Protein profiles in zebrafish (Danio rerio) embryos exposed to perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and in wildlife, and it has the potential for developmental toxicity. However, the molecular mechanisms that lead to these toxic effects are not well known. In the present study, proteomic analysis has been performed to investigate the proteins that are differentially expressed in zebrafish embryos exposed to 0.5 mg/l PFOS until 192 h postfertilization. Two-dimensional electrophoresis coupled with mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that 69 proteins showed altered expression in the treatment group compared to the control group with either increase or decrease in expression levels (more than twofold difference). Of the 69 spots corresponding to the proteins with altered expression, 38 were selected and subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 18 proteins were identified in this analysis. These proteins can be categorized into diverse functional classes such as detoxification, energy metabolism, lipid transport/steroid metabolic process, cell structure, signal transduction, and apoptosis. Overall, proteomic analysis using zebrafish embryos serves as an in vivo model in environmental risk assessment and provides insight into the molecular events in PFOS-induced developmental toxicity. © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.link_to_subscribed_fulltex

DE-71-induced apoptosis involving intracellular calcium and the bax-mitochondria-caspase protease pathway in human neuroblastoma cells in vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2, 5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+]i) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3μM) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of [Ca2+]i in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway. © The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.link_to_subscribed_fulltex

Relationships between tissue concentrations of paralytic shellfish toxins and antioxidative responses of clams, Ruditapes philippinarum

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Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L -1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L -1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L -1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L -1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. © 2009 Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell line

Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo-p-dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genes - cytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3β- hydroxysteroid dehydrogenase (3βHSD2), 17β-hydroxysteroid dehydrogenase (17βHSD1 and 17βHSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) - was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3βHSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo-p-dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption. © 2007 SETAC.link_to_subscribed_fulltex

Effects of fifteen PBDE metabolites, DE71, DE79 and TBBPA on steroidogenesis in the H295R cell line

Polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA) are brominated flame retardants that are produced in large quantities and are commonly used in construction materials, textiles, and as polymers in electronic equipment. Environmental and human levels of PBDEs have been increasing in the past 30 years, but the toxicity of PBDEs is not fully understood. Studies on their effects are relatively limited, and show that PBDEs are neurotoxins and potential endocrine disrupters. Hydroxylated (OH{single bond}) and methoxylated (MeO{single bond}) PBDEs have also been reported in the adipose tissue, blood and milk of wild animals and humans. In the present study, 15 PBDE metabolites, two BDE mixtures (DE71 and DE79), and TBBPA were studied individually to determine their effects on ten steroidogenic genes, aromatase activity, and concentrations of two steroid hormones (testosterone and 17β-estradiol) in the H295R human adrenocortical carcinoma cell line. Exposure to 0.05 μM 2′-OH-BDE-68 significantly induced the expression of CYP11A, CYP11B2, CYP17, CYP21, 3βHSD2, 17βHSD1, and 17βHSD4, and the expression of StAR was induced by 6-OH-BDE-90 at the three exposure concentrations. Exposure to DE71 and DE79 resulted in dose-dependent trend towards induction, but these effects were not significant. Exposure to 0.5 μM 2-OH-BDE-123 and 2-MeO-BDE-123 resulted in significantly greater aromatase activity. However, none of the compounds affected sex hormone production at the concentrations tested. Generally, OH-BDEs had a much stronger ability to affect steroidogenic gene expression than MeO-BDEs. © 2008 Elsevier Ltd. All rights reserved.link_to_subscribed_fulltex