Environmental and Molecular Mutagenesis最新文献

{"title":"Mitochondria-Nuclear Crosstalk: Orchestrating mtDNA Maintenance.","authors":"Ghazal Darfarin, Janice Pluth","doi":"10.1002/em.70013","DOIUrl":"https://doi.org/10.1002/em.70013","url":null,"abstract":"<p><p>The mitochondria (mt) and nucleus engage in a dynamic bidirectional communication to maintain cellular homeostasis, regulating energy production, stress response, and cell fate. Anterograde signaling directs mt function, while retrograde signaling conveys metabolic and stress-related changes from mt to the nucleus. Central to this crosstalk is mitochondrial DNA (mtDNA), which encodes key oxidative phosphorylation components. MtDNA integrity is preserved through quality control mechanisms, including fusion and fission dynamics, mitophagy, and nuclear-encoded DNA repair. Disruption in these pathways contributes to mt dysfunction, oxidative stress, and genetic instability-hallmarks of aging and diseases. Additionally, redox signaling and NAD+ homeostasis integrate mt and nuclear responses, modulating transcriptional programs that support mt biogenesis and stress adaptation. This review explores the molecular mechanisms coordinating mito-nuclear interactions, emphasizing their role in maintaining mtDNA integrity and cellular equilibrium. Understanding these processes provides insights into how mt dysfunction drives aging and disease, paving the way for targeted therapeutic strategies.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144141638","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":"Application of Biological Modifiers to a Multiplexed, Human Cell-Based DNA Damage Assay Provides Mechanistic Information on Genotoxicity and Molecular Targets.","authors":"Steven M Bryce, Svetlana L Avlasevich, Adam Conrad, Nikki E Hall, Kyle Tichenor, Erica Briggs, Stephen D Dertinger, Jeffrey C Bemis","doi":"10.1002/em.70017","DOIUrl":"https://doi.org/10.1002/em.70017","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144127001","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":"Fertilized Avian Egg Fetal Liver Assays for Assessing DNA Damaging Potential of Chemicals: A Comparative Analysis With In Vitro and In Vivo Genotoxicity Assays and Rodent Carcinogenicity.","authors":"Tetyana Kobets, Gary M Williams","doi":"10.1002/em.70016","DOIUrl":"https://doi.org/10.1002/em.70016","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144109742","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":"Applicability of In Silico New Approach Methods for the Risk Assessment of Tattoo Ink Ingredients","authors":"Prachi Pradeep,&nbsp;Stefanie Seifert,&nbsp;Ajay Vikram Singh,&nbsp;Peter Laux,&nbsp;Ralph Pirow","doi":"10.1002/em.70010","DOIUrl":"https://doi.org/10.1002/em.70010","url":null,"abstract":"<p>Tattoo inks contain several substances, including organic and inorganic pigments, additives, and solvents, which may pose a health risk to not only the tattooed skin but also to other parts of the human body due to intradermal exposure. Substances in tattoo inks are regulated by entry 75 in Annex XVII of REACH Regulation (EC) No. 1907/2006. However, despite these legal requirements, a well-defined criterion for the safety assessment of tattoo inks remains lacking. In this context, 2021 BfR opinion titled “Tattoo inks: minimum requirements and test methods” proposed a comprehensive risk assessment of pigments using in vitro/in-chemico data in accordance with OECD Guidelines and CLP Regulations. In the absence of experimental data, new approach methodologies (NAMs) may be used for data-gap filling. Therefore, this work evaluates the applicability of in silico NAMs for data-gap filling for a list of tattoo ink ingredients identified by the Joint Research Centre (JRC) and BfR for genotoxicity assessment. Experimental in vitro genotoxicity data were acquired from the International Uniform Chemical Information Database (IUCLID) which makes non-confidential REACH Study Results publicly accessible. The specific aims of this analysis were the evaluation of in silico genotoxicity predictions from publicly available QSAR tools and structural alerts, the development and validation of new QSAR models specific to tattoo ink ingredients, and the application of in silico models for categorization and prioritization of data-poor ingredients for further screening. Based on the workflow developed in this study, 4 high priority, 18 medium priority, and 2 low priority substances were identified for further assessment.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 4","pages":"199-209"},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/em.70010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144091823","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":"The Chicken Egg Genotoxicity Assay (CEGA): Assessing Target Tissue Exposure and Metabolism in the Embryo-Fetal Chicken Livers.","authors":"Yax Thakkar, T Kobets, Anne Marie Api, J D Duan, G M Williams","doi":"10.1002/em.70015","DOIUrl":"https://doi.org/10.1002/em.70015","url":null,"abstract":"<p><p>The Chicken Egg Genotoxicity Assay (CEGA) is an avian egg-based model that utilizes the livers of developing chicken embryo-fetuses to assess the ability of chemicals to produce direct DNA damage. The main goal of the study was to evaluate target tissue exposure and metabolism in the CEGA to assess its suitability as a biologically relevant new approach methodology (NAM) for detecting the genotoxic potential of chemicals. An imaging study using two-photon excitation microscopy after the administration of a fluorescent dye (acridine orange) verified that chemicals following administration into the air sac of the fertilized chicken egg reach the target organ, liver. A metabolism study using liquid chromatography with high resolution mass spectrometry (LC/MS), conducted after the administration of benzo(a)pyrene (B(a)P) according to the CEGA protocol, confirmed the formation of sufficient amounts of reactive metabolite(s) responsible for the genotoxic effects of a parent compound upon reaching the target tissue. Moreover, an RNA sequencing study revealed that B(a)P in embryo-fetal chicken livers significantly upregulated several genes responsible for the activity of the CYP1A1 enzyme, which is critical for the bioactivation of B(a)P. These findings, along with the previously reported DNA damage (i.e., DNA adducts and single-strand breaks) produced by B(a)P in CEGA, support sufficient target tissue exposure to B(a)P and the ability of avian fetal livers to bioactivate B(a)P to a reactive intermediate. Overall, the findings in the study support the conclusion that the CEGA can be considered a robust potential alternative to the animal testing strategy for assessing the genotoxic potential of chemicals.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143970973","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":"Adverse Outcome Pathway-Informed Integrated Testing to Identify Chemicals Causing Genotoxicity Through Oxidative DNA Damage: Case Study on 4-Nitroquinoline 1-Oxide","authors":"Elizabeth Huliganga,&nbsp;Eunnara Cho,&nbsp;Carol D. Swartz,&nbsp;Andrew Williams,&nbsp;Leslie Recio,&nbsp;Jesse J. Salk,&nbsp;Francesco Marchetti,&nbsp;Carole L. Yauk","doi":"10.1002/em.70011","DOIUrl":"10.1002/em.70011","url":null,"abstract":"<p>Adverse outcome pathways (AOPs) provide a framework to organize and weigh evidence linking molecular interactions of toxicants in cells to adverse outcomes relevant to risk assessment or regulatory decision-making. Applying this framework facilitates the interpretation of data produced using new test methods. We used an existing AOP (AOP #296) that describes how oxidative DNA damage leads to mutations and chromosomal aberrations to develop an integrated testing strategy to evaluate whether a chemical operates through this pathway. We exposed human TK6 cells to increasing concentrations of 4-nitroquinoline 1-oxide (4NQO), a tobacco mimetic that causes oxidative DNA damage, in a time-series design. We measured oxidative DNA damage and strand breaks using the high-throughput CometChip assay with and without formamidopyrimidine DNA glycosylase (Fpg), alongside analyses of micronucleus (MN) frequency by flow cytometry, and mutations by error-corrected sequencing (duplex sequencing—DS). Our analysis shows how these methods can be combined to quantify 4NQO-induced, concentration- and time-dependent increases in: (a) oxidative DNA damage (occurred early and at low concentrations); (b) strand breaks (remained elevated to 6 h post-exposure); (c) MN frequency (at 24 h); (d) mutation frequency (at 48 h); and (e) C &gt; A transversions consistent with expected substitutions induced by oxidative DNA lesions. The time series shows the repair of oxidative DNA damage with persistent strand breaks remaining at 6 h. Overall, we provide an example of an AOP-informed testing strategy and contribute to the quantitative understanding of AOP #296. We also demonstrate the value of DS as an effective approach for mutagenicity assessment.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 4","pages":"185-198"},"PeriodicalIF":2.3,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/em.70011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143970486","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":"Aflatoxin B1-Induced Hepatic Mutagenesis in Mice Expressing Gene-Edited Neil1","authors":"Irina G. Minko,&nbsp;Vladimir L. Vartanian,&nbsp;Michael M. Luzadder,&nbsp;Yingming Wang,&nbsp;Lev M. Fedorov,&nbsp;Amanda K. McCullough,&nbsp;R. Stephen Lloyd","doi":"10.1002/em.70014","DOIUrl":"10.1002/em.70014","url":null,"abstract":"<div>\u0000 \u0000 <p>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 <i>Neil1</i>^<i>A51V</i> and <i>Neil1</i>^<i>G83D</i> 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 <i>Neil1</i>^{<i>−/−</i>} mice revealed that although mutation frequencies in <i>Neil1</i>^<i>A51V</i> and <i>Neil1</i>^<i>G83D</i> 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.</p>\u0000 </div>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 4","pages":"144-154"},"PeriodicalIF":2.3,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143987432","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":"Using N-Nitrosodiethanolamine (NDELA) and N-Nitrosopiperidine (NPIP) Transgenic Rodent Gene Mutation Data and Quantum Mechanical Modeling to Derive Potency-Based Acceptable Intakes for NDSRIs Lacking Robust Carcinogenicity Data","authors":"Jason M. Roper,&nbsp;Troy R. Griffin,&nbsp;George E. Johnson,&nbsp;Jakub Kostal,&nbsp;Raphael Nudelman,&nbsp;Gregory R. Ott,&nbsp;Adelina Voutchkova-Kostal,&nbsp;Valerie Niddam-Hildesheim","doi":"10.1002/em.70012","DOIUrl":"10.1002/em.70012","url":null,"abstract":"<div>\u0000 \u0000 <p>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. <i>N</i>-nitrosodiethanolamine (NDELA) and <i>N</i>-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 <i>N</i>-nitrosodiethylamine (NDEA) and <i>N</i>-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.</p>\u0000 </div>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 4","pages":"155-171"},"PeriodicalIF":2.3,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144003967","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":"Benchmark Response (BMR) Values for In Vivo Mutagenicity Endpoints","authors":"Paul A. White,&nbsp;Guangchao Chen,&nbsp;Nikolai Chepelev,&nbsp;Madison A. Bell,&nbsp;Lauren R. Gallant,&nbsp;George E. Johnson,&nbsp;Andreas Zeller,&nbsp;Marc A. Beal,&nbsp;Alexandra S. Long","doi":"10.1002/em.70006","DOIUrl":"10.1002/em.70006","url":null,"abstract":"<p>The benchmark dose (BMD) approach constitutes the most effective and pragmatic strategy for the derivation of a point of departure (PoD) for comparative potency analysis, risk assessment, and regulatory decision-making. There is considerable controversy regarding the most appropriate benchmark response (BMR) for genotoxicity endpoints. This work employed the Slob (2017) Effect Size (ES) theory to define robust BMR values for the in vivo transgenic rodent (TGR) and <i>Pig-a</i> mutagenicity endpoints. An extensive database of dose–response data was prepared and curated; BMD analyses were used to determine endpoint-specific maxima (i.e., parameter <i>c</i>) and within-group variance (i.e., <i>var</i>). Detailed analyses investigated the dependence of <i>var</i> on experimental factors such as tissue, administration route, treatment duration, and post-exposure tissue sampling time. The overall lack of influence of these experimental factors on <i>var</i> permitted the determination of typical values for the endpoints investigated. Typical <i>var</i> for the TGR endpoint is 0.19; the value for the <i>Pig-a</i> endpoint is 0.29. Endpoint-specific <i>var</i> values were used to calculate endpoint-specific BMR values; the values are 47% for TGR and 60% for <i>Pig-a</i>. Endpoint-specific BMR values were also calculated using the trimmed distribution of study-specific standard deviation (SD) values for concurrent controls. Those analyses yielded endpoint-specific BMR values for the TGR and <i>Pig-a</i> endpoints of 33% and 58%, respectively. Considering the results obtained, and the in vivo genetic toxicity BMR values noted in the literature, we recommend a BMR of 50% for in vivo mutagenicity endpoints. The value can be employed to interpret mutagenicity dose–response data in a risk assessment context.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 4","pages":"172-184"},"PeriodicalIF":2.3,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/em.70006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788162","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":"An Enhanced Metabolization Protocol for In Vitro Genotoxicity Assessment of N-Nitrosamines in Mammalian Cells","authors":"M. E. Geijer,&nbsp;A. M. Gernaat,&nbsp;N. Moelijker,&nbsp;I. Brandsma,&nbsp;G. Hendriks","doi":"10.1002/em.70009","DOIUrl":"10.1002/em.70009","url":null,"abstract":"<p>N-Nitrosamines (NAs) are probable human carcinogens and were detected as impurities in pharmaceuticals, which led to a concern for human health. NAs require metabolic activation before they become mutagenic, and not all NAs are mutagenic since their reactivity is related to their structure. While some NAs are potent mutagens in vivo, in vitro metabolization with exogenous S9 liver extract is generally less efficient. While an enhanced bacterial mutagenicity protocol was recently developed, which uses increased concentrations of S9 liver extracts, there presently is not an improved metabolization protocol suitable for mammalian cell genotoxicity assays. Therefore, we optimized a hamster S9 liver extract-based protocol for in vitro NA metabolization and assessed the genotoxic potential of various NAs using ToxTracker. With this enhanced metabolization protocol (EMP), the genotoxic potency of N-nitrosodimethylamine (NDMA) increased approximately 200-fold compared with the standard S9 liver extract-based exposure protocol in ToxTracker. The EMP was further validated with seven additional mutagenic NAs to which humans are commonly exposed: N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDELA), N-nitrosodibutylamine (NDBA), N-nitrosofluoxetine (NF), 1-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), and 1-cyclopentyl-4-nitrosopiperazine (CPNP), and two non-mutagenic NAs: N-nitrosobupropion (NBuPRO) and N-nitrosoproline (NPRO). Genotoxicity could be confirmed for six NAs using the EMP, demonstrating that mammalian cells and the new approach methodology (NAM) ToxTracker may have potential when investigating NA-related genotoxicity.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 4","pages":"210-220"},"PeriodicalIF":2.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/em.70009","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728764","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}