DNA repair plays an important role in genomic integrity. Major DNA repair pathways for substance-induced DNA damage are base excision and nucleotide excision repair, as well as the repair of double strand breaks by homologous recombination and non-homologous end joining. However, the quantitative measurement of cellular DNA repair capacity remains a challenge. Here we applied a modified comet assay in which DNA repair capacity can be tracked using DNA repair inhibitors combined with selected DNA-damaging substances. The combinations of the alkylating agent methylmethansulfonate + olaparib, UV-C (causing pyrimidine dimers) + aphidicolin, and the topoisomerase II inhibitor etoposide + SCR7 were used to reflect base excision repair, nucleotide excision repair, and non-homologous end joining. Although neither the agents nor the inhibitors are by themselves completely selective for these DNA repair pathways, the combinations and also the missing reaction to other combinations of the same substances indicated a certain degree of preference. By using all of them within one experiment, a pattern of DNA repair activity of cell lines was established. A comparison of five cell lines/types differing in their sensitivity to the damaging agents demonstrated its applicability. Although each of the damaging agents and inhibitors had been used in the comet assay before, this combination within one experimental setup was described here for the first time and the resulting characterization of the phenotypic pattern of DNA repair capacity of cell lines is more extensive than previously achieved. Its applicability to human peripheral blood mononuclear cells (PBMCs) suggest that the assay may in the future be useful to investigate interindividual variability in DNA repair capacity in humans, for example in relation to disease or exposures.
Abstract Our body is continuously exposed to endogenous and exogenous sources of oxidative stress arising from physiological metabolism and environmental factors such as ultraviolet (UV) radiation. These stresses generate reactive oxygen species (ROS) that can induce genotoxicity through oxidative DNA damage. To evaluate the contribution of NRF2, which is a master transcription factor regulating cellular antioxidant responses, to the suppression of oxidative stress-induced genotoxicity in the skin, we examined Nrf2-deficient mice regarding mutagenic responses to two separate biological situations: acute UVB exposures and the chronic process of aging. Analyses were performed using transgenic mice carrying the bacterial lacZ gene as a mutational reporter. No significant differences were detected between Nrf2-deficient and wild-type mice in mutagenicity, induced mutation spectrum, and mutation distribution within the reporter gene after acute UVB exposure, whereas the dose-response kinetics of UVB-induced mutation in the phase called “mutation induction suppression” (MIS) differed significantly between genotypes, with a more pronounced MIS response observed in the Nrf2-/- epidermis. Little contribution of ROS to genotoxicity was observed after the acute exposure. Notably, however, the age-dependent accumulation of mutations was significantly elevated in the Nrf2-deficient epidermis compared to the wild-type. These findings indicate that NRF2 plays a protective role for the skin, not by directly preventing genotoxicity, but by promoting tissue recovery after acute UVB damage and, more importantly, by suppressing the genotoxic effects of physiologically produced ROS in a chronic aging process.
Abstract The comet assay is a simple method to assess DNA damage. Although standardized protocols exist, there is no standard protocol for comet image analysis, representing a potential source of inter-laboratory variability. Several scoring programs exist, including commercially available (e.g. Comet IV) and freeware (e.g. CaspLab, CometScore and OpenComet). As costs frequently drive the selection of freeware, it is essential to evaluate their performance when compared to Comet IV (considered the reference method). This study compared these programs using comet images with varying levels of DNA damage, categorized into three data sets: (A) the full data set comprising comets with an extensive range of % Tail DNA, (B) a subset comprising comets with <15% Tail DNA (low damage), and (C) a subset with >50% Tail DNA (high damage). Across the full dataset and at high levels of DNA damage, most freeware programs differed significantly from Comet IV. CometScore was the only freeware that did not significantly differ from Comet IV in all three subsets. Correlation and agreement analyses revealed weaker associations for subset B, especially for OpenComet. CaspLab and OpenComet exhibited systematic and/or proportional biases, whereas CometScore consistently showed strong correlations and minimal bias relative to CometIV. Collectively, these findings demonstrate that freeware programs can provide acceptable performance, but they may deviate from Comet IV. Our results highlight the importance of informed software selection and the need for standardized and validated image analysis workflows, to improve the accuracy, reproducibility, and inter-laboratory comparability of comet assay data.
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 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.
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.
