| Polybrominated diphenyl ethers (PBDEs) are a new class of persistent organic pollutants that have a potential for thyroid toxicity, reproductive toxicity, and neurotoxicity. Of the various PBDEs,2,2′,4,4′-tetraBDE (BDE-47) is predominantly found in the biota and in human samples. Perchlorates (PER) are another class of persistent inorganic pollutants. Several studies report that PER have a potential to cause thyroid toxicity, reproductive toxicity, and endocrine toxicity following exposure in various organisms. PBDEs and PER are recognized endocrine disruptors that can potentially exist in an aquatic environment. The objective of the present study was to characterize the combined effects of BDE-47and PER on developmental toxicity, to examine the disruptive effects of short-term exposure on the thyroid hormone levels and gene expression in the HPT axis of zebrafish larvae; to investigate joint effects of long-term exposure on the reproduction system in whole life of zebrafish and potential molecular mechanism.The current paper thoroughly examines the combined effects of BDE-47and PER on the development of zebrafish embryos, by conducting the following experiments. Embryos were exposed to (BDE-470.004+PER3.5),(BDE-470.04+PER35), and (BDE-470.4+PER350) mg/L for96h to study acute toxicity; to (BDE-470.001+PER35),(BDE-470.01+PER35),(BDE-470.1+PER35) mg/L groups for14days to study oxidative stress; embryos were exposed to (BDE-470.004+PER3.5),(BDE-470.04+PER35), and (BDE-470.4+PER350) mg/L groups for96h to observe apoptotic effects. For analysis of the combined effects of BDE-47and PER on thyroid toxicity, the studies were conducted in two parts. Embryos were exposed to BDE-47(1μg/L,3μg/L,5μg/L,10μg/L,20μg/L)+3.5mg/L PER for14days for analyzing the effects of PER on BDE-47-induced thyrotoxicity; embryos were exposed to (BDE-470.0002+PER0.175),(BDE-470.002+PER1.75),(BDE-470.02+PER17.5) mg/L groups for the analysis of the interaction between the two substances in thyroid toxicity and the associated molecular mechanisms. Fertilized zebrafish embryos were exposed to mixtures of (BDE-470.001+PER35),(BDE-470.01+PER35),(BDE-470.1+PER35) mg/L for141days to investigate the combined effects of BDE-47and PER on reproductive toxicity and offspring quality. Based on the above analyses, the following conclusions were reached:(1) The96-LC50values of BDE-47and PER were4.18mg/L and3502.78mg/L, respectively. Based on the results of an acute experiment, an equivalent effect acute experiment of the combination of BDE-47and PER was developed. The results showed that the combined effects of developmental toxicity on zebrafish embryos were higher than the effects of the two chemicals individually. The main morphological abnormalities observed following treatment with the mixtures were pericardial edema and the lack of swim bladder. The possible mechanism of malformations was the abnormal hatching induced by retardation of voluntary movement.(2) BDE-47and PER induced oxidative stress in embryos/larvae. PER could exacerbate the induction of ROS after BDE-47treatment, and the levels of ROS increased with an extension of the exposure time. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (Gpx) and exposure concentrations exist in a concentration-and time-dependent manner. The following genes were upregulated after a coexposure to BDE-47and PER: Mn-Sod, Cu/Zn-Sod, Cat and Gpx1a.(3) Staining with acridine orange (AO) indicated that co-exposure to BDE-47and PER induced a dose-dependent apoptosis in the embryos, and a considerable number of apoptotic cells appeared around the spine. The genes p53and Bax were up-regulated and the enzyme activity of Caspase-3and Caspase-9increased following treatment with the BDE-47and PER mixtures. Olive tail moment increased significantly with exposure to increasing concentrations of the mixtures, indicating that PER could enhance the DNA damage-induced by BDE-47.(4) Compared with only BDE-47treatment, we observed that the transcription of CRH, TSHβ, TTR was significantly down-regulated, whereas the transcription of NIS and Nkx2.1a was significantly up-regulated after treatment with PER. PER can strengthen the effects of BDE-47induction on TG and reduction on TTR, both at the transcription and translation levels, thus exacerbating the thyroid interference effects of BDE-47on zebrafish.(5) Co-exposures can significantly decrease the survival rate, induce malforma-tion rate, and inhibit the growth of embryos/larvae. Equivalent effect mixtures displayed a synergistic effect on malformations. Co-exposure to BDE-47and PER significantly reduced T4levels and BDE-47and PER showed a synergistic effect in the (BDE-470.002+PER1.75) and (BDE-470.02+PER17.5) mg/L groups. Co-exposures significantly up-regulated the expression of CRH, TSHβ, NIS, Nkx2.1a, TG, Dio2and down-regulated the expression of TRα and TRβ. BDE-47and PER showed a synergistic effect on the expression of CRH and an antagonistic effect on the expression of TTR.(6) A chronic co-exposure to BDE-47and PER resulted in decreased testoste-rone (T) levels in males and increased estradiol (E2) levels in females. Co-exposure to BDE-47and PER significantly up-regulated the expression of FSHβ in the fish brain, indicating that BDE-47and PER were likely to promote a germ cell formation and growth. In addition, a significant up-regulation in the expression of LHβ was observed, suggesting that BDE-47and PER inhibited sperm maturation. The development of gonads was inhibited and mature sperm and egg cells decreased. A high dose mixture reduced the survival rate, hatching rate, and fertility rate and retarded growth in the offspring, thus impairing reproduction in zebrafish.In summary, the mixture of BDE-47and PER could induce developmental toxicity, thyroid toxicity, and reproductive toxicity in the embryos. The complex mechanisms of interaction between the two contaminants maintain a close relationship with the regulation of hormones and genes at the transcriptional and translational level of regulation. |
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