Endocrine disrupting effects of waterborne PBDE on the brain-pituitary-gonad axis and reproduction of zebrafish (Danio rerio)

Abstract

Polyborminated diphenyl ethers (PBDEs) are found ubiquitously in the environment and biota. Since their molecular structure and physicochemical properties are similar to those of polychlorinated biphenyl (PCBs) and dioxins, there is increasing concern that PBDEs may disrupt endocrine systems, leading to reproductive impairments in aquatic animals. In this thesis, a commercial low brominated PBDEs mixture, DE-71 was selected as a model chemical, and zebrafish (Danio rerio) employed as a model species, to test the hypothesis that PBDE may affect fish reproduction by affecting multiple target sites along the brain-pituitary-gonadal axis. Zebrafish were exposed to 5 ng/L, 1 µg/L and 50 µg/L of DE-71 for the whole life cycle (120 days, from eggs to adults), and experiments were carried out to investigate the effects of PBDE on: (a) gonadal development and reproductive processes; (b) level and balance of sex steroids, as well as the various key enzymes regulating steroidogenesis and/or sex steroid hormone receptors, and (c) the gene expression controlling the hypothalamic and pituitary hormones and their receptors. A significantly higher percentage of malformation was found in embryos when exposed to the 1 µg/L (9.1%, p <0.05) and 50 µg/L (11.7%, p <0.01) of DE-71 at 96 hpf. At the same time, estradiol (E2) level in embryos was reduced at 48 hpf and 120 hpf (up to -48%, p <0.001). Effects of DE-71 on testosterone (T) level were variable: the level of T increased at 48 hpf (+26%, p <0.05), while decreased at 120 hpf (at least -42%, p <0.05) and 12 dpf (up to -70%, p <0.01). The level of 11-ketotestosterone (11KT) was decreased at all three time-points (at least -27% and up to - 62%, p <0.01) upon to exposure of DE-71. The expression of selected steroidogenic genes was also significantly affected at these key time-points even at the lowest DE-71 treatment (5 ng/L). At 48 hpf, expression of StAR was down-regulated (by 0.4-fold, p <0.05) in the 1 µg/L treatment, but up-regulated (by 3.0-fold, p <0.001) in the 50 µg/L treatment group. Exposure to 5 ng/L and 1 µg/L DE-71 at 48 hpf upregulated expression of CYP11a by 13.6-fold (p <0.01) and 18.2-fold (p <0.001), respectively. Expression of CYP19a was down-regulated by 0.4-fold in the 5 ng/L (p <0.05) and 0.5-fold in the 50 µg/L (p <0.05) treatment group. Growth, however, was not affected in any treatment. At 60 dpf, E2 level of males significantly increased in the 1 µg/L (+73%, p <0.001) and 50 µg/L (+58%, p <0.01) treatment groups. In females, E2 level increased by 32% in the 1 µg/L treatment group (p<0.05) and by 45% in the 50 µg/L treatment group (p<0.05). T level of male fish was increased significantly in the 1 µg/L group (+80%, p <0.05) and 11KT level was greatly increased by 375% in the minimum exposure of 5 ng/L group (p <0.05). In female fish, T level increased only in the 50 µg/L (+61%, p <0.05) treatment group, and 11KT level was decreased by 36% only in the 5 ng/L (p <0.05) treatment group. Correspondingly, up-regulation (3-3.7 fold) of GnRH-2 was observed in both males and females in the minimum 5 ng/L treatment (p <0.001). FSH-R in males was upregulated (+1.9-fold) upon exposure to the 5 ng/L DE-71 (p <0.001), but downregulated in the 1 µg/L and 50 µg/L treatments (p <0.01). Only the 50 µg/L treatment led to 2-fold (p <0.01) of up-regulation of LH-R. At 60dpf in testis, the lowest treatment 5 ng/L DE-71 stimulated the expression of StAR (1.5-fold, p <0.05), while the highest 50 µg/L treatment depressed it (0.5-fold, p <0.05). The expression of 3βHSD in male fish was down-regulated in all treatments (0.2-0.4-fold, p <0.001). CYP19a was up-regulated by 1.7-fold (p <0.05) in the 5 ng/L treatment group and down-regulated by 0.4-fold (p <0.05) in the 1 µg/L treatment group. There was no significant difference in expression of CYP11a in all PBDE treatments. In females, all selected genes were down-regulated at 60 dpf. FSH-R and LH-R were down-regulated at higher concentrations of DE-71 (FSH-R: 0.4-0.5 fold, p <0.05; LH-R: 0.6-fold, p <0.05). The expression of 3βHSD in female fish was down-regulated by 0.5-fold in both the 5 ng/L (p <0.05) and 1 µg/L (p <0.05) treatment groups. Ovarian CYP19a was down-regulated by 0.6-fold (p <0.05) only in the 1 µg/L treatment group. At 120 dpf, the level of E2 in both male and female fish was reduced upon exposure to DE-71 (at least -30%, p <0.05). In male fish, both T and 11KT levels increased significantly in the 1µg/L (at least +80%, p <0.05) and 50 µg/L treatment groups (up to +150%, p <0.001). 11KT level was increased greatly by 87% even in the minimum exposure of 5 ng/L group (p <0.01). In female fish, T and 11KT levels remained unchanged in all treatment groups, except a 24% decrease in 11KT level in the 5 ng/L (p <0.05) treatment at 120dpf. At 120 dpf, the mRNA level of GnRH-2 was up-regulated in both male and female fish in the minimum 5 ng/L DE-71 treatment: 5.6-fold in males (p <0.001) and 4.7- fold (p <0.001) in females. Both FSHβ and LHβ in both males and females were upregulated in all concentrations after 120 days. In males, the 1 µg/L DE-71 exposure led to 1.9-fold up-regulation in FSHβ (p <0.01) and LHβ was up-regulated by 2.9- fold in the 1 µg/L (p <0.01) and 2.5-fold in the 50 µg/L (p <0.01) treatments. In females, the 5 ng/L exposure caused 2.2-fold up-regulation in FSHβ (p <0.01), while LHβ was up-regulated by 5-fold in the 5 ng/L (p <0.001) and 2.5-fold in the 50 µg/L (p <0.01) treatment group. This study provides the first evidence that whole life cycle exposure to PBDE (DE- 71) increased the transcripts of brain GnRH-2 and pituitary FSHβ and LHβ in both male and female zebrafish. At 120 dpf the expression of TH and TPH of female were more variable than male, they both were depressed by PBDE treatments, especially at the highest concentration of 50 µg/L. At 120 dpf, no significant difference was observed in StAR among all DE-71 treatments for both male and female fish. Only in males was CYP11a downregulated by all three DE-71 treatments at 120 dpf. In male fish, the expression of 3βHSD was down-regulated by 0.3-fold in the 50 µg/L (p <0.01) treatment group at 120 dpf, but 2.4-fold up-regulation (p <0.01) was found in ovary in the same treatment. The only up-regulated effect was found in testicular CYP19a expression in the 50 µg/L (1.9-fold, p <0.05) treatment group. Our study demonstrated, for the first time, the disruption of low brominated PBDEs on fish steroidogenesis in vivo after long-term exposure. The effects on the expression of key genes and hence sex hormone levels eventually led to the impairment of gonadal development and reproductive processes. At 120 dpf, the observed disruption of sex hormones was associated with a reduction in spawning, fertilization and hatching success when fish were exposed to environmentally realistic concentrations of DE-71. Exposure to 5 ng/L DE-71 reduced egg production by 60% (p <0.05), while exposure to 50 µg/L increased it by 40% (p <0.05). Significantly lower fertilization was also found in the 5 ng/L DE-71 group (-47%, p <0.05). Only 22% and 50% of fertilized eggs survived after 120 hpf (p <0.05), following exposure to 5 ng/L and 50 µg/L respectively. Interestingly, the percentage of males in the F1 generation significantly increased when exposed to the 1 µg/L and 50 µg/L of DE-71 (by 68% and 69%, respectively; p <0.05). Taken together, an increase in percentage of malformation, a lower production of eggs, fertilization success, larval survival rate and a male-biased F1 generation, suggested that low brominated PBDEs may pose a significant threat to natural fish populations. Since hormonal regulation along the BPG axis is highly conserved in vertebrates, the effects observed in zebrafish may also occur in other vertebrates.