Mutagenesis最新文献

Micronuclei (MNi) are critical biomarkers for pathological conditions, yet their manual scoring is inherently laborious and prone to significant inter-observer variability, limiting the reliability and scalability of genotoxicity assessments. Recent advancements in deep learning and computer vision have revolutionised automated MNi detection in various assay samples, enhancing accuracy, efficiency, and reducing human bias. While these AI-powered techniques have been demonstrated in in vitro genotoxicity testing, their application to the minimally invasive Buccal Micronucleus Cytome (BMCyt) assay for human biomonitoring remains largely unexplored. The BMCyt assay, invaluable for assessing genotoxic damage in environmentally exposed populations, presents unique challenges, including sample variability, confounding factors, and the complexity of scoring multiple cytogenetic endpoints. This review covers the evolution of AI-based MNi detection, analysing key methodologies and advancements. It highlights the untapped potential of integrating AI into the BMCyt assay to overcome current analytical limitations, improve reproducibility, increase throughput, and eliminate observer bias. By facilitating more robust and scalable genomic damage monitoring, AI integration will significantly enhance the utility of the BMCyt assay in large-scale epidemiological studies and human biomonitoring.

The standard comet assay detects DNA strand breaks (SBs) and alkali-labile sites (ALS), but these lesions are nonspecific. They may result directly from genotoxic agents or arise as intermediates during the repair of other DNA damage, such as oxidised bases or DNA bulky adducts. Different approaches have been developed to generate or trap these repair intermediates, making them detectable with the comet assay. Recently, the combination of the comet assay with DNA repair inhibitors like hydroxyurea (HU) and cytosine arabinoside (Ara-C) has been proposed to detect bulky DNA adducts. These lesions are mainly repaired through nucleotide excision repair (NER), a process that transiently produces SBs when damaged oligonucleotides are excised. Normally, these intermediates are rapidly repaired by DNA resynthesis and ligation. However, by inhibiting this repair step, SBs persist and can be detected by the comet assay. This strategy has been applied in various fields, including genotoxicity testing, environmental toxicology, human biomonitoring, and studies on DNA repair kinetics. This review focuses specifically on the use of HU, Ara-C, and aphidicolin (APC) in in vitro experiments to evaluate the utility and specificity of this method for detecting different types of DNA lesions. Notably, in approximately 70% of studies reviewed, the inclusion of DNA repair inhibitors led to a significant increase in DNA damage, highlighting the added value of this approach. However, although the method enhances sensitivity to bulky adducts, it also responds to other types of damage, such as those induced by alkylating or oxidative agents.

The Buccal Micronucleus Cytome (B-MNcyt) assay is used world-wide to study chromosomal abnormalities and environmental genotoxicity and cytotoxicity in humans. The aim of this paper is to discuss the strengths and limitations of the B-MNcyt assay and to identify emerging opportunities to further improve and validate its use. This can be achieved by innovating and evolving the B-MNcyt assay by identifying and solving important knowledge and technological gaps that hinder its utility. The cells examined in the B-MNcyt assay are squamous epithelial cells that can be easily collected from the inside of the mouth. These cells are post-mitotic cells generated from the proliferative basal layer and may contain micronuclei (MN). MN can be generated during mitosis of the basal cells prior to their differentiation into squamous cells. The B-MNcyt assay is increasingly being used to measure DNA damage induced in vivo by environmental genotoxins. Results with this assay have been shown to correlate positively with MN frequency measured using the well-validated lymphocyte cytokinesis-block micronucleus cytome (L-CBMNcyt) assay. However, the B-MNcyt assay has some important limitations that need to be addressed to achieve a similar level of validation and applicability as the L-CBMNcyt assay. These include: (i) lack of evidence that the buccal MN frequency predicts disease risk in prospective studies; (ii) no automated scoring system to score MN in buccal cells which is essential to achieve statistically robust results and to improve the feasibility of the assay in population studies; (iii) kinetics of MN expression in buccal cells needs more research to define optimal time frames to score MN after acute exposure or during chronic genotoxin exposure; (iv) studies are also required to test the suitability of using the B-MNcyt assay for radiation exposure bio-dosimetry. This paper discusses these issues and provides some suggestions how to address them.

The mutagenicity of chemical compounds is a key consideration in toxicology, drug development, and environmental safety. Traditional methods such as the Ames test, while reliable, are time-intensive and costly. With advances in imaging and machine learning (ML), high-content assays like cell painting offer new opportunities for predictive toxicology. Cell painting captures extensive morphological features of cells, which can correlate with chemical bioactivity. In this study, we leveraged cell painting data to develop ML models for predicting mutagenicity and compared their performance with structure-based models. We used two datasets: a Broad Institute dataset containing profiles of over 30 000 molecules and a U.S.-Environmental Protection Agency dataset with images of 1200 chemicals tested at multiple concentrations. By integrating these datasets, we aimed to improve the robustness of our models. Among three algorithms tested-Random Forest, Support Vector Machine, and Extreme Gradient Boosting-the third showed the best performance for both datasets. Notably, selecting the most relevant concentration per compound, the phenotypic altering concentration, significantly improved prediction accuracy. Our models outperformed traditional quantitative structure activity relationship (QSAR) tools such as the Virtual models for property Evaluation of chemicals within a Global Architecture (VEGA) and the CompTox Dashboard for the majority of compounds, demonstrating the utility of cell painting features. The cell painting-based models revealed morphological changes related to DNA and RNA perturbation, especially in mitochondria, endoplasmic reticulum and nuclei, aligning with mutagenicity mechanisms. Despite this, certain compounds remained challenging to predict due to inherent dataset limitations and inter-laboratory variability in cell painting technology. The findings highlight the potential of cell painting in mutagenicity prediction, offering a complementary perspective to chemical structure-based models. Future work could involve harmonizing cell painting methodologies across datasets and exploring deep learning techniques to enhance predictive accuracy. Ultimately, integrating cell painting data with QSAR descriptors in hybrid models may unlock novel insights into chemical mutagenicity.

The comet assay is a widely used method for measuring DNA damage and DNA repair. When DNA strand breaks happen, the supercoiling of DNA is relaxed, and after alkaline or neutral electrophoresis, depending on the type of performed comet assay, DNA moves toward the anode, forming a comet tail. Thus, with increasing frequency of DNA strand breaks, an increase in the percentage of DNA in the tail is observed. The aim of this study was to compare systematically various steps like lysis, duration of electrophoresis, and pH of the electrophoresis solution and their effect on the comet tail with regard to sensitivity for detection and quantification of DNA damage. We treated human lymphoblastoid TK6 cells with known genotoxic substances with a different mode of action and then performed both standard and modified alkaline and neutral comet assays. The modifications included Fpg- and MspI-modified comet assays. Several aspects of this comparison are investigated for the first time here. The results obtained from these experiments showed a higher %DNA in tail in the alkaline comet assay compared to the neutral comet assay. Additionally, the lysis step was not critical in the alkaline comet assay, whereas it was essential for the neutral comet assay. Results from alkaline Fpg-modified comet assay showed higher sensitivity in detecting single strand breaks and the neutral MspI-modified comet assay was better in detecting DNA double-strand breaks. Overall, our findings provided valuable insight into the differences between alkaline and neutral electrophoresis conditions in the comet assay and indicated that the alkaline comet assay is more sensitive for measuring total DNA damage.

Evaluating the genotoxic potential of nanomaterials (NMs) presents unique challenges not associated with traditional toxicological assessment. A key question in any NM focused toxicity study is whether the material has reached the target cell and what its subsequent subcellular localization is. This current study aimed to assess the potential of a panel of industrially relevant NMs; TiO2-NM102, TiO2-NM105, TiO2-E171, silica, polyethylene, polystyrene, carbon black, gold nanorods, tungsten carbide/cobalt, and tungsten carbide, to undergo cellular uptake in mouse embryonic stem cells, which are applied in the ToxTracker genotoxicity assay. Ultrastructural cellular analysis by transmission electron microscopy was undertaken following 100 μg/ml treatment with the test NMs for 24 h; any observed uptake was confirmed by energy-dispersive X-ray spectroscopy. Induction of DNA damage, cytotoxicity, p53 activation, protein stress, and oxidative stress was evaluated by the ToxTracker assay following 24-h treatment with the test NMs (0-100 μg/ml) in the absence of S9. TiO2-NM105, silica, polystyrene, carbon black, and tungsten carbide were all shown to undergo cellular uptake, localized in membrane-bound vesicles within the cytoplasm. None of the internalized NMs promoted a genotoxic response in ToxTracker, and similarly, no DNA damage was observed by the materials not internalized. Interestingly, of the internalized NMs, only polystyrene caused a slight cytotoxic response at 100 μg/ml treatment (10% loss in cell viability). Of the NMs not internalized, cytotoxicity was observed in mES cells treated with 100 μg/ml TiO2-NM102 (15%), polyethylene (15%), gold nanorods (35%), and tungsten carbide/cobalt (45%). In summary, this study demonstrated that TiO2-NM105, silica, polystyrene, carbon black, and tungsten carbide are non-genotoxic in vitro despite undergoing cell uptake in the ToxTracker cells. A continued focus is needed to supplement NM genotoxicity studies with cellular uptake analysis.

Aristolochic acid I (AAI) is a carcinogen associated with various human cancers. However, its causal relationship with hepatocellular carcinoma remains controversial, and inconsistent results from rodent studies have suggested species-specific differences. Here we evaluated AAI genotoxicity using functional human-induced hepatocyte-like cells (hiHep cells), a model that closely mimics primary human hepatocytes in gene expression and function, thereby shedding light on its potential hepatocarcinogenic risk in humans. First, we assessed AAI genotoxicity by evaluating AAI-DNA adducts and micronucleus frequency. In hiHep cells, AAI (0.7-2.5 µM) induced up to 105 adducts per 108 nucleotides, indicating high metabolic activation of AAI. A concentration-dependent increase in micronucleus frequency indicated a significant increase in chromosomal aberrations in hiHep cells. Considering the evidence of AAI inducing oxidative stress, we assessed 8-hydroxy-2'-deoxyguanosine and reactive oxygen species levels to evaluate DNA oxidative damage. For both indicators, significantly elevated levels were observed. A mechanism involving oxidative damage was further supported by observations of mitochondrial dysfunction, including changes in mitochondrial membrane potential and mitochondrial complex activity. Ascorbate treatment decreased AAI-induced oxidative DNA damage and DNA adduct formation, providing direct cellular evidence for free radical intermediates in AAI metabolic activation-a mechanism previously hypothesized but not experimentally validated in a human-relevant hepatocyte model. Our study findings revealed the genotoxic effects of AAI on hiHep cells and implicated oxidative stress as the key mechanism. These findings strengthen the association between AAI exposure and liver disease and highlight the potential role of antioxidant therapies in mitigating AAI-associated carcinogenesis.

Aneuploidy and polyploidy have a major impact in congenital and acquired diseases, in particular in cancer development. This mini-review highlights the use of exfoliated buccal cells as a non-invasive tool for monitoring aneuploidy. It offers a mechanistic overview and illustrative diagrams addressing five key areas: i) the causes of aneuploidy; ii) cell kinetics and aneuploidy induction in epithelial buccal cells; iii) the methods for the detecting of aneuploidy; iv) the scientific and medical domains applying aneuploidy detection in exfoliated buccal cells; v) the knowledge gaps and future research perspectives. Although well validated protocols, automated systems and specific probes allowing discrimination between chromosome aberration, aneuploidy and polyploidy in exfoliated buccal cells are available, large-scale cohort human studies remain lacking. These studies are crucial for evaluating aneuploidy in populations exposed to genotoxic agents or at risk for buccal dysplasia. Future validation and predictivity studies should compare FISH and total DNA content methods such as densitometry or flow cytometry, in exposed individuals and control groups with careful control of confounding factors and adherence to standardized reporting guidelines. In addition, assessment of aneuploidy frequencies in parallel in lymphocytes and exfoliated buccal cells in the same populations would allow to compare their predictive value in both tissues. Moreover, mechanistic studies are also needed to better understand the sources of variability in aneuploidy and how buccal cell biology compares to other cell types. In conclusion, due to their ease of collection and non-invasive nature, exfoliated buccal cells represent a promising tool for aneuploidy testing for risk assessment of environmental or occupational exposures and disease prediction and monitoring.