In the event of a large-scale incident involving radiological or nuclear exposures, there is a potential for large numbers of individuals to have received doses of radiation sufficient to cause adverse health effects. It is imperative to quickly identify these individuals in order to provide information to the medical community to assist in making decisions about their treatment. The cytokinesis-block micronucleus assay is a well-established method for performing biodosimetry. This assay has previously been adapted to imaging flow cytometry and has been validated as a high-throughput option for providing dose estimates in the range of 0-10 Gy. The goal of this study was to test the ability to further optimize the assay by reducing the time of culture to 48 h from 68 h as well as reducing the volume of blood required for the analysis to 200 μL from 2 mL. These modifications would provide efficiencies in time and ease of processing impacting the ability to manage large numbers of samples and provide dose estimates in a timely manner. Results demonstrated that either the blood volume or the culture time could be reduced while maintaining dose estimates with sufficient accuracy for triage analysis. Reducing both the blood volume and culture time, however, resulted in poor dose estimates. In conclusion, depending on the needs of the scenario, either culture time or the blood volume could be reduced to improve the efficiency of analysis for mass casualty scenarios.

OBJECTIVE: To determine the genetic effects of panoramic radiography on the epithelial cells of the buccal mucosa by examining the micronucleus formation in these cells.
METHODS: In this cross-sectional study, exfoliative cytology samples were prepared from the buccal mucosa of 36 patients immediately before and 10 days after panoramic radiography. The samples were prepared using liquid-based cytology with Papanicolaou staining. The slides were simultaneously evaluated by two expert pathologists and the ratio of the number of cells with micronuclei to the total number of cells on the slide was reported as a percentage. Data analysis was done using paired-samples T test, Pearson\'s correlation coefficient, and covariance analysis (α = 0.05).
RESULTS: The study sample consisted of 24 (66.67%) males and 12 females (33.33%) with a mean (SD) age of 27.36 (8.19) years. The frequency of cells with micronucleus before and after panoramic radiography was not statistically different (p = 0.468). Additionally, the frequency of micronucleated cells was not correlated with age (p = 0.737) and sex (p = 0.211).
CONCLUSIONS: Panoramic exposure slightly increased the frequency of cells with micronucleus in epithelial cells of the buccal mucosa. However, this increase was not statistically significant.

Metformin is a biguanide currently used in the treatment of diabetes mellitus type 2. Besides its anti-glycemic effects, metformin has been reported to induce different cellular pleiotropic effects, depending on concentration and time of treatment. Here we report one administration of metformin (0.5 mM) has radioprotective effects in vitro on BJ human fibroblasts, increasing DNA damage repair and increasing SOD1 expression in the nucleus. Importantly, metformin (200 mg/kg) pre-administration for only 3 days in wild type 129/sv mice, decreases the formation of micronuclei in bone marrow cells and DNA damage in colon and lung tissues compared to control irradiated mice at sub-lethal and lethal doses, increasing the overall survival fraction by 37% after 10Gy total body irradiation. We next pre-treated with metformin and then exposed 129/sv mice, to a galactic cosmic rays simulation (GCRsim), at the NASA Space Radiation Laboratory (NSRL). We found metformin pre-treatment decreases the presence of bone marrow micronuclei and DNA damage in colon and lung tissues and an increase of 8-oxoguanine DNA glycosylase-1 (OGG1) expression. Our data highlight a radioprotective effect of metformin through an indirect modulation of the gene expression involved in the cellular detoxification rather than its effects on mitochondria.

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer\'s software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay\'s ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

OBJECTIVE: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo.
METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE.
RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment.
CONCLUSIONS: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.

Glufosinate-ammonium (GLA), an organophosphate herbicide, is released at high concentrations in the environment, leading to concerns over its potential genotoxic effects. However, few articles are available in the literature reporting the possible cellular and nuclear effects of this compound. We assessed, by in vitro and in vivo micronucleus assays, the genotoxicity of GLA on cultured human lymphocytes and Lymnaea stagnalis hemocytes at six concentrations: 0.010 (the established acceptable daily intake value), 0.020, 0.050, 0.100, 0.200, and 0.500 µg/mL. In human lymphocytes, our results reveal a significant and concentration-dependent increase in micronuclei frequency at concentrations from 0.100 to 0.500 μg/mL, while in L. stagnalis hemocytes, significant differences were found at 0.200 and 0.500 μg/mL. A significant reduction in the proliferation index was observed at all tested concentrations, with the only exception of 0.010 μg/mL, indicating that the exposure to GLA could lead to increased cytotoxic effects. In L. stagnalis, a significant reduction in laid eggs and body growth was also observed at all concentrations. In conclusion, we provided evidence of the genomic and cellular damage induced by GLA on both cultured human lymphocytes and a model organism\'s hemocytes; in addition, we also demonstrated its effects on cell proliferation and reproductive health in L. stagnalis.

Ivermectin (IVM) is an anti-parasitic drug which is used for treating parasitic infestations. It has been used in humans for treating intestinal strongyloidiasis and onchocerciasis however, currently researchers are investigating its potential for treating coronavirus SARS-CoV-2. Due to its broad-spectrum activities, IVM is being used excessively in animals which has generated an interest for researchers to investigate its toxic effects. Cytotoxic and genotoxic effects have been reported in animals due to excessive usage of IVM. Therefore, this study aims to evaluate the cytotoxic and genotoxic effects of IVM on the Madin-Darby-Bovine-Kidney (MDBK) cell line by examining the expression of a DNA damage-responsive gene (OGG1). Cytotoxicity of IVM was tested using an assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereas the genotoxicity was evaluated using comet assay along with micronucleus assay. Moreover, the gene expression of DNA damage response gene (OGG1) was measured by qRT-PCR, after extraction of RNA from the MDBK cell line using the TRIzol method and its conversion to cDNA by reverse-transcriptase PCR. During the experiment, cell viability percentage was measured at different doses of IVM i.e., 25%, 50%, 75%, along with LC50/2, LC50 and LC50*2. It was observed that the gene expression of OGG1 increased as the concentration of IVM increased. It was concluded that IVM has both cytotoxic and genotoxic effects on the MDBK cell line. Furthermore, it is recommended that studies related to the toxic effects of IVM at molecular level and on other model organisms should be conducted to combat its hazardous effects.

Zearalenone (ZEN) and deoxynivalenol (DON) and their derivatives are well-known mycotoxins, which can occur not only in crops but also in water bodies, including drinking water sources. In vitro bioassays can be used to detect biological effects of hazardous compounds in water. To this, when studying biological effects and toxicity in vitro, metabolism is important to consider. In this study, ZEN, α-zearalenol (α-ZEL), DON, 3-acetyl DON, and 15-acetyl DON were evaluated in vitro for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and genotoxicity (micronucleus assay) in the presence of an exogenous metabolic activation system (MAS). The ER bioassay proved to be a highly sensitive method to detect low concentrations of the ZEN compounds (EC10 values of 31.4 pM for ZEN, 3.59 pM for α-ZEL) in aqueous solutions. In the presence of the MAS, reduced estrogenic effects were observed for both ZEN compounds (EC10 values of 6.47 × 103 pM for ZEN, 1.55 × 102 pM for α-ZEL). Of the DON compounds, only 3-acetyl DON was estrogenic (EC10 of 0.31 µM), and the effect was removed in the presence of the MAS. Anti-androgenic effects of the ZEN compounds and androgenic effects of the DON compounds were detected in the micromolar range. No induction of genotoxicity was detected for ZEN or DON in the presence of the MAS. Our study highlighted that inclusion of exogenous MAS is a useful tool to detect biological effects of metabolites in in vitro bioassays.

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.

Micronuclei, detected through the cytokinesis-block micronucleus assay, are valuable indicators of ionizing radiation exposure, especially in short-term lymphocyte cultures. The peripheral human blood lymphocyte assay is recognized as a prime candidate for automated biodosimetry. In a prior project at the Columbia University Center for Radiological Research, we automated this assay using the 96-well ANSI/SLAS microplate standard format and relied on established biotech robotic systems named Rapid Automated Biodosimetry Tool (RABiT). In this study, we present the application of a similar automated biotech setup at an external high-throughput facility (RABiT-III) to implement the same automated cytokinesis-block micronucleus assay. Specifically, we employed the Agilent BRAVO liquid-handling system and GE IN Cell Analyzer 6000 imaging system in conjunction with the PerkinElmer Columbus image data storage and analysis system. Notably, this analysis system features an embedded PhenoLOGIC machine learning module, simplifying the creation of cell classification algorithms for CBMN assay image analysis and enabling the generation of radiation dose-response curves. This investigation underscores the adaptability of the RABiT-II CBMN protocol to diverse RABiT-III biotech robotic platforms in non-specialized biodosimetry centers. Furthermore, it highlights the advantages of machine learning in rapidly developing algorithms crucial for the high-throughput automated analysis of RABiT-III images.