title: Activity of binary mixtures of drospirenone with progesterone and 17α-ethinylestradiol in vitro and in vivo.
abstract: 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.
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Nadine Madeleine
authors.initials: NM
authors.lastname: Rossier
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Chew
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Environmental Biomarkers Unit, Department of Environmental Health Sciences, Via La Masa 19, I-20156 Milan, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Riva
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland; Swiss Federal Institute of Technology (ETH Zürich), Institute of Biogeochemistry and Pollution Dynamics, Department of Environmental System Sciences, CH-8092 Zürich, Switzerland. Electronic address: karl.fent@fhnw.ch.
authors.forename: Karl
authors.initials: K
authors.lastname: Fent
mesh_terms: D000736:Androstenes / Q000633:toxicity*; D000818:Animals; D047628:Estrogen Receptor alpha / Q000235:genetics; D004997:Ethinyl Estradiol / Q000633:toxicity*; D029941:Fish Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D006801:Humans; D011374:Progesterone / Q000633:toxicity*; D011944:Receptors, Androgen / Q000235:genetics; D011980:Receptors, Progesterone / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity; D015027:Zebrafish / Q000196:embryology / Q000235:genetics
chemical_list: D000736:Androstenes; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D029941:Fish Proteins; D011944:Receptors, Androgen; D011980:Receptors, Progesterone; D014874:Water Pollutants, Chemical; D004997:Ethinyl Estradiol; D011374:Progesterone; C035144:drospirenone
keywords: Ethinylestradiol; Mixture activity; Progestins; Recombinant yeast assays; Steroid mixtures; Transcriptional effects; Zebrafish embryos