cancer, residence, smoking, number of pregnancies, miscarriages, and abortions. The analyzed sample consisted of 60 individuals, 30 endometrial cancer patients with an average age of 64.37 ± 7.08, and 30 healthy control women with an average age of 60.23 ± 11.55. Chromosomal instability was evaluated by the cytokinesis-block micronucleus (CBMN) assay, and the level of DNA damage by the single-cell gel electrophoresis (comet) assay in PBMCs. The average frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) as well as nuclear buds (NBUDs) were significantly higher in cancer patients compared to controls (P < .0005). There was no difference in the nuclear division index (NDI) among the analyzed samples. The comet assay showed that the patients had a significantly increased genetic damage index (GDI) compared with controls (P < .0005). Using linear regression analysis, we found that health status (diagnosis) had the strongest influence on the MN frequency as well as GDI (P < .0005). Our results indicated that there is a high level of genetic damage in both the level of DNA and the level of chromosomes in the PBMCs of newly diagnosed patients with endometrial cancer, where the frequency and level of damage were significantly affected by health status, grade of cancer, residence, number of pregnancies, miscarriages, and abortions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The risk of generating false positive in vivo comet assay results can be increased when procedural bias and/or technical variability is poorly controlled. This has been an ongoing concern since comet was first introduced into regulatory safety testing. But the proprietary nature of regulated studies and the 3Rs have limited the ability to conduct and publish the comparative in vivo studies necessary to determine the effect these factors can have on comet assay results when substances other than well characterized positive control compounds are evaluated in multiple tissues. That changed when Helix3 was asked to repeat for regulatory submission three independent in vivo comet studies with positive results generated by three other laboratories evaluating the effects of three different test substances on the liver, duodenum, and stomach. We repeated each study using the same test substance and experimental design as the original labs but with our standard quality control methods implemented to reduce procedural bias and variability. In every case, we generated negative results that regulatory authorities accepted over the initial positive results due to evidence of high technical variability and procedural bias in the original labs and studies. Meanwhile, the International Workshop on Genotoxicity (IWGT) compared >14 years of Helix3 comet historical control data (HCD) to HCD from 6 other experienced comet laboratories and concluded that our data exhibited the highest overall background % tail DNA levels with the lowest inter-study variability resulting in the highest quality HCD of all the labs evaluated. These case studies and the IWGT report suggest that our enhanced quality control methods and higher (>2 % mean of slide median tail DNA) background levels can effectively mitigate the nuisance factors that can generate false positive in vivo comet assay results. To facilitate a better understanding of the technical parameters that can significantly influence the comet results, we describe our enhanced procedures with justifications and examples., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marie Z. Vasquez reports a relationship with Helix3 Inc. that includes: employment and equity or stocks. Marie Z. Vasquez has patent #5624360315217 licensed to Helix3 Inc. Marie Z. Vasquez is on the editorial board of Mutation Research, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study compared genetic damage and immunological markers between surgical patients who underwent inhalational anesthesia with isoflurane or sevoflurane. Blood samples were collected from surgical patients (n = 18 in the isoflurane group and n = 17 in the sevoflurane group) at baseline (before the anesthesia procedure) and the day after anesthesia. DNA damage was detected using an alkaline comet assay; proinflammatory interleukin (IL)-6 was detected by flow cytometry, and white blood cells were detected via an automatic hematology analyzer. The characteristics of both groups were similar, and neither of the two anesthetics induced DNA damage. Similarly, mild neutrophilia was observed after anesthesia in both groups. Increased IL-6 levels were observed 1 day after anesthesia regardless of the type of anesthetic, but this increase was greater in the isoflurane group. Our study suggested that isoflurane and sevoflurane administration may contribute to changes in the immune parameters measured, though no genotoxic hazard was identified, in healthy adult patients who undergo low-stress surgery., (© 2024 Environmental Mutagenesis and Genomics Society.)

The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different test items. The corresponding OECD Test guideline 489 highlights the importance of statistical analyses and historical control data (HCD) but does not provide detailed procedures. Therefore, the working group "Statistics" of the German-speaking Society for Environmental Mutation Research (GUM) collected HCD from five laboratories and >200 comet assay studies and performed several statistical analyses. Key results included that (I) observed large inter-laboratory effects argue against the use of absolute quality thresholds, (II) > 50% zero values on a slide are considered problematic, due to their influence on slide or animal summary statistics, (III) the type of summarizing measure for single-cell data (e.g., median, arithmetic and geometric mean) may lead to extreme differences in resulting animal tail intensities and study outcome in the HCD. These summarizing values increase the reliability of analysis results by better meeting statistical model assumptions, but at the cost of information loss. Furthermore, the relation between negative and positive control groups in the data set was always satisfactorily (or sufficiently) based on ratio, difference and quantile analyses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic exposure to high (20,000 ppm) concentrations of tert-butyl alcohol (TBA) in drinking water, equivalent to ~2100 mg/kg bodyweight per day, is associated with slight increases in the incidence of thyroid follicular cell adenomas and carcinomas in mice, with no other indications of carcinogenicity. In a recent toxicological review of TBA, the U.S. EPA determined that the genotoxic potential of TBA was inconclusive, largely based on non-standard studies such as in vitro comet assays. As such, the potential role of genotoxicity in the mode of action of thyroid tumors and therefore human relevance was considered uncertain. To address the potential role of genotoxicity in TBA-associated thyroid tumor formation, CD-1 mice were exposed up to a maximum tolerated dose of 1500 mg/kg-day via oral gavage for two consecutive days and DNA damage was assessed with the comet assay in the thyroid. Blood TBA levels were analyzed by headspace GC-MS to confirm systemic tissue exposure. At study termination, no significant increases (DNA breakage) or decreases (DNA crosslinks) in %DNA tail were observed in TBA exposed mice. In contrast, oral gavage of the positive control ethyl methanesulfonate significantly increased %DNA tail in the thyroid. These findings are consistent with most genotoxicity studies on TBA and provide mechanistic support for non-linear, threshold toxicity criteria for TBA. While the mode of action for the thyroid tumors remains unclear, linear low dose extrapolation methods for TBA appear more a matter of policy than science., (© 2024 Environmental Mutagenesis and Genomics Society.)

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hydroxycoumarins are an important source of biologically active compounds. Previous studies have shown that the number and position of the hydroxyl substituents in the scaffold play an important role for the observed biological activity. In the present study, 3-(3-hydroxyphenyl)-7-hydroxycoumarin was synthesized, and potential cytogenotoxic effects determined in human HepG2/C3A cells displaying phase 1 and phase 2 enzymes (metabolizing cell ability) and compared to human peripheral blood mononuclear cells (PBMC) without xenobiotics metabolizing capacity. Cell viability was determined with concentrations between 0.01 and 10 µg/ml of 3-(3-hydroxyphenyl)-7-hydroxycoumarin using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and trypan blue tests. Genotoxicity was determined utilizing the comet assay, and the clastogenic/aneugenic potential employing the micronucleus (MN) test. The results of the in vitro cytotoxicity assays showed a significant decrease in cell viability of PBMC following exposure to 10 µg/ml concentration of the studied compound after 48 and 72 hr. Comet assay observations noted significant DNA damage in PBMC after 4 hr treatment. No marked cytogenotoxic effects were found in HepG2/C3A cells. No chromosomal mutations were observed in both cell lines. It is important to note that 3-(3-hydroxyphenyl)-7-hydroxycoumarin may exert beneficial pharmacological actions at the low micromolar range and with half-life less than 24 hr. Therefore, the results obtained encourage the continuation of studies on this new molecule for medicinal purposes, but its potential toxicity at higher concentrations and longer exposure times needs to be investigated in further studies.

Genotoxicity of styrene monomer was evaluated in male Fischer 344 rats using the alkaline comet assay for DNA damage, micronucleus assay for cytogenetic damage and the Pig-a assay for gene mutations. In a dose range finding (DRF) study, styrene was administered by oral gavage in corn oil for 28 consecutive days at 0, 100, 500, and 1000 mg/kg/day. The bioavailability of styrene was confirmed in the DRF by measuring its plasma levels at approximately 7- or 15-min following dosing. The 1000 mg/kg/day group exceeded the maximum tolerated dose based on body weight and organ weight changes and signs of central nervous system depression. Based on these findings, doses of 0, 100, 250, and 500 mg/kg/day (for 28 or 29 days) were selected for the genotoxicity assays. Animals were sacrificed 3-4 h after treatment on Day 28 or 29 for assessing various genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in peripheral blood erythrocytes. The comet assay was conducted in the glandular stomach, duodenum, liver, lung, and kidney. These studies were conducted in accordance with the relevant OECD test guidelines. Oral administration of styrene did not lead to genotoxicity in any of the investigated endpoints. The adequacy of the experimental conditions was assured by including animals treated by oral gavage with the positive control chemicals ethyl nitrosourea and ethyl methane sulfonate. Results from these studies supplement to the growing body of evidence suggesting the lack of in vivo genotoxic potential for styrene., (© 2024 Styrene Information and Research Center. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

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 3 , 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 (H 2 O 2, 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., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The preservation of the nuclear genome's integrity is paramount for the viability and overall health of cells, tissues, and organisms. DNA, being susceptible to damage under physiological conditions and vulnerable to both endogenous and environmental factors, faces constant threats. To assess DNA damage and repair within individual eukaryotic cells, the comet assay presents itself as a versatile, gel electrophoresis-based, relatively simple, and highly sensitive method. Originally designed to monitor DNA damage and repair within populations of mammalian cells, the comet assay has now found applications across diverse domains, including yeast, protozoa, plants, and invertebrates. This technique has proven invaluable in cryopreservation studies, serving as a valuable adjunct for determining suitable cryopreservation protocols. These protocols encompass choices related to cryoprotectants, sample preparation, as well as storage conditions in terms of time and temperature. In the realm of animal cryopreservation research, the comet assay stands as a gold-standard method for assessing DNA integrity. Nevertheless, when applied in plant-oriented investigations, additional efforts are essential due to the distinct nature of plant cells and associated technical challenges. This review elucidates the fundamental principles underlying the comet assay, discusses its current iterations, and delineates its applications in the cryopreservation of both animal and plant specimens. Moreover, we delve into the primary challenges confronting the comet assay's utility as a monitoring tool in the context of plant sample cryopreservation. https://doi.org/10.54680/fr24110110112.

Background and Objectives: In order to detect genetic damage, different methods have been developed, such as micronuclei and comet assay. The comet assay presents some advantages when compared to the other aforementioned methods, including wide versatility, as any eukaryotic cell can be evaluated at an individual cellular level. In this context, the aim of this systematic review was designed to help further elucidate the following question: is the comet assay a suitable biomarker of in vivo oral carcinogenesis?, Material and Methods: The present systematic review was performed in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. Full manuscripts from 18 studies were carefully selected in this setting., Results: A total of 15 studies demonstrated positive findings for genotoxicity in peripheral blood or oral cells in patients with pre-malignant lesions or oral cancer. In the quality assessment of studies, 1 was classified as Strong, 5 were considered as Moderate, and 12 were classified as Weak., Conclusion: In summary, the comet assay can be a useful biomarker for oral carcinogenesis. However, further studies with more strict parameters are suggested (with less uncontrolled confounders) in order to increase findings reliability for diagnosis of oral potentially malignant lesions.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents., (© 2023 Environmental Mutagenesis and Genomics Society.)

There is concern about human exposure to nanoplastics from intentional use or degradation of plastics in the environment. This review assesses genotoxic effects of nanoplastics, defined as particles with a primary size of less than 1000 nm. The majority of results on genotoxicity come from studies on polystyrene (PS) particles in mammalian cell cultures. Most studies have measured DNA strand breaks (standard comet assay), oxidatively damaged DNA (Fpg-modified comet assay) and micronuclei. Twenty-nine out of 60 results have shown statistically significant genotoxic effects by PS exposure in cell cultures. A statistical analysis indicates that especially modified PS particles are genotoxic (odds ratio = 8.6, 95 % CI: 1.6, 46) and immune cells seems to be more sensitive to genotoxicity than other cell types such as epithelial cells (odds ratio = 8.0, 95 % CI: 1.6, 39). On the contrary, there is not a clear association between statistically significant effects in genotoxicity tests and the primary size of PS particles, (i.e. smaller versus larger than 100 nm) or between the type of genotoxic endpoint (i.e. repairable versus permanent DNA lesions). Three studies of PS particle exposure in animals have shown increased level of DNA strand breaks in leukocytes and prefrontal cortex cells. Nanoplastics from polyethylene, propylene, polyvinyl chloride and polyethylene terephthalate have been investigated in very few studies and it is currently not possible to draw conclusion about their genotoxic hazard. In summary, there is some evidence suggesting that PS particles may be genotoxic in mammalian cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

Male B6C3F1 mice were administered styrene monomer by oral gavage for 29 consecutive days at dose levels of 0, 75, 150, or 300 mg/kg/day. The highest dose level represented the maximum tolerated dose based on findings in a 28-day dose range-finding study, in which the bioavailability of orally administered styrene was also confirmed. The positive control group received ethyl nitrosourea (ENU; 51.7 mg/kg/day) on Study Days 1-3 and ethyl methanesulfonate (EMS; 150 mg/kg/day) on Study Days 27-29 by oral gavage. Approximately 3 h following the final dose, blood was collected to assess erythrocyte Pig-a mutant and micronucleus frequencies. DNA strand breakage was assessed in glandular stomach, duodenum, kidney, liver, and lung tissues using the alkaline comet assay. The %tail DNA for stomach, liver, lung, and kidney in the comet assay among the styrene-treated groups was neither significantly different from the respective vehicle controls nor was there any dose-related increasing trend in any of the tissues; results for duodenum were interpreted to be inconclusive because of technical issues. The Pig-a and micronucleus frequencies among styrene-treated groups also did not show significant increases relative to the vehicle controls and there was also no evidence for a dose-related increasing trend. Thus, orally administered styrene did not induce DNA damage, mutagenesis, or clastogenesis/aneugenesis in these Organization of Economic Co-operation and Development test guideline-compliant genotoxicity studies. Data from these studies can contribute to the overall assessment of genotoxic hazard and risk posed to humans potentially exposed to styrene., (© 2023 Styrene Information and Research Center. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets.

The in vitro micronucleus (MN) assay is a component of most test batteries used in assessing potential genotoxicity. Our previous study adapted metabolically competent HepaRG cells to the high-throughput (HT) flow-cytometry-based MN assay for genotoxicity assessment (Guo et al. in J Toxicol Environ Health A 83:702-717, 2020b, https://doi.org/10.1080/15287394.2020.1822972 ). We also demonstrated that, compared to HepaRG cells grown as two-dimensional (2D) cultures, 3D HepaRG spheroids have increased metabolic capacity and improved sensitivity in detecting DNA damage induced by genotoxicants using the comet assay (Seo et al. in ALTEX 39:583-604, 2022, https://doi.org/10.14573/altex.22011212022 ). In the present study, we have compared the performance of the HT flow-cytometry-based MN assay in HepaRG spheroids and 2D HepaRG cells by testing 34 compounds, including 19 genotoxicants or carcinogens and 15 compounds that show different genotoxic responses in vitro and in vivo. 2D HepaRG cells and spheroids were exposed to the test compounds for 24 h, followed by an additional 3- or 6-day incubation with human epidermal growth factor to stimulate cell division. The results demonstrated that HepaRG spheroids showed generally higher sensitivity in detecting several indirect-acting genotoxicants (require metabolic activation) compared to 2D cultures, with 7,12-dimethylbenzanthracene and N-nitrosodimethylamine inducing higher % MN formation along with having significantly lower benchmark dose values for MN induction in 3D spheroids. These data suggest that 3D HepaRG spheroids can be adapted to the HT flow-cytometry-based MN assay for genotoxicity testing. Our findings also indicate that integration of the MN and comet assays improved the sensitivity for detecting genotoxicants that require metabolic activation. These results suggest that HepaRG spheroids may contribute to New Approach Methodologies for genotoxicity assessment., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility., (© 2023. Springer Nature Limited.)

The comet assay is an effective method for identifying DNA breaks and alkali-labile sites induced by genotoxins. Performed as a single-cell electrophoresis, this assay is especially simplistic, and the results are easily reproducible. DNA breakage can be quantitatively assessed by the induced comet tail regions, which can be measured using a variety of comet software. This protocol will finish within approximately two hours with adequate preparation, and digitized images can be taken using a confocal or standard fluorescence microscopes after staining the cell nucleus with a DNA dye., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful., Competing Interests: Conflict of interest All authors declare no conflict of interest., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

The purpose of this chapter is to evaluate DNA damage during axolotl tail regeneration using an alkaline comet assay. Our method details the isolation of cells from regenerating and non-regenerating tissues and the isolation of peripheral blood for single-cell gel electrophoresis. Also, we detail each of the steps for the development of the comet assay technique which includes mounting the isolated cells on an agarose matrix, alkaline electrophoresis, and DNA damage detection., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bisguaiacols, lignin-derivable bisphenols, are considered promising and possibly safer alternatives to bisphenol A (BPA), but comprehensive toxicity investigations are needed to ensure safety. Most toxicity studies of BPA and its analogues have focused on potential estrogenic activity, and only limited toxicological data are available on other toxicity aspects, such as genotoxicity at low exposure levels. In this study, the genotoxicity of six lignin-derivable bisguaiacols with varying regioisomer contents and degrees of methoxy substitution was investigated using a multi-tiered method, consisting of in silico simulations, in vitro Ames tests, and in vivo comet tests. The toxicity estimation software tool, an application that predicts toxicity of chemicals using quantitative structure-activity relationships, calculated that the majority of the lignin-derivable bisguaiacols were non-mutagenic. These results were supported by Ames tests using five tester strains (TA98, TA100, TA102, TA1535, and TA1537) at concentrations ranging from 0.5 pmol/plate to 5 nmol/plate. The potential genotoxicity of bisguaiacols was further evaluated using in vivo comet testing in fetal chicken livers, and in addition to the standard alkaline comet assay, the formamidopyrimidine DNA glycosylase enzyme-modified comet assay was employed to investigate oxidative DNA damage in the liver samples. The oxidative stress analyses indicated that the majority of lignin-derivable analogues showed no signs of mutagenicity (mutagenic index < 1.5) or genotoxicity, in comparison to BPA and bisphenol F, likely due to the methoxy groups on the lignin-derivable aromatics. These findings reinforce the potential of lignin-derivable bisphenols as safer alternatives to BPA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Single-cell gel electrophoresis (SCGE or Comet assay) and the Fast Halo assay, also known as the Halo assay, are powerful tools to generate DNA damage measurements with single-cell resolution. Though these techniques are prone to have variability, they can be robust tools for quantifying DNA damage when planned and executed carefully. Here, we present both assays and highlight each technique's advantages and challenges in measuring DNA damage in cells with limiting cell number, such as hematopoietic stem cells (HSCs). The Comet assay is highly sensitive at the cost of increased variability. The Halo assay attenuates some of the effects of variability present in the Comet assay but does not eliminate them entirely and is less sensitive. Overall, the Comet and Halo assays are powerful means of directly measuring DNA damage. We recommend the below methods for detecting damage in hematopoietic stem cells, but the methods can easily be adjusted for measuring damage in any type of single cells in suspension., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Carboplatin is amongst the most commonly used anticancer drugs for the management of several human malignancies. However, it has displayed genotoxic properties against normal cells. Evaluation of natural products for their protective effects against chemotherapeutic drug induced toxicity has been growing in recent years. A naturally occurring flavonoid, chrysin, has strong antioxidant abilities and protects against DNA impairment. This study used multiple assays to evaluate the levels of damage to DNA in normal cells and to examine any possible protective role of chrysin against such damage. Male BALB/c mice were administered chrysin orally in two doses of 20 and 40 mg/kg for 10 consecutive days and then a single injection of carboplatin [90 mg/kg body weight (b.w.)] was administered intraperitoneally to induce carboplatin toxicity. 24 h after the carboplatin injection, mice were sacrificed. DNA damage was evaluated using several genotoxicity tests (8-Hydroxydeoxy-guanosine marker, comet assay, micronucleus test, and chromosomal aberration assay) to identify diverse types of damage to the DNA. The results suggest that pretreatment with chrysin significantly decreased the level of DNA damage caused by carboplatin probably due to its potent antioxidant traits. Therefore, chrysin can be considered to be developed as a chemoprotective agent against chemotherapy associated side-effects.

DNA damage is an established initiating event in the mutagenicity and carcinogenicity of genotoxic chemicals. Accordingly, assessment of this endpoint is critical for chemicals which are being developed for use in humans. To assess the ability of the Chicken Egg Genotoxicity Assay (CEGA) to detect genotoxic pharmaceuticals, a set of 23 compounds with different pharmacological and reported genotoxic effects was tested for the potential to produce nuclear DNA adducts and strand breaks in the embryo-fetal livers using the 32 P-nucleotide postlabeling (NPL) and comet assays, respectively. Due to high toxicity, two aneugens, colchicine and vinblastine, and an autophagy inhibitor, hydroxychloroquine, could not be evaluated. Out of the 20 remaining pharmaceuticals, 10 including estrogen modulators, diethylstilbestrol and tamoxifen, antineoplastics cyclophosphamide, etoposide, and mitomycin C, antifungal griseofulvin, local anesthetics lidocaine and prilocaine, and antihistamines diphenhydramine and doxylamine, yielded clear positive outcomes in at least one of the assays. The antihypertensive vasodilator hydralazine and antineoplastics streptozotocin and teniposide, produced only DNA strand breaks, which were not dose-dependent, and thus, the results with these 3 pharmaceuticals were considered equivocal. No DNA damage was detected for 7 compounds, including the purine antagonist 6-thioguanine, antipyretic analgesics acetaminophen and phenacetin, antibiotic ciprofloxacin, antilipidemic clofibrate, anti-inflammatory ibuprofen, and sedative phenobarbital. However, low solubility of these compounds limited dosages tested in CEGA. Overall, results in CEGA were largely in concordance with the outcomes in other systems in vitro and in vivo, indicating that CEGA provides reliable detection of DNA damaging activity of genotoxic compounds. Further evaluations with a broader set of compounds would support this conclusion.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

Bisphenol A (BPA) is one of the environmental endocrine disrupting toxicants and is widely used in the industry involving plastics, polycarbonate, and epoxy resins. This study was designed to investigate the toxicological effects of BPA on hematology, serum biochemistry, and histopathology of different organs of common carp ( Cyprinus carpio ). A total of 60 fish were procured and haphazardly divided into four groups. Each experimental group contained 15 fish. The fish retained in group A was kept as the untreated control group. Three levels of BPA 3.0, 4.5, and 6 mg/L were given to groups B, C, and D for 30 days. Result indicated significant reduction in hemoglobin (Hb), lymphocytes, packed cell volume (PCV), red blood cells (RBC), and monocytes in a dose-dependent manner as compared to the control group. However, significantly higher values of leucocytes and neutrophils were observed in the treated groups ( P < 0.05). Results on serum biochemistry revealed that the quantity of glucose, cholesterol, triglycerides, urea, and creatinine levels was significantly high ( P < 0.05). Our study results showed significantly ( P < 0.05) increase level of oxidative stress parameters like reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and lower values of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) in treated groups (4.5 mg/L and 6 mg/L)) in the brain, liver, gills, and kidneys. Our study depicted significant changes in erythrocytes (pear shaped erythrocytes, leptocytes, microcytes, spherocytes, erythrocytes with broken, lobed, micronucleus, blabbed, vacuolated nucleus, and nuclear remnants) among treated groups (4.5 mg/L and 6 mg/L). Comet assay showed increased genotoxicity in different tissues including the brain, liver, gills, and kidneys in the treated fish group. Based on the results of our experiment, it can be concluded that the BPA exposure to aquatic environment is responsible for deterioration of fish health, performance leading to dysfunction of multiple vital organs., Competing Interests: All authors declare no potential conflict and no competing interest., (Copyright © 2022 Gulnaz Afzal et al.)

Background: Nowadays bodybuilders use anabolic steroids frequently. Abuse of these substances can cause significant side effects; therefore, we aim to investigate the effect of anabolic steroids on DNA damage in bodybuilders' blood lymphocytes., Methods and Materials: This case-control study was performed on 36 male bodybuilders in Gonabad. The case group included bodybuilders with a history of taking anabolic-androgenic steroids ( n  = 18), and the control group composed of bodybuilders who did not use anabolic-androgenic steroids ( n  = 18). Intravenous blood samples were obtained and then the lymphocytes, cells and electrophoresis of blood were extracted. Afterward, the coloured slides and DNA damage were measured using a fluorescent microscope and CometScore software. The DNA damage was compared using t -tests ., Results: Results showed that there was no significant difference between age, marital status, BMI, systolic and diastolic blood pressure in the case and control group. However, parameters related to the DNA damage including tail length, percent tail DNA, and tail moment were significantly higher in the case group., Conclusion: The use of anabolic-androgenic steroids increases DNA damage in the bodybuilders' blood lymphocytes.

We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The in vivo comet assay can evaluate the genotoxic potential of a chemical in theoretically any tissue that can be processed to a single cell suspension. This flexibility enables evaluation of point-of-contact tissues using a relevant route of test material administration; however, assessing cytotoxicity is essential for the interpretation of comet results. Histopathological evaluation is routinely utilized to assess cytotoxicity, but temporal- and cell-specific considerations may compromise applicability to the comet assay. In the present study, 1,1'-methylenebis(4-isocyanatobenzene) (4,4'-MDI) was administered to rats for 6 h by nose-only inhalation, and the comet assay was conducted to evaluate genotoxicity in the site-of-contact tissue (bronchoalveolar lavage cells) and distal tissues (liver and glandular stomach). Given the reactive nature of MDI, cellular and molecular metrics at the site-of-contact- including inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress- were used to set appropriate exposure concentrations, in addition to the standard systemic measures of toxicity. In the range-finding study, a concentration of 4 mg/m 3 was considered the maximum noninflammatory concentration; hence target concentrations of 2, 5, and 11 mg/m 3 were selected for the comet study. In the lung lavage, MDI exposure substantially increased total protein and β-glucuronidase, along with cellular apoptosis. Although MDI did not increase the comet assay response (% tail DNA) in any of the tissues examined, the positive control (ethyl methanesulfonate, EMS) significantly increased % tail DNA in all tissues. In total, these data indicate that appropriate cellular and molecular measurements may facilitate dose selection to discern cellular status in the comet assay., (© 2021 The Dow Chemical Company. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society.)

DNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities., (Copyright © 2021. Published by Elsevier B.V.)

The response to DNA damage is the mechanism that allows the interaction between stress signals, inflammatory secretions, DNA repair, and maintenance of cell and tissue homeostasis. Adipocyte dysfunction is the cellular trigger for various disease states such as insulin resistance, diabetes, and obesity, among many others. Previously, our group demonstrated that adipogenesis per se, from mesenchymal/stromal stem cells derived from human adipose tissue (hASCs), involves an accumulation of DNA damage and a gradual loss of the repair capacity of oxidative DNA damage. Therefore, our objective was to identify whether healthy adipocytes differentiated for the first time from hASCs, when receiving inflammatory signals induced with TNFα, were able to persistently activate the DNA Damage Response and thus trigger adipocyte dysfunction. We found that TNFα at similar levels circulating in obese humans induce a sustained response to DNA damage response as part of the Senescence-Associated Secretory Phenotype. This mechanism shows the impact of inflammatory environment early affect adipocyte function, independently of aging.

Comet assay is a standard approach for studying DNA damage in malaria, but high-throughput options are not available. The CometChip was previously developed using mammalian cells as a high-throughput version of the comet assay. It is based on the same principle as the comet assay but provides greater efficacy, automated data processing, and improved consistency between experiments. In this protocol, we present MalariaCometChip to quantitatively assess drug-induced DNA damage in Plasmodium falciparum. For complete details on the use and execution of this protocol, please refer to Xiong et al. (2020)., Competing Interests: B.P.E. is a co-inventor on a patent for the CometChip., (© 2021 The Authors.)

The abusive consumption of thermogenic supplements occurs worldwide and deserves special attention due to their use to stimulate weight loss and prevent obesity. Thermogenic formulations usually contain Synephrine (SN) and Caffeine (CAF), stimulating compounds extracted from natural sources, but no genetic toxicology studies have predicted this hazardous combination potential. This study examined the toxicogenomic responses induced by SN and CAF, either alone or in combination, in the human hepatic cell line HepG2 in vitro. SN (0.03-30 μM) and CAF (0.6-600 μM) alone did neither decrease cell viability nor induce DNA damage, as assessed using the MTT and comet assays, respectively. SN (3 μM) and CAF (30-600 μM) were combined at concentrations similar to those found in commercial dietary supplements. SN/CAF at 3:90 and 3:600 μM ratios significantly decreased cell viability and increased DNA damage levels in HepG2 cells. CAF (600 μM) and the SN/CAF association at 3:60, 3:90, and 3:600 μM ratios promoted cell death by apoptosis, as demonstrated by flow cytometry. Similar results were observed in gene expression (RT-qPCR): SN/CAF up-regulated the expression of apoptosis- (BCL-2 and CASP9) and DNA repair-related (XPC) genes. SN/CAF at 3:90 μM also downregulated the expression of cell cycle control (CDKN1A) genes. In conclusion, the SN/CAF combination reduces cell viability by inducing apoptosis, damages DNA, and modulates the transcriptional expression of apoptosis-, cell cycle-, and DNA repair-related genes in human hepatic (HepG2) cells in vitro. These effects can be worrisome to consumers of thermogenic supplements., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage., (© 2021. The Author(s).)

Aloe-emodin, one of the molecules belonging to the group of hydroxyanthracene derivatives, was recently described as genotoxic in vivo. Indeed, the EFSA judged that aloe-emodin, together with other similar molecules (emodin and danthron) and extracts from the leaf of Aloe species containing hydroxyanthracene derivatives, could represent a risk factor for colorectal cancer mediated by a genotoxic effect. Given the marked uncertainty regarding the conclusions in the opinion of the EFSA ANS Panel and conflicts in the epidemiological data on which the opinion is based, a new in vivo study (in vivo alkaline comet assay in mice - OECD 489) was conducted to test the potential genotoxicity of aloe-emodin at doses of 250, 500, 1000 and 2000 mg/kg bw/day on preparations of single cells from the kidney and colon of treated male mice. Following treatment with the test item, no clinical signs were observed in animals in any treatment group. Slight body-weight loss was randomly observed in all groups treated with the test item and was more evident in the groups dosed at 1000 and 2000 mg/kg bw/day. Under these experimental conditions, aloe-emodin showed no genotoxic activity. Possible oxidative damage to colon tissues could not be excluded based on the results obtained after repair enzyme treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

DNA integrity is considered an important parameter of semen quality and is of significant value as a predictor of male fertility. Currently, there are several methods that can assess sperm DNA integrity. One such assay is the comet assay, or single-cell gel electrophoresis, which is a simple, sensitive, reliable, quick and low-cost technique that is used for measuring DNA strand breaks and repair at the level of individual cells. Although the comet assay is usually performed with somatic cells from different organs, the assay has the ability to detect genotoxicity in germ cells at different stages of spermatogenesis. Since the ability of sperm to remove DNA damage differs between the stages, interpretation of the results is dependent on the cells used. In this paper we give an overview on the use and applications of the comet assay on mature sperm and its ability to detect sperm DNA damage in both animals and humans. Overall, it can be concluded that the presence in sperm of significantly damaged DNA, assessed by the comet assay, is related to male infertility and seems to reduce live births. Although there is some evidence that sperm DNA damage also has a long-term impact on offspring's health, this aspect of DNA damage in sperm is understudied and deserves further attention. In summary, the comet assay can be applied as a useful tool to study effects of genotoxic exposures on sperm DNA integrity in animals and humans., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mining has a direct impact on the environment and on the health of miners and is considered one of the most hazardous occupations worldwide. Miners are exposed to several occupational health risks, including genotoxic substances, which may cause adverse health effects, such as cancer. This review summarizes the relation between DNA damage and mining activities, focusing on coal and uranium miners. The search was performed using electronic databases, including original surveys reporting genetic damage in miners. Additionally, a temporal bibliometric analysis was performed using an electronic database to create a map of cooccurrence terms. The majority of studies were performed with regard to occupational exposure to coal, whereas genetic damage was assessed mainly through chromosomal aberrations (CAs), micronuclei (MNs) and comet assays. The bibliometric analysis demonstrated associations of coal exposure with silicosis and pneumoconiosis, uranium miners with lung cancer and tumors and some associated factors, such as age, smoking, working time and exposure to radiation. Significantly higher DNA damage in miners compared to nonexposed groups was observed in most of the studies. The timeline reveals that classic biomarkers (comet assay, micronucleus test and chromosomal aberrations) are still important tools to assess genotoxic/mutagenic damage in occupationally exposed miners; however, newer studies concerning genetic polymorphisms and epigenetic changes in miners are being conducted. A major challenge is to investigate further associations between miners and DNA damage and to encourage further studies with miners of other types of ores., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Comet assay, applied to in vitro, in vivo and ex vivo systems, is a quick, simple, and sensitive method for the detection of genotoxicity. In general, fresh whole blood or peripheral blood mononuclear cells (PBMCs) are used in the assay for the determination of DNA damage and repair. In this study, the effects of storage conditions on genotoxicity assessed by Comet assay in human whole blood and lymphocyte samples, were evaluated. Whole blood and lymphocyte samples were stored at 4 °C for 1, 2, 3, 4, 5 and 7 days; at -20 °C for 1 month and at -80 °C for 3, 6 and 12 months. 1% DMSO was used as cryoprotectant. No significant differences in DNA damage were demonstrated in all of the storage conditions and durations, and the results were similar according to the median values (p < 0.05). According to Spearman or Pearson correlations, an important correlation was found between the DNA damage of the fresh samples and the samples which were kept at -80 °C for 6 months with temperature and time (p < 0.01 for Pearson and p < 0.05 for Spearman). The results of this study indicated that blood and lymphocyte samples stored in +4 °C, -20 °C and -80 °C up to 12 months can be used instead of fresh samples especially in human biomonitoring studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

About 40 million workers are occupationally exposed to crystalline silica (CS) which was classified as a human carcinogen by the IARC. It is assumed that damage of the genetic material via inflammation and reactive oxygen species by CS lead to formation of malignant cells. We conducted a systematic literature search to find out if inhalation of CS containing dusts at workplaces causes damage of the genetic material. Thirteen studies were found eligible for this review, in most of them (n = 9) micronuclei (MN) which reflect structural/numerical chromosomal aberrations were monitored in lymphocytes and/or in exfoliated buccal cells. In 5 investigations DNA damage was measured in blood cells in single cell gel electrophoresis (comet) experiments. Frequently studied groups were potters, stone cutters, miners and construction workers. Results of meta-analyses show that exposure to CS causes formation of MN and DNA breaks, the overall ratio values were in exposed workers 2.06- and 1.96-fold higher than in controls, respectively. Two studies reported increased levels of oxidized guanine, and higher levels of DNA adducts with malondialdehyde indicating that exposure to CS leads to oxidative damage. The exposure of the workers to CS was quantified only in two studies, information concerning the size and chemical structures of the particles is lacking in most investigations. Therefore, it is not possible to use the results to derive occupational exposure limits of workers to CS which vary strongly in different countries. Nevertheless, the evaluation of the current state of knowledge shows that biomonitoring studies in which damage of the genetic material is measured in CS exposed workers can contribute to assess adverse health effects as consequence of DNA instability in specific occupations., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The enzyme-modified comet assay was developed in order to detect DNA lesions other than those detected by the standard version (single and double strand breaks and alkali-labile sites). Various lesion-specific enzymes, from the DNA repair machinery of bacteria and humans, have been combined with the comet assay, allowing detection of different oxidized and alkylated bases as well as cyclobutane pyrimidine dimers, mis-incorporated uracil and apurinic/apyrimidinic sites. The enzyme-modified comet assay has been applied in different fields - human biomonitoring, environmental toxicology, and genotoxicity testing (both in vitro and in vivo) - as well as in basic research. Up to now, twelve enzymes have been employed; here we describe the enzymes and give examples of studies in which they have been applied. The bacterial formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII) have been extensively used while others have been used only rarely. Adding further enzymes to the comet assay toolbox could potentially increase the variety of DNA lesions that can be detected. The enzyme-modified comet assay can play a crucial role in the elucidation of the mechanism of action of both direct and indirect genotoxins, thus increasing the value of the assay in the regulatory context., (Copyright © 2020 Elsevier Ltd. All rights reserved.)