Liqin Yu

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.

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.

Prenatal Transfer of Polybrominated Diphenyl Ethers (PBDEs) Results in Developmental Neurotoxicity in Zebrafish Larvae

Parental exposure to polybrominated diphenyl ethers (PBDEs) in animals has been found to be transferred to the offspring. The environmental health risk and toxicity to the offspring are still unclear. The objective of the present study was to identify environmentally relevant concentrations of PBDEs for parental exposure that would cause developmental neurotoxicity in the offspring. Adult zebrafish were exposed to environmentally relevant concentrations of DE-71 (0.16, 0.8, 4.0 μg/L) via water. The results showed that PBDE exposure did not affect larvae hatching, malformation, or survival. The residue of PBDEs was detected in F1 eggs upon parental exposure. Acetylcholinesterase (AChE) activity was significantly inhibited in F1 larvae. Genes of central nervous system development (e.g., myelin basic protein, synapsin IIa, α1-tubulin) were significantly downregulated in larvae. Protein levels of α1-tubulin and synapsin IIa were also reduced. Decreased locomotion activity was observed in the larvae. This study provides the first evidence that parental exposure to environmentally relevant concentrations of PBDEs could cause adverse effects on neurodevelopment in zebrafish offspring.

Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic–pituitary–gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0mg/l 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ERalpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity.

Endocrine disruption and reproductive impairment in zebrafish by exposure to 8:2 fluorotelomer alcohol

The fluorotelomer alcohols (FTOHs) have been detected in the aquatic environment and shown potential estrogenic activity, but the effects on fish reproduction remain unknown. In the present study, 4-month-old zebrafish were exposed to different concentrations of 8:2 FTOH (0, 10, 30, 90, 270microg/L) for 4 weeks, and the impact on reproduction was investigated. The plasma testosterone (T) and estradiol (E2) levels were significantly increased in the females, while T and E2 levels were decreased and increased in the males, respectively. The average number of eggs spawned and sperm production were reduced upon exposure to the chemical, which also resulted in thinning of eggshell, and reduced protein content and egg diameter. Histological examination showed promotion of oocyte maturation and retarded spermiation in female and male fish, respectively. The gene transcription of follicle-stimulating hormone beta (FSHbeta) and luteinizing hormone beta (LHbeta) in the pituitary were up- and down-regulated in female and male fish, respectively. Up-regulation of vitellogenin (VTG1) and zona pellucida protein 2 (ZP2a) gene transcription was observed in the males, indicating estrogenic activity. However, down-regulation of gene transcription of VTG1 and ZP2a was measured in the females, associated with decreased fecundity. In addition, exposure of adult zebrafish to 8:2 FTOH caused reduced hatching rates in the offspring. The results demonstrated that waterborne exposure to 8:2 FTOH caused disruption of sex hormone biosynthesis and impaired reproduction in adult zebrafish, ultimately resulting in decreased hatching rates in the offspring.

Effects of Tris(1,3-dichloro-2-propyl) Phosphate on Growth, Reproduction, and Gene Transcription of Daphnia magna at Environmentally Relevant Concentrations

The synthetic flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been frequently detected in natural waters, and its maximum concentration ever reported is 377 ng/L. However, information on the adverse effects of environmentally relevant concentrations of TDCIPP on aquatic organisms are totally unknown. In this study, <12-h old water fleas, D. magna, were exposed to concentrations of 0, 65±7.1, 550±33, or 6500±1400 ng/L TDCIPP, and dose- and time-dependent effects on reproduction and development were evaluated. Sequences of genes of D. magna were obtained from the National Center for Biotechnology Information and were used to develop PCR arrays for D. magna. Arrays were then used to study transcriptional responses of D. magna to TDCIPP. Exposure to environmentally relevant concentrations of TDCIPP significantly decreased fecundity as well as length of F0 and F1 generations. Transcriptional responses showed that, of the 155 genes tested, expressions of 57 genes were significantly changed, and some changes occurred following exposure to environmentally relevant concentrations (i.e., 65±7.1 and 550±23 ng/L). Furthermore, pathways related to protein synthesis and metabolism and endocytosis were considered to be significantly affected in a dose- and time-dependent manner and might be responsible for TDCIPP-induced reproductive and developmental toxicities.

Acute Exposure to Tris(1,3-dichloro-2-propyl) Phosphate (TDCIPP) Causes Hepatic Inflammation and Leads to Hepatotoxicity in Zebrafish.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been frequently detected in environmental media and has adverse health effect on wildlife and humans. It has been implicated to have hepatotoxicity, but its molecular mechanisms remain unclear. In the present study, adult male zebrafish were exposed to TDCIPP and global hepatic gene expression was examined by RNA-Seq and RT-qPCR in order to understand the molecular mechanisms of TDCIPP-induced hepatotoxicity. Our results indicated that TDCIPP exposure significantly up-regulated the expression of genes involved in endoplasmic reticulum stress and Toll-like receptor (TLR) pathway, implying an inflammatory response, which was supported by up-regulation of inflammation-related biomaker genes. Hepatic inflammation was further confirmed by histological observation of increase of infiltrated neutrophils and direct observation of liver recruitment of neutrophils labeled with Ds-Red fluorescent protein of Tg(lysC:DsRed) zebrafish upon TDCIPP exposure. To further characterize the hepatotoxicity of TDCIPP, the expression of hepatotoxicity biomarker genes, liver histopathology and morphology were examined. The exposure to TDCIPP significantly up-regulated the expression of several biomarker genes for hepatotoxicity (gck, gsr and nqo1) and caused hepatic vacuolization and apoptosis as well as increase of the liver size. Collectively, our results suggest that exposure to TDCIPP induces hepatic inflammation and leads to hepatotoxicity in zebrafish.