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Chemical risk assessment has historically focused on single compounds, neglecting the implications of combined exposures. To bridge this gap, several methodologies, such as concentration addition (CA) and independent action (IA), have been developed. However, a systematic, consistent, and integrated approach across various legislative frameworks is still lacking. The assessment of combined effects of genotoxicants is even more challenging, as genotoxicity data are typically evaluated qualitatively, without considering the effect size. This study aimed to develop a quantitative approach for evaluating the combined effects of genotoxic compounds with both similar and dissimilar modes of action (MoA), based on the benchmark concentration (BMC) principle. A proof-of-concept study was conducted using the in vitro micronucleus (MNvit) test to examine two types of binary mixtures: ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS), which share similar MoA, and MMS and etoposide (ETP), which have dissimilar MoA. The methodology involved collecting data for individual compounds, calculating BMC values, composing mixtures with different ratios and inducing various effect levels, testing these mixtures, and comparing the experimental results with the modelled data to verify additivity. The findings indicated that for both mixtures, the experimental responses aligned with the predicted additive effects, supporting the validity of the additivity principle. This study highlights the potential of an optimized BMC-based approach as a robust framework for testing chemical mixtures. It should be adopted in future studies to evaluate a wider range of genotoxic compounds, offering a more comprehensive and quantitative strategy for assessing combined chemical exposures.

Methyl methanesulfonate (MMS), a genotoxic impurity, poses potential risks to human health due to its residues in pharmaceutical production, making its detection crucial. This study was based on the property that MMS can enhance the blue fluorescence of Eu-MOF. For the first time, we developed a molecular imprinting ratio fluorescent probe and test strip. The color change of the probe solution and test strip from pink to blue enables specific visual detection of MMS. Under optimal conditions, the fluorescence intensity ratio (I 453 /I 588 ) exhibited a good linear relationship with MMS concentration in the range of 1.0–100.0 μM, with a detection limit of 87.5 nM. Studies on sensing mechanism have revealed that the coordination between Eu and the sulfonic acid in MMS resulted in a weakened interaction between the ligand 2-aminoterephthalic acid and Eu metal clusters, thereby inhibiting photoinduced electron transfer (PET) and ultimately leading to enhanced fluorescence. Notably, this work provides a novel method for fluorescence-based quantitative detection of MMS in pharmaceutical wastewater, soil, and simulated human serum, as well as on-site visual detection using test strips. Graphical Abstract

The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H2O2) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20–30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H2O2 or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.

Retinal ex vivo systems have been widely used to investigate diverse physiological and pathological processes in the retina. Although retinal ex vivo systems are generally considered to approximate in vivo environment of the retina, careful evaluation of how ex vivo conditions affect cellular function is necessary for accurate interpretation of such data. Here we analyzed the responses of retinal Müller glia to photoreceptor injury in vivo and ex vivo to assess the effects of ex vivo culture on the phenotypes of Müller glia in pathological contexts. A rat model of photoreceptor injury was generated by intraperitoneal injection of methyl methanesulfonate (MMS) and retinal ex vivo cultures were prepared 1 or 2 days after MMS treatment. A substantial number of Müller glia die by apoptosis after injury-induced proliferation in vivo, but not ex vivo. While the pattern of Müller glia proliferation was similar between the models, the extent of phagocytic activity, migration, DNA damage response, and neurogenic factor expression varied across the models. These findings reveal previously unrecognized effects of ex vivo culture on Müller glia in disease contexts, highlighting the need for caution when assessing injury-induced phenotypes of Müller glia using retinal ex vivo systems.

Bat white-nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans , has decimated North American hibernating bats since its emergence in 2006. Here, we utilize comparative genomics to examine the evolutionary history of this pathogen in comparison to six closely related nonpathogenic species. P. destructans displays a large reduction in carbohydrate-utilizing enzymes (CAZymes) and in the predicted secretome (~50%), and an increase in lineage-specific genes. The pathogen has lost a key enzyme, UVE1, in the alternate excision repair (AER) pathway, which is known to contribute to repair of DNA lesions induced by ultraviolet (UV) light. Consistent with a nonfunctional AER pathway, P. destructans is extremely sensitive to UV light, as well as the DNA alkylating agent methyl methanesulfonate (MMS). The differential susceptibility of P. destructans to UV light in comparison to other hibernacula-inhabiting fungi represents a potential “Achilles’ heel” of P. destructans that might be exploited for treatment of bats with WNS. White-nose syndrome, caused by the fungus Pseudogymnoascus destructans , is decimating North American bats. Here, Palmer et al. use comparative genomics to examine the evolutionary history of this pathogen, and show that it has lost a crucial DNA repair enzyme and is extremely sensitive to UV light.

Micronuclei originate from DNA damage generated by clastogenic and/or by aneugenic effects. Depending on the pattern of damage, they may have distinct genomic origin and composition. Sequences of the centromere, telomere and rDNA have been identified in plant micronuclei. However, other DNA sequences may also be present in the micronuclei, as well as their DNA contents may be different. Here, we investigate the DNA content, genomic composition and origin of micronuclei induced in Zea mays by methyl methanesulfonate (MMS). DNA contents showed a wide range of distribution, suggesting their diverse genomic origins and illustrating how much of the nuclear genome can be lost due to mutagen effects. Micronuclei diversity was also evidenced by in situ probing with different DNA sequences (5S and 18S rDNAs, 180-bp knob and Grande LTR-retrotransposon) and by 6-diamidino-2 phenylindole (DAPI) fluorochrome. Perhaps these sequences are hotspots for MMS damage, especially the Grande LTR-retrotransposon, 5S and 18S rDNAs, which are rich in guanine. In addition, probe pools were constructed from individual genomic DNA of two microdissected micronuclei. These probe pools hybridized on all Z. mays chromosomes. However, the centromere, knob and secondary constriction were hybridized by only one probe pool, evidencing the distinct genomic composition of the micronuclei. We illustrate the micronuclei genomic diversity as they originated from several different chromosomes following the MMS treatment, and demonstrate the extent of the genotoxic damage to the genome. We provide some insights into micronuclei structure and diversity, and show that they can be further explored in mutagenesis research.

Programmed necrosis is important in many (patho)physiological settings. For specific therapeutic intervention, however, a better knowledge is required whether necrosis occurs through one single “core program” or through several independent pathways. Previously, the poly(ADP-ribose) polymerase (PARP) pathway has been suggested as a crucial element of tumor necrosis factor (TNF)-mediated necroptosis. Here, we show that TNF-induced necroptosis and the PARP pathway represent distinct and independent routes to programmed necrosis. First, DNA-alkylating agents such as 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or methyl methanesulfonate rapidly activate the PARP pathway, whereas this is a late and secondary event in TNF-induced necroptosis. Second, inhibition of the PARP pathway does not protect against TNF-induced necroptosis, e.g., the PARP-1 inhibitor 3-AB prevented MNNG- but not TNF-induced adenosine-5′-triposphate depletion, translocation of apoptosis-inducing factor, and necrosis. Likewise, olaparib, a more potent and selective PARP-1 inhibitor failed to block TNF-induced necroptosis, identical to knockdown/knockout of PARP-1, pharmacologic and genetic interference with c-Jun N-terminal kinases and calpain/cathepsin proteases as further components of the PARP pathway. Third, interruption of TNF-induced necroptosis by interference with ceramide generation, RIP1 or RIP3 function or by the radical scavenger butylated hydroxyanisole did not prevent programmed necrosis through the PARP pathway. In summary, our results suggest that the currently established role of the PARP pathway in TNF-induced necroptosis needs to be revised, with consequences for the design of future therapeutic strategies.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25–5.0 μg/mL) and/or carbendazim (0.8–1.6 μg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the “DeepFlow” neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for ‘mononucleates’, ‘binucleates’, ‘mononucleates with MN’ and ‘binucleates with MN’, respectively. Successful classifications of ‘trinucleates’ (90%) and ‘tetranucleates’ (88%) in addition to ‘other or unscorable’ phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

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

Observing cellular responses to perturbations is central to generating and testing hypotheses in biology. We developed a massively parallel microchemostat array capable of growing and observing 1,152 yeast-GFP strains on the single-cell level with 20 min time resolution. We measured protein abundance and localization changes in 4,085 GFP-tagged strains in response to methyl methanesulfonate and analyzed 576 GFP strains in five additional conditions for a total of more than 10,000 unique experiments, providing a systematic view of the yeast proteome in flux. We observed that processing bodies formed rapidly and synchronously in response to UV irradiation, and in conjunction with 506 deletion-GFP strains, identified four gene disruptions leading to abnormal ribonucleotide-diphosphate reductase (Rnr4) localization. Our microchemostat platform enables the large-scale interrogation of proteomes in flux and permits the concurrent observation of protein abundance, localization, cell size, and growth parameters on the single-cell level for thousands of microbial cultures in one experiment.