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Double strand break repair and γ-H2AX formation in erythrocytes of medaka (Oryzias latipes) after γ-irradiation
2017
Sayed, Alaa El-Din Hamid | Igarashi, Kento | Watanabe-Asaka, Tomomi | Mitani, Hiroshi
The study of the DNA damage response in erythrocytes after γ-irradiation may provide evidence for its effectiveness as a biomarkers for genotoxic environmental stress. We previously reported various malformations in erythrocytes of medaka irradiated with10 Gy, but not in their micronuclei. In this study, we optimized an assay method for γ-H2AX and double strand breaks in erythrocytes of adult medaka fish after 15 Gy of γ-irradiation. The highest level of apoptosis and nuclear abnormalities, including in micronuclei, were recorded 4 h after γ-irradiation, as was the highest level of γ-H2AX foci in erythrocytes. These results suggest that recognition and repair processes occur as a response to DNA damage in erythrocytes in medaka.
Show more [+] Less [-]Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM2.5
2017
Leclercq, B. | Platel, A. | Antherieu, S. | Alleman, L.Y. | Hardy, E.M. | Perdrix, E. | Grova, N. | Riffault, V. | Appenzeller, B.M. | Happillon, M. | Nesslany, F. | Coddeville, P. | Lo-Guidice, J-M. | Garçon, G.
Even though clinical, epidemiological and toxicological studies have progressively provided a better knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects, further in vitro studies on relevant cell systems are still needed. Hence, aiming of getting closer to the human in vivo conditions, primary human bronchial epithelial cells derived from normal subjects (NHBE) or sensitive chronic obstructive pulmonary disease (COPD)-diseased patients (DHBE) were differentiated at the air-liquid interface. Thereafter, they were repeatedly exposed to air pollution-derived PM2.5 to study the occurrence of some relevant genetic and/or epigenetic endpoints. Concentration-, exposure- and season-dependent increases of OH-B[a]P metabolites in NHBE, and to a lesser extent, COPD-DHBE cells were reported; however, there were more tetra-OH-B[a]P and 8-OHdG DNA adducts in COPD-DHBE cells. No increase in primary DNA strand break nor chromosomal aberration was observed in repeatedly exposed cells. Telomere length and telomerase activity were modified in a concentration- and exposure-dependent manner in NHBE and particularly COPD-DHBE cells. There were a global DNA hypomethylation, a P16 gene promoter hypermethylation, and a decreasing DNA methyltransferase activity in NHBE and notably COPD-DHBE cells repeatedly exposed. Changes in site-specific methylation, acetylation, and phosphorylation of histone H3 (i.e., H3K4me3, H3K9ac, H3K27ac, and H3S10ph) and related enzyme activities occurred in a concentration- and exposure-dependent manner in all the repeatedly exposed cells. Collectively, these results highlighted the key role played by genetic and even epigenetic events in NHBE and particularly sensitive COPD-DHBE cells repeatedly exposed to air pollution-derived PM2.5 and their different responsiveness. While these specific epigenetic changes have been already described in COPD and even lung cancer phenotypes, our findings supported that, together with genetic events, these epigenetic events could dramatically contribute to the shift from healthy to diseased phenotypes following repeated exposure to relatively low doses of air pollution-derived PM2.5.
Show more [+] Less [-]Exposure scenario: Another important factor determining the toxic effects of PM2.5 and possible mechanisms involved
2017
Zhou, Wei | Tian, Dongdong | He, Jun | Zhang, Li | Tang, Xiuli | Zhang, Lijun | Wang, Yimei | Li, Lizhong | Zhao, Jun | Yuan, Xiaoyan | Peng, Shuangqing
Worsening air pollution is a serious threat to public health in many urban and heavily industrialized areas. Particle size and chemical composition are well known determinants of the pathological response to air pollution. In addition, pathological responses may depend on the exposure profile (or scenario) of air pollution. For instance, we previously demonstrated that repeated exposure to low levels of fine airborne particulate matter (PM2.5) induced distinct epigenetic changes compared to acute high-doses exposure. In the present study, we evaluated the differential pathological responses of BEAS-2B human bronchial epithelial cells to two distinct PM2.5 exposure scenarios: 24-h exposure to high-doses PM2.5 (0, 6, 12, 24, 48, 96 μg/cm2) and 10 days’ repeated exposure to low levels of PM2.5 (0, 1.5, 3, 6 μg/cm2). Acute exposure to high concentrations of PM2.5 caused ROS burst, marked DNA damage, dysfunction of the endoplasmic reticulum (ER) stress response, autophagy and necrotic cell death. In contrast, repeated low levels of PM2.5 led to sustained low-grade ROS accumulation, milder DNA damage, ER stress/unfolded protein response (UPR), S-phase arrest, apoptosis, and autophagy. Notably, most cells surviving repeated low-level exposure showed a series of abnormal adaptive responses, such as inhibition of mitochondria biogenesis and epigenetic dysregulation. These results indicate that different PM2.5 exposure scenarios induce distinct forms cytotoxicity and adaptive response. In addition to particle size and chemical composition, exposure scenario may be a critical factor determining the toxic health effects of PM2.5.
Show more [+] Less [-]ATM signals to AMPK to promote autophagy and positively regulate DNA damage in response to cadmium-induced ROS in mouse spermatocytes
2017
Li, Renyan | Luo, Xue | Zhu, Yijian | Zhao, Letian | Li, Lianbing | Peng, Qiang | Ma, Mingfu | Gao, Yanfei
Cadmium (Cd) is a toxic heavy metal and harmful to human health due to its ability to accumulate in organs. Previous studies have shown that Cd can induce DNA damage and autophagy. Autophagy can stabilize genetic material and DNA integrity. The aim of the present study was to determine the exact mechanism and role of autophagy induced by Cd in spermatozoa cells. Mouse spermatocyte-derived cells (GC-2) were treated with 20 μM Cd chloride for 24 h. The level of reactive oxygen species (ROS), DNA damage, autophagy and the expression of the molecular signaling pathway ATM/AMP-activated protein kinase (AMPK)/mTOR were determined. The results showed that Cd induced autophagy and DNA damage in GC-2 cells via ROS generation, and the autophagy signal pathway AMPK/mTOR was activated by ATM which is a DNA damage sensor. Melatonin, a well-known antioxidant, ameliorated DNA damage, and inhibited autophagy via the AMPK/mTOR signal pathway. Furthermore, after inhibition of autophagy by knockdown of AMPKα, increased DNA damage by Cd treatment was observed in GC-2 cells. These findings demonstrated the protective role of autophagy in DNA damage and suggested that the mechanism of autophagy induced by Cd was through the ATM/AMPK/mTOR signal pathway in spermatozoa cells.
Show more [+] Less [-]Evaluation of cytotoxic and genotoxic activity of fungicide formulation Tango® Super in bovine lymphocytes
2017
Schwarzbacherová, Viera | Wnuk, Maciej | Lewinska, Anna | Potocki, Leszek | Żebrowski, Jacek | Koziorowski, Marek | Holečková, Beáta | Šiviková, Katarína | Dianovský, Ján
Tango® Super is a two-compound fungicide formulation widely employed in grain protection. However, details of Tango® Super effects on cell cultures have not been fully investigated. In this study, bovine lymphocytes were exposed to a concentration range 0.5; 1.5; 3; 6; and 15 μg mL⁻¹ for 4 h to assess the cytotoxicity and genotoxicity of the fungicide. Our experiments revealed that this fungicide treatment reduced cell viability, decreased cell proliferation and provoked apoptotic cell death. Cell cycle analysis showed predominant accumulation of cells in the G0/G1 phase of the cell cycle. The fungicide was able to induce mitochondrial superoxide production accompanied by elevated levels of carbonylated proteins and changes in the lipid membrane composition. The fungicide did not induce micronuclei production, but stimulated both DNA double-strand breaks and the formation of p53 binding protein, which is accumulated during the DNA repair process at the site of double-strand breaks. Based on the obtained data we suppose that the fungicide-induced DNA damage is the result of oxidative stress, which may contribute to higher occurrence of apoptotic cell death. Because ergosterol biosynthesis-inhibiting fungicides are widely used in agriculture to ensure higher crop yields and may cause health impairment of animals and humans, there is a need for further testing to elucidate their potential genotoxic effects using in vivo and/or in vitro systems.
Show more [+] Less [-]Toxic organic substances and marker compounds in size-segregated urban particulate matter - Implications for involvement in the in vitro bioactivity of the extractable organic matter
2017
Besis, Athanasios | Tsolakidou, Alexandra | Balla, Dimitra | Samara, Constantini | Voutsa, Dimitra | Pantazaki, Anastasia | Choli-Papadopoulou, Theodora | Lialiaris, Theodore S.
Toxic organic substances and polar organic marker compounds, i.e. polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs) and their nitro-derivatives (N-PAHs), as well as dicarboxylic acids (DCAs) and sugars/sugar anhydrites (S/SAs) were analyzed in size-segregated PM samples (<0.49, 0.49–0.97, 0.97–3 and >3 μm) collected at two urban sites (urban traffic and urban background) during the cold and the warm season. The potential associations between the organic PM determinants and the adverse cellular effects (i.e. cytotoxicity, genotoxicity, DNA damage, oxidative DNA adduct formation, and inflammatory response) induced by the extractable organic matter (EOM) of PM, previously measured in Velali et al. (2016b), were investigated by bivariate correlations and Principal Component Analysis (PCA). Partial Least Square regression analysis (PLS) was also employed in order to identify the chemical classes mainly involved in the EOM-induced toxicological endpoints in the various particle size fractions. Results indicated that particle size range <0.49 μm was the major carrier of PM mass and organic compounds at both sites. All toxic organic compounds exhibited higher concentrations at the urban traffic site, except PCBs and OCPs that did not exhibit intra-urban variations. Conversely, wintertime levels of levoglucosan were significantly higher at the urban background site as a result of residential biomass burning. The PLS regression analysis allowed quite good prediction of the EOM-induced cytotoxicity and genotoxicity based on the determined organic chemical classes, particularly for the finest size fraction of PM. Nevertheless, it is expected that other chemical constituents, not determined here, also contribute to the measured toxicological responses.
Show more [+] Less [-]TRIM36 hypermethylation is involved in polycyclic aromatic hydrocarbons-induced cell transformation
2017
He, Zhini | Li, Daochuan | Ma, Junxiang | Chen, Liping | Duan, Huawei | Zhang, Bo | Gao, Chen | Li, Jie | Xing, Xiumei | Zhao, Jian | Wang, Shan | Wang, Fangping | Zhang, Haiyan | Li, Huiyao | Chen, Shen | Zeng, Xiaowen | Wang, Qing | Xiao, Yongmei | Zheng, Yuxin | Chen, Wen
Long term exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with the increasing risk of lung cancer. To identify differentially hypermethylated genes associated with PAHs-induced carcinogenicity, we performed genome-wide DNA methylation analysis in 20 μM benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells at different stages of cell transformation. Several methylated genes (CNGA4, FLT1, GAREM1, SFMBT2, TRIM36) were differentially hypermethylated and their mRNA was suppressed in cells at both pre-transformed and transformed stages. Similar results were observed in HBE cells transformed by 20 μg/mL coke oven emissions (COEs) mixture collected from a coking manufacturing facility. In particular, hypermethylation of TRIM36 and suppression of TRIM36 expression were gradually enhanced over the time of COEs treatment. We developed bisulfite pyrosequencing assay and assessed TRIM36 methylation quantitatively. We found that hypermethylation of TRIM36 and reduced gene expression was prevalent in several types of human cancers. TRIM36 hypermethylation appeared in 90.0% (23/30) of Non-Small Cell Lung Cancer (NSCLCs) tissues compared to their paired adjacent tissues with an average increase of 1.32 fold. Furthermore, an increased methylation rate (5.90% v.s 7.38%) and reduced levels of TRIM36 mRNA were found in peripheral lymphocytes (PBLCs) of 151 COEs-exposed workers. In all subjects, TRIM36 hypermethylation was positively correlated with the level of urinary 1-hydroxypyrene (P < 0.001), an internal exposure marker of PAHs, and the DNA damage (P = 0.013). These findings suggest that aberrant hypermethylation of TRIM36 might be involved in the acquisition of malignant phenotype and could be served as a biomarker for risk assessment of PAHs exposure.
Show more [+] Less [-]Imidacloprid induces various toxicological effects related to the expression of 3β-HSD, NR5A1, and OGG1 genes in mature and immature rats
2017
Abdel-Rahman Mohamed, Amany | Mohamed, Wafaa A.M. | Khater, Safaa I.
This study aimed to evaluate the adverse effects of the insecticide imidacloprid (IMI) on male spermatogenesis, steroidogenesis, and DNA damage in sexually mature and immature rats. Forty male rats (mature and immature) were equally divided into four groups: two mature and two immature groups. IMI groups of both ages were orally administered IMI in corn oil at a concentration of 1 mg/mL for kg BW/day, whereas their respective controls were orally administered corn oil only (1 mL/kg of body weight) daily for 65 days. On day 66, the rats were lightly anesthetized and then euthanized by cervical dislocation. Whole blood was collected for hemogram, serum for hormonal profile, semen for sperm profile, and testes for gene expression and histopathological, and immunohistochemical examinations. The obtained results revealed that both sexually mature and immature rats orally exposed to IMI showed serious abnormalities in sperm morphology and concentrations, with an imbalance of sexual hormones. There were increases in the level of serum 8-hydroxy-2′-deoxyguanosine and in the percentage of comet (tailed) sperm DNA in the IMI-treated groups. The results exhibited the upregulation of a DNA damage tolerance gene (8-oxoguanine glycosylase 1) and downregulation of the activity of steroidogenic genes (nuclear receptor subfamily 5, group A, member 1 and 3β-hydroxysteroid dehydrogenase). Immunohistochemical examination of the B-cell lymphoma 2-associated X apoptotic protein in testicular sections showed various degrees of apoptosis in the spermatogonial cells of the IMI-treated rats compared to the control groups. These damaging effects of IMI were more pronounced in the sexually mature rats than in the immature rats. In conclusion, despite using a low dose of IMI in the present study, there were noticeable harmful consequences on the reproductive system at different stages of sexual maturity in male rats.
Show more [+] Less [-]Effect of substituted phenylamine antioxidants on three life stages of the freshwater mussel Lampsilis siliquoidea
2017
Prosser, R.S. | Gillis, P.L. | Holman, E.A.M. | Schissler, D. | Ikert, H. | Toito, J. | Gilroy, E. | Campbell, S. | Bartlett, A.J. | Milani, D. | Parrott, J.L. | Balakrishnan, V.K.
Substituted phenylamines (SPAs) are incorporated into a variety of consumer products (e.g., polymers, lubricants) in order to increase the lifespan of the products by acting as a primary antioxidant. Based on their physicochemical properties, if SPAs were to enter the aquatic environment, they would likely partition into sediment. No studies to date have investigated the effect of sediment-associated SPAs on aquatic organisms. The current study examined the effect of four SPAs (diphenylamine (DPA); N-phenyl-1-napthylamine (PNA); N-(1,3-dimethylbutyl)-N’-phenyl-1,4-phenylenediamine (DPPDA); 4,4’-methylene-bis[N-sec-butylaniline] (MBA)) on three different life stages of the freshwater mussel, Lampsilis siliquoidea. The viability of larvae (glochidia) of L. siliquoidea and Lampsilis fasciola was assessed after 48 h of exposure to SPAs in water. The 48-h EC50s for glochidia viability of L. siliquoidea were 5951, 606, 439, and 258 μg/L for DPA, PNA, DPPDA, and MBA, respectively, and 7946, 591, 137, and 47 μg/L, respectively, for L. fasciola. Juvenile (7–15 months) and adult L. siliquoidea were exposed to sediment-associated SPAs for 28 d. LC50s for juvenile mussels were 18, 55, 62, and 109 μg/g dry weight (dw) of sediment for DPA, PNA, DPPDA, and MBA, respectively. Adult mussels were exposed to sub-lethal concentrations of sediment-associated SPAs in order to investigate reactive oxygen species (ROS), lipid peroxidation and total glutathione in the gill, gonad, and digestive gland tissue, and viability and DNA damage in hemocytes. No significant concentration-dependent trend in any of these biochemical and cellular endpoints relative to the concentration of sediment-associated SPAs was observed in any tissues. Investigations into the concentration of SPAs in the aquatic environment are required before a conclusion can be made on whether these compounds pose a hazard to the different life stages of freshwater mussels.
Show more [+] Less [-]Multigenerational effects and DNA alterations of QDs-Indolicidin on Daphnia magna
2017
Maselli, Valeria | Siciliano, Antonietta | Giorgio, Antonella | Falanga, Annarita | Galdiero, Stefania | Guida, Marco | Fulgione, Domenico | Galdiero, Emilia
The complex QDs-Indolicidin (QDs-Ind) has been previously shown to be a good antimicrobial system with a low acute toxicity on Daphnia magna (D. magna). However, multigenerational effects caused by exposure to QDs-Ind and after subsequent recovery are still unknown. In this study, we performed multigenerational exposure tests and we evaluated individual fitness, population growth, DNA alteration, expression of Dhb (haemoglobin), Vtg (vitellogenin), CYP4 (cytochrome P450s CYP4 family), and CYP314 (cytochrome P450s mitochondrial family 314) genes on three generation of D. magna. Results showed that the total amount of eggs produced per female and total number of brood per female and body lengths were significantly decreased, Dhb, CYP4 were upregulated while Vtg was down-regulated except at reproduction days when it was slightly up-regulated under QDs-Ind exposure. Random Amplification of Polymorphic DNA (RAPD) method has proven to be useful to qualitative assess of DNA damage during generation and to underline modification in somatic or germinal cells. The results of the study suggest that effects of chronic exposure cannot be ignored.
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