Environmental and Molecular Mutagenesis最新文献

There is growing recognition across broad sectors of the toxicology community that gene expression biomarkers have the potential to identify genotoxic and nongenotoxic carcinogens through a weight-of-evidence approach, providing opportunities to reduce reliance on the 2-year bioassay to identify carcinogens. In August 2022, a workshop within the International Workshops on Genotoxicity Testing (IWGT) was held to critically review current methods to identify genotoxicants using various 'omics profiling methods. Here, we describe the findings of a workshop subgroup focused on the state of the science regarding the use of biomarkers to identify chemicals that act as genotoxicants in vivo. A total of 1341 papers were screened to identify those that were most relevant. While six published biomarkers with characterized accuracy were initially examined, four of the six were not considered further, because they had not been tested for classification accuracy using additional sets of chemicals or other transcript profiling platforms. Two independently derived biomarkers used in conjunction with standard computational techniques can identify genotoxic chemicals in vivo (rat liver or both rat and mouse liver) on different gene expression profiling platforms. The biomarkers have predictive accuracies of ≥92%. These biomarkers have the potential to be used in conjunction with other biomarkers in integrated test strategies using short-term rodent exposures to identify genotoxic and nongenotoxic chemicals that cause cancer.

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

This interlaboratory evaluation of HepaRG CometChip was conducted to assess transferability and reproducibility of this new approach methodology (NAM) across four laboratories. Concentrations inducing up to ~70% relative cytotoxicity were determined by the organizing laboratory, and frozen chemical formulation blocks were sent to each participant. When noncytotoxic, 10 mM was the maximum dose. Cultures were exposed once daily for three consecutive days, and both cytotoxicity assessment, via ATP quantification, and comet analysis, commenced 3–4 h after initiation of the final exposure. Positive response was statistical pairwise significance (p < 0.05) with concentration-related increases in %Tail DNA across ≥ 2 consecutive exposures. For 8 of 11 compounds, all four labs generated unanimous test results, with four negative compounds (2-acetylaminofluorene [2-AAF], 2,4-dichlorophenol, eugenol and hydroquinone) and four positive compounds (azidothymidine, benzo(a)pyrene [BP], cyclophosphamide [CP], ethyl methanesulfonate).For the remaining chemicals, three of four labs generated negative calls for amitrole, cadmium chloride, and DMBA. In cases where bulky lesions were anticipated, the magnitude of %Tail DNA was low, due to the inherent insensitivity of the alkaline comet assay (not the CometChip per se) to detect bulky adducts repaired by nucleotide excision repair. This is supported by the small magnitude in %Tail DNA induced by BP and CP. Taken together, for all compounds there was majority agreement in CometChip results across participating laboratories supporting that the endpoint is readily transferable to new labs. Overall, this platform is a promising human-relevant NAM, with a physiologically relevant detoxification process that could be incorporated into rodent replacement strategies.

Water buffalo (Bubalus bubalis) skin exhibits unique morphological and physiological adaptations compared to other domestic bovines to enhance thermoregulation facilitated by abundant dermal melanin. Melanin synthesis and distribution, governed by the melanogenesis pathway and associated genes underpin coat color phenotypes and determine thermotolerance. The molecular mechanisms regulating pigmentation in buffalo skin remain underexplored as well as the reliable internal control genes (ICGs) for qPCR normalization. This study addresses the critical gap by systematically evaluating a panel of nine candidate reference genes for their expression stability in skin tissues from contrastingly pigmented buffaloes, jet-black Murrah and Nili Ravi (CC1) versus extensively white-spotted Nili Ravi (CC2). From the application of comprehensive statistical algorithms (deltaC_q, NormFinder, geNorm, BestKeeper), the combination of GAPDH, EEF1A1, and RPS23 emerged as the most stable reference gene set for normalization. Validation using key melanogenesis genes (KIT and TYR) confirmed that normalization with this trio yields consistent and biologically relevant expression profiles, with KIT robustly downregulated in white versus black skin. The recommended reference gene combination ensures robust normalization in gene expression profiling of melanogenesis-related genes in buffalo skin tissue.

Gene mutations can be detected in mammalian cells in vitro using indicator genes such as the hypoxanthine-guanine-phosphoribosyltransferase (HPRT) gene. These assays have been adopted as OECD test guidelines (TG, e.g., OECD TG no. 476) and are used for regulatory purposes. The in vitro transgenic rodent assay (TGRA) in primary MutaMouse hepatocytes is a novel approach for the detection and quantification of gene mutations. Its methodology follows the same principles as the in vivo TGRA, an in vivo gene mutation assay with regulatory adoption (OECD TG no. 488). Although the potential of the in vitro TGRA to identify mutagens has been reported, its performance compared to an established in vitro gene mutation assay has not been reported. This study compared the in vitro TGRA with the HPRT assay using 10 known in vivo mutagens. The in vitro TGRA correctly identified all 10 mutagens, whereas the HPRT assay identified only nine. Benchmark concentration (BMC) modeling for the nine substances detected by both assays revealed overlapping confidence intervals for six compounds, indicating comparable sensitivity. For three mutagens, the HPRT assay yielded lower BMC intervals. Additionally, eight substances known to be non-mutagenic in vivo tested negative in the in vitro TGRA. While increased cytotoxicity did not induce increased mutant frequencies, it reduced DNA yield, thereby impairing mutagenicity assessment. The results of this study contribute to the understanding of the sensitivity and robustness of the in vitro TGRA and provide essential information for the validation of the assay.

Breast cancer is the most prevalent cancer in women and has been linked to exposure to environmental chemicals. However, many chemicals have not been evaluated for relationships with this outcome. In this study, we analyzed RNA sequencing data from human breast cancer-derived MCF7 cells exposed to hundreds of individual chemicals. These chemicals were binned into three categories: (1) chemicals with known associations to breast cancer (BCs); (2) chemicals with a lack of relationship to breast cancer (NBCs); and (3) chemicals that remain understudied for breast cancer risk (UCs). Machine learning models were trained to discriminate between BCs and NBCs based on transcriptomic and physicochemical property data. The best model yielded a balanced accuracy of 80% and was applied to the UCs. A total of 170 genes were found to contribute to model performance, including Claspin (CLSPN), Runt-related Transcription Factor 2 (RUNX2), and Ubinuclein 2 (UBN2). These genes further informed enriched pathways relevant to inflammation, ferroptosis signaling, and cell proliferation. Additionally, 97 UCs were predicted to be more analogous to BCs, including select biocides and dyes. To ground results in human population data, expression profiles for the 170 genes were assessed in tumor samples from The Cancer Genome Atlas, yielding overlap in human cancer-relevant alterations and in vitro chemical-induced alterations. Collectively, this study addresses a gap related to understanding which chemicals may be of interest for further characterization of breast cancer risk by prioritizing chemicals and underlying mechanisms using high-throughput transcriptomic screening data.

Long-duration spaceflight exposes astronauts to various stressors that can alter human physiology, potentially causing immediate and long-term health effects. These stressors can damage biomolecules, cells, tissues, and organs, leading to adverse outcomes. Developing adverse outcome pathways (AOPs) relevant to radiation exposure can guide research priorities and inform risk assessments of future space exploration activities. Through expert consultation, we developed an AOP network linking 18 key events (KEs) to four non-cancer outcomes: learning and memory impairment, bone loss, abnormal vascular remodeling, and cataract development. A novel scoping review methodology informed the evidence evaluation and supported causal linkages between two KEs. The AOP network begins with the molecular initiating event (MIE) of energy deposition onto cells, which may trigger oxidative stress and DNA damage. If DNA damage is misrepaired, it could lead to gene mutations or chromosomal aberrations. In cases where these occur in critical cell cycle genes, there is a possibility of uncontrolled cellular proliferation. Persistent KEs may contribute to the activation of tissue-resident cells, suppression of anti-inflammatory processes, and promotion of chronic inflammation. This inflammatory cycle, potentially driven by mitochondrial dysfunction and immune cell activation, could lead to cell death and tissue damage. Over time, this accumulation of damage might contribute to organ-specific adverse outcomes associated with radiation exposure. This AOP network consolidates knowledge across biological levels and identifies gaps in understanding causal relationships. It aims to guide research for space traveler risk models and can also apply to other radiation exposure scenarios, such as in medical or occupational settings.

Millions of healthcare professionals are occupationally exposed to waste anesthetic gases (WAGs) worldwide; however, their effect on DNA methylation is unknown. Thus, for the first time, global DNA methylation analysis was performed in addition to the comet assay and gene expression analysis in this case–control study to reveal possible molecular alterations associated with WAG exposure. A total of 86 participants were included (43 healthcare professionals exposed to WAGs and 43 matched-controls). Mononuclear cells were isolated for the comet assay, and DNA was extracted from whole blood for global DNA methylation analysis. RNA was isolated for the gene expression of NRF2, SIRT1, and HO1. Increased DNA damage was observed in the exposed group (p = 0.02). No differences in global DNA methylation or HO1 expression were observed (p > 0.05), but SIRT1 (p = 0.01) and NRF2 (p = 0.02) upregulation were observed in the exposed group. These findings highlight the molecular effects of WAG exposure.