Activity of binary mixtures of drospirenone with progesterone and 17α-ethinylestradiol in vitro and in vivo.

Despite potential exposure of aquatic organisms to mixtures of steroid hormones, very little is known on their joint activity in fish. Drospirenone (DRS) is a new synthetic progestin used in contraceptive pills in combination with 17α-ethinylestradiol (EE2). Here we systematically analyzed effects of DRS in binary mixtures with progesterone (P4) and EE2. First, we determined the in vitro activity of single compounds in recombinant yeast assays that express the human progesterone, androgen, or estrogen receptor, followed by determination of mixture activities of DRS and P4, DRS and EE2, as well as medroxyprogesterone acetate (MPA) and dydrogesterone (DDG). Mixtures of DRS and P4, as well as of DRS and EE2 showed additive progestogenic and androgenic activities. However, DDG and MPA showed non-additive progestogenic and androgenic activities. We then analyzed the in vivo activity of single compounds and mixtures of DRS and P4, as well as DRS and EE2, by assessing transcriptional changes of up to 14 selected target genes in zebrafish embryos at 48h post fertilization (hpf), and in eleuthero-embryos at 96hpf and 144hpf. DRS, P4, and EE2 led to significant transcriptional alteration of genes, including those encoding hormone receptors (pgr, esr1), a steroidogenic enzyme (hsd17b3), and estrogenic markers (vtg1, cyp19b), in particular at 144 hpf. In general, DRS showed stronger transcriptional changes than P4. In mixtures of DRS and P4, they were mainly non-additive (antagonistic interaction). In mixtures of DRS and EE2, transcriptional responses of esr1, vtg1 and cyp19b were dominated by EE2, suggesting an antagonistic interaction or independent action. Equi-effective mixtures of DRS and EE2, based on progesterone receptor transcripts, showed antagonistic interactions. Our data suggest that interactions in mixtures assessed in vitro in recombinant yeast cannot be translated to the in vivo situation. The receptor-based responses did not correspond well to the transcriptional responses in embryos which are much more complex due to the interplay between hormonal pathways, receptor crosstalk, and hormonal feedback loops.

Benzotriazole UV-stabilizers and benzotriazole: Antiandrogenic activity in vitro and activation of aryl hydrocarbon receptor pathway in zebrafish eleuthero-embryos.

Benzotriazole UV-stabilizers (BUVs) are applied in materials for protection against UV-irradiation. They are widely used, bioaccumulate and share structural similarities to benzotriazole. Benzotriazole (1HBT) finds application as corrosion inhibitor in dishwashing detergents, antifreeze (vehicles) and aircraft de-icing agent. BUVs and 1HBT are persistent and ubiquitous in the aquatic environment, but there is little understanding of the ecotoxicological implications. Here, we comparatively analyze the hormonal activity in vitro and effects in zebrafish eleuthero-embryos in vivo. 2-(2-Hydroxy-5-methylphenyl)benzotriazole (UV-P), 2-(3-t-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole (UV-326), UV-327, UV-328, UV-329 and UV-320 showed no estrogenicity (YES assay) and androgenicity (YAS assay). However, UV-P and 1HBT showed significant antiandrogenic activity. We assessed the transcription profiles of up to 26 genes associated with different toxicological pathways in zebrafish eleuthero-embryos to elucidate potential modes of action of UV-P, UV-326 and 1HBT. Embryos were experimentally exposed for 144hpf to three measured concentrations of 15.8, 70.8, and 690µg/L UV-P, 7.5, 31.7, and 84.3µg/L UV-326 and 7.9, 97.3 and 1197.3µg/L 1HBT. Among the 26 transcripts, the induction of the aryl hydrocarbon receptor (AHR) pathway by UV-P and UV-326 was the most significant finding. UV-P led to dose-related induction of AHR1, ARNT2 and cyp1a1, as well as of phase II enzymes glutathione-S-transferase (gstp1) and ugt1a. UV-326 led to a significant induction of cyp1a1 and AHR2, but down-regulation of gstp1 at 84µg/L. Only little transcriptional alterations occurred in genes related to apoptosis, oxidative stress, hormone receptors, and steroidogenesis including aromatase. 1HBT led to only a few expressional changes at 1197µg/L. Our data lead to the conclusion that UV-P and UV-326 activate the AHR-pathway, whereas 1HBT shows only little transcriptional alterations. It should be noted, however, that effects have been observed at concentration much higher than those occurring in the environment. Forthcoming studies should show whether the observed antiandrogenic activities and transcriptional changes translate into physiological effects .

Accumulation and effects of the UV-filter octocrylene in adult and embryonic zebrafish (Danio rerio).

Wide application of the UV-filter octocrylene (OC) in cosmetics leads to contamination of the aquatic environment, but effects of OC remain unclear. Here we determine bioaccumulation and molecular effects of OC. Adult male zebrafish were exposed to 22, 209 and 383 µg/L and embryos to 69, 293 and 925 µg/L OC. OC accumulated in fish up to 17 µg/g. Calculated BCF varied between 41 and 136. Microarray analysis in brain and liver following exposure to 383 µg/L OC revealed alteration of 628 and 136 transcripts, respectively. Most prominent GO processes included developmental processes, organ development, hematopoiesis, formation of blood vessels, blood circulation, fat cell differentiation and metabolism. Validation by RT-qPCR in brain and liver of adult fish and embryos included a series of genes. Blood levels of 11-ketotestosterone were not altered. The transcriptomics data suggest that OC mainly affects transcription of genes related to developmental processes in the brain and liver as well as metabolic processes in the liver.