Levels Of DNA Strand Breaks Research Articles

Validation of coffee silverskin extract as a food ingredient by the analysis of cytotoxicity and genotoxicity

The aim of the present study was to validate the food safety of CSE, by studying its effect on cytotoxicity (100–20000μg/ml) and genotoxicity (10, 100 and 1000μg/ml) and also to investigate its preventive potential (1, 10 and 100μg/ml) against B(a)P induced DNA damage. Prior to analyses, the antioxidant capacity and the microbiological quality of CSE were tested. DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. HepG2 cells were pre-treated with CSE (1, 10 and 100μg/ml) for 24h followed by the addition of 100μM B(a)P in presence of CSE for other 24h. Detection of oxidized purines and pyrimidines was carried out using Formamidopyrimidine DNA glycosylase or Endonuclease III enzymes, respectively. Chlorogenic acid (CGA), the major antioxidant present in coffee, was used as a control. Treatment with 100 μM B(a)P significantly increased (p<0.05) levels of DNA strand breaks and oxidized purine and pyrimidine bases. Treatment of HepG2 cells with CSE did not induce either cytotoxicity or genotoxicity. CSE significantly inhibited (p<0.05) genotoxicity induced by B(a)P and the observed effect may be associated to its antioxidant capacity. CGA alone at the concentration present in CSE was effective against B(a)P. Thus, CGA seems to be a contributor to the preventive effect of CSE against B(a)P induced DNA damage in HepG2 cells. In conclusion, CSE presents potential as a natural sustainable chemoprotective agent against the chemical carcinogen B(a)P.

Effects of β-Carotene and Its Cleavage Products in Primary Pneumocyte Type II Cells.

β-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET) and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC). Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs) and the major product apo-8′carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of β-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of β-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ) exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, β-carotene in the presence of DMNQ did not cause DNA damage. Instead, β-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of β-carotene in the two cell types.

Оценка генотоксичности парацетамола и его комбинации с верапамилом &lt;i&gt;in vivo&lt;/i&gt;

Парацетамол в дозах 30 и 60 мг/кг (внутрь) отдельно или в сочетании с известным комутагеном верапамилом (0,25 – 10 мг/кг, внутрь) при однократном или 5-кратном ежедневном введении не проявил цитогенетической активности в клетках костного мозга мышей. Цитогенетический эффект отсутствовал также после однократного введения парацетамола в дозе 300 мг/кг. В тесте ДНК-комет парацетамол при однократном введении в дозах 30 и 60 мг/кг на срок 3 или 24 ч не проявил ДНК-повреждающей активности в клетках костного мозга, печени, почек, селезенки и головного мозга мышей. В дозе 300 мг/кг при экспозиции в течение 3 ч парацетамол индуцировал ДНК-кометы типа «ghost cells» в клетках печени, что было расценено как проявление гепатотоксического эффекта препарата.

Assessment of sperm quality in palaemonid prawns using Comet assay: methodological optimization

The aim of this study was to adapt the Comet assay in spermatozoa of the marine prawn Palaemon serratus to use it as a marker of sperm quality. Indeed, due to the characteristics of their spermatozoa, the measurement of DNA integrity is one of the few markers which can be transferred to crustaceans to assess the quality of their semen. In the first step, the methods of collecting and maintaining spermatozoa were optimized. Cell survival was estimated during kinetics of preservation (i.e. 1, 2, 4 and 8h) in various suspension media to define artificial seawater (ASW) as optimal. Several methods in the releasing of spermatozoa from the spermatophore of prawns were estimated with regard to their incidence both on the efficiency of extraction and the survival of cells. Pipetting up and down turned out to be the most successful and the least invasive technique. Secondly, the transfer of Comet assay was optimized by studying various times in both cell lysis (i.e. 1, 6, 18h) and DNA denaturation (i.e. 15, 30 and 45min), after in vitro exposure of spermatozoa to an H2O2 gradient as model genotoxicant. Results revealed that a minimum of 1h in cell lysis and 15min of DNA denaturation were sufficient to obtain valuable results, linked with a low compaction of DNA in spermatozoa of Palaemon sp. Finally, the sensitivity of P. serratus spermatozoa was assessed after in vitro exposures to model genotoxicants displaying various modes of interaction with DNA (i.e. UV-C, 13.3-79.5Jm-2; H2O2, 5-10μM and MMS, 0.5-5mM) and some environmental contaminants known or suspected to be genotoxic (i.e. cadmium and diuron, 0.015-1.5μgL-1; carbamazepine, 0.1-10μgL-1) for invertebrates. The low variability of the baseline level of DNA strand breaks recorded in controls highlighted the robustness of the method. P. serratus spermatozoa displayed significant DNA damage from the lowest doses tested for all model genotoxicants, but conversely, no genotoxic effect of tested environmental contaminants was observed. These results, which are discussed according to the protocol tested in the present study and the comparison with literature data, could suggest a difference in the response or sensitivity of spermatozoa to environmental genotoxicity between invertebrate species, and therefore the interest of Palaemonidae prawns in ecogenotoxicology. In conclusion, the present study underlines the potential of the Comet assay as a marker to assess the contamination impact on the sperm quality in Palaemonidae prawns in view to a potential application for in situ biomonitoring surveys.

Evaluation of the cytotoxicity, genotoxicity and mucus permeation capacity of several surface modified poly(anhydride) nanoparticles designed for oral drug delivery

The main concerns with drugs designed for oral administration are their inactivation or degradation in the harsh conditions of the gastrointestinal tract, their poor solubility through the gastrointestinal mucus gel layer, the poor intestinal epithelium permeability that limits their absorption, and their toxicity. In this context, poly(anhydride) nanoparticles are capable of protecting the drug from the harsh environment, reduce the drug’s toxicity and, by virtue of surface modification, to enhance or reduce their mucus permeability and the bioadhesion to specific target cells.The copolymer between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN 119) are part of the poly(anhydride) nanoparticles. These biocompatible and biodegradable nanoparticles (NPs) can be modified by using different ligands. Their usefulness as drug carriers and their bioadhesion with components of the intestinal mucosa have been described. However, their toxicity, genotoxicity and mucus permeation capacity has not been thoroughly studied.The aim of this work was to evaluate and compare the in vitro toxicity, cell viability and in vitro genotoxicity of the bioadhesive empty Gantrez® AN 119 NPs modified with dextran, aminodextran, 2-hydroxypropyl-β-cyclodextrin, mannosamine and poly-ethylene glycol of different molecular weights.Results showed that, in general, coated NPs exhibit better mucus permeability than the bare ones, those coated with mannosamine being the most permeable ones. The NPs studied did not affect cell metabolism, membrane integrity or viability of Caco-2 cells at the different conditions tested. Moreover, they did not induce a relevant level of DNA strand breaks and FPG-sensitive sites (as detected with the comet assay).

Health risk evaluation in a population exposed to chemical releases from a petrochemical complex in Thailand

Emissions from petrochemical industries may contain toxic and carcinogenic compounds that can pose health risk to human populations. The scenario may be worse in developing countries where management of such exposure-health problems is typically not well-implemented and the public may not be well-informed about such health risk. In Thailand, increasing incidences of respiratory diseases and cancers have been reported for the population around a major petrochemical complex, the Map Ta Phut Industrial Estate (MTPIE). This study aimed to systematically investigate an exposure-health risk among these populations. One-hundred and twelve healthy residents living nearby MTPIE and 50 controls located approximately 40km from MTPIE were recruited. Both external and internal exposure doses to benzene and 1,3-butadiene, known to be associated with the types of cancer that are of concern, were measured because they represent exposure to industrial and/or traffic-related emissions. Health risk was assessed using the biomarkers of early biological effects for cancer and inflammatory responses, as well as biomarkers of exposure for benzene and 1,3-butadiene. The exposure levels of benzene and 1,3-butadiene were similar for both the exposed and control groups. This was confirmed by a non-significant difference in the levels of specific urinary metabolites for benzene (trans,trans-muconic acid, t,t-MA) and 1,3-butadiene (monohydroxy-butyl mercapturic acid, MHBMA). Levels of 8-hydroxydeoxyguanosine (8-OHdG) and DNA strand breaks between the two groups were not statistically significantly different. However, functional biomarkers, interleukin-8 (IL-8) expression was significantly higher (p<0.01) and DNA repair capacity was lower (p<0.05) in the exposed residents compared to the control subjects. This suggests that the exposed residents may have a higher risk for development of diseases such as cancer compared to controls. However, the increased expression of IL-8 and lower DNA repair capacity were not associated with recent and excessive exposure to benzene and 1,3-butadiene, which were at the similar levels as those in the controls. The data would indicate that previous exposure to the two chemicals together with exposure to other toxic chemicals from the MTPIE may be responsible for the elevated functional biomarkers and health risk. Further studies are required to determine which other pollutants from the industrial complex could be causing these functional abnormalities.

Integrated biological responses and tissue-specific expression of p53 and ras genes in marine mussels following exposure to benzo(α)pyrene and C60 fullerenes, either alone or in combination.

We used the marine bivalve (Mytilus galloprovincialis) to assess a range of biological or biomarker responses following exposure to a model-engineered nanoparticle, C60 fullerene, either alone or in combination with a model polycyclic aromatic hydrocarbon, benzo(α)pyrene [B(α)P]. An integrated biomarker approach was used that included: (i) determination of 'clearance rates' (a physiological indicator at individual level), (ii) histopathological alterations (at tissue level), (iii) DNA strand breaks using the comet assay (at cellular level) and (iv) transcriptional alterations of p53 (anti-oncogene) and ras (oncogene) determined by real-time quantitative polymerase chain reaction (at the molecular/genetic level). In addition, total glutathione in the digestive gland was measured as a proxy for oxidative stress. Here, we report that mussels showed no significant changes in 'clearance rates' after 1 day exposure, however significant increases in 'clearance rates' were found following exposure for 3 days. Histopathology on selected organs (i.e. gills, digestive glands, adductor muscles and mantles) showed increased occurrence of abnormalities in all tissues types, although not all the exposed organisms showed these abnormalities. Significantly, increased levels of DNA strand breaks were found after exposure for 3-days in most individuals tested. In addition, a significant induction for p53 and ras expression was observed in a tissue and chemical-specific pattern, although large amounts of inter-individual variability, compared with other biomarkers, were clearly apparent. Overall, biological responses at different levels showed variable sensitivity, with DNA strand breaks and gene expression alterations exhibiting higher sensitivities. Furthermore, the observed genotoxic responses were reversible after a recovery period, suggesting the ability of mussels to cope with the toxicants C60 and/or B(α)P under our experimental conditions. Overall, in this comprehensive study, we have demonstrated mussels as a suitable model marine invertebrate species to study the potential detrimental effects induced by possible genotoxicants and toxicants, either alone or in combinations at different levels of biological organisation (i.e. molecular to individual levels).

DNA damage induced by NADPH cytochrome P450 reductase-activated idarubicin in sensitive and multidrug resistant MCF7 breast cancer cells.

Idarubicin (IDA) is one of clinically important anticancer drugs belonging to the anthracycline antibiotic family. The aim of this study was to examine DNA damage induced by NADPH cytochrome P450 reductase (CPR)-activated IDA in human sensitive MCF7 and multidrug resistant MCF7/DOX500 (overexpressing P-gp) breast adenocarcinoma cells. The evaluation of DNA fragmentation caused by single strand breaks (SSB) and double strand breaks (DSB) was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) test. Additionally, DSB formation was examined using H2AX histone phosphorylation assays. It was found that IDA alone and CPR-activated used at IC90 caused a higher level of DNA strand breaks in sensitive MCF7 cells detected by TUNEL assessments (p=0.0011 for IDA alone and p=0.0109 for IDA reductively activated, Kruskal-Wallis test) and γ-H2AX-positive staining (p=0.0003 for IDA alone and p=0.0193 for IDA reductively activated, Kruskal-Wallis test) than in multidrug resistant MCF7/DOX500 cells. However, no changes were observed in the percentage of TUNEL-positive and DSB-positive cells for MCF7 as well as MCF7/DOX500 cells in the case of IDA alone and the drug pretreated in the presence of the activating system. The obtained results suggest that CPR-activation of IDA does not significantly change the cellular DNA damage response of studied sensitive MCF7 and multidrug resistant MCF7/DOX500 breast cancer cells, even if the results concerning the interaction of IDA undergoing CPR activation with naked DNA showed the important differences in comparison with the drug alone (non-activated).

Surface modification does not influence the genotoxic and inflammatory effects of TiO2nanoparticles after pulmonary exposure by instillation in mice

The influence of surface charge of nanomaterials on toxicological effects is not yet fully understood. We investigated the inflammatory response, the acute phase response and the genotoxic effect of two different titanium dioxide nanoparticles (TiO2 NPs) following a single intratracheal instillation. NRCWE-001 was unmodified rutile TiO2 with endogenous negative surface charge, whereas NRCWE-002 was surface modified to be positively charged. C57BL/6J BomTac mice received 18, 54 and 162 µg/mouse and were humanely killed 1, 3 and 28 days post-exposure. Vehicle controls were tested alongside for comparison. The cellular composition and protein concentration were determined in bronchoalveolar lavage (BAL) fluid as markers for an inflammatory response. Pulmonary and systemic genotoxicity was analysed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. The pulmonary and hepatic acute phase response was analysed by Saa3 mRNA levels in lung tissue or Saa1 mRNA levels in liver tissue by real-time quantitative polymerase chain reaction. Instillation of NRCWE-001 and -002 both induced a dose-dependent neutrophil influx into the lung lining fluid and Saa3 mRNA levels in lung tissue at all assessed time points. There was no statistically significant difference between NRCWE-001 and NRCWE-002. Exposure to both TiO2 NPs induced increased levels of DNA strand breaks in lung tissue at all doses 1 and 28 days post-exposure and NRCWE-002 at the low and middle dose 3 days post-exposure. The DNA strand break levels were statistically significantly different for NRCWE-001 and -002 for liver and for BAL cells, but no consistent pattern was observed. In conclusion, functionalisation of reactive negatively charged rutile TiO2 to positively charged did not consistently influence pulmonary toxicity of the studied TiO2 NPs.

The detoxification responses, damage effects and bioaccumulation in the scallop Chlamys farreri exposed to single and mixtures of benzo[a]pyrene and chrysene

This study aimed to investigate the detoxification responses, damage effects and biotransformation in scallop Chlamys farreri exposed to benzo[a]pyrene (BaP) (0.1, 1μg/L), chrysene (CHR) (0.1, 1μg/L) and BaP+CHR (0.1+0.1, 1+1μg/L) for 15days. Results demonstrated that BaP and CHR concentration (BaP<CHR) in tissues increased rapidly in a time and dose effect. The mRNA expression of aryl hydrocarbon receptor (AhR), cytochrome P450 1A1 (CYP1A1), CYP1B1, multidrug resistance protein 1 (MRP1/ABCC1), breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp) were induced especially in the mixtures of BaP and CHR. Heat shock protein 90 (HSP90) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA expression was significantly elevated at days 1, 10 and 15. Detoxification enzymes of 7-ethoxyresorufin O-deethylase (EROD), uridine-diphosphate-glucuronyl-transferase (UGT) and sulfotransferase (SULT) were significantly induced and then became stable gradually while glutathione-S-transferase (GST) was inhibited in the mixtures of BaP and CHR at days 10 and 15. Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH) were all stimulated especially in the mixtures of BaP and CHR. The levels of DNA strand breaks, lipid peroxidation (LPO) and protein carbonyl (PC) contents showed damage effects exposed BaP and CHR. All the results indicated that BaP and CHR have similar induced effect and a majority of the biomarkers pointed to a more toxic effect when BaP and CHR were mixed. These will provide a solid foundation for the study of PAHs detoxification mechanism in bivalves and valuable information for marine pollution monitoring.

Granular Activated Carbon Treatment May Result in Higher Predicted Genotoxicity in the Presence of Bromide.

Certain unregulated disinfection byproducts (DBPs) are more of a health concern than regulated DBPs. Brominated species are typically more cytotoxic and genotoxic than their chlorinated analogs. The impact of granular activated carbon (GAC) on controlling the formation of regulated and selected unregulated DBPs following chlorine disinfection was evaluated. The predicted cyto- and genotoxicity of DBPs was calculated using published potencies based on the comet assay for Chinese hamster ovary cells (assesses the level of DNA strand breaks). Additionally, genotoxicity was measured using the SOS-Chromotest (detects DNA-damaging agents). The class sum concentrations of trihalomethanes, haloacetic acids, and unregulated DBPs, and the SOS genotoxicity followed the breakthrough of dissolved organic carbon (DOC), however the formation of brominated species did not. The bromide/DOC ratio was higher than the influent through much of the breakthrough curve (GAC does not remove bromide), which resulted in elevated brominated DBP concentrations in the effluent. Based on the potency of the haloacetonitriles and halonitromethanes, these nitrogen-containing DBPs were the driving agents of the predicted genotoxicity. GAC treatment of drinking or reclaimed waters with appreciable levels of bromide and dissolved organic nitrogen may not control the formation of unregulated DBPs with higher genotoxicity potencies.

Effects of thermal stress on the immune and oxidative stress responses of juvenile sea cucumber Holothuria scabra.

Holothuria scabra is the most valued and cultured tropical sea cucumber, given the great demand of this species for human consumption. However, despite its ecological and economic relevance, little is known regarding its immune responses under thermal stress. Here, the main goal was to study the response of sea cucumbers to temperature stress, assessing sub-organismal alterations and acclimation capacities of juveniles to temperature changes. After changing temperature (1°C/day) for 6days, organisms were exposed to temperature conditions of 21°C (cold), 27°C (control), and 33°C (warm) over a 30 day period. At each 15-day interval (T0, T15, and T30), six replicates per condition were killed for biochemical analysis. Immune responses were addressed by studying the activity of phenoloxidase (PO) and prophenoloxidase (ProPO) in the coelomic fluid. Antioxidant defence responses-catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) enzymatic activities-were measured in the muscle and respiratory tree tissues, whereas oxidative damage was evaluated by measuring levels of superoxide radicals (ROS), DNA-strand breaks and lipid peroxidation (LPO). Juvenile H. scabra increased SOD and PO activities when temperature was elevated, and revealed low levels of ROS and damage in both cold and warm treatments throughout the experiment, confirming the organism's moderate thermal stress. After the short acclimation period, the immune and antioxidant responses prevented damage and maintained homeostasis. This multi-biomarker approach highlights its usefulness to monitor the health of H. scabra and to gain insight concerning the use of this high-valued species in global-scale aquaculture from different temperature regions.

Effects of pyrogallic acid on Microcystis aeruginosa: oxidative stress related toxicity

Pyrogallic acid (PA) is used in various industrial and consumer products. The molecular mechanisms underlying PA’s toxicity was not fully understood. In this study, toxicity of PA on Microcystis aeruginosa with reactive oxygen species (ROS) generation as an end point was investigated. The results showed an increase in the percentage of cells with loss of membrane integrity and enhanced intracellular ROS production. Exposure to 50mgL−1 PA for 48h caused the highest percentage of loss of membrane integrity (56.7%), and a 2.54-fold higher intracellular ROS level compared to control. Further investigation revealed that PA caused a dose-dependent increase in DNA strand breaks (DSB) of M. aeruginosa at exposure concentration from 2 to 50mgL−1. The incubation of cells with ROS scavengers ascorbic acid, N-acetyl-l-cysteine (NAC) and tocopherol markedly alleviated the level of PA-induced DSB. Analysis of PA autoxidized products in culture solution showed that PA was quickly converted to purpurogallin (PG), and PG was further autoxidized to other polyphenolic compounds. PA and PG might participate a futile redox cycle, which mediated ROS production in M. aeruginosa. These results suggested DNA strands and cell membrane were two targets of ROS induced by PA, and oxidative damage was an important mechanism for the toxicity of PA against M. aeruginosa.

Nitroglycerin induces DNA damage and vascular cell death in the setting of nitrate tolerance

Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10-1000µM; in vivo: 10, 20 and 50mg/kg/day for 3days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O (6)-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O (6)-methylguanine-DNA methyltransferase displayed more vascular O (6)-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O (6)-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction.

PARP-1 inhibition influences the oxidative stress response of the human lens

Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects.

Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens.

Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens. The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software. Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls. Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation.

DNA damage and oxidative stress response to selenium yeast in the non-smoking individuals: a short-term supplementation trial with respect to GPX1 and SEPP1 polymorphism.

PurposeSelenium, both essential and toxic element, is considered to protect against cancer, though human supplementation trials have generated many inconsistent data. Genetic background may partially explain a great variability of the studies related to selenium and human health. The aim of this study was to assess whether functional polymorphisms within two selenoprotein-encoding genes modify the response to selenium at the level of oxidative stress, DNA damage, and mRNA expression, especially in the individuals with a relatively low selenium status.MethodsThe trial involved 95 non-smoking individuals, stratified according to GPX1 rs1050450 and SEPP1 rs3877899 genotypes, and supplemented with selenium yeast (200 µg) for 6 weeks. Blood was collected at four time points, including 4 weeks of washout.ResultsAfter genotype stratification, the effect of GPX1 rs1050450 on lower GPx1 activity responsiveness was confirmed; however, in terms of DNA damage, we failed to indicate that individuals homozygous for variant allele may especially benefit from the increased selenium intake. Surprisingly, considering gene and time interaction, GPX1 polymorphism was observed to modify the level of DNA strand breaks during washout, showing a significant increase in GPX1 wild-type homozygotes. Regardless of the genotype, selenium supplementation was associated with a selectively suppressed selenoprotein mRNA expression and inconsistent changes in oxidative stress response, indicating for overlapped, antioxidant, and prooxidant effects. Intriguingly, DNA damage was not influenced by supplementation, but it was significantly increased during washout.ConclusionsThese results point to an unclear relationship between selenium, genotype, and DNA damage.Electronic supplementary materialThe online version of this article (doi:10.1007/s00394-015-1118-4) contains supplementary material, which is available to authorized users.

Long-term exposure to diesel engine exhaust induces primary DNA damage: a population-based study

ObjectivesDiesel engine exhaust (DEE) is a ubiquitous environmental pollutant and is carcinogenic to humans. To seek early and sensitive biomarkers for prediction of adverse health effects, we analysed the components...

DNA oxidation and DNA repair in gills of zebra mussels exposed to cadmium and benzo(a)pyrene.

Freshwater bivalve molluscs are considered as effective indicators of environmental pollution. The comet assay allows the detection of DNA damage such as DNA strand breaks and alkali-labile sites. The main oxidative lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a pre-mutagenic lesion, can be detected by the comet assay coupled with the hOGG1 DNA repair enzyme. With this modified assay we recently observed that BaP induced 8-oxodG lesions and with the modified comet-Fpg assay we observed that Cd induced oxidative DNA damage. The aim of this study was to determine the stability of DNA lesions in Cd and BaP exposed zebra mussels using the comet-hOGG1 assay. Mussels were exposed for 24 h to these two chemicals and then placed in clean water for 6 days. We observed that BaP (7, 12 and 18 µg/L) induced an increase of DNA strand break levels as soon as 6 h of exposure and that the two highest concentrations of BaP induced a low level of hOGG1-sensitive sites. After 2 days of depuration, BaP induced DNA lesions returned to the basal level, indicating an effective DNA repair. Cd (3, 32 and 81 µg/L) induced an increase of the DNA strand break levels and a low level of hOGG1-sensitive sites. This study revealed that BaP-induced DNA lesions are repaired more efficiently than Cd-induced DNA lesions. As the level of hOGG1 sensitive sites was increased in Cd and BaP exposed mussels, it seems that these chemicals induce 8-oxo-dG.

Genotoxicity and gene expression modulation of silver and titanium dioxide nanoparticles in mice

Recently, we showed that silver nanoparticles (AgNPs) caused apoptosis, necrosis and DNA strand breaks in different cell models in vitro. These findings warranted analyses of their relevance in vivo. We investigated the genotoxic potential and gene expression profiles of silver particles of nano- (Ag20, 20 nm) and submicron- (Ag200, 200 nm) size and titanium dioxide nanoparticles (TiO2-NPs, 21 nm) in selected tissues from exposed male mice including the gonades. A single dose of 5 mg/kg bw nanoparticles was administered intravenously to male mice derived from C57BL6 (WT) and 8-oxoguanine DNA glycosylase knock-out (Ogg1−/− KO). Testis, lung and liver were harvested one and seven days post-exposure and analyzed for DNA strand breaks and oxidized purines employing the Comet assay with Formamidopyrimidine DNA glycosylase (Fpg) treatment, and sperm DNA fragmentation by the sperm chromatin structure assay (SCSA). Based on an initial screening of a panel of 21 genes, seven genes were selected and their expression levels were analyzed in all lung and testis tissues sampled from all animals (n = 6 mice/treatment group) using qPCR. AgNPs, in particular Ag200, caused significantly increased levels of DNA strand breaks and alkali labile sites in lung, seven days post-exposure. Fpg-sensitive lesions were significantly induced in both testis and lung. The transcript level of some key genes; Atm, Rad51, Sod1, Fos and Mmp3, were significantly induced compared to controls, particularly in lung samples from Ag200-exposed KO mice. We conclude that the Ag200 causes genotoxicity and distinct gene expression patterns in selected DNA damage response and repair related genes.