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.
