Mutation research. Genetic toxicology and environmental mutagenesis最新文献

{"title":"Artificial intelligence-driven advancements in radiation biodosimetry","authors":"Karthik Prathaban,&nbsp;Wilner Martinez-Lopez,&nbsp;Manoor Prakash Hande","doi":"10.1016/j.mrgentox.2026.503961","DOIUrl":"10.1016/j.mrgentox.2026.503961","url":null,"abstract":"<div><div>Radiation dose estimation is fundamental to the effective management of radiological emergencies, where prompt and accurate assessment is critical for medical intervention. Conventional biodosimetry methods, including the dicentric chromosome assay (DCA), premature chromosome condensation (PCC), γ-H2AX foci detection, cytokinesis-block micronucleus (CBMN) assay, and fluorescence in situ hybridisation (FISH) translocation analysis, remain important approaches for biological dose estimation. However, their conventional implementation can involve labour-intensive sample preparation and manual or semi-automated analysis, require specialised expertise, and have limited scalability for mass-casualty events. Recent advances in Artificial Intelligence (AI), particularly machine learning (ML) and deep learning (DL), offer considerable potential to enhance biodosimetry through automated image analysis, multimodal data integration, and accelerated dose assessment. DL approaches, including Convolutional Neural Networks (CNNs), enable high-throughput image analysis and automated identification of radiation-induced cytogenetic damage. Furthermore, integrating explainable AI (XAI) approaches may improve model transparency, interpretability, and confidence in clinical applications. This review examines current advances, challenges, and prospects of AI in radiation biodosimetry, highlighting its potential to support rapid, accurate, reproducible, and scalable dose assessment in radiological emergencies.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"915 ","pages":"Article 503961"},"PeriodicalIF":2.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149161025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Micronuclei and other nuclear anomalies in urothelial cells of women with type 1 and type 2 diabetes mellitus","authors":"Gohar Parsadanyan,&nbsp;Gayane Zalinyan,&nbsp;Elena Agajanova,&nbsp;Lusine Araqelyan,&nbsp;Armen Nersesyan","doi":"10.1016/j.mrgentox.2026.503962","DOIUrl":"10.1016/j.mrgentox.2026.503962","url":null,"abstract":"<div><div>Chronic hyperglycemia in diabetes mellitus (DM) may promote oxidative stress, cytotoxicity, and chromosomal instability. However, evidence regarding exfoliated urothelial cells remains limited. This study evaluated micronucleus (MN) frequency and other nuclear anomalies in urothelial cells from women with type 1 DM (T1DM) and type 2 DM (T2DM) and investigated predictors of MN formation. Twenty women with T1DM, 25 with T2DM, and 22 controls were included. Second-void urine samples were collected, and urothelial cells were Feulgen-stained. MN and other nuclear anomalies were assessed in 1000 cells per participant. Clinical and metabolic characteristics were compared, and multivariable binary logistic regression identified predictors of MN formation. Age and BMI differed significantly among T1DM, T2DM, and control groups. T2DM participants had significantly longer diabetes duration than T1DM participants. HbA1c was significantly higher in both DM groups than in controls. Total cholesterol, HDL, and triglycerides also differed significantly among groups, whereas LDL did not. MN frequency was higher in diabetic participants overall but this increase was not statistically significant. Nuclear buds and degenerative changes were significantly more frequent in both DM groups. In direct comparisons of the DM patient groups, NB, KR, KL, and CC frequencies were significantly higher in T2DM than in T1DM participants. In multivariable analysis, longer DM duration and higher HbA1c levels independently predicted MN formation. DM was associated with increased nuclear anomalies in urothelial cells, although MN frequency was not significantly elevated. Longer disease duration and higher HbA1c independently predicted MN formation, suggesting that chronic metabolic exposure may contribute to urothelial cellular damage.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"915 ","pages":"Article 503962"},"PeriodicalIF":2.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149161030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Copper (II) complexes involving quinazolinone Schiff base ligands: In vitro study of cytotoxic, genotoxic and DNA-protective potential in human hepatoma HepG2 cell line","authors":"Eva Horváthová,&nbsp;Peter Hergott,&nbsp;Terézia Zajičková,&nbsp;Jana Hricovíniová,&nbsp;Eliška Gálová,&nbsp;Michal Hricovíni,&nbsp;Andrea Ševčovičová,&nbsp;Zuzana Hricovíniová","doi":"10.1016/j.mrgentox.2026.503964","DOIUrl":"10.1016/j.mrgentox.2026.503964","url":null,"abstract":"<div><div>DNA oxidation damage contributes to carcinogenesis and limits the efficacy of many chemotherapeutics. Although platinum‑based drugs remain crucial in cancer therapy, their clinical use is restricted by toxicity and acquired resistance. However, quinazolinone derivatives have recently attracted attention as scaffolds with promising biological activities. Among these, Schiff bases are of particular interest because they can provide suitable ligands for the complexation with metals, making them a potential substitute for cisplatin. Therefore, in the search for new anticancer agents, Cu(II) complexes based on 2,3-substituted quinazolinones have been studied in detail. The radical scavenging/reducing efficiency and DNA-protective effects were monitored using DPPH, FRAP and DNA topology assays. Ligands and Cu(II) complexes, were screened for their <em>in vitro</em> cytotoxic, genotoxic, and protective activity in human hepatocarcinoma (HepG2) cells using the MTT and Comet assays. The results showed that compounds studied are redox-active agents. Cu(II) complexes exhibited higher DPPH radical-scavenging activity in comparison with their corresponding free ligands; they manifested comparable effects in ferric reducing power as well as in DNA topology assays. The cellular assays revealed markedly higher cytotoxicity of Cu(II) complexes compared with the corresponding free ligands. In the presence of H₂O₂, C1 significantly enhanced DNA damage, whereas C2 did not alter the level of DNA damage induced by H₂O₂ alone. On the contrary, ligands exhibited much lower toxicity and significant protection of HepG2 cells against H₂O₂. The incorporation of Cu(II) ions into quinazolinone Schiff base ligands substantially alters their biological activity, shifting their profile from DNA‑protective to cytotoxic/pro‑oxidant. DFT calculations were performed for both Cu(II) complexes to investigate molecular features that may contribute to their different biological activity. These results highlight the potential of quinazolinone‑Cu(II) complexes as anticancer candidates and support further studies into their mechanisms of action.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"915 ","pages":"Article 503964"},"PeriodicalIF":2.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149212426","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanoplastic-induced DNA damage in mammalian cells","authors":"Mohammad Alzaben,&nbsp;Martin Roursgaard,&nbsp;Peter Møller","doi":"10.1016/j.mrgentox.2026.503963","DOIUrl":"10.1016/j.mrgentox.2026.503963","url":null,"abstract":"<div><div>Plastic pollution is a growing public health concern. Nanoplastics (NPs) are defined as plastic particles &lt; 1000 nm diameter. Microplastics and NPs have been detected in foods, water, and air. In this mini-review, we assess the genotoxic effects of primary (polystyrene) and secondary (‘true-to-life’) NPs in cultured mammalian cells. Studies were identified in the PubMed, Scopus, and Web of Science databases. Narrative review of the literature was supported by statistical analysis of associations between reported genotoxic effects (positive or null effects, or semi-quantitative indicators of effect size) and cell type (immune <em>vs.</em> non-immune cells), genotoxicity endpoint (repairable <em>vs.</em> permanent DNA lesions), maximal exposure concentration, particle size (&gt; the defined size of nanoparticles, 100 nm) and surface modification in polystyrene NP studies. Results from 36 studies show that, as determinants of the effects of polystyrene NPs, biological differences (cell type and genotoxicity endpoint) are more important than the exposure concentration or the particle characteristics (particle size and surface modification). There is higher consistency or stronger effect in studies on immune cells vs. non-immune cells, and some evidence of stronger effect on permanent DNA lesions (mainly micronuclei, MN) vs. repairable damages (mainly, comet assay endpoints). Results from nine studies on true-to-life NPs from polyethylene terephthalate (PET) plastics suggest genotoxic potential, although differences in plastic degradation methods may affect the level of genotoxicity. Overall, the analysis shows that primary (polystyrene) and true-to-life (PET) NPs can generate DNA damage in cultured mammalian cells.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"915 ","pages":"Article 503963"},"PeriodicalIF":2.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149160735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Limitations of a uniform two-fold criterion in the Ames test: Insights from historical control distributions in frameshift-detecting Salmonella typhimurium strains TA97, TA97a, and TA1537","authors":"Tsuneo Hashizume,&nbsp;Yoko Inoue,&nbsp;Yuka Sakai,&nbsp;Toru Ishii,&nbsp;Yasunori Takahashi,&nbsp;Yasuyoshi Miura","doi":"10.1016/j.mrgentox.2026.503955","DOIUrl":"10.1016/j.mrgentox.2026.503955","url":null,"abstract":"<div><div>In the bacterial reverse mutation (Ames) test, <em>Salmonella typhimurium</em> strains TA1537, TA97, and TA97a are generally regarded as interchangeable for detection of frameshift mutations. A uniform two‑fold increase criterion, vs. the concurrent negative control, is commonly applied when using these strains, without consideration of strain-specific background characteristics. To investigate control criteria applicability, we analyzed negative-control revertant counts for TA97, TA97a, and TA1537, using data from 20 independent experiments, conducted with and without metabolic activation. TA97 and TA97a showed higher mean negative-control counts than TA1537 but exhibited narrower variability, when assessed using historical control ranges defined as the mean ± 2 standard deviations. The upper limits of these ranges corresponded to approximately 1.12–1.45‑fold × the mean for TA97 and TA97a, whereas wider relative ranges were observed for TA1537. These findings indicate that the applicability of a two‑fold increase criterion differs among frameshift‑detecting strains and that interpretation of TA97 and TA97a data may benefit from strain‑specific historical-control data rather than relying solely on a uniform threshold.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"914 ","pages":"Article 503955"},"PeriodicalIF":2.8,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148745446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An O-prenylated benzylidene-thiazolidinedione derivative suppresses NF-κB signaling in a breast cancer cell line: Possible implications for therapy","authors":"Somnath Singha Roy,&nbsp;Subhadip Hajra,&nbsp;Abhishek Basu,&nbsp;Sudin Bhattacharya","doi":"10.1016/j.mrgentox.2026.503951","DOIUrl":"10.1016/j.mrgentox.2026.503951","url":null,"abstract":"<div><div>Nuclear factor-kappa B (NF-κB), a proinflammatory transcription factor, plays a pivotal role in chemotherapeutic resistance and chemotherapy-induced organ injury. Consequently, combining conventional chemotherapy with an NF-κB inhibitor may optimize therapeutic outcomes by enhancing tumor cell cytotoxicity while mitigating systemic toxicity.</div><div>In this study, we synthesized a novel thiazolidinedione-based small molecule, <em>O</em>-prenylated benzylidene-thiazolidinedione (PBT), and evaluated its efficacy in combination with cisplatin (CDDP). Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice received oral PBT (4 mg/kg b.w.,) either alone or in combination with a single intraperitoneal dose of cisplatin (CDDP; 5 mg/kg b.w.,).</div><div>PBT effectively suppressed NF-κB expression in both tumor cells and renal tissue. PBT effectively inhibited tumor cell proliferation, leading to significant suppression of in vivo tumor growth and prolonged host survival. Mechanistic analysis confirmed that PBT triggered tumor cell apoptosis by upregulating Bax, cytochrome-c, and caspases, while downregulating the anti-apoptotic protein Bcl-2. Furthermore, PBT treatment significantly reduced vascular endothelial growth factor (VEGF) expression and matrix metalloproteinase-9 (MMP-9) levels, underscoring its tumor sensitization potential.</div><div>Concurrently, PBT attenuated CDDP-induced nephrotoxicity and genotoxicity by modulating inflammatory mediators and antioxidant enzymes. While CDDP monotherapy induced a significant increase (<em>p</em> &lt; 0.05) in chromosomal aberrations and DNA damage in bone marrow cells, PBT co-treatment significantly (<em>p</em> &lt; 0.05) mitigated these genotoxic effects. These findings demonstrate that NF-κB inhibition by PBT represents a promising strategy to improve the efficacy and safety profile of conventional cancer chemotherapy.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"914 ","pages":"Article 503951"},"PeriodicalIF":2.8,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148745445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The lizard Phyllopezus pollicaris (Spix, 1825) as a bioindicator of exposure to natural background radiation in a semi-arid region of Brazil","authors":"Leticia Gurgel Bezerra Medeiros Cavalcanti,&nbsp;Julio Alejandro Navoni,&nbsp;Viviane Souza do Amaral,&nbsp;Raul Fernandes Dantas Sales,&nbsp;Eliza Maria Xavier Freire","doi":"10.1016/j.mrgentox.2026.503958","DOIUrl":"10.1016/j.mrgentox.2026.503958","url":null,"abstract":"<div><div>This study investigated the potential of the lizard <em>Phyllopezus pollicaris</em> as a bioindicator of genotoxic damage caused by exposure to ionizing radiation in uranium-rich areas of Rio Grande do Norte State, Brazil. The hypothesis tested was that <em>P. pollicaris</em> is sensitive to ionizing radiation due to its wide distribution and generalist ecological behavior and may exhibit cytogenetic effects in areas with high natural background radiation. Blood samples were collected from specimens in three localities with medium to high natural background radiation levels in the Seridó Caatinga and were compared with samples from a low-radiation area, the Mata Estrela Reserve in the Atlantic Forest. Cytogenetic analyses were conducted by assessing the frequency of micronuclei and other nuclear abnormalities in erythrocytes using the micronucleus test. The results indicated a significant increase in the frequency of micronuclei and other nuclear abnormalities in specimens from areas with high natural background radiation compared to the absence of micronuclei (MN) in individuals from the Mata Estrela reference (non-exposed) area. Thus, this study suggests a link between ionizing radiation and genotoxic damage, highlighting the potential of <em>P. pollicaris</em> as a bioindicator of environmental quality in areas with high natural background radiation, such as the Seridó region of Rio Grande do Norte, Brazil.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"914 ","pages":"Article 503958"},"PeriodicalIF":2.8,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148779397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modulation of X-irradiation-induced γ-H2AX focus stability in human blood lymphocytes: A technical note","authors":"Anushka Regil,&nbsp;Revanth Shankar,&nbsp;Divya K. Mohan,&nbsp;Venkateswarlu Raavi,&nbsp;Venkatachalam Perumal","doi":"10.1016/j.mrgentox.2026.503957","DOIUrl":"10.1016/j.mrgentox.2026.503957","url":null,"abstract":"<div><div>Histones, essential building blocks of the nucleosome, undergo dynamic changes during the cell cycle. Phosphorylation of histone protein H2AX occurs at sites of DNA double-strand breaks (DSBs) and is a sensitive marker of DNA damage. Detection of phosphorylated H2AX, the γ-H2AX assay, has been widely used in experimental models, including human tissues and cell lines. The assay has been used in research on DNA repair, drug development, genotoxicity assessment, radiation biology, diagnostic radiology, and radiotherapy protocol optimization. Among these applications, the γ-H2AX assay has gained particular attention in radiation biodosimetry due to its rapid turnaround time. Despite its potential for radiation triage, the assay's utility in cases of delayed sample collection/processing remains limited, because quantitation is constrained by rapid dephosphorylation, catalyzed by phosphatases such as Protein Phosphatase 2 A (PP2A). Potential stabilizing agents, such as fostriecin, okadaic acid, calyculin A, and LiCl, have been suggested to preserve γ-H2AX by modulating phosphatase activity. We evaluated the possible stabilising effects of calyculin A and LiCl on γ-H2AX foci in irradiated human blood lymphocytes. Pre-treatment with either calyculin A (10 nM) or LiCl (20 mM) yielded higher levels of γ-H2AX foci, 0.5 h post-X-irradiation; however, the stability of the γ-H2AX foci declined, 24 h post-irradiation.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"914 ","pages":"Article 503957"},"PeriodicalIF":2.8,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148745447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vivo micronucleus assay: Increased efficiency with an automated imaging system","authors":"Seng-Min Back,&nbsp;Da Yeon Kim,&nbsp;Ho Bin Yi,&nbsp;Na-Young Jeong,&nbsp;Nan-Young Kim,&nbsp;Seong-Hoon Park","doi":"10.1016/j.mrgentox.2026.503959","DOIUrl":"10.1016/j.mrgentox.2026.503959","url":null,"abstract":"<div><div>The <em>in vivo</em> erythrocyte micronucleus (MN) test is a standard assay for detecting chromosomal damage; however, conventional manual scoring is labor-intensive and subject to inter-scorer variability. Although automated image-based scoring offers a practical alternative, its application to bone marrow analysis requires effective enrichment of erythrocytes, to reduce interference from non-target nucleated cells. We have established and validated an automated bone marrow MN scoring workflow, using the Metafer slide-scanning system and associated software platform. We have compared a conventional cellulose column-based enrichment method with a syringe-filter-based method. The filter method reduced residual non-erythroid cellular components more effectively than the column method, although both methods yielded preparations suitable for automated analysis. Automated scoring showed no significant differences from manual microscopy in MN frequency or polychromatic erythrocyte ratio, in either negative- or positive-control groups. Repeated analysis of the same slide demonstrated high repeatability. Automated and manual MN frequencies showed positive linear correlations in both column- and filter-enriched preparations, and Bland-Altman analysis showed overall agreement between the two scoring methods. Automated historical control data (HCD) generated from male and female rats and mice were comparable to manually established laboratory HCD. These findings demonstrate that automated image-based scoring combined with appropriate erythrocyte enrichment is a reliable and practical alternative to conventional manual scoring in the <em>in vivo</em> bone marrow MN assay and may improve analytical efficiency, objectivity, and reproducibility in genotoxicity testing.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"914 ","pages":"Article 503959"},"PeriodicalIF":2.8,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148846114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effect of aging on the response to 2-acetamidofluorene in the repeated-dose rat-liver micronucleus assay","authors":"Kensuke Satomoto,&nbsp;Akiko Okada,&nbsp;Eito Ikeda,&nbsp;Naoko Hongo,&nbsp;Hisateru Yoshida,&nbsp;Akari Kaganomiya,&nbsp;Yuta Baba,&nbsp;Mika Nagaike,&nbsp;Shuichi Hamada","doi":"10.1016/j.mrgentox.2026.503954","DOIUrl":"10.1016/j.mrgentox.2026.503954","url":null,"abstract":"<div><div>The repeated-dose liver micronucleus (RDLMN) assay is being developed toward an International Workshop on Genotoxicity Testing (IWGT)-recommended protocol, for potential adoption as an Organisation for Economic Co-operation and Development (OECD) guideline. During this development, the possible effect of animal age has been identified as a key issue, since, in general rodent toxicity studies, agent administration is typically initiated at 6–8 weeks of age (OECD Test Guideline 407, “Repeated-Dose 28-Day Oral Toxicity Study in Rodents”). To address this issue, we have conducted an RDLMN assay in rats treated with 2-acetamidofluorene (2-AAF), with dosing initiated at either 6 or 8 weeks of age. 2-AAF was selected as a representative compound known to produce weak positive responses in genotoxicity studies. The results demonstrated comparable increases in micronucleus frequency between the two age groups, indicating no clear impact of age. These findings, obtained with a weakly positive compound, provide further supporting evidence that the RDLMN assay can be reliably applied to animals when dosing is initiated at either 6 or 8 weeks of age, thereby supporting its integration into repeated-dose toxicity studies. Using PCNA- and Ki-67-positive cell counts as indicators of cell proliferative activity, we examined the suppressive effect of 2-AAF on cell proliferation; such suppression may affect the sensitivity of the assay. Our findings support the use of both PCNA and Ki-67 as indicators of cell proliferative activity in the RDLMN assay.</div></div>","PeriodicalId":18799,"journal":{"name":"Mutation research. Genetic toxicology and environmental mutagenesis","volume":"914 ","pages":"Article 503954"},"PeriodicalIF":2.8,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148779551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}