Levels Of DNA Strand Breaks Research Articles

Impact of indoor air pollution on DNA damage and chromosome stability: a systematic review.

Indoor air pollution is becoming a rising public health problem and is largely resulting from the burning of solid fuels and heating in households. Burning these fuels produces harmful compounds, such as particulate matter regarded as a major health risk, particularly affecting the onset and exacerbation of respiratory diseases. As exposure to polluted indoor air can cause DNA damage including DNA sd breaks as well as chromosomal damage, in this paper, we aim to provide an overview of the impact of indoor air pollution on DNA damage and genome stability by reviewing the scientific papers that have used the comet, micronucleus, and γ-H2AX assays. These methods are valuable tools in human biomonitoring and for studying the mechanisms of action of various pollutants, and are readily used for the assessment of primary DNA damage and genome instability induced by air pollutants by measuring different aspects of DNA and chromosomal damage. Based on our search, in selected studies (in vitro, animal models, and human biomonitoring), we found generally higher levels of DNA strand breaks and chromosomal damage due to indoor air pollutants compared to matched control or unexposed groups. In summary, our systematic review reveals the importance of the comet, micronucleus, and γ-H2AX assays as sensitive tools for the evaluation of DNA and genome damaging potential of different indoor air pollutants. Additionally, research in this particular direction is warranted since little is still known about the level of indoor air pollution in households or public buildings and its impact on genetic material. Future studies should focus on research investigating the possible impact of indoor air pollutants in complex mixtures on the genome and relate pollutants to possible health outcomes.

Antagonistic effects of a COX1/2 inhibitor drug in human HepG2 cells exposed to an environmental carcinogen

Understanding interactions between legacy and emerging environmental contaminants has important implications for risk assessment, especially when mutagens and carcinogens are involved, whose critical effects are chronic and therefore difficult to predict. The current work aimed to investigate potential interactions between benzo[a]pyrene (B[a]P), a carcinogenic polycyclic aromatic hydrocarbon and legacy pollutant, and diclofenac (DFC), a non-steroidal anti-inflammatory drug and pollutant of emerging concern, and how DFC affects B[a]P toxicity. Exposure to binary mixtures of these chemicals resulted in substantially reduced cytotoxicity in human HepG2 cells compared to single-chemical exposures. Significant antagonistic effects were observed in response to high concentrations of B[a]P in combination with DFC at IC50 and ⅕ IC50. While additive effects were found for levels of intracellular reactive oxygen species, antagonistic mixture effects were observed for genotoxicity. B[a]P induced DNA strand breaks, γH2AX activation, and micronuclei formation at ½ IC50 concentrations or lower, whereas DFC induced only low levels of DNA strand breaks. Their mixture caused significantly lower levels of genotoxicity by all three endpoints compared to those expected based on concentration additivity. In addition, antagonistic mixture effects on CYP1 enzyme activity suggested that the observed reduced genotoxicity of B[a]P was due to its reduced metabolic activation as a result of enzymatic inhibition by DFC. Overall, the findings further support the growing concern that co-exposure to environmental toxicants and their non-additive interactions may be a confounding factor that should not be neglected in environmental and human health risk assessment.

Pleural inflammatory response, mesothelin content and DNA damage in mice at one-year after intra-pleural carbon nanotube administration

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

The goby fish Sicydium spp. as valuable sentinel species towards the chemical stress in freshwater bodies of West Indies

Implementation of the European Water Framework Directive in tropical areas such as the French West Indies (FWI) requires to select relevant aquatic sentinel species for investigating the ecological status of surface waters. The present work aimed to study the biological response of the widespread fish Sicydium spp. towards river chemical quality in Guadeloupe island through a set of proper biomarkers. During a 2-year survey, the hepatic EROD activity, the micronucleus formation and the level of primary DNA strand breaks in erythrocytes were measured respectively as an enzymatic biomarker of exposure and genotoxicity endpoints in fish living upstream and downstream of two chemically-contrasted rivers. Hepatic EROD activity was shown to be variable along the time but always significantly higher in fish from the most contaminated river (Rivière aux Herbes) compared to the low contaminated one (Grande Rivière de Vieux-Habitants). Fish size did not influence EROD activity. Female fish exhibited a lower EROD activity compared to males depending on the catching period. We observed significant temporal variation in micronucleus frequency and primary DNA damage level measured in fish erythrocytes that did not depend on the fish size. Micronucleus frequency and to a lesser extent DNA damage were significantly higher in fish from the Rivière aux Herbes compared to the Grande Rivière de Vieux-Habitants. Our results argue for the interest of using Sicydium spp. as sentinel species to assess river quality and chemical pressures in FWI.

Age-related DNA damage in stallions: an ongoing investigation

Foals sired by younger stallions record significantly higher racing speeds (Sharman.et.al. Royal Society Open Science. 2022;9:220691), and more race wins than foals sired by older stallions (Brazil.et.al. Honours Dissertation. University of Limerick. 2016). We hypothesize that this may be due to an age-associated increase in sperm DNA damage, as has been demonstrated in other species. Therefore, this study aimed to investigate the relationship between stallion age and sperm DNA damage. Post-coital dismount semen samples (n=75) were collected weekly from 46 commercial Thoroughbred stallions in the Hunter Valley (Australia). Samples were immediately diluted (2:1, EquiPlus:semen), and the high-density spermatozoa were isolated using EquiPure centrifugation to reduce contaminants (somatic cells and other debris). Any samples that were contaminated with urine were excluded from the study. Sperm count and motilities were recorded onsite (iSperm™), and samples were snap frozen for DNA damage analyses. Management information, including pregnancy scan results, were also collected. For this study, stallions aged ≤9 years were categorized as “young” while those aged ≥12 were categorized as “old”. Stallions between 9 and 12 years were excluded to improve the likelihood of observing an age-related difference. Mare age and fertility status were similar across both groups. All data were checked for normality (Shapiro-Wilk) and analyzed using unpaired t-tests. Sperm count and motility did not differsignificantly between age cohorts. DNA strand breaks were significantly higher in spermatozoa from older stallions compared to younger stallions (alkaline comet assay: 19.7±0.55% vs 15.4±0.57% mean tail intensity; P≤0.05, and sperm chromatin dispersion or ‘Halo’ assay; 0.48±0.045in vs 0.70±0.074in ‘halo’ area; P≤0.05, respectively). These breaks may be attributed to poor chromatin packaging, as older stallions had a concomitant deficiency of sperm protamines compared to young stallions (11.6±1.53 vs 6.9±1.07 chromomycin A3 positive cells; P≤0.05, respectively). Although younger stallions had lower levels of DNA strand breakage, they had more oxidized DNA adducts (8-hydroxy-2′-deoxyguanosine (8-OHdG) assay; P≤0.05). Interestingly, regardless of age, positive correlations between the proportion of oxidized DNA adducts and fertility were observed (R2=0.269 for younger, R2=0.483 for older stallions; P≤0.05). This is likely a reflection of the increased use of oxidative phosphorylation, and subsequent ROS production, in higher fertility ejaculates (Gibb.et.al. Biology of Reproduction. 2014;91(3):77). In conclusion, while stallion sperm DNA damage rises with age, it does not appear to influence fertility outcomes. However, further research is needed to determine if age-related sperm DNA damage compromises the next generation's genome. In addition, we must identify the most vulnerable genomic loci for sperm DNA damage and determine how these DNA lesions may contribute to reduced racing performance and health in offspring.

Genotoxicity by rapeseed methyl ester and hydrogenated vegetable oil combustion exhaust products in lung epithelial (A549) cells.

Biofuel is an attractive substitute for petrodiesel because of its lower environmental footprint. For instance, the polycyclic aromatic hydrocarbons (PAH) emission per fuel energy content is lower for rapeseed methyl ester (RME) than for petrodiesel. This study assesses genotoxicity by extractable organic matter (EOM) of exhaust particles from the combustion of petrodiesel, RME, and hydrogenated vegetable oil (HVO) in lung epithelial (A549) cells. Genotoxicity was assessed as DNA strand breaks by the alkaline comet assay. EOM from the combustion of petrodiesel and RME generated the same level of DNA strand breaks based on the equal concentration of total PAH (i.e. net increases of 0.13 [95% confidence interval (CI): 0.002, 0.25, and 0.12 [95% CI: 0.01, 0.24] lesions per million base pairs, respectively). In comparison, the positive control (etoposide) generated a much higher level of DNA strand breaks (i.e. 0.84, 95% CI: 0.72, 0.97) lesions per million base pairs. Relatively low concentrations of EOM from RME and HVO combustion particles (<116 ng/ml total PAH) did not cause DNA strand breaks in A549 cells, whereas benzo[a]pyrene and PAH-rich EOM from petrodiesel combusted using low oxygen inlet concentration were genotoxic. The genotoxicity was attributed to high molecular weight PAH isomers with 5-6 rings. In summary, the results show that EOM from the combustion of petrodiesel and RME generate the same level of DNA strand breaks on an equal total PAH basis. However, the genotoxic hazard of engine exhaust from on-road vehicles is lower for RME than petrodiesel because of lower PAH emission per fuel energy content.

Maternal exposure to genistein during pregnancy and oxidative DNA damage in testes of male mouse offspring.

BackgroundGenistein is a dietary supplement with phyto-estrogenic properties. Therefore, high intake of genistein during pregnancy may have adverse effects on the genetic integrity of testes and germ cells of male offspring. In this study, we examined whether maternal exposure to genistein during pregnancy induced oxidative DNA damage in the male germline at adolescence.MethodsAtm-ΔSRI mice have lower glucose-6-phosphate dehydrogenase (G6PDH) activity, which is important for maintaining levels of reduced glutathione and therefore these mice have an increased susceptibility to oxidative stress. Parental heterozygous Atm-ΔSRI mice received a genistein-rich or control diet, after which they were mated to obtain offspring. During pregnancy, mothers remained on the respective diets and after delivery all animals received control diets. Redox status and oxidative DNA damage were assessed in testes and sperm of 12 weeks old male offspring. Gene expression of Cyp1b1, Comt, and Nqo1 was assessed in testes, and DNA methylation as possible mechanism for transmission of effects to later life.ResultsIntake of genistein during pregnancy increased oxidative DNA damage in testes of offspring, especially in heterozygous Atm-ΔSRI mice. These increased DNA damage levels coincided with decreased expression of Comt and Nqo1. Heterozygous Atm-ΔSRI mice had higher levels of DNA strand breaks in sperm compared to wild type littermates, and DNA damage was further enhanced by a genistein-rich maternal diet. G6PDH activity was higher in mice with high maternal intake of genistein compared to control diets, suggesting compensation against oxidative stress. A positive correlation was observed between the levels of DNA methylation and oxidative DNA damage in testes.ConclusionThese data indicate that prenatal exposure to genistein altered gene expression and increased DNA damage in testes and sperm of adolescent male offspring. These effects of genistein on DNA damage in later life coincided with alterations in DNA methylation.

Impact of P-Chloroaniline on Oxidative Stress and Biomacromolecules Damage in the Clam Ruditapes philippinarums: A Simulate Toxicity Test of Spill Incident.

As a hazardous chemical, p-chloroaniline (PCA) shows intensive adsorption and accumulation after entering the aquatic ecosystem, which can be enriched in organisms and cause damage. With the objective of achieving an integrated and mechanistic view of the toxic effects of PCA in the marine sentinel organism Ruditapes philippinarum, Manila clams were exposed to different concentration of PCA (0.5, 2 and 5 mg/L) for 15 days. Focusing on the gills, first targeting the toxic and digestive gland, the metabolic detoxification organ, we detected dose- and time-related changes inantioxidase activities and biomacromolecular damages in treated clams. Glutathione S-transferase (GST) activity and glutathione (GSH) contents were significantly induced, and superoxide dismutase (SOD) activity increased at the beginning of exposure and then decreased. The malondialdehyde (MDA) and protein methylation (PC) contents which represent lipid peroxidation and carbonylation of proteins, increased first with exposure time and then decreased in the digestive gland. DNA strand break levels were consistently higher than those in the control group. The digestive gland showed more sensitivity to the stress of PCA than the gills. GST and MDA in the gill and GST, GSH, SOD, DNA strand break level in the digestive gland showed significant correlation with PCA exposure, which indicated that these parameters can be used as sensitive biomarkers to indicate toxic effects from chloraniline leakage.

Integration of Genotoxic Biomarkers in Environmental Biomonitoring Analysis Using a Multi-Biomarker Approach in Three-Spined Stickleback (Gasterosteus aculeatus Linnaeus, 1758).

Water is impacted by a variety of increasing pressures, such as contaminants, including genotoxic pollutants. The proposed multi-biomarker approach at a sub-individual level gives a complementary indicator to the chemical and ecological parameters of the Water Framework Directive (WFD, 2000/60/EC). By integrating biomarkers of genotoxicity and erythrocyte necrosis in the sentinel fish species the three-spined stickleback (Gasterosteus aculeatus) through active biomonitoring of six stations of the Artois-Picardie watershed, north France, our work aimed to improve the already existing biomarker approach. Even if fish in all stations had high levels of DNA strand breaks, the multivariate analysis (PCA), followed by hierarchical agglomerative clustering (HAC), improved discrimination among stations by detecting an increase of nuclear DNA content variation (Etaing, St Rémy du Nord, Artres and Biache-St-Vaast) and erythrocyte necrosis (Etaing, St Rémy du Nord). The present work highlighted that the integration of these biomarkers of genotoxicity in a multi-biomarker approach is appropriate to expand physiological parameters which allow the targeting of new potential effects of contaminants.

Parvovirus B19 induces cellular senescence in human dermal fibroblasts: putative role in systemic sclerosis-associated fibrosis.

ObjectiveEmerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in ageing or in SSc. Recently we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/pro-inflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis.MethodsPrimary cultures of fibroblasts were infected with B19V and analysed for the acquisition of senescence markers, such as morphological modifications, senescence-associated β-galactosidase (SA-β-gal) activity, DNA damage response and expression of senescence-associated secretory phenotype (SASP)-related factors.ResultsWe demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-β-gal activity similar to that observed in SSc skin fibroblasts. They also developed an SASP-like phenotype characterized by mRNA expression and release of some pro-inflammatory cytokines, along with activation of the transcription factor nuclear factor κB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significantly higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. An increased level and nuclear localization of γH2AX, a hallmark of DNA damage response, were also found.ConclusionsB19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process.

Mammalian MutY Homolog (MYH or MUTYH) is Critical for Telomere Integrity under Oxidative Stress.

Telomeres consist of special features and proteins to protect the ends of each chromosome from deterioration and fusion. The telomeric DNA repeats are highly susceptible to oxidative damage that can accelerate telomere shortening and affect telomere integrity. Several DNA repair factors including MYH/MUTYH DNA glycosylase, its interacting partners Rad9/Rad1/Hus1 checkpoint clamp, and SIRT6 aging regulator, are associated with the telomeres. MYH prevents C:G to A:T mutation by removing adenine mispaired with a frequent oxidative DNA lesion, 8-oxoguanine. Here, we show that hMYH knockout (KO) human HEK-293T cells are more sensitive to H2O2 treatment, have higher levels of DNA strand breaks and shorter telomeres than the control hMYH+/+ cells. SIRT6 foci increase at both the global genome and at telomeric regions in H2O2-treated hMYH+/+ cells. However, in untreated hMYH KO HEK-293T cells, SIRT6 foci only increase at the global genome, but not at the telomeric regions. In addition, the hMYH KO HEK-293T cells have increased extra-chromosomal and intra-chromosomal telomeres compared to the control cells, even in the absence of H2O2 treatment. After H2O2 treatment, the frequency of extra-chromosomal telomeres increased in control HEK-293T cells. Remarkably, in H2O2-treated hMYH KO cells, the frequencies of extra-chromosomal telomeres, intra-chromosomal telomeres, and telomere fusions are further increased. We further found that the sensitivity to H2O2 and shortened telomeres of hMYH KO cells, are restored by expressing wild-type hMYH, and partially rescued by expressing hMYHQ324H mutant (defective in Hus1 interaction only), but not by expressing hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions). Thus, MYH interactions with SIRT6 and Hus1 are critical for maintaining cell viability and telomeric stability. Therefore, the failure to coordinate 8-oxoG repair is detrimental to telomere integrity.

Nanoparticles: Weighing the Pros and Cons from an Eco-genotoxicological Perspective.

The exponential growth of nanotechnology and the industrial production have raised concerns over its impact on human and environmental health and safety (EHS). Although there has been substantial progress in the assessment of pristine nanoparticle toxicities, their EHS impacts require greater clarification. In this review, we discuss studies that have assessed nanoparticle eco-genotoxicity in different test systems and their fate in the environment as well as the considerable confounding factors that may complicate the results. We highlight key mechanisms of nanoparticle-mediated genotoxicity. Then we discuss the reliability of endpoint assays, such as the comet assay, the most favored assessment technique because of its versatility to measure low levels of DNA strand breakage, and the micronucleus assay, which is complementary to the former because of its greater ability to detect chromosomal DNA fragmentation. We also address the current recommendations on experimental design, including environmentally relevant concentrations and suitable exposure duration to avoid false-positive or -negative results. The genotoxicity of nanoparticles depends on their physicochemical features and the presence of co-pollutants. Thus, the effect of environmental processes (e.g., aggregation and agglomeration, adsorption, and transformation of nanoparticles) would account for when determining the actual genotoxicity relevant to environmental systems, and assay procedures must be standardized. Indeed, the engineered nanoparticles offer potential applications in different fields including biomedicine, environment, agriculture, and industry. Toxicological pathways and the potential risk factors related to genotoxic responses in biological organisms and environments need to be clarified before appropriate and sustainable applications of nanoparticles can be established.

Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells.

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.

Inflammatory Response, Reactive Oxygen Species Production and DNA Damage in Mice After Intrapleural Exposure to Carbon Nanotubes.

Carbon nanotubes (CNTs) are speculated to cause mesothelioma by persistent inflammation, oxidative stress, tissue injury, and genotoxicity. To investigate the pleural response to CNTs, we exposed C57BL/6 mice by intrapleural injection of 0.2 or 5 µg multiwalled CNTs (MWCNT-7, NM-401, and NM-403) or single-walled CNTs (NM-411). Inflammatory response, cellular reactive oxygen species (ROS) production of pleural lavage cells, and genotoxicity in cells from the mesothelial surface were assessed at days 1 and 90 after the exposure. Long and rigid types of MWCNTs (MWCNT-7 and NM-401) caused acute inflammation, characterized by influx of macrophages, neutrophils, and eosinophils into the pleural cavity. The inflammation was still evident at 90 days after the exposure, although it had reduced dramatically. The cellular ROS production was increased at day 90 after the exposure to MWCNT-7 and NM-401. The short and tangled type of MWCNT (ie NM-403) did not cause pleural inflammation or ROS production in pleural fluid cells. The exposure to NM-411 did not cause consistent inflammation responses or cellular ROS production. Levels of DNA strand breaks and DNA oxidation damage were unaltered, except for NM-411-exposed mice that had increased levels of DNA strand breaks at 90 days after the exposure. In conclusion, the long and rigid CNTs caused prolonged inflammatory response and increased ROS production in pleural lavage cells, yet it was not reflected in higher levels of DNA damage in pleural tissue.

DNA damage in leukocytes and serum nitrite concentration are negatively associated in type 1 diabetes

Chronic hyperglycaemia leads to DNA damage in diabetes and might be associated with nitrosative stress. In this study, we aimed at assessing the level of DNA strand breaks in leukocytes, serum nitrite and nitrate in patients with type 1 diabetes and healthy controls and associations of these parameters with diabetes-related outcomes in a prospective study. The level of DNA damage was determined in 71 patients with type 1 diabetes and 57 healthy controls by comet assay and scored with arbitrary units (AU). The chemiluminescence method was used to measure nitrite and nitrate. Clinical information and data on consumption of alcohol, physical activity and smoking were collected. Progression of complications in patients with diabetes was assessed after a follow-up time of 4-5 years. We observed a higher level of DNA damage in leukocytes of patients with type 1 diabetes compared with healthy subjects [type 1 diabetes AU 50 (36-74.5); control AU 30 (24.1-43), P < 0.001]. According to regression, type 1 diabetes leads to a 2-fold increase in DNA damage. In the group of type 1 diabetes, DNA damage correlated positively with total cholesterol (R = 0.262, P = 0.028) and negatively with serum glucose level (R = -0.284; P = 0.018) and serum nitrite (R = -0.335; P = 0.008). DNA damage was not significantly associated with HbA1c, diabetes duration, complications and lifestyle factors. However, DNA damage > 57 AU was associated with statistically significantly lower serum nitrite and 1.52 higher risk of progression of complications of diabetes over the follow-up period. The latter result was not statistically significant due to insufficient study power [relative risk 1.52 (95% confidence interval = 0.68, 3.42, P = 0.31)]. Our results confirm that type 1 diabetes is associated with a higher level of DNA strand breaks in leukocytes when compared with the reference group and demonstrate the negative association between DNA damage and serum nitrite concentration.

Sublethal effects of terbium and praseodymium in juvenile rainbow trout

Abstract The increasing extraction of rare earth elements (REEs) and their subsequent release to freshwater ecosystems are expected to threaten aquatic organisms, yet little is known about the scope of this issue. The purpose of this study was to identify key molecular responses associated with exposure and toxicity of two data-poor REEs, terbium (Tb, heavy REE) and praesodymium (Pr, light REE) in rainbow trout juveniles. Fish were exposed for 96 h to increasing concentrations of Tb and Pr to determine impacts on survival and variation in biomarkers involved in oxidative stress, inflammation, DNA damage and metal homeostasis (metallothioneins). In surviving fish, a suite of 14 gene expression endpoints were also examined as well as genes associated with protein stability, calcium binding, immune response, protein damage, cell division, iron homeostasis and mitochondrial activity. The data showed that Tb was approximately two times more toxic than Pr, with estimated LC50 of 5.8 and 11 mg/L respectively. In addition, Tb and Pr exhibit respectively anti and pro-inflammatory responses relative to controls as demonstrated by the significant variation in the arachidonate cycloocygenase (COX) activity. However only Pr displayed a genotoxic effect due to the increase in the level of DNA strand breaks. Exposure to these two elements lead to the expression of genes involved in DNA repair activity, inflammation, protein denaturation, calcium binding, oxidative stress and divalent metal handling, ATP synthesis and hemoprotein activity. Based on the observed responses to both Tb and Pr, metal homeostasis, protein denaturation and DNA repair activity were commonly affected. The most toxic element (Tb) produced more important changes in the expression of genes involved in oxidative stress, calcium binding, hemoprotein activity and protein folding and turnover. These genes represents potential physiological targets involved in the mode of action for Tb and Pr in fish toxicity.

The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection.

The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1–2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.

Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals

The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations.

3-Aminobenzamide alleviates elevated DNA damage and DNA methylation in a BTBR T+Itpr3tf/J mouse model of autism by enhancing repair gene expression

Little is known about genetic and epigenetic alterations in autism spectrum disorder. Moreover, the efficiency of DNA repair in autism must be improved to correct these alterations. We examined whether 3-aminobenzamide (3-AB) could reverse these alterations. We conducted experiments to clarify the molecular mechanism underlying these ameliorations. An assessment of genetic and epigenetic alterations by a modified comet assay showed elevated levels of oxidative DNA strand breaks and DNA hypermethylation in BTBR T+Itpr3tf/J (BTBR) mice used as a model of autism. Oxidative DNA strand breaks and DNA methylation were further quantified fluorometrically, and the results showed similar changes. Conversely, 3-AB treated BTBR mice showed a significant reduction in these alterations compared with untreated mice. The expressions of 43 genes involved in DNA repair were altered in BTBR mice. RT2 Profiler PCR Array revealed significantly altered expression of seven genes, which was confirmed by RT-PCR analyses. 3-AB treatment relieved these disturbances and significantly improved Ogg1 and Rad1 up-regulation. Moreover, autism-like behaviors were also mitigated in BTBR animals by 3-AB treatment without alterations in locomotor activities. The simultaneous effects of reduced DNA damage and DNA methylation levels as well as the regulation of repair gene expression indicate the potential of 3-AB as a therapeutic agent to decrease the levels of DNA damage and DNA methylation in autistic patients. The current data may help in the development of therapies that ultimately provide a better quality of life for individuals suffering from autism.