Smoking is a cause of serious diseases in adult smokers. Next generation products (NGP) can provide potentially reduced harm alternatives of nicotine delivery, due to the reductions in numbers and levels of toxicants associated with their use. These include heated tobacco and herbal products (HTPs/ HHPs) and electronic vapour products (EVPs). This study assessed the effects of a range of NGP (an HTP, an HHP and two EVPs) and a reference cigarette (1R6F) in the ToxTracker Antioxidant (AO) and ToxProfiler Mitotox Assessment Xtension (MAX) assays, which provide mechanistic insights via a range of cell stress endpoints. Aqueous extracts of smoke/ aerosol were generated by bubbling through phosphate-buffered saline solution, which was analysed for nicotine and carbonyl content. In the ToxTracker AO assay, 1R6F induced oxidative stress, genotoxicity and protein damage responses. Addition of N-acetyl cysteine (NAC) and glutathione (GSH) partially reduced selected responses, indicating oxidative stress contributes but does not fully account for all observed effects. Oxidative stress responses followed a similar trend for HTP; but was not classed as genotoxic. The HHP induced one oxidative stress marker, which was reduced by the addition of NAC/GSH. In the ToxProfiler MAX assay, 1R6F induced oxidative, endoplasmic reticulum and cell cycle stress. HTP induced the same stress pathways but was less potent than 1R6F. The HHP only induced an oxidative stress response, and at higher concentrations than 1R6F. The EVPs did not induce any of the cell stress markers across both assays and none of the test products induced mitochondrial toxicity. Overall, biological activity followed a decreasing gradient from 1R6F to HTP, HHP and EVPs. The findings of the assays used in this study reflect the proposed placement of such products on the relative reduced risk scale based on their smoke/aerosol chemistry and thus highlights the tobacco harm reduction potential of NGP.
The comet assay is a widely used tool for evaluating DNA damage in various biological samples, with frozen whole blood (WB) as an attractive biomatrix, due to its ease of collection, handling, and storage. Although frozen WB has been successfully applied in the comet assay to study DNA damage, its application in the comet assay to study potential protective mechanisms, such as DNA repair or antioxidant capacity, has not been fully established yet. In this study, we set up a protocol using frozen WB for assessing base excision repair (BER) activity and further optimized the protocol to assess the cellular antioxidant defense capacity against oxidation-induced DNA damage. White blood cells (WBCs) were isolated to extract proteins, and a protein concentration of 2 mg/mL enabled reliable detection of BER activity, even in samples stored for up to 27 months. This optimized assay was subsequently applied in a pilot study involving 28 participants with either a family tumor history (FTH, n = 10) or not (NFTH, n = 18). The FTH group exhibited significantly less BER activity compared to the NFTH group (p = 0.005). In addition, the hydrogen peroxide (H₂O₂) challenge comet assay was further optimized to assess the cellular antioxidant defense capacity against oxidation-induced DNA damage in frozen WB and subsequently applied to a cohort study (n = 140). Frequent intake of seeds (p = 0.025) and vegetables (p = 0.003) was observed to protect against H2O2-induced DNA damage. Together, these innovations expand the applications of the comet assay in frozen blood samples, providing a more comprehensive biomonitoring toolbox. By enabling reliable measurements of DNA damage, antioxidant capacity, and DNA repair activity from a single frozen blood sample, these assays are ideally suited for large-scale human biomonitoring and clinical studies.
Inflammatory bowel disease (IBD) is associated with alterations in the intestinal luminal environment that may modify the composition of fecal water (FW) and its capacity to induce DNA damage. The aim of this study was to identify FW metabolites associated with DNA damage and aryl hydrocarbon receptor (AhR) activity and to investigate whether shared metabolic pathways may link these responses in patients with IBD. FW samples from 80 IBD patients and 20 healthy controls were analyzed. DNA damage in Caco-2 cells following FW exposure was assessed using the comet assay, AhR activity using the ethoxyresorufin-O-deethylase (EROD) assay, and FW metabolites by using liquid chromatography-mass spectrometry. Metabolite signatures associated with DNA damage and AhR activity were identified separately, and subsequent pathway analysis revealed overlapping metabolic pathways, prompting further investigation of AhR-associated tryptophan metabolites. Kynurenine was inversely associated with AhR activity but positively associated with DNA damage in IBD, whereas serotonin was positively related to AhR activity and was protective against FW-induced DNA damage. Indoles did not significantly contribute to FW-induced DNA damage. These findings suggest that inflammation-associated alterations in tryptophan metabolism are associated with altered AhR signaling and epithelial DNA damage in IBD.
Human exposure scenarios to emerging pollutants, micro- and nanoplastics are increasing. However, in vivo reports defining their acute genotoxic hazard, particularly under combined (microplastics (MPs) + nanoplastics (NPs)) exposure are limited. Polystyrene (PS) MPs and NPs are among the most prevalent and are being detected in food and environmental matrices. Hence, we investigated the acute genotoxic effects of PS MPs, NPs, and MPs+NPs treatment following oral administration at 10, 100, and 1000 mg/kg body weight doses in female Wistar rats. Particle characterization confirmed polymer integrity and size distribution, with hydrodynamic diameters of 1422 nm for MPs and 112.3 nm for NPs in Milli-Q water. A dose-dependent hepatic and renal injury was observed in histopathological studies and the effect was more prominent at the 1000 mg/kg dose in rats treated with PS MPs+NPs. Comet assay results showed significant DNA damage in peripheral blood leukocytes, bone marrow, liver, and kidney cells with PS NPs and MPs+NPs compared with PS MPs treatment alone. Notably, PS MPs+NPs exposure induced DNA damage even at the lowest dose (10 mg/kg) tested. Cell cycle analysis in rats treated with PS MPs and NPs depicted arrest in G0/G1, S, and G2/M phases which was consistent with activation of DNA damage checkpoints. A dose dependent elevation in Thiobarbituric acid reactive substances (TBARS) levels levels and depletion of reduced glutathione in plasma samples of rats indicate disruption of redox homeostasis. Overall, the findings of this study provide invaluable insights on PS plastic particles in vivo effects after acute oral exposure indicating oxidative stress mediated DNA damage, cell cycle perturbation, and tissue injury. The overall toxicity trend followed the order MPs+NPs > NPs > MPs, suggesting enhanced toxicity following combined exposure and highlighting the need for long-term toxicity studies of MPs+NPs mixtures to inform regulatory risk assessment.
Osteosarcoma is an aggressive malignant bone tumor that often requires chemotherapy with agents such as doxorubicin (Dox), which is known for its potent cytotoxic and genotoxic effects. To manage cancer-related pain, morphine is widely used due to its strong analgesic properties. The concurrent administration of these drugs in clinical practice might affect cancer therapy, since Dox is a well-established inducer of genomic instability, whereas the potential contribution of morphine to this process remains poorly understood. This study evaluated the cytotoxicity, genotoxicity, and chromosomal damage induced by morphine, both alone and in combination with Dox. Cytotoxicity was assessed in MG63 osteosarcoma cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Genotoxicity and chromosomal damage were evaluated in MG63 and non-tumor V79 fibroblast cells using the comet assay and the cytokinesis-block micronucleus (CBMN) assay. Morphine alone induced DNA damage in both MG63 and V79 cells in the comet assay and increased micronucleus frequency only in MG63 cells. In co-treatment, morphine significantly reduced Dox-induced DNA damage and chromosomal instability parameters in the CBMN assay in MG63 cells. Conversely, micronucleus frequency increased in co-treatment in V79 cells. Overall, morphine alone increased chromosomal damage in MG63 tumor cells but not in V79 non-tumor cells. However, in combination with Dox, morphine attenuated Dox-induced genomic instability in osteosarcoma cells while enhancing chromosomal damage in non-tumor fibroblasts. These findings suggest that morphine may differentially modulate the genomic effects of Dox, potentially impacting both antitumor efficacy and genomic stability in normal cells.
The indoor environment changes based on a variety of different factors, therefore changing the genotoxic potential of that environment. Despite the associations of exposure to outdoor air pollution and links to health impacts, enough is not currently known about the impacts to human health indoor air may have. Due to the variable nature of the indoor environment (multiple things can change this environment, including but not limited to, the use, occupants, materials within the environment, animals, the outside environment and ventilation) this can prove difficult. Indoor particulate matter (PM) of various sizes (PM2.5-PM10 for example) has been shown to induce reactive oxygen species, increase markers in oxidative stress, double strand DNA breaks, and cause 8-oxoguanine lesions in both in vitro and in vivo models (including humans). The particle type and source influence these responses, highlighting heating and cooking fuel sources that may be more genotoxic than others. A similar pattern has also been observed with volatile organic compounds (VOCs) with various mixtures of VOCs and different VOC compositions inducing DNA damage differently, with diet also being a contributing factor to genotoxic potential. A significant knowledge gap in the potential genotoxicity of indoor air pollution is the exposure of various biologicals found indoors. Moulds, other fungi, and allergens are an emerging health risk and are of significant public interest. Little is known about the inhalation toxicology and mechanisms of action of these components, and this is becoming increasing important to understand due to public deaths linked to them. There is overlap with the health effects of outdoor air pollution where there has been much more research focus. Therefore, the aim of this review is to establish what is already known, the potential limitations of the current work and where we should focus research in the future.
Maternal nutrition can influence genetic health during pregnancy, increasing the risk of DNA damage in both the mother and fetus. This study investigated the effect of maternal nutritional factors on DNA damage biomarkers in the mother-infant dyad. A cross-sectional study was conducted with pregnant women residing in Northeast Brazil. Participants included women aged ≥18 years in the third trimester, excluding those with non-communicable chronic diseases and smokers. Data collection included sociodemographic, lifestyle, anthropometric information, and a 76-item Food Frequency Questionnaire. The micronucleus cytome assay was performed on oral mucosa cells, with DNA damage biomarkers expressed per thousand cells (‰). Statistical analysis involved hierarchical clustering and Generalized Linear Models using Jamovi 2.5.6. The median age of the pregnant women (n = 209) was 26 years. Only 44.5% had adequate pre-pregnancy nutritional status, with a 9.5% increase in low birth weight. Total energy intake was 1842 (588-5709) kcal, with 72% consuming less than 90% of their caloric needs. The diet was predominantly hyperproteic and hyperlipidic, with over 60% showing inadequate intake of magnesium and folic acid. Among 209 participants, 80 consented to oral mucosa cell collection, and 59 mother-infant pairs were analyzed. Three maternal nutritional profiles were identified: Cluster 1 ("Multiple Burdens of Malnutrition"), Cluster 2 ("Low Burdens of Malnutrition"), and Cluster 3 ("High Nutritional Intake"). While maternal nutritional profiles did not affect nuclear abnormalities in mothers, they influenced newborns. Infants of mothers in Clusters 1 and 2 exhibited more nuclear abnormalities than those in Cluster 3. Maternal nutritional inadequacy was associated with increased nuclear abnormalities in newborns, suggesting an impact on fetal genomic stability.
Potassium bromate (KBrO₃), classified as a probable human carcinogen, has long been used as a flour-improving and oxidizing agent in baking and food processing industries. It is also generated as a by-product during ozonation of drinking water, raising concerns about widespread, low-dose exposure. Experimental studies consistently demonstrate that KBrO₃ induces multiple-organ toxicity primarily through oxidative stress-mediated mechanisms. Exposure to KBrO₃ leads to excessive generation of reactive oxygen species, resulting in depletion of glutathione reserves, enhanced lipid peroxidation, and oxidative DNA damage. Such genotoxic effects include DNA strand breaks, micronuclei formation, and chromosomal aberrations, which collectively contribute to its well-documented carcinogenic and mutagenic potential. Consequently, these molecular alterations promote renal, hepatic, and thyroid tumor formation reported in experimental animal models, while emerging epidemiological evidence suggests possible associations between chronic dietary exposure to KBrO₃ and gastrointestinal malignancies in humans. However, recent research has thoroughly investigated the chemopreventive potential of dietary antioxidants in mitigating KBrO₃-induced toxicity, highlighting their ability to attenuate oxidative damage through enhancement of endogenous antioxidant defenses, modulation of the key cytoprotective signaling pathways and restoration of cellular redox homeostasis. With continued usage of KBrO₃ and the inconsistency of regulatory enforcement worldwide, strengthening risk assessment, surveillance and preventive strategies-particularly the substitution of KBrO₃ with safer alternatives and the promotion of antioxidant-rich diets is crucial to reduce long term carcinogenic risk and safeguard global public health.
Previously, tetrazole-based Schiff base ligands (TBSL) and their Pt(II) complexes were synthesized and evaluated for cytotoxic activity. The results revealed that low cytotoxicity for the ligands and moderate effects for the Pt(II) complexes compared to cisplatin. Given these promising findings, the genotoxic potential of the Pt(II) complexes was further investigated. Genotoxicity was assessed using in vitro chromosomal aberration, micronucleus, and comet assays. The TBSL exhibited low genotoxicity, whereas the Pt(II) complexes induced higher genotoxic effects; however, these effects were lower than those observed for cisplatin. The low cytotoxic and genotoxic profiles of TBSL support their consideration for further pharmaceutical evaluation. In addition, the genotoxicity findings suggest that the Pt(II) complexes merit further investigation in the context of antitumor drug development.
Diet quality can be a potential modifier of post-acute COVID-19 syndrome (PACS), yet the relationship between dietary patterns, symptom burden, and biological markers remains poorly understood. We hypothesized that a low-quality diet would positively associate with PACS symptom burden, DNA damage, inflammatory and neurotrophic markers, and with overall health quality. A total of 186 individuals were classified into three groups: control (n = 64), acute PACS (n = 66), and chronic PACS (n = 55). Dietary intake was assessed by a food frequency questionnaire, and diet quality was quantified via the Protective and Risk Eating Score (PRES). Blood samples were analyzed for inflammatory (tumor necrosis factor-α, transforming growth factor-β, interleukin-6, interleukin-1β) and neurotrophic (brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, S100 calcium-binding protein B) markers. DNA damage was assessed by micronuclei frequency, telomere length, and comet assay parameters. Health index (HI) was assessed by daily habits (alcohol consumption, sleeping patterns, hydration, exercise frequency, smoking habits, anthropometric parameters, and PRES). No significant differences were found among groups in ultra-processed foods or sugary products intake, PRES scores, inflammatory or neurotrophic biomarkers, DNA damage indicators, and HI. Of note, poorer diet quality was associated with higher symptom burden in PACS women, mainly in fatigue, memory, mental health, circulatory, and skin symptom domains. Few associations among diet quality, HI, and biomarkers emerged. Of note, principal component analysis shows that PACS relies on the convergence of genomic instability, systemic inflammation, and low diet quality. These findings suggest that diet may represent a modifiable factor in PACS management, warranting longitudinal and interventional studies to determine causality and therapeutic potential.
Detection of N-nitrosamines (NA) in pharmaceuticals became a point of interest due to the mutagenic and carcinogenic potential of some compounds of this class, and their identification as nitrosated forms of marketed drugs, otherwise known as NA Drug Substance-Related Impurities (NDSRIs). The Ames test is used to assess the mutagenic potential of drug impurities, including NAs. Concerns over the sensitivity of the Ames test, as recommended in Organization for Economic Co-operation and Development Test Guideline 471, in predicting the rodent carcinogenic potential of NAs has prompted optimization of several test parameters used for detecting the mutagenicity of NAs (e.g. methods used for metabolic activation and the selection of tester strains). In order to discuss optimal Ames test conditions for the evaluation of NAs, including NDSRIs, the Office of New Drugs in the US Food and Drug Administration's Center for Drug Evaluation and Research and the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee co-organized and co-sponsored a workshop entitled "Nitrosamines: Ames Data Review and Method Development Workshop". The workshop featured five sessions addressing charge questions pertinent to the Ames test conditions and performance through the presentation of data and panel discussions. This report outlines the key takeaway points from the workshop.
This study aimed to establish a novel BRCA1 mutant cell line from Chinese hamster V79 cells and clarify the role of BRCA1 in the DNA damage response (DDR) by comparison with a BRCA2 mutant line, V-C8. Using CRISPR/Cas9 editing, we generated a hypomorphic BRCA1 mutant, designated B1-21, carrying a 27-bp in-frame deletion in exon 4. This mutation deletes nine amino acids within the RING domain. B1-21, V79, and V-C8 cells were analyzed for DDR phenotypes. Both mutants showed impaired RAD51 foci formation, defective homologous recombination repair, and increased sensitivity to DNA-damaging agents. B1-21 cells were particularly sensitive to camptothecin and the PARP inhibitor NU1025, whereas V-C8 cells showed higher sensitivity to etoposide, cisplatin, mitomycin C, and bleomycin. Although γH2AX and FANCD2 focus responses were similar between V79 and B1-21, RAD51 recruitment was only partially reduced in B1-21 and completely absent in V-C8. B1-21 also displayed chromosomal instability (19-20 chromosomes), while V79 and V-C8 maintained a stable karyotype. After gamma irradiation, V-C8 cells accumulated substantially more chromatid-type aberrations and retained unrepaired chromatin longer than B1-21. Neither mutant showed normal RAD51 foci formation, radiation-induced sister chromatid exchange or an effective G2/M checkpoint arrest, unlike wild-type cells. Mitotic index measurements further confirmed checkpoint failure: V79 cells suppressed mitotic entry after irradiation, while B1-21 and V-C8 continued to enter mitosis, with V-C8 showing the most complete checkpoint breakdown. These findings indicate that partial disruption of the BRCA1 RING domain results in a hypomorphic phenotype with impaired homologous recombination, defective checkpoint control, and enhanced genotoxic sensitivity. The isogenic BRCA1 and BRCA2 mutant V79 lines offer a valuable model for dissecting DDR pathway differences and developing mutation-specific therapeutic strategies targeting BRCA-mutant cancers.
Cardiovascular diseases (CVD) are the leading cause of death globally, posing a major public health challenge due to their increasing prevalence and complex etiology. Natural products from medicinal mushrooms have gained attention for their cardioprotective properties. To evaluate whether the aqueous extract of Lenzites betulinus can protect mononuclear cells of acute coronary syndrome (ACS) and heart failure (HF) patients from therapy-induced DNA damage and to determine its polyphenolic composition. The study included 30 healthy controls, 30 ACS, and 30 HF patients. The genoprotective potential of aqueous mushroom extract at concentrations of 50, 100, 150, and 200 mg/mL was examined in vitro using the comet assay on human blood mononuclear cells. Total phenolic and flavonoid contents were determined spectrophotometrically, while polyphenolic profiling was performed using UHPLC-DAD-MS/MS. The mean level of DNA damage in treated cell cultures from healthy donors increased in a concentration-dependent manner compared with the negative control, but without statistical significance. The extract significantly reduced therapy-induced level of DNA damage in mononuclear cells of ACS and HF patients (P < .0005). DNA damage level was significantly higher in HF patients. In ACS patients, %DNA damage was significantly reduced at the three highest concentrations, while genetic damage index (GDI) decreased at all concentrations. In HF patients, %DNA damage decreased at the two highest concentrations, and GDI at all except the lowest. Phytochemical analysis revealed high levels of phenolics and flavonoids, with chlorogenic acid and quercetin as dominant compounds. L. betulinus possesses significant nutritional and functional value. Its rich phytochemical profile likely underlies its chemoprotective effects against therapy-induced DNA damage, suggesting potential as a natural agent for genome stability support in CVD patients.
Popular ergogenic supplements for weight loss raise safety concerns due to potential cardiovascular, liver, and kidney toxicity. Their genotoxicity remains insufficiently studied, highlighting the need for more research on their long-term effects. The Brazilian health surveillance and regulatory system does not include dietary supplements under OECD genotoxicity regulatory guidelines. This study evaluates the mutagenic, cytotoxic, and genotoxic effects of a multi-herbal ergogenic supplement using the Ames test, WST-1 cell viability assay, and cytokinesis-block micronucleus tests in HepG2 and HEK293 cell lines. The Ames test revealed dose-dependent mutagenicity, particularly with metabolic activation, while the WST-1 assay showed hepatocytotoxic and nephrocytotoxic potential with dose- and time-dependent response. The cytokinesis-block micronucleus assay further confirmed genotoxicity, with significant formation of micronuclei and other cellular damage indicators. Our study emphasizes the need for careful safety monitoring of ergogenic supplements, highlighting the toxicity risks associated with their mutagenic, cytotoxic, and genotoxic potential.
This article summarizes the scientific contributions of Professor Sachi Prasad Ray-Chaudhuri and his unwavering commitment to advancing research and teaching in mutagenesis and genetics in post-independence India, following his postgraduate training in the United Kingdom under the mentorship of Professor H. J. Muller. His contributions in basic mutagenesis, radiation protection, animal cytogenetics, and population genetics using a range of model and native species laid the foundation of scientific research in India in these contemporary fields. This played a pivotal role in advancing genetics and biological sciences in India at a time when scientific infrastructure and resources were limited. He also took the initiative to modernize the curriculum of biology teaching in India in line with the developments in the subject globally. He expanded these efforts by teaching as a visiting professor in different, often remote parts of the country, where he inspired the students. The seeds of his relentless effort to connect the advances in Indian science with those in the rest of the world have blossomed, and Indian science today contributes significantly to the global knowledge base. His pioneering scientific contributions and inspiring legacy continue unabated.
Reactive oxygen species (ROS) play a dual role in cancer biology, contributing to both tumor progression and therapeutic responses. Many chemotherapeutic agents exert their cytotoxic effects through ROS generation, although the extent and biological relevance of this process remain influenced by cellular context, including the functional status of tumor suppressor genes such as TP53. In this study, we investigated the induction of genomic damage and oxidative stress in two isogenic human colorectal cancer cell lines-HCT116TP53+/+ and HCT116TP53-/--after exposure to four commonly used anticancer agents: oxaliplatin (OXA), irinotecan (IRI), paclitaxel (PAC), and 5-fluorouracil (5-FU). Drug concentrations were selected to ensure cell viability while inducing genotoxicity. Genome damage was assessed by micronucleus (MN) assay. ROS production was measured using the BODIPY581/591 C11 lipid peroxidation fluorescent probe. Our results showed that OXA and IRI induced both significant MN formation and robust ROS production in a dose-dependent manner, while PAC predominantly triggered genomic damage with limited ROS generation. Conversely, 5-FU exhibited marginal (or no) effect on both endpoints. Notably, with regard to the extent of ROS accumulation and MN induction, no significant differences were detected under the tested conditions between the two HCT116 cell lines. This suggests that under non-cytotoxic conditions, p53 is not a critical modulator of oxidative responses to these agents. Overall, our data reveal a drug-specific pattern of genome and oxidative damage in HCT116 colorectal cancer cells, emphasizing the importance of considering drug mechanism of action in evaluating redox responses. These insights may contribute to the development of targeted combinatorial therapies aimed at modulating oxidative stress, especially in TP53-mutated tumors.
According to Organisation for Economic Co-operation and Development test guideline no. (TG) 487 for the in vitro mammalian cell micronucleus test (MNT), short (pulse) treatment of primary human lymphocytes should be followed immediately by a sampling period of 1.5-2.0 cell cycle lengths in the presence of cytochalasin B. However, TG 487 allows extending the sampling period "if it is known or suspected that the test chemical affects the cell cycling time." It is often unknown in advance whether a test substance induces cell cycle delays, and therefore whether an extension of the sampling period is warranted. Here, we investigate whether extending the sampling time after pulse treatment-by including a recovery period prior to cytochalasin B treatment-affects the sensitivity of the in vitro MNT. Primary human lymphocytes were exposed to 11 mutagens and assessed using two protocols ("recovery" and "no recovery") in parallel from the same treatment culture. Including a recovery period after pulse treatment generally allowed testing of higher concentrations without excessive cytotoxicity. In addition, cultures subjected to a recovery period showed generally higher micronucleus rates at concentrations with acceptable levels of cytotoxicity, compared to cultures not having undergone a recovery period. To compare the sensitivity of both protocols at non-cytotoxic concentrations, benchmark concentration analysis was performed. Here, the "recovery method" proved to be at least as sensitive as the "no recovery method." The reproducibility of these findings was demonstrated in an interlaboratory ring trial using a subset of up to five mutagens. In conclusion, the inclusion of a recovery period after pulse treatment per se covers potential substance-induced cell cycle delays without any loss in sensitivity toward the induced mutagenic effect. The "recovery" protocol can be used for assessing the mutagenic potential of test substances affecting and not affecting the cell cycle.
Nitrosamine drug substance-related impurities (NDSRIs) have raised significant concern among the pharmaceutical industry and regulatory agencies, prompting a need to establish methods to determine the mutagenic risks associated with these substances. As human, metabolically competent HepaRG cells previously showed promise in evaluating the mutagenicity of a small-molecule nitrosamine impurity, N-nitroso-dimethylamine (NDMA), this study evaluated the suitability of HepaRG cells for mutagenesis assessment of NDSRIs. HepaRG cells, cultured in both two-dimensional (2D) and three-dimensional (3D) spheroid formats, were exposed to N-nitroso-fluoxetine and N-nitroso-varenicline for either 3 days or 14 days. DNA damage was evaluated using the comet assay, clastogenicity/aneugenicity was measured with the micronucleus (MN) assay, and mutagenesis was assessed using high-fidelity sequencing (HiFi-seq). N-Nitroso-fluoxetine and N-nitroso-varenicline were cytotoxic, produced DNA damage, and increased mutation frequency in both 2D and 3D HepaRG models. Both compounds increased MN formation only at the highest concentration in 3D spheroids after the 14-day exposure. Benchmark concentration (BMC) analysis indicated that, despite overlapping confidence intervals between 2D and 3D HepaRG cultures, in most instances BMC values tended to be lower in 3D than in 2D cultures after both 3-day and 14-day treatments. In addition, the 14-day treatments of both 2D and 3D HepaRG cultures with N-nitroso-fluoxetine resulted in higher levels of mutagenesis than the 3-day treatments, while 3-day and 14-day treatments with N-nitroso-varenicline produced similar mutagenesis responses. These findings demonstrate the feasibility of employing HepaRG cells with HiFi-seq as a mammalian cell assay for assessing NDSRI mutagenesis.
We describe the impact of lifestyle related factors on the formation of micronuclei (MN) in buccal cells. It is known that consumption of alcohol and of other drugs including tobacco and other plants cause damage of the genetic material and leads to adverse health effects. Overall, 235 articles were evaluated in total. Tobacco smoking, i.e., consumption of cigarettes (75 publications), water pipes (11 publications) and crack smoking (5 publications) caused mainly positive effects, while negative results were obtained with pure marijuana (2 publications). Chewing habits (smokeless tobacco (27 publications) and betel with and without tobacco (23 publications) caused induction of MN in cells from the oral cavity (numbers indicate the number of publications). A study concerning khat chewing yielded a positive finding while reduction of MN was seen in consumers of coca leaves. Investigations concerning the effects of alcohol (11 publications), meat consumption (3 publications) and studies concerning the impact of the body mass index and body fat (4 publications) led to inconsistent results. No evidence of MN induction was observed in studies on continuous physical exercise (2 publications). Furthermore, exhaustive sporting activities also showed no indications of chromosomal damage (2 publications). A number of studies (in total 22 publications) were realized to investigate the effects of mobile phone specific radiation. The exposure was not adequately assessed in these investigations with questionnaires, furthermore, frequently inadequate stains (11 publications). The findings are controversial, and a clear negative result was obtained in a recent controlled intervention study. Overall the results indicate that the MN assay with buccal cells can be used to monitor chromosomal damage caused by smoking and chewing habits and to predict adverse health effects in the respiratory system and on the upper gastrointestinal tract. To draw firm conclusions about the effects of nutrition, mobile phone use, physical exercise, and body weight, further well-controlled trials are needed.
Chromosomal instability (CIN), impaired telomere biology, and aberrant DNA methylation are implicated in colorectal cancer (CRC) development. Tracking these alterations from precancerous lesions through tumors to metastases may reveal biomarkers of CRC initiation and progression. Tissue samples from 44 patients with either high-grade colorectal dysplasia (HGA; n = 13) or advanced metastatic CRC (n = 31) were analyzed. CIN was assessed in all patients using either low-coverage whole-exome sequencing or microarray-based comparative genomic hybridization. In a subset of patients, genome-wide CpG methylation profiling (n = 19) and telomere length measurements (n = 15) were performed. CIN was detected in 85% of HGA patients, spanning focal CNVs in MALAT1 (46%) to recurrent alterations on chromosomes 11, 13, and 20, with PTK6 being the most frequently amplified (61%). CIN was comparable between primary tumors and synchronous metastases but was significantly elevated in metachronous cases. DEK was amplified in all metastases but the aberration was absent in primaries, irrespective of tissue chronicity. Methylation profiling distinguished HGA from adjacent non-dysplastic mucosa (9859 differentially methylated CpGs) and unrelated tumor tissues (17 638 CpGs), whereas primary tumors and metastases differed at only five CpG sites. Both primary tumors and metastases appeared epigenetically younger than colonic mucosa. Metastases exhibited significantly shorter telomeres than both primary tumors (P = .019) and colonic mucosa (P = .001). The amplification of PTK6 may serve as an early biomarker detectable at the HGA stage, while DEK amplification appears crucial for metastatic progression and may represent a therapeutic target. Further validation is needed.
