Environmental and Molecular Mutagenesis最新文献

Under the influence of multiple anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as particularly sensitive ecosystems to contamination. The present study aimed at investigating the genotoxic potential of a contaminated sediment sample from an urban and industrial area of the Sado Estuary, by combining the analysis of multiple endpoints in the LacZ plasmid-based transgenic mouse model exposed for 28 days to contaminated estuarine sediment extracts through drinking water. The DNA and chromosome damaging effects were monitored in peripheral blood at 7-day intervals using the standard and enzyme-modified Comet assay, as well as the micronucleus assays in peripheral blood cells. After euthanasia, DNA damage was analyzed in several mouse tissues, and LacZ mutant frequencies were determined in the liver. Livers were also surveyed for histopathological analysis. A time-dependent increase in micronuclei frequency was seen at all tested doses, in spite of no induction of DNA damage in any organ or mutation induction in the liver of exposed mice. The liver from mice exposed to sediment extracts did not reveal major alterations besides evidence of inflammation. Overall, the integration of the endpoints analyzed in the mice is suggestive of potential chronic, rather than acute, adverse effects in vivo, and points to the need for further research in the resident human population in the area. This experimental design can be used to assess the genotoxicity of complex environmental mixtures, understand how they work, and reduce costs and resources while speeding up data collection and interpretation.

The Ames test is a fundamental assay for evaluating chemical mutagenicity, particularly for nitrosamines, which are widespread environmental and pharmaceutical contaminants. To improve sensitivity, regulatory agencies have endorsed the enhanced Ames test (EAT), which incorporates five tester strains, a 30-min pre-incubation step, and metabolic activation using both rat and hamster liver S9 fractions. While Salmonella typhimurium TA102 is known for its sensitivity to oxidative mutagens, its performance under EAT conditions has not been fully characterized. This study evaluated the mutagenic response of TA102 using two nitrosamine positive controls: N-nitrosodimethylamine (NDMA) and 1-cyclopentyl-4-nitrosopiperazine (CPNP). E. coli WP2 uvrA(pKM101) showed consistent mutagenic responses to both NDMA and CPNP, consistent with existing EAT data. TA102 demonstrated a robust response to NDMA but not to CPNP, suggesting limited sensitivity to certain nitrosamines. These findings support the continued use of WP2 uvrA(pKM101) in EAT protocols and highlight the limited utility of TA102 for comprehensive nitrosamine mutagenicity assessment.

This laboratory has reported that the combined use of In Vitro MicroFlow and MultiFlow assays provides information regarding chemicals' genotoxic mode of action (MoA). In an effort to go beyond MoA assessments, we incorporated a panel of biological response modifiers that elicit specific effects on the assays' biomarker response profiles. This was done to pursue our hypothesis that such perturbation signatures would reveal information on clastogenic mechanisms and molecular targets. For this proof-of-concept study, we exposed TK6 cells to 20 known clastogens. Cells were exposed in 96-well plates in the presence and absence of each of four modifying agents at one optimized concentration: talazoparib (PARP inhibitor), MK-8776 (CHK1 inhibitor), AZD-7648 (DNA-PK inhibitor), or a cocktail of reactive oxygen species scavengers. In parallel, cells were also exposed to each of the test chemicals for 4 h, at which time cells were washed and allowed to recover for an additional 20 h. For each of these treatment conditions, sample processing and flow cytometric analyses were performed using standard In Vitro MicroFlow and MultiFlow procedures to measure micronuclei, γH2AX, phosphohistone-H3 (p-H3), p53 activation, and relative nuclei counts. The resulting biomarker response data were processed with PROAST benchmark dose (BMD) software, with modifying agent as a covariate. Unsupervised hierarchical clustering of the collective potency metrics for various combinations of biomarkers showed that clastogens with similar genotoxic mechanisms grouped together. Overall, this study shows that in combination with biological response modifiers, MultiFlow and In Vitro MicroFlow biomarkers can provide mechanistic insights into chemical-induced genotoxicity.

The ability to produce direct DNA damage (genotoxicity), which underlies the carcinogenicity of various chemicals, is typically evaluated in a regulatory-approved battery of in vitro tests with potential in vivo follow-up. Growing concerns for animal welfare and implementation of regulations restricting the use of animal testing necessitate the introduction of New Approach Methodologies (NAMs). The avian egg-based (in ovo) models were developed as metabolically competent NAMs capable of bioactivation, detoxication, and elimination of xenobiotics to potentially replace short-term in vivo genotoxicity assays for chemicals that are genotoxic in vitro. These models utilize avian (chicken or turkey) fetal livers for the evaluation of endpoints indicative of DNA damage produced by either direct or indirect mechanisms, the formation of nuclear DNA adducts and strand breaks. Avian embryos have genetic and morphologic resemblance to mammals and can be used for the evaluation of other endpoints including histopathology and genomic profiling. A concordance analysis of 87 and 59 chemicals assessed in the chicken and turkey models, respectively, revealed a stronger correlation with the results from in vivo genotoxicity assays (76% and 67% sensitivity, 79% and 72% specificity for chicken and turkey, respectively) compared to in vitro assays (58% and 56% sensitivity, 45% and 63% specificity for chicken and turkey, respectively). These results demonstrate that in ovo models detect the genotoxic potential of a broader range of compounds compared to in vitro assays with S9 supplementation. In conclusion, fertilized avian egg fetal liver assays offer a promising alternative to traditional in vivo genotoxicity assays.

Acceptable intake (AI) limits for nitrosamine drug substance related impurities (NDSRIs) that lack carcinogenicity data could be estimated from mutagenic potency relative to anchor nitrosamines with carcinogenicity data. This approach integrates points of departure (PoDs) derived from in vivo mutagenicity studies with in silico predictions generated by a validated quantum-mechanical (QM) model. N-nitrosodiethanolamine (NDELA) and N-nitrosopiperidine (NPIP), with AIs derived from robust carcinogenicity data, were tested in the transgenic rodent (TGR) gene mutation assay. Liver mutant frequency and benchmark dose (BMD) modeling provided a suitable, robust, and precise PoD metric. BMD confidence intervals (CIs) calculated from mutant frequency expanded the potency range of previously reported BMD CIs for other anchor nitrosamines. Cancer-protective AIs for mutagenic NDSRIs can be pragmatically calculated on a potency basis by comparing their lower bound TGR BMD CIs with the BMD CIs and AIs derived from model/anchor nitrosamines that have results for in vivo gene mutation and cancer bioassays. In vivo modeling was supported by the Computer-Aided Discovery and RE-design (CADRE) program, a validated QM model for predicting NDSRI carcinogenic potency based on the underlying mechanism of mutagenicity. CADRE distinguished between anchor nitrosamines N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) and the less potent NDELA and NPIP. Scrutiny of underlying reactivity indices and relevant physicochemical properties rationalized the observed trend in metabolic activity and thus predicted carcinogenic potency. Leveraging the in vivo–in silico approach is valuable in gaining confidence in the proposed AIs, whereby the QM model serves as mechanistic validation of in vivo results.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-associated mortality, correlating with obesity, alcohol consumption, hepatitis B and C infections, and dietary exposure to aflatoxin B₁ (AFB₁). The etiology of AFB₁-induced HCC involves the formation of highly mutagenic guanine lesions that can be repaired by a branch of the base excision repair pathway initiated by the DNA glycosylase, NEIL1. In a murine model, NEIL1 deficiency results in increased AFB₁-induced mutagenesis and carcinogenesis. Previous analyses identified several defective NEIL1 variants in human populations, including the temperature-sensitive A51V and glycosylase-deficient G83D variants. Herein, we report AFB₁-induced mutagenesis in mice expressing the A51V and G83D NEIL1 variants. Cohorts of 6-day-old Neil1^A51V and Neil1^G83D homozygous mice were injected with a single dose of AFB₁, and frequencies and spectra of mutations were assessed in liver genomes 2.5 months post-exposure using duplex sequencing. Comparisons of these data with previously generated data on AFB₁-induced mutagenesis in wild-type (WT) and Neil1^−/− mice revealed that although mutation frequencies in Neil1^A51V and Neil1^G83D animals were comparable to those measured in WT, elevated proportions of base substitutions at A/T sites were consistent with NEIL1 deficiency in both of these models. These findings suggest that individuals carrying these NEIL1 variants could be at an elevated risk for the development of AFB₁-induced HCC.