The dangerous link between coal dust exposure and DNA damage: unraveling the role of some of the chemical agents and oxidative stress.

Exposure to coal mining dust poses a substantial health hazard to individuals due to the complex mixture of components released during the extraction process. This study aimed to assess the oxidative potential of residual coal mining dust on human lymphocyte DNA and telomeres and to perform a chemical characterization of coal dust and urine samples. The study included 150 individuals exposed to coal dust for over ten years, along with 120 control individuals. The results revealed significantly higher levels of DNA damage in the exposed group, as indicated by the standard comet assay, and oxidative damage, as determined by the FPG-modified comet assay. Moreover, the exposed individuals exhibited significantly shorter telomeres compared to the control group, and a significant correlation was found between telomere length and oxidative DNA damage. Using the PIXE method on urine samples, significantly higher concentrations of sodium (Na), phosphorus (P), sulfur (S), chlorine (Cl), potassium (K), iron (Fe), zinc (Zn), and bromine (Br) were observed in the exposed group compared to the control group. Furthermore, men showed shorter telomeres, greater DNA damage, and higher concentrations of nickel (Ni), calcium (Ca), and chromium (Cr) compared to exposed women. Additionally, the study characterized the particles released into the environment through GC-MS analysis, identifying several compounds, including polycyclic aromatic hydrocarbons (PAHs) such as fluoranthene, naphthalene, anthracene, 7H-benzo[c]fluorene, phenanthrene, pyrene, benz[a]anthracene, chrysene, and some alkyl derivatives. These findings underscore the significant health risks associated with exposure to coal mining dust, emphasizing the importance of further research and the implementation of regulatory measures to safeguard the health of individuals in affected populations.

Evaluation of imazethapyr-induced DNA oxidative damage by alkaline Endo III- and Fpg-modified single-cell gel electrophoresis assay in Hypsiboas pulchellus tadpoles (Anura, Hylidae).

Imazethapyr (IMZT) is a selective postemergent herbicide with residual action. Available data analyzing its effects in aquatic vertebrates are scarce. In previous studies, we demonstrated that IMZT induces lesions into the DNA of Hypsiboas pulchellus tadpoles using the single-cell gel electrophoresis (SCGE) assay as a biomarker for genotoxicity. Currently, this assay can be modified by including incubation with lesion-specific endonucleases, e.g., endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which detect oxidized pyrimidine and purine bases, respectively. The aim of this study was to evaluate the role of oxidative stress in the genotoxic damage in circulating blood cells of H. pulchellus tadpoles exposed to the IMZT-based Pivot H® formulation (10.59% IMZT) at a concentration equivalent to 25% of the LC50 (96h) value (0.39mg/L IMZT) during 48 and 96h. Our results demonstrate that the herbicide induces oxidative DNA damage on H. pulchellus tadpoles at purines bases but not at pyrimidines. Our findings represent the first evidence of oxidative damage caused by IMZT on anuran DNA using the alkaline restriction enzyme-modified SCGE assay.

Modulating effects of pycnogenol® on oxidative stress and DNA damage induced by sepsis in rats.

The aim of this study was to evaluate the protective effects of Pycnogenol® (Pyc), a complex plant extract from the bark of French maritime pine, on oxidative stress parameters (superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and total glutathione (GSH) and malondialdehyde (MDA) levels), an inflammatory cytokine (tumor necrosis factor alpha (TNF-α) level) and also DNA damage in Wistar albino rats. Rats were treated with 100 mg/kg intraperitonally Pyc following the induction of sepsis by cecal ligation and puncture. The decreases in MDA levels and increases in GSH levels, and SOD and GPx activities were observed in the livers and kidneys of Pyc-treated septic rats. Plasma TNF-α level was found to be decreased in the Pyc-treated septic rats. In the lymphocytes, kidney, and liver tissue cells of the sepsis-induced rats, Pyc treatment significantly decreased the DNA damage and oxidative base damage using standard alkaline assay and formamidopyrimidine DNA glycosylase-modified comet assay, respectively. In conclusion, Pyc treatment might have a role in the prevention of sepsis-induced oxidative damage not only by decreasing DNA damage but also increasing the antioxidant status and DNA repair capacity in rats.

Level of primary DNA damage in the early stage of metabolic syndrome.

Metabolic syndrome (MetS) is a multi-component disease, characterised by abdominal obesity, hypertension, hyperglycaemia and dyslipidaemia. Since the number of MetS patients has significantly increased over the past two decades and because MetS may lead to development of cardiovascular diseases, diabetes type-2, and cancer, it has become important to extend the knowledge on the pathogenesis of MetS and to establish its possible early biomarkers. Studies on MetS and DNA damage are few and are inconclusive. The aim of this study was to elucidate the involvement of DNA damage in the development of MetS and to establish if DNA damage can serve as early biomarker of MetS. A total of 121 subjects participated in the study: 56 healthy controls and 65 MetS patients who were diagnosed with MetS for the first time. The amount of primary DNA damage in peripheral leukocytes of the subjects was assessed with three types of comet assay: the alkaline, the hOGG1-modified, and the neutral comet assay. In addition, the extent of oxidative DNA damage was monitored in urine by assessing 8-oxo-dG. The parameters of the three types of comet assay did not differ between the control and the MetS group. Interestingly, urinary 8-oxo-dG level in the control group was higher than in the MetS group. Our results imply that DNA damage is not involved in the early stage of MetS and, therefore, DNA damage cannot serve as an early marker of MetS.

The small molecule Erk1/2 signaling pathway inhibitor PD98059 improves DNA repair in an experimental autoimmune encephalomyelitis SJL/J mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disorder in which the myelin sheath covering the central nervous system axons is damaged or lost, disrupting action potential conduction and leading to various neurological complications. The pathogenesis of MS remains unclear, and no effective therapies are currently available. MS is triggered by environmental factors in genetically susceptible individuals. DNA damage and DNA repair failure have been proposed as MS genetic risk factors; however, inconsistent evidence has been found in multiple studies. Therefore, more investigations are needed to ascertain whether DNA damage/repair is altered in this disorder. In this context, therapies that prevent DNA damage or enhance DNA repair could be effective strategies for MS treatment. The overactivation of the extracellular-signal-related kinase 1 and 2 (Erk1/2) pathway can lead to DNA damage and has been linked to MS pathogenesis. In our study, we observed substantially elevated oxidative DNA damage and slower DNA repair rates in an experimentally autoimmune encephalomyelitis animal model of MS (EAE). Moreover, statistical decreases in oxidative DNA strand breaks and faster repair rates were observed in EAE animals injected with the Erk1/2 inhibitor PD98059 (PD). Moreover, the expression of several genes associated with DNA strand breaks and repair changed in EAE mice at both the mRNA and protein levels, as revealed by the RT2 Profiler PCR array and verified by RT-PCR and protein analyses. The treatment with PD mitigated these changes and improved DNA repair gene expression. Our results demonstrate clear associations between Erk1/2 activation, DNA damage/repair, and MS pathology, and further suggest that PD therapy may be a promising adjuvant therapeutic strategy.

Application of the Fpg-modified comet assay on three-spined stickleback in freshwater biomonitoring: toward a multi-biomarker approach of genotoxicity.

Aquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment.

DNA Oxidative Damage as a Sensitive Genetic Endpoint to Detect the Genotoxicity Induced by Titanium Dioxide Nanoparticles.

The genotoxicity of nanomaterials has attracted great attention in recent years. As a possible occupational carcinogen, the genotoxic effects and underlying mechanisms of titanium dioxide nanoparticles (TiO2 NPs) have been of particular concern. In this study, the effect of TiO2 NPs (0, 25, 50 and 100 µg/mL) on DNA damage and the role of oxidative stress were investigated using human bronchial epithelial cells (BEAS-2B) as an in vitro model. After detailed characterization, the cytotoxicity of TiO2 NPs was detected. Through transmission electron microscopy (TEM), we found that TiO2 NPs entered the cytoplasm but did not penetrate deep into the nucleus of cells. The intracellular levels of reactive oxygen species (ROS) significantly increased in a dose-dependent manner and the ratios of GSH/GSSG also significantly decreased. The results of the normal comet assay were negative, while the Fpg-modified comet assay that specifically detected DNA oxidative damage was positive. Meanwhile, N-acetyl-L-cysteine (NAC) intervention inhibited the oxidative stress and genotoxicity induced by TiO2 NPs. Therefore, it was suggested that TiO2 NPs could induce cytotoxicity, oxidative stress and DNA oxidative damage in BEAS-2B cells. DNA oxidative damage may be a more sensitive genetic endpoint to detect the genotoxicity of TiO2 NPs.

Genotoxicity and Toxicity Assessment of a Formulation Containing Silver Nanoparticles and Kaolin: An In Vivo Integrative Approach.

A new material composed of a kaolin base with silver nanoparticles (AgNPs) attached to its surface was developed, as an alternative to antibiotics used as supplements in animal feed. As part of its safety assessment, an in vivo geno-toxicological evaluation of this material was conducted in rats. First, a preliminary dose finding study was carried out to decide the doses to be tested in the main study: 50, 300 and 2000 mg/kg b.w. For the main study, a combined strategy composed of the MN test (TG 474) and the comet assay (TG 489), integrated in a repeated dose 28-day oral toxicity study (TG 407), was performed. A No Observed Adverse Effect Level (NOAEL) of 2000 mg of the silver-kaolin formulation/kg b.w. by oral route, for 28 days, was determined. The silver-kaolin formulation did not induce micronuclei in bone marrow, or DNA strand breaks (SBs) or alkali labile sites (ALS) in liver, spleen, kidney or duodenum at any dose. The modified Fpg comet assay did not reveal oxidized bases in the same tissues at the dose of 2000 mg/kg b.w. Silver was quantified by ICP-MS in all the target organs, confirming the negative results obtained under these conditions.

Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats.

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 µg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

The role of the enzyme-modified comet assay in in vivo studies.

The in vivo comet assay is an established genotoxicity test, with an OECD test guideline, but in its standard form it measures only DNA strand breaks. Including in the assay an additional step, in which the DNA is incubated with a lesion-specific enzyme, can provide important information about the nature of the DNA damage. Formamidopyrimidine DNA glycosylase, 8-oxoguanine DNA glycosylase or endonuclease III are commonly used in the in vitro genotoxicity test and in human biomonitoring to detect oxidised bases, but in vivo applications are rarer. A systematic literature search has identified a total of 60 papers that report such in vivo experiments, testing a variety of agents. In many cases, strand breaks were not seen, but significant levels of enzyme-sensitive sites were induced - indicating a mechanism of action involving oxidative stress. Compounds such as methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS) could be used as positive controls in both the standard and the enzyme-modified in vivo comet assays.

Elevated oxidative DNA damage in patients with coronary artery disease and its association with oxidative stress biomarkers.

OBJECTIVE: The objective of the present study was to evaluate oxidative DNA damage in peripheral blood leukocytes (PBLs) of patients with coronary artery disease (CAD) and to explore the relationship of oxidised purine and pyrimidine with oxidative stress. METHODS: The study participants (n = 100) included 50 patients and unrelated 50 age-, sex- and population-subgroup (Jat Sikhs)-matched healthy controls. Oxidative DNA damage using the modified enzymatic comet in PBLs, and malondialdehyde (MDA) levels, total oxidant status (TOS) and total antioxidant status (TAS) in blood serum samples using spectrophotometric methods was determined. RESULTS: The basal DNA damage of percent tail DNA (T-DNA%) was increased as were tail moment (TM) and olive tail moment (OTM). Oxidative DNA damage in terms of oxidised purines and oxidised pyrimidines was also significantly (p < .001) elevated in patients. Rather the advanced stages of CAD, unstable angina and acute myocardial infarction had significantly more basal and oxidative DNA damage (p < .05) compared to stable angina. MDA levels (p < .01) and TOS (p < .001) were increased significantly in patients with significant (p < .001) decrease in TAS. There was positive correlation of oxidised purines (T-DNA% r = 0.399, p = .004; TM r = 0.623, p = .001; OTM r = 0.456, p= .001) and of total oxidative damage (TM r = 0.515, p = .001; OTM r = 0.463, p = .001) with disease severity, and, with TOS (r = 0.279, p = .050) and negative with TAS (r = -0.341, p = .015). Multiple linear regression analysis revealed TOS and disease severity as independent predictors of oxidative DNA damage. CONCLUSIONS: There was significant increase in oxidative DNA damage and oxidative stress in CAD patients compared to levels in healthy controls.

Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies.

The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage - strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the duration of alkaline unwinding; the duration of electrophoresis and the voltage gradient applied; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored - and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter-laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology.

Auxinic herbicides induce oxidative stress on Cnesterodon decemmaculatus (Pisces: Poeciliidae).

Pesticides might increase the production of reactive oxygen species (ROS). Dicamba (DIC) and 2,4-dichlorophenoxyacetic acid (2,4-D) are auxinic herbicides commonly applied in agroecosystems to control unwanted weeds. We analysed the oxidative damage exerted on the fish Cnesterodon decemmaculatus by an acute exposure to DIC- and 2,4-D-based herbicides formulations Banvel® and DMA®, respectively. The Endo III- and Fpg-modified alkaline comet assay was employed for detecting DNA damage caused by oxidative stress, whereas enzymatic and non-enzymatic biomarkers such as the activities of catalase (CAT), glutathione-S-transferase (GST), acetylcholinesterase (AChE), and glutathione content (GSH) were used to assess antioxidant response to these two herbicides. At the DNA level, results demonstrate that both auxinic herbicides induce oxidative damage at purines level. An increase on CAT and GST activities were detected in 48 h- and 96 h-treated specimens with both auxinics. GSH content decreased in fish exposed to DIC during 48 h and to 2,4-D after 96 h of exposure. Additionally, a diminished AChE activity in specimens treated with DIC and 2,4-D was observed only after 96 h. Total protein content decreased in fish exposed to both auxinics during 96 h. These results represent the first evaluation of oxidative damage related to DIC and 2,4-D exposure on a fish species as the Neotropical freshwater teleost C. decemmaculatus.

Investigation of genotoxicity risk and DNA repair capacity in breast cancer patients using anastrozole.

OBJECTIVE: Breast cancer is the most common cancer in women worldwide and the incidence increases in postmenopausal women. Anastrozole is a non-steroidal (type II), third-generation aromatase inhibitor (AI) that is used in the treatment of postmenopausal estrogen-related breast cancer. Several studies have been conducted to assess the efficacy, safety, and superiority of AIs to tamoxifen; however, a literature search did not reveal a study that investigated the genotoxic potential of AIs. The aim of this study was to investigate the possible DNA damage risk profile and individual DNA repair capacity of patients using anastrozole with the modified alkaline comet assay in order to contribute to public health and health economics. METHODS: Women diagnosed with breast cancer after menopause comprised the study group. Six patients who had taken anastrozole for at least 6 months were retrospectively enrolled, and 12 patients who had not yet received treatment were prospectively enrolled as a control group. Peripheral blood lymphocytes were used to measure oxidized DNA damage using formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (endo III) in a modified comet assay. Individual DNA repair capacity was evaluated with the comet assay after a hydrogen peroxide (H2O2) challenge to examine the difference in DNA damage susceptibility. RESULTS: Analysis of DNA damage, oxidative base damage, susceptibility to DNA damage, and repair capacity revealed no significant difference between the control group and the patients taking anastrozole (p>0.05). Susceptibility to H2O2 damage was observed to increase with age (p<0.05). CONCLUSION: According to the results obtained in this study, anastrozole did not contribute to oxidative DNA damage. An H2O2 challenge with the comet assay is useful to evaluate circumstances of increased vulnerability to damage, such as aging and cancer.

Evaluation of the mutagenicity and genotoxic potential of carvacrol and thymol using the Ames Salmonella test and alkaline, Endo III- and FPG-modified comet assays with the human cell line Caco-2.

Currently, direct antimicrobial and antioxidant additives derived from essential oils are used in food packaging and are perceived by consumers as low-health-risk compounds. In this study, we investigated the potential mutagenicity and genotoxicity of carvacrol and thymol, major compounds in several essential oils, using the Ames Salmonella test and the alkaline, Endo III- and formamidopyrimidine glycosylase (FPG)-modified comet assays, respectively. Thymol did not show any mutagenic activity at any concentration assayed (0-250 µM), whereas carvacrol exhibited mutagenic potential, displaying greater activity in presence of the metabolic fraction (29-460 µM). The genotoxic effects were evaluated in the human colon carcinoma cell line Caco-2, and the standard comet assay revealed that neither carvacrol (0-460 µM) nor thymol (0-250 µM) had any affects at 24 and 48 h. The FPG-modified comet assay showed that the highest concentration of carvacrol (460 µM) caused DNA damage, indicating damage to the purine bases. These results should be used to identify the appropriate concentrations of carvacrol and thymol as additives in food packaging. Moreover, further studies are necessary to explore the safety and/or the toxicity mechanisms of these compounds.

Genotoxic potential of several naphthenic acids and a synthetic oil sands process-affected water in rainbow trout (Oncorhynchus mykiss).

The exploitation of oil sands has raised major environmental concerns, particularly regarding the presence of high concentration in contaminants such as polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) in oil sands process-affected water (OSPW). The purpose of this study was, first to evaluate the genotoxic impact of OSPW-related compounds such as NAs and PAHs in a salmonid species and secondly to assess if OSPW exposure leads to genotoxicity. For this purpose, rainbow trout hepatocytes were exposed in vitro to environmentally relevant concentrations of synthetic NAs, naphtalene, benzo(a)pyrene, and extracts of synthetic OSPW (generated by a laboratory bitumen extraction) and of oil sands leaching water (OSLW, mimicking leaching of oil sands in river water). Primary DNA damage was assessed by the formamidopyrimidine-DNA glycolyase (Fpg)-modified comet assay. Genotoxicity was observed in hepatocytes exposed to several NAs, mixture of them, OSPW and OSLW extracts. The chemical structure of NAs influences the genotoxicity potential: among the NAs tested, the most cyclic NA was the most genotoxic. It also appears that genotoxicity was more marked for OSPW than for OSLW. Because exposure to OSPW led to oxidative DNA damage, while after exposure to several NAs, these types of DNA damage were limited, the NAs tested in this study could not be qualified as the only major contaminants responsible for OSPW genotoxicity. Notwithstanding, it should be noteworthy that exposure to NAs resulted in genotoxic impact at concentrations lower than those documented by literature for fresh OSPW. Further research is needed to explore the relationships between the chemical structure of NAs and their genotoxicity in the light of the distribution of NAs in fresh OSPW samples as well as in surface waters.