Transcriptional effects of androstenedione and 17α-hydroxyprogesterone in zebrafish embryos.

Steroid hormones in the aquatic environment may pose a risk to fish health. Here we evaluated effects of two different class steroids that frequently occur in the aquatic environment, the androgen androstenedione (A4) and the progestin 17α-hydroxyprogesterone (17-OHP4). Zebrafish embryos were exposed to four concentrations of A4 and the positive control testosterone and to 17-OHP4, and transcriptional changes were determined at 96 h post fertilization (hpf) and 120 hpf. Transcriptional changes of 18 selected genes were assessed upon exposure to measured concentrations of 0.004, 0.046, 0.62 and 6.56 µg/L A4. Significant induction of the genes encoding sulfotransferase (sult2st3) and aromatase (cyp19b) occurred in 120 hpf embryos at 6.56 µg/L A4 and 1 µg/L testosterone. Additionally, cyp2k7 was significantly induced in two of three independent experiments. 17-OHP4 did not induce physiological effects (muscle contraction, heart rate, hatching success, swimming activity) at concentrations between 0.01 and 10 µg/L. Of the analyzed 15 genes, slight transcriptional alterations occurred for the genes encoding progesterone receptor, aromatases (cyp19a) and (cyp19b) and cyp2k7 at 10 µg/L. Our study highlights sult2st3, cyp19b and cyp2k7 as potential markers of androgen exposure in fish and indicates that 17-OHP4 is not likely to pose a risk for fish at environmental concentrations.

Reproductive and transcriptional effects of the antiandrogenic progestin chlormadinone acetate in zebrafish (Danio rerio).

Chlormadinone acetate (CMA) is a frequently used progestin with antiandrogenic activity in humans. Residues may enter the aquatic environment but potential adverse effects in fish are unknown. While our previous work focused on effects of CMA in vitro and in zebrafish eleuthero-embryos, the present study reports on reproductive and transcriptional effects in adult female and male zebrafish (Danio rerio). We performed a reproductive study using breeding groups of zebrafish. After 15 days of pre-exposure, we exposed zebrafish to different measured concentrations between 6.4 and 53,745 ng/L CMA for 21 days and counted produced eggs daily to determine fecundity. Additionally, transcriptional effects of CMA in brains, livers, and gonads were analyzed. CMA induced a slight but statistically significant reduction in fecundity at 65 ng/L and 53,745 ng/L compared to pre-exposure. Furthermore, we observed differential expression for gene transcripts of steroid hormone receptors, genes related to the hypothalamic-pituitary-gonadal axis, and steroidogenesis. In particular, we found a significant decrease of transcript levels of vitellogenin (vtg1) in ovaries and liver, and of cyp2k7 in the liver of males, as well as a significant increase of transcripts of the progesterone receptor (pgr) in testes, and cyp2k1 in the liver of females. The observed effects were weaker than those of other very potent progestins, which is probably related to the lack of interaction of CMA with the zebrafish progesterone receptor.

Effects of antiandrogenic progestins, chlormadinone and cyproterone acetate, and the estrogen 17α-ethinylestradiol (EE2), and their mixtures: Transactivation with human and rainbowfish hormone receptors and transcriptional effects in zebrafish (Danio rerio) eleuthero-embryos.

Synthetic progestins act as endocrine disrupters in fish but their risk to the environment is not sufficiently known. Here, we focused on an unexplored antiandrogenic progestin, chlormadinone acetate (CMA), and the antiandrogenic progestin cyproterone acetate (CPA). The aim was to evaluate whether their in vitro interaction with human and rainbowfish (Melanotaenia fluviatilis) sex hormone receptors is similar. Furthermore, we investigated their activity in zebrafish (Danio rerio) eleuthero-embryos. First, we studied agonistic and antagonistic activities of CMA, CPA, and 17α-ethinylestradiol (EE2), in recombinant yeast expressing either the human progesterone (PGR), androgen (AR), or estrogen receptor. The same compounds were also investigated in vitro in a stable transfection cell system expressing rainbowfish nuclear steroid receptors. For human receptors, both progestins exhibited progestogenic, androgenic and antiestrogenic activity with no antiandrogenic or estrogenic activity. In contrast, interactions with rainbowfish receptors showed no progestogenic, but antiandrogenic, antiglucocorticoid, and some antiestrogenic activity. Thus, interaction with and transactivation of human and rainbowfish PGR and AR were distinctly different. Second, we analyzed transcriptional alterations in zebrafish eleuthero-embryos at 96 and 144h post fertilization after exposure to CPA, CMA, EE2, and binary mixtures of CMA and CPA with EE2, mimicking the use in oral contraceptives. CMA led to slight down-regulation of the ar transcript, while CPA down-regulated ar and pgr transcripts. EE2 exposure resulted in significant transcriptional alterations of several genes, including esr1, pgr, vtg1, cyp19b, and gonadotropins (fshb, lhb). The mixture activity of CMA and EE2 followed the independent action model, while CPA and EE2 mixtures showed additive action in transcriptional alterations. Third, we analyzed the interactions of binary mixtures of CMA and CPA, and of CMA and EE2 for their joint activity in vitro and in eleuthero-embryos. Both mixtures behaved according to the concentration addition model in their in vitro interaction with human and rainbowfish receptors, often showing antagonism. In zebrafish eleuthero-embryos, binary mixtures of CMA and EE2 showed the same expression patterns as EE2 alone, indicating an independent action in vivo. Our study demonstrates that CMA and CPA interact distinctly with human and rainbowfish receptors, suggesting that activities of these and possibly additional environmental steroids determined with yeast expressing human receptors cannot simply be translated to fish. The lack of agonistic activities of both progestins to rainbowfish PGR and AR is the probable reason for the low activity found in zebrafish eleuthero-embryos.