The in vivo 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 in vivo bone marrow MN assay and may improve analytical efficiency, objectivity, and reproducibility in genotoxicity testing.
Genetic instability has been reported in several neurodegenerative diseases, such as Alzheimer's and Parkinson's, but only a few studies have addressed sclerosis. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Micronuclei (MNi) and nuclear buds (NBUDs) are established markers of chromosomal instability, yet no data are available regarding a possible link between genetic instability and ALS. The novelty of this case-control study lies in the assessment of genetic instability in oral exfoliated cells from ALS patients (n = 20) and matched controls (n = 20), contributing to the identification of potential novel markers related to the molecular pathogenesis of this severe disorder. Groups were matched for age, sex, and lifestyle (p > 0.05). No significant differences were observed in MNi or NBUD frequencies between groups (p > 0.05). These findings suggest no association between ALS and MN/NBUD frequencies in oral cells.
The in vitro genotoxicity testing battery comprising a bacterial gene mutation test (Ames test) and an in vitro micronucleus (MNvit) test is widely recommended by regulatory authorities since it detects the 3 key modes of genotoxic action (gene mutations, clastogenicity and aneuploidy). The recommendations that this battery is acceptable for regulatory use are based on its ability to effectively detect in vivo genotoxins and carcinogens. However, substances inducing gene mutations are a particular concern (representing stable genetic change and a key step in the carcinogenic process), and more in vivo gene mutation data have been published in recent years due to the wider use of transgenic rodent gene mutation (TGR) and Pig-a assays. It is appropriate to question whether the Ames + MNvit battery is efficient at detecting substances that induce gene mutations in vivo. A review of several compilations of genotoxicity studies revealed 26 substances that were reported positive in TGR and/or Pig-a but negative in the Ames test. A detailed analysis showed that 25 of these results were questionable for various reasons, and not robust examples of the failure of the 2-test in vitro battery. Only procarbazine was convincingly positive in vivo but predominantly negative in vitro, and this is mainly due to its complex metabolism which is probably not optimal with standard in vitro metabolic activation systems. The analysis shows no evidence that adding a mammalian cell gene mutation test to the in vitro battery would improve the detection of in vivo gene mutagens.
In the bacterial reverse mutation (Ames) test, Salmonella typhimurium 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.
The health consequences of cigarette smoking and its commercial variants have been extensively investigated for more than three decades. However, despite substantial evidence demonstrating the detrimental effects of tobacco use, including respiratory and cardiovascular diseases and several types of cancer, the global number of users remains alarmingly high. This scientometric review mapped the scientific literature on the health impacts of cigarettes and commercial tobacco products, focusing on studies employing biomarkers of toxicological effects, including the comet assay, micronucleus test, redox status assessment, and telomere length analysis. Of 1990 retrieved records, 155 articles met the inclusion criteria and were analyzed. Redox status assessment and the comet assay were the most frequently applied biomarkers. In vivo studies primarily analyzed blood samples, whereas in vitro investigations exposed diverse cell types to cigarette smoke. Although the adverse effects of conventional cigarettes are well established, the health impacts of alternative products, including e-cigarettes, pods, and vapes, remain insufficiently investigated. This review provides a focused scientometric overview of biomarker-based research on tobacco-related toxicological effects and highlights the need for broader future studies incorporating additional biomarkers, health outcomes, and emerging tobacco and nicotine products.
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.In this study, we synthesized a novel thiazolidinedione-based small molecule, O-prenylated benzylidene-thiazolidinedione (PBT), and evaluated its efficacy in combination with cisplatin (CDDP). Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice received oral PBT (4mg/kg b.w.,) either alone or in combination with a single intraperitoneal dose of cisplatin (CDDP; 5mg/kg b.w.,).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.Concurrently, PBT attenuated CDDP-induced nephrotoxicity and genotoxicity by modulating inflammatory mediators and antioxidant enzymes. While CDDP monotherapy induced a significant increase (p < 0.05) in chromosomal aberrations and DNA damage in bone marrow cells, PBT co-treatment significantly (p < 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.
This article reviews the evolution of radioprotection research in major Indian laboratories over the past seventy years, tracing a systematic progression in the exploration of various protective agents. The significant contribution of premier Indian institutions dedicated to ‘Radioprotection Development Programme’ is highlighted. Early investigations focused on synthetic radiopro-tectors, particularly sulphydryl compounds, which demonstrated strong radioprotective effects in laboratory settings. However, their clinical application was limited due to severe tissue toxicity. Subsequent research shifted toward natural thiols, novel synthetic compounds, and antioxidant nutrients. While these approaches produced encouraging experimental results, only a few showed real promise in clinical contexts. In recent decades, there has been a marked increase in studies on phytochemicals and herbal extracts, especially in Asian countries, driven by rich biodiversity and traditional medicinal systems such as Ayurveda in India. These phyto-products have shown high potential as effective radioprotectors with minimal toxicity. India has emerged as a key contributor in this domain, with extensive indigenous research efforts. Using databases such as PubMed and Google Scholar, this review identifies and compiles 46 Indian medicinal plants investigated for their radioprotective properties. Extract-based studies reveal promising efficacy through multiple mechanisms, including antioxidant activity and tissue protection. Preclinical findings demonstrate the ability of plant-derived bioactive compounds to mitigate radiation-induced damage. Notable examples include orientin and vicinin (Ocimum sanctum), podophyllin (Podophyllum hexandrum), curcumin (Curcuma longa), phyllanthin and flavonoids (Phyllanthus niruri) and polyphenol rich Rhodiola imbricata. Despite promising efficacy, clinical translation remains limited due to issues like poor bioavailability, low solubility, and formulation instability. This review concludes addressing the various pharmacological limitations, advanced delivery strategies, and the need for validated biomarkers for radiation damage to improve therapeutic effectiveness.
Acromegaly is characterized by the hypersecretion of growth hormone (GH) and insulin-like growth factor (IGF-1). Individuals with acromegaly have a higher risk of developing certain cancers, and the mitogenic and anti-apoptotic effects of GH and IGF-1 may heighten their predisposition to tumorigenesis. The presence of micronuclei (MN) and nuclear buds (NBs) in buccal mucosa cells is a marker of DNA damage, and high levels have been linked to carcinogenesis. The aims of this study were i) to comparatively analyze the proportions of buccal mucosa cells with MN and NBs in patients with acromegaly and controls, and ii) to identify associations between increased levels of such cells and patient and clinical factors. MN- and NB-containing cells were quantified using fluorescence microscopy. Data on the following demographic and clinical characteristics were collected: age, sex, smoking, alcohol consumption, time since acromegaly, GH and IGF-1 levels, radiation exposure, diabetes, body mass index, blood pressure, and cancer history. The patients with acromegaly (n = 42; 17 male, 25 female) had higher proportions of MN- and NB-containing cells than the controls, although only the levels of the MN-containing cells significantly differed (p = 0.023). The levels of MN-containing cells significantly differed with age (< 45 vs. ≥ 45 years). A significant inverse correlation was observed between the frequency of MN-containing cells and the IGF-1/upper limit of normal ratio. In conclusion, the patients with acromegaly exhibited relatively higher proportions of buccal mucosa cells with MN, a marker of genomic instability.
This paper is part of a series of publications developed by the Latin America-Comet assay (LA-COMET) group, which emerged in 2021, during the Asociacion Latinoamericana de Mutagenesis, Carcinogenesis y Teratogenesis Ambiental Congress, to organize the LA-COMET initiative. A total of 104 alkaline comet assay publications in animal (aquatic and terrestrial) and plant models, authored by members of this group, were analyzed to determine how DNA damage under experimental and environmental exposure conditions is assessed. The manuscript reflects the broad use and versatility of the comet assay in diverse taxonomic groups (invertebrates and vertebrates), in vivo research models, and cell types (e.g., erythrocytes, branchia cells, retinal epithelial cells, peripheral blood, liver, kidney, lung, bone marrow, testicle and nasal cells). Application of the comet assay in diverse biological systems requires careful methodological standardization to ensure reproducibility and allow for comparability of results. In order to unify the evaluations of the papers, a quality score system was developed, the 'quality score comet assay' (QSca), providing values reflecting the methodological rigor of the execution of the assay. The group identified key elements when performing the comet assay and uses them as the focus for the QSca score. The LA-COMET initiative will provide opportunities to strengthen collaborative networks among Latin American countries, promoting more integrative and regionally connected research where the comet assay can be used as a reliable and reproducible tool for the assessment of DNA damage in diverse biological models.
The increasing application of nanotechnology has raised concerns regarding the genotoxic potential of engineered nanoparticles due to their unique physicochemical properties and biological interactions. The present study evaluated the genotoxic effects of four oxide nanoparticles-aluminium oxide (Al₂O₃NPs), iron oxide (Fe₃O₄NPs), silicon dioxide (SiO₂NPs), and titanium dioxide (TiO₂NPs)-in human peripheral blood cells using the alkaline comet and cytokinesis-block micronucleus (CBMN) assays. Sublethal concentrations were selected based on IC₅₀ values determined by a resazurin-based cell viability assay. The comet assay revealed a significant dose-dependent increase in DNA strand breaks for all nanoparticles, with TiO₂NPs inducing the highest levels of primary DNA damage, as reflected by the percentage of tail DNA. In contrast, SiO₂NPs produced the highest frequency of micronuclei in the CBMN assay, indicating pronounced chromosomal instability. Fe₃O₄NPs showed a significant increase in nucleoplasmic bridge formation at higher concentrations, while Al₂O₃NPs exhibited comparatively lower genotoxic effects. Overall, these findings demonstrate differential genotoxic responses among oxide nanoparticles, influenced by particle size and composition, and highlight their potential to induce genome instability even at sublethal exposure levels.
Microplastics (MPs) and nanoplastics (NPs) are environmental pollutants with paramount implications for aquatic ecosystems and, through that route, human health, particularly due to their oxidative stress-mediated genotoxic potential. This review is a synthesis of findings from recent studies, with emphasis on the scenario in India, on the bioavailability, toxicological risks, and cellular mechanisms of MPs and NPs (MNPs) in various organisms, separately addressing evidence from aquatic models, including marine mussels, common carp, zebrafish, rotifers, etc., and mammalian systems relying essentially on in vitro studies. Key evidence indicates that MPs adsorb persistent organic pollutants like Polycyclic Aromatic Hydrocarbons (PAHs), enhancing their bioavailability and inducing oxidative stress, immunological alterations, and developmental toxicity, which are closely associated with DNA damage and chromosome instability. As regards aquatic organisms, combined exposure to MPs and heavy metals to fish models exacerbates biochemical disruptions and immune suppression, along with oxidative stress-linked genotoxic responses such as DNA strand breaks and micronucleus formation. Zebrafish embryos exhibit microcirculation dysfunction and pathological angiogenesis upon NP exposure. Mammalian cell studies reveal size-dependent cytotoxicity, with smaller NPs causing greater oxidative damage and membrane disruption, which triggers mitochondrial dysfunction, excessive ROS production, cell-cycle arrest, and activation of DNA damage response pathways, evidenced by micronucleus formation, chromosomal abnormalities, and oxidative DNA lesions. Overall, toxicity is influenced by particle size, charge and co-contaminants, with oxidative stress emerging as the central mechanism that connects cellular toxicity to genetic damage. This review underscores the urgent need for integrated, multidisciplinary approaches to assess the environmental and toxicological risks of MNPs with special emphasis on standardized genotoxicity assessment, while informing regulatory and mitigation strategies for the future.
The repeated-dose liver micronucleus (RDLMN) assay is an in vivo genotoxicity test that is useful for detecting hepatocarcinogens and is anticipated to be incorporated into general toxicity studies. Although many RDLMN studies have been reported, further investigation is necessary to demonstrate age-related sensitivity and to evaluate compounds with cytostatic properties.In this study, a repeated oral dose study of 2,6-dinitrotoluene (2,6-DNT), a substance known to have cytostatic properties, was conducted in Crl:CD(SD) rats starting at 6 or 8 weeks of age for 28 days. Liver micronucleus (LMN) frequency, mitotic index (MI), and cell proliferation markers, Ki-67 and PCNA, were subsequently evaluated. As a result, LMN frequency increased significantly at all dose levels, showing a reverse dose-response relationship, and there were little differences between age groups. The number of Ki-67-positive cells increased in both age groups, and it was considered compensatory cell proliferation following toxicity at higher doses. Histopathological evaluation revealed prominent anisokaryosis, particularly at 8 weeks of age at start of dosing, and DNA content analysis confirmed that these cells were polyploid, suggesting endoreduplication.This study confirmed that LMN detection sensitivity in the RDLMN assay did not differ between rats aged 6 or 8 weeks at the start of dosing, and that micronucleus-inducing ability was detected even in compounds with cytostatic properties. For appropriate interpretation of the results, histopathological assessment, the analysis of cell proliferation markers and DNA content analysis are useful.
India's rapid population growth positively correlates with solid waste generation. Indiscriminate disposal in unsanitary landfills is dominant, and releases hazardous contaminants, that pose genotoxic risks to humans and biota. This study evaluated DNA damage and genome instability induced by solid waste emissions of Indian origin. Following PRISMA guidelines, 36 peer-reviewed studies published until 2025 were selected. These studies reported 40 bioindicators and 56 genotoxic biomarkers, with cell lines and comet assay most frequently reported. Significant DNA strand breaks, micronucleus formation, chromosomal aberrations, nuclear abnormalities, and sperm defects in exposed models were consistently reported, often in dose- and time-dependent patterns. Random-effects meta-analysis using pooled standardized mean differences showed significant positive effect sizes for micronucleus and comet endpoints (p ≤ 0.0001), confirming increased genetic damage in exposed groups. Although substantial heterogeneity was observed (I² = 95.73% for micronucleus; 87.66% for comet), the direction of effect remained consistently positive. Risk-of-bias assessment indicated overall moderate to high methodological quality, with common reporting gaps in exposure characterization and blinding procedures. Oxidative stress and ROS generation are dominant drivers of genome instability. Signaling pathways causing DNA adduct, DNA repair impairment, epigenetic dysregulation, and mitochondrial-mediated apoptosis were other mechanisms. Despite strong evidence of somatic and germline genotoxicity, epidemiological data and quantitative exposure assessments remain inadequate. Integrated exposure monitoring, advanced molecular and epigenetic biomarkers, dose-response characterization and long-term population-level biomonitoring are needed to strengthen causal inference and human health risk assessment. Emissions from solid waste facilities in India represent a credible genotoxic hazard warranting improved waste management, and regulatory enforcement.
On a global scale, the versatility and high sensitivity of the comet assay have promoted its use from different perspectives to determine genotoxic damage. With this in mind and through the formation of a Latin American group denominated "LA-COMET", a review of the manuscripts published by the group is proposed from the perspective of in vitro studies, using the alkaline comet assay. To facilitate the analysis of the 81 manuscripts published between 1996 and 2022, they were classified into four categories based on the type of agents evaluated by the assay: 1) environmental pollutants; 2) pesticides and their mixtures; 3) radiation and drugs; and 4) natural products. The objective of this review was to describe the strengths and versatility of in vitro studies reflected by cellular models, putative DNA damaging agents, and the use of specific enzymes to identify particular DNA lesions. In addition, an in vitro QS (quality score) was established, taking into consideration the data provided in the publications regarding the cellular model, the method of disaggregation and the cell suspension obtention. Other considerations included the clarity of the description of the treatments, the use of negative and positive reference controls, and the use of specific enzymes and/or the functional evaluation of DNA repair. The analysis demonstrated that the publications of the LA-COMET group of in vitro models exhibited a high score with respect to the important parameters established in the OECD agreements.
Technological and chemical advancements have heightened human exposure to synthetic and natural substances, necessitating precise toxicological assessments through sensitive biomarkers. The comet assay has emerged as a robust, minimally invasive tool for biomonitoring. This study reviews 64 articles published between 1997 and 2022 by the LA-COMET group—a collaborative network of 17 research teams across seven Latin American countries (Argentina, Bolivia, Brazil, Colombia, Mexico, Paraguay, and Uruguay).The analysis reveals that research primarily focuses on occupational and environmental exposures, specifically evaluating the impact of pesticides, heavy metals, organic solvents, and particulate matter. The assay proved highly effective in identifying genotoxic effects in exposed populations compared to reference groups, including vulnerable cohorts such as children, women, and patients with clinical conditions like breast cancer and malaria. To enhance future research rigor, this work utilizes and proposes standardized scoring systems, namely QSca (assay quality score) and hmQS (human monitoring quality score). Ultimately, this review underscores the comet assay’s pivotal role in characterizing regional health risks and seeks to establish evidence-based practices to inform public health policies in Latin America.
Cisplatin and its analogues are a cornerstone of cancer chemotherapy; however, their therapeutic potential is limited by severe side effects and the development of drug resistance. To address these issues, nanocarriers and/or platinum prodrugs are being evaluated. Previous in vitro studies showed that ultra-small (< 5 nm) iron oxide nanoparticles coated with tartaric and adipic acids (FeAT-NPs) are an efficient delivery system for the cisplatin prodrug cis-diamminetetrachloroplatinum(IV) (cisplatin(IV)). In this study, the in vivo behavior of cisplatin (IV) conjugated with the FeAT-NPs (FeAT-NPs-Pt(IV)) was evaluated using Drosophila melanogaster as a preliminary non-mammalian animal model. The resulting FeAT-NPs-Pt(IV) nanoconjugate was compared to free cisplatin and cisplatin(IV) regarding platinum incorporation into DNA, measured using inductively coupled plasma mass spectrometry (ICP-MS), as well as toxicity and genotoxicity, which were assessed using the alkaline comet and the w/w+ eye-spot SMART assays. Results revealed that the nanoconjugate achieved over eightfold higher DNA platination than free cisplatin, which is consistent with efficient cellular uptake and the subsequent intracellular release and activation of the prodrug. Despite this high platination, the nanoconjugate induced DNA strand-break levels similar to cisplatin and lower mutation and recombination frequencies than both cisplatin and cisplatin(IV), in both nucleotide excision repair (NER) efficient and deficient conditions. No significant toxicity was detected at the tested concentrations. These findings suggest that, in vivo, FeAT-NPs-Pt(IV) acts as an effective delivery system, potentially enabling the slow intracellular formation of active cisplatin. This would increase bioavailability to its target (DNA), while maintaining a significantly lower genotoxic stress profile compared to free cisplatin. Nonetheless, additional studies in tumor mammalian models are required to further explore these observations.
The in vitro micronucleus (IVMN) assay is a genetic toxicity assay routinely conducted in early drug development to evaluate clastogenicity and aneugenicity. At this discovery stage, synthetic routes for potential active pharmaceutical ingredients (API) are not optimized, which can lead to incomplete purification or solvation. API in genetic toxicity screens often contain residual solvent(s) in higher quantities than will be present in the GMP material. Understanding when solvents used either in API synthesis or as a vehicle may interfere with genetic toxicity screening results of APIs is important. In this work, twenty solvents commonly used in API synthesis were analyzed in the IVMN assay. These twenty solvents include acetonitrile, formic acid, anisole, dichloromethane, heptane, isopropyl amine, dimethyl sulfoxide, sodium hexafluorophosphate, N,N-Dimethylethylamine, N,N-Dimethylformamide, cyclopentyl methyl ether, 2-propanol, 1-butanol, 1-Methyl-2-pyrrolidinone, methanol, tetrahydrofuran, 2-Methyltetrahydrofuran, ethyl acetate, trifluoroacetic acid, and 2,6-Di-tert-butyl-4-methylphenol. When the in vitro data was correlated to available in vivo genotoxicity data published in the literature, only 5 of the 14 positively correlated. However, when a 10 mM limit of exposure was applied to the solvents, 13 of the 14 correlated to the in vivo literature results. While the results do not consider vehicle interaction within the assay, the data demonstrates that residual solvent carryover from API or other sources is generally not of concern. The 10 mM limit also reduces the false-positive risk of this assay. Baseline cytotoxicity and micronucleus formation from the solvents in this system provide a ranking on which solvents may produce confounding results in the assay.
