Bingsheng Zhou

Effects of Prochloraz or Propylthiouracil on the Cross-Talk between the HPG, HPA, and HPT Axes in Zebrafish

The objective of this study was to assess chemical-induced effects on cross-talk among the hypothalamic-pituitary-gonad (HPG), hypothalamic-pituitary-adrenal (HPA), and hypothalamic-pituitary-thyroid (HPT) axes of fish. Adult female zebrafish were exposed to 300 μg/L prochloraz (PCZ) or 100 mg/L propylthiouracil (PTU), and the transcriptional profiles of the HPG, HPA, and HPT axes were examined. Exposure to PCZ decreased plasma testosterone (T) and 17β-estradiol (E2) concentrations and affected HPA and HPT axes by down-regulating corticotrophin-releasing hormone (CRH) after 12 and 48 h. By using correlation analyses, it was found that the decrease in E2 plasma concentrations caused by PCZ was correlated with the down-regulation of CRH mRNA expression. Exposure to PTU resulted in lesser concentrations of thyroxine (T4) and triiodothyronine (T3), greater concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) peptides, and increase in steroidogenic gene expression after 12 and 48 h. Concentrations of FSH and LH were negatively correlated with concentrations of T4 and T3. These results are consistent with the hypothesis that increased steroidogenic gene expression after PTU exposure resulted from a reduction in T4 and T3 concentrations, which resulted in greater secretion of FSH and LH.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity.

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.

Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis of zebrafish larvae

Polybrominated diphenyl ethers (PBDEs) have the potential to disrupt thyroid hormone homeostasis, but the molecular mechanisms underlying this process have not yet been clarified. In the present study, zebrafish (Danio rerio) embryos were exposed to a low concentration (0, 1, 3, and 10microg/L) of DE-71 from fertilization to 14 days thereafter. The whole-body content of thyroid hormone and transcription of genes in the hypothalamic-pituitary-thyroid (HPT) axis were analyzed. Exposure to up to 10microg/L of DE-71 significantly reduced thyroxine (T4) levels and significantly upregulated the transcription of corticotrophin-releasing hormone (CRH) and thyroid-stimulating hormone (TSHbeta) genes in a concentration-dependent manner. The transcription of genes involved in the synthesis of TH proteins, sodium/iodide symporter (Slc5a5), and thyroglobulin (TG) and the transcription of marker genes associated with early thyroid development (Pax8 and Nkx2.1) were significantly upregulated upon DE-71 exposure. The expression of thyronine deiodinase (Deio1 and Deio2) mRNAs was also significantly upregulated, possibly as a compensatory response to the decreased T4 levels. However, DE-71 exposure resulted in the downregulation of transthyretin (TTR) gene transcription and did not affect the transcription of thyroid hormone receptors (TRs). Exposure to DE-71 significantly induced the transcription of the uridinediphosphate-glucuronosyltransferase (UGT1ab) gene. The results of our study confirmed the reliability of the zebrafish larvae as models for assessment of the developmental toxicity of PBDEs and transcription of genes of the HPT axis can evaluate the potential mechanisms of thyroid disruption.

The Role of Nrf2 and MAPK Pathways in PFOS-Induced Oxidative Stress in Zebrafish Embryos

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and causes oxidative stress, apoptosis, and developmental toxicity in zebrafish embryos. In the present study, we examined nuclear factor erythroid 2-related factor 2 (Nrf2)- and mitogen-activated protein kinases (MAPKs)-mediated oxidative stress pathways in zebrafish embryos upon exposure to PFOS. Four-hour postfertilization (hpf) zebrafish embryos were exposed to 0.2, 0.4, and 1.0 mg/l PFOS until 96 hpf. PFOS enhanced production of reactive oxygen species (ROS) in a concentration-dependent manner. Activity of antioxidative enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, was significantly induced in zebrafish larvae in all PFOS-treated groups relative to the control. Exposure to 1.0 mg/l PFOS significantly increased malondialdehyde production in zebrafish larvae. The Nrf2 and heme oxygenase-1 (HO-1) gene expressions were both significantly upregulated compared with the control group. For MAPKs, we investigated gene expression profiles of extracellular signal-regulated protein kinase (ERK), c-Jun NH (2)-terminal kinase (JNK), and p38. The ERK gene expression levels were unchanged, whereas JNK and p38 gene expressions were significantly upregulated, which could be linked to PFOS-induced cell apoptosis in zebrafish larvae. In addition, we found that coexposure with sulforaphane, an Nrf2 activator, could significantly protect against PFOS-induced ROS generation, whereas inhibition of MAPKs did not exhibit significant effects on PFOS-induced HO-1 gene expression and ROS production. Furthermore, we showed that morpholino-mediated knockdown of Nrf2 reduced PFOS-induced HO-1 gene expression. These findings demonstrate that Nrf2 is protective against PFOS-induced oxidative stress in zebrafish larvae.

Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: A partial life-cycle test

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and wildlife. The main aim of this study was to investigate the impact of long-term exposure to low concentrations of PFOS in zebrafish. Zebrafish fry (F(0), 14 d post-fertilization, dpf) were exposed via the water for 70 d to 0 (control), 10, 50 and 250 microg L(-1) PFOS, followed by a further 30 d to assess recovery in clean water. The effects on survival and growth parameters and liver histopathology were assessed. Although growth suppression (weight and length) was observed in fish treated with high concentrations PFOS during the exposure period, no mortality was observed throughout the 70 d experiment. Embryos and larvae (F(1)) derived from maternal exposure suffered malformation and mortality. Exposure to 50 and 250 microg L(-1) PFOS could inhibit the growth of the gonads (GSI) in the female zebrafish. Histopathological alterations, primary with lipid droplets accumulation, were most prominently seen in the liver of males and the changes were not reversible, even after the fish were allowed to recover for 30 d in clean water. The triiodothyronine (T(3)) levels were not significantly changed in any of the exposure groups. Hepatic vitellogenin (VTG) gene expression was significantly up-regulated in both male and female zebrafish, but the sex ratio was not altered. The overall results suggested that lower concentrations of PFOS in maternal exposure could result in offspring deformation and mortality.

Waterborne exposure to PFOS causes disruption of the hypothalamus–pituitary–thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 microgL(-1)) and gene expression patterns were examined 15d post-fertilization. The expression of several genes in the HPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), iodothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TRalpha and TRbeta), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 microg L(-1) PFOS. A significant increase in NIS and Dio1 gene expression was observed at 200 microg L(-1) PFOS exposure, while TG gene expression was down-regulated at 200 and 400 microg L(-1) PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TRalpha and TRbeta gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T(4)) content remained unchanged, whereas triiodothyronine (T(3)) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

Bioconcentration and metabolism of decabromodiphenyl ether (BDE-209) result in thyroid endocrine disruption in zebrafish larvae

Polybrominated diphenyl ethers (PBDEs) have the potential to disturb the thyroid endocrine system, but little is known of such effects or underlying mechanisms of BDE-209 in fish. In the present study, bioconcentration and metabolism of BDE-209 were investigated in zebrafish embryos exposed at concentrations of 0, 0.08, 0.38 and 1.92 mg/L in water until 14 days post-fertilization (dpf). Chemical analysis revealed that BDE-209 was accumulated in zebrafish larvae, while also metabolic products were detected, including octa- and nona-BDEs, with nona-BDEs being predominant. The exposure resulted in alterations of both triiodothyronine (T3) and thyroxine (T4) levels, indicating thyroid endocrine disruption. Gene transcription in the hypothalamic-pituitary-thyroid (HPT) axis was further examined, and the results showed that the genes encoding corticotrophin-releasing hormone (CRH) and thyroid-stimulating hormone (TSHβ) were transcriptionally significantly up-regulated. Genes involved in thyroid development (Pax8 and Nkx2.1) and synthesis (sodium/iodide symporter, NIS, thyroglobulin, TG) were also transcriptionally up-regulated. Up-regulation of mRNA for thyronine deiodinase (Dio1 and Dio2) and thyroid hormone receptors (TRα and TRβ) was also observed. However, the genes encoding proteins involved in TH transport (transthyretin, TTR) and metabolism (uridinediphosphate-glucuronosyl-transferase, UGT1ab) were transcriptionally significantly down-regulated. Furthermore, protein synthesis of TG was significantly up-regulated, while that of TTR was significantly reduced. These results suggest that the hypothalamic-pituitary-thyroid axis can be evaluated to determine thyroid endocrine disruption by BDE-209 in developing zebrafish larvae.

Pharmaceuticals in Tap Water: Human Health Risk Assessment and Proposed Monitoring Framework in China

Background: Pharmaceuticals are known to contaminate tap water worldwide, but the relevant human health risks have not been assessed in China. Objectives: We monitored 32 pharmaceuticals in Chinese tap water and evaluated the life-long human health risks of exposure in order to provide information for future prioritization and risk management. Methods: We analyzed samples (n = 113) from 13 cities and compared detected concentrations with existing or newly-derived safety levels for assessing risk quotients (RQs) at different life stages, excluding the prenatal stage. Results: We detected 17 pharmaceuticals in 89% of samples, with most detectable concentrations (92%) at < 50 ng/L. Caffeine (median–maximum, nanograms per liter: 24.4–564), metronidazole (1.8–19.3), salicylic acid (16.6–41.2), clofibric acid (1.2–3.3), carbamazepine (1.3–6.7), and dimetridazole (6.9–14.7) were found in ≥ 20% of samples. Cities within the Yangtze River region and Guangzhou were regarded as contamination hot spots because of elevated levels and frequent positive detections. Of the 17 pharmaceuticals detected, 13 showed very low risk levels, but 4 (i.e., dimetridazole, thiamphenicol, sulfamethazine, and clarithromycin) were found to have at least one life-stage RQ ≥ 0.01, especially for the infant and child life stages, and should be considered of high priority for management. We propose an indicator-based monitoring framework for providing information for source identification, water treatment effectiveness, and water safety management in China. Conclusion: Chinese tap water is an additional route of human exposure to pharmaceuticals, particularly for dimetridazole, although the risk to human health is low based on current toxicity data. Pharmaceutical detection and application of the proposed monitoring framework can be used for water source protection and risk management in China and elsewhere.

Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are frequently detected in biota, including fish. However, knowledge of the toxicological and molecular effects of these currently used flame retardants is limited. In the present study, an in vivo screening approach was developed to evaluate effects of TDCPP and TPP on developmental endpoints and receptor-associated expression of mRNA in zebrafish embryos/larvae. Exposure to TDCPP or TPP resulted in significantly smaller rates of hatching and survival, in dose- and time-dependent manners. The median lethal concentration (LC(50)) was 7.0 mg/L for TDCPP and 29.6 mg/L for TPP at 120 hour post-fertilization (hpf). Real-time PCR revealed alterations in expression of mRNAs involved in aryl hydrocarbon receptors (AhRs)-, peroxisome proliferator-activated receptor alpha (PPARα)-, estrogenic receptors (ERs)-, thyroid hormone receptor alpha (TRα)-, glucocorticoid receptor (GR)-, and mineralocorticoid receptor (MR)-centered gene networks. Exposure to positive control chemicals significantly altered abundances of mRNA in corresponding receptor-centered gene networks, a result that suggests that it is feasible to use zebrafish embryos/larvae to evaluate effects of chemicals on mRNA expression in these gene networks. Exposure to TDCPP altered transcriptional profiles in all six receptor-centered gene networks, thus exerting multiple toxic effects. TPP was easily metabolized and its potency to change expression of mRNA involved in receptor-centered gene networks was weaker than that of TDCPP. The PPARα- and TRα-centered gene networks might be the primary pathways affected by TPP. Taken together, these results demonstrated that TDCPP and TPP could alter mRNA expression of genes involved in the six receptor-centered gene networks in zebrafish embryos/larvae, and TDCPP seemed to have higher potency in changing the mRNA expression of these genes.