Transcriptional and Physiological Responses Induced by Binary Mixtures of Drospirenone and Progesterone in Zebrafish (Danio rerio) Sara Zucchi, †,∥ Leda Mirbahai, †,∥,⊥ Sara Castiglioni, ‡ and Karl Fent* ,†,§ † University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Grü ndenstrasse 40, CH−4132 Muttenz, Switzerland ‡ IRCCS − Istituto di Ricerche Farmacologiche “Mario Negri”, Environmental Biomarkers Unit, Department of Environmental Health Sciences, Via La Masa 19, I-20156, Milan, Italy § Swiss Federal Institute of Technology (ETH Zü rich), Institute of Biogeochemistry and Pollution Dynamics, Department of Environmental Systems Science, CH−8092 Zü rich, Switzerland * S Supporting Information ABSTRACT: Drospirenone (DRS) is a synthetic progestin increasingly used in oral contraceptives with similar effects to progesterone (P4). Wild fish are exposed to DRS and P4 through wastewater. However, the effects of DRS on fish, both as an individual compound and in mixtures, have not been extensively studied. Therefore, in this study, global gene expression profiles of ovary and brain of female zebrafish (Danio rerio) were characterized after exposure to 55, 553, and 5442 ng/L DRS for 14 days. The effects were then compared to the observed responses after exposure to mixtures of DRS and P4 (DRS+P4: 27 + 0.8, 277 + 8 and 3118 + 123 ng/L). Transcriptomics findings were related to the changes in vitellogenin protein concentrations in the blood, morphology, and histology of gonads. Multivariate analysis indicated tissue-, dose-, and treatment-dependent expression profiles. Genes involved in steroid hormone receptor activity and circadian rhythm were enriched in DRS and mixture groups, among other pathways. In mixtures, the magnitude of response was dose- and transcript-dependent, both at the molecular and physiological levels. Effects of DRS and P4 were additive for most of the investigated parameters and occurred at environmentally relevant concentrations. They may translate to adverse reproductive effects in fish. ■ INTRODUCTION Natural and synthetic steroid hormones are among the most active endocrine disrupters, entering aquatic systems via excretion by humans and livestock. They may adversely affect reproduction of fish at environmental levels. 1,2 Less investigated synthetic progestins (gestagens) used in oral contraceptives and hormone replacement therapies equally display strong hormonal activities in fish. 3 Adverse outcomes in reproduction have been found in fathead minnows (Pimephales promelas) 4 and sticklebacks 5 exposed to levonorgestrel or norethindrone, 6 gestodene, 3 and desogestrel. 3 Adverse effects were also observed in zebrafish exposed to ng/L concentrations of progesterone (P4) 7,8 and the antiprogestin mifepristone. 8,9 To date, knowledge on the modes of action (MOA) of progestins is scarce, and the effects of progestin mixtures are unknown. The consumption of P4 in medicine was 500 kg in Switzerland in 2010, and it is higher than the sum of all synthetic progestins (213 kg) mainly used in contraceptives (www.imshealth.com). Of the synthetic progestins, drospir- enone (DRS) shows the highest consumption of over 100 and 153 kg in Switzerland in 2009 and in the U.K. in 2006, respectively (www.imshealth.com). 10 Residues of P4 and metabolites as well as synthetic progestins originating from sewage are detected at ng/L in aquatic environments. P4 also originates from livestock farms, where up to 350 ng/L P4 was detected in surface- and ground-waters located downstream. 11 Although DRS levels have not been reported in aquatic environments, it is thought that their levels are similar to other steroid hormones in surface water (low ng/L range). An increase in DRS levels is expected due to its increased usage in the new generation of contraceptives (e.g., Yasmin). As a derivative of spironolactone, DRS exhibits antimineralcorticoid (antialdosterone) and slight antiandrogenic activity in mam- mals 12−14 and androgenic activity in recombinant yeast. 3 Furthermore, DRS altered fecundity and gonad histology in fathead minnows at 6.5 and 70 μg/L, 4 but not at 100 ng/L. 3 Received: December 17, 2013 Revised: February 21, 2014 Accepted: February 25, 2014 Article pubs.acs.org/est © XXXX American Chemical Society A dx.doi.org/10.1021/es405584f | Environ. Sci. Technol. XXXX, XXX, XXX−XXX