Article pubs.acs.org/est
Synthetic Progestins Medroxyprogesterone Acetate and Dydrogesterone and Their Binary Mixtures Adversely Affect Reproduction and Lead to Histological and Transcriptional Alterations in Zebrafish (Danio rerio) Yanbin Zhao,† Sara Castiglioni,‡ and Karl Fent*,†,§ †
University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 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 Zürich), Institute of Biogeochemistry and Pollution Dynamics, Department of Environmental System Sciences, CH−8092 Zürich, Switzerland S Supporting Information *
ABSTRACT: Medroxyprogesterone acetate (MPA) and dydrogesterone (DDG) are synthetic progestins widely used in human and veterinary medicine. Although aquatic organisms are exposed to them through wastewater and animal farm runoff, very little is known about their effects in the environment. Here we provide a comprehensive analysis of the responses of zebrafish (Danio rerio) to MPA, DDG, and their binary mixtures at measured concentrations between 4.5 and 1663 ng/L. DDG and both mixtures impaired reproductive capacities (egg production) of breeding pairs and led to histological alterations of ovaries and testes and increased gonadosomatic index. Transcriptional analysis of up to 28 genes belonging to different pathways demonstrated alterations in steroid hormone receptors, steroidogenesis enzymes, and specifically, the circadian rhythm genes, in different organs of adult zebrafish and eleuthero-embryos. Alterations occurred even at environmentally relevant concentrations of 4.5−4.8 ng/L MPA, DDG and the mixture in eleuthero-embryos and at 43−89 ng/L in adult zebrafish. Additionally, the mixtures displayed additive effects in most but not all parameters in adults and eleuthero-embryos, suggesting concentration addition. Our data suggest that MPA and DDG and their mixtures induce multiple transcriptional responses at environmentally relevant concentrations and adverse effects on reproduction and gonad histology at higher levels.
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reproduction of fish at environmental and higher concentrations5−9 and lead to endocrine effects.10−13 Progesterone was the most widely consumed progestins for medical applications with annual consumption of about 500 kg in Switzerland in 2010, followed by several synthetic progesterone analogs, such as drospirenone (90 kg), dydrogesterone (DDG; 34 kg), medroxyprogesterone acetate (MPA; 25 kg), norethindrone (17 kg) and levonorgestrel (3 kg) (www.imshealth.com). In U.K., MPA showed highest prescription (530 kg) of all progestins, followed by norethindrone (440 kg) and DDG (209 kg).2 Progesterone and synthetic progestins mediate their activities mainly through the nuclear and membrane progesterone receptors. Progesterone has been proven to be involved in many processes
INTRODUCTION
Numerous pharmaceuticals used in human and veterinary medicine enter the aquatic environment via wastewater effluents and agriculture run offs. They may pose hazards on aquatic organisms. Natural and synthetic steroid hormones are among the most active endocrine disruptive compounds and therefore of ecotoxicological concern.1,2 Besides rather wellstudied estrogens, progestins (also called progestogens or gestagens) came into increasing attention in the past few years. Progesterone and synthetic progestins are widely used for contraception and in hormone replacement therapy, prevention of preterm labor and in different hormone-related diseases (e.g., endometriosis). They show higher prescription than (synthetic) estrogens in Switzerland and in the U.K., where in 2006, in total 1700 kg were prescribed compared to 500 kg estrogens.2 Progestins show incomplete elimination in wastewater treatment plants and occur in surface and ground waters in the range of ng/L.3,4 Recent studies demonstrated that progesterone and synthetic progestins adversely affect fecundity and © 2015 American Chemical Society
Received: Revised: Accepted: Published: 4636
November 14, 2014 February 26, 2015 March 5, 2015 March 5, 2015 DOI: 10.1021/es505575v Environ. Sci. Technol. 2015, 49, 4636−4645
Article
Environmental Science & Technology
histological and transcriptional responses of zebrafish (Danio rerio) to MPA and DDG as single compounds and as binary mixtures. Thereby we tested the hypothesis that the combined action of both progestins is additive. By a systematic analysis of their effects in adult zebrafish as well as their transcriptional changes in different tissues of adults and in embryos, we contribute to the environmental hazard and risk assessment of these progestins.
including meiotic oocyte maturation, secretory activity of the endometrium, ovulation, spermatogenesis, sperm motility, and acrosome reaction.14,15 Therefore, the widespread occurrences of natural and synthetic progestins in aquatic ecosystems raise concerns about their potential hormonal activities and endocrine disruptions to aquatic organisms, in particular to fish. Recent studies have demonstrated such adverse effects in fathead minnows,6,9 zebrafish,10−13 medaka,7 three-spined stickleback8 and roach.16 For instance, the reduced fecundity and adverse effects on reproduction have been reported for fathead minnows after exposure to several synthetic progestins, including norethindrone and gestodene at concentrations as low as 1 ng/L7,9 and to levonorgestrel at 2 ng/L.6 Desogestrel reduced fecundity at 1000 ng/L9 and drospirenone at 6500 ng/L and higher.6 Additionally, we demonstrated that low ng/L levels of progesterone, norethindrone and levonorgestrel could alter the expression levels of genes involved in hormone signaling and steroidogenesis in zebrafish eleutheroembryos.10 In addition to the reproductive and physiological effects, transcriptomics revealed more responses to progestin exposures, including the transcriptional alteration of genes involved in circadian rhythm pathway and cell cycle among others.11,13 Currently, there is still limited knowledge about potential effects of other frequently used progestins. MPA and DDG are among the mostly prescribed synthetic progestins in Switzerland and the U.K. and probably other countries. They are used in contraception and in hormone replacement therapy, often in combination with an estrogen. DDG is also used for therapy of menstrual disorders and endometriosis.17 Despite their frequent use, information about their environmental occurrence is very limited and their ecotoxicological implications are unknown. Residues of MPA were detected up to 15 ng/L in municipal wastewater effluents and at 1 ng/L in surface waters in the U.S.,18 up to 17 ng/L in wastewater effluents and 34 ng/L in rivers in Beijing, China.19 Similarly, DDG was detected at 35 ng/L in municipal wastewater influent and 10 ng/L in rivers in Xintang, China.20 In swine farm flush water samples, MPA and DDG levels can reach 330 and 2188 ng/L, respectively.20 In general, little is known about the bioactivity of MPA and DDG. Both compounds bind to the human progesterone receptor, although at different affinities. Several human nuclear receptor cell-based in vitro studies demonstrated the androgenic (230 times lower than methyltrienolone) and glucocorticoid activities (110 times lower than dexamethasone) of MPA, while almost no such activities (70%) and pH value (6.7−7.2) were continuously measured to ensure water quality. The light: dark photoperiod was 14:10 h. After an acclimatization of 4 days (including spawning trays in tanks), the experiment started with a pre-exposure period of 14 days to establish the baseline rate of fecundity for each tank (and spawning group), followed by 1 day of equilibration when chemical-dosing started, and finally 21 days of exposure. During the whole exposure period, appearance, mortality and abnormal behavior of fish were recorded daily, and fish were fed twice daily with a combination of frozen brine shrimps (A. salina), white mosquito larvae and D. magna. Eggs were collected during the whole experimental period. Each breeding tank was equipped with a spawning tray of stainless steel placed on the bottom of the aquaria, which was covered by a fine net with an appropriate mesh size for eggs to fall through. Each morning, at about 1.5 h after the beginning of the light period, eggs were collected carefully and transferred to Petri dishes for counting. The study was conducted based on OECD Guideline 229/230. At the end of the exposure, fish were anesthetized by the KoiMed Sleep (1.5 mL/L water). Before blood collection, all fish were measured for wet weight (g) and length (mm), which was used to calculate the condition factor. Four fish from each of the four replicates were dissected immediately. Brain 4637
DOI: 10.1021/es505575v Environ. Sci. Technol. 2015, 49, 4636−4645
Article
Environmental Science & Technology
Sex Hormone Analysis. Blood concentrations of 17βestradiol (17β-E2) in adult females and 11-ketotestosterone (11-KT) in adult males were measured using the commercial hormone detection kits (Cayman Chemical Company, Ann Arbor, MI) (detection limits: 19 pg/mL for 17β-E2; 1.3 pg/mL for 11-KT), following the manufacturer’s instructions. All blood samples were analyzed in duplication and reassayed if the coefficient of variation exceeded 20%. RNA Isolation and RT-PCR Analysis. RNA isolation, firststrand cDNA synthesis and the relative quantitation in real time RT-PCR were performed according to methods described previously10,11 with slight modifications. The details are provided in the SI. For RT-PCR analyses, five biological replicates were analyzed in case of eleuthero-embryos. Four and eight biological replicates were analyzed for adult fish brain, liver and testis and adult fish ovary, respectively. Each biological replicate was analyzed with two technical replicates. Melting curves were analyzed to ensure that only a single product was amplified. Primer details are presented in the SI (Table S6), and the efficiencies were calculated to ensure no significant change between the primer efficiencies of the target genes and the reference gene, RpL13a. RpL13a is ribosomal protein gene, which showed high gene expression stabilities in zebrafish embryos and adults under the different treatments of progestins and the different zebrafish tissues in our previous studies. It was verified and employed as the housekeeping gene for normalization for several progestins and antiprogestin, including progesterone, norethindrone, levonorgestrel, drospirenone, and mifepristone.10−13 In the present study, the stabilities of RpL13a gene expressions were also demonstrated; they displayed very little variation in different treatments and tissue categories (SI Figure S1). Threshold cycle (CT) values were recorded in the linear phase of amplification and the data were analyzed using the delta−delta CT method of relative quantification.23 Investigation of Mixture Effects. Additive effects in the mixtures were investigated by following the same concept as with mixtures of P4 and drospirenone.13 Reproductive and selected transcriptional responses (nr1d1, nr1d2b, per1b, cry5, cdc20, cyp11b, hsd11b, and cyp17) were investigated. In brief, the measured response was plotted against the concentrations of individual compounds. A logarithmic curve was fitted to the data, and the response for the concentrations used in individual exposures was predicted based on the equation. The difference between the observed and predicted responses for individual compounds was quantified and used as margin of error (MOE, the difference between observed and predicted response) for the equations. The expected response was predicted for the concentrations used in the mixture studies ± MOE. The expected additive effect (A+B) was calculated based on the predicted response and compared to the observed response (described in detail in SI Tables S7, S8). Data Analysis and Statistics. Hierarchical clustering (HAC) maps were constructed using the MultiExperimental Viewer v4.9 (http://www.tm4.org/mev.html; Dana-Farber Cancer Institute, Boston, MA, USA). Data from the gene expressions were illustrated graphically with GraphPad Prism 5 (GraphPad Software, San Diego, CA). The significance of differences between the solvent control and progestin exposed zebrafish (adults and embryos) in transcript levels (determined by RT-qPCR), egg production, condition factors, GSI, blood content of 17β-E2 and 11-KT, and ovaries and testis stages (histology) were analyzed by one-way analysis of variance
(including pituitary gland), liver and testis of four males were pooled, respectively, transferred to RNAlater and stored at −80 °C for subsequent RNA extraction. Ovaries were collected from individual females and weighed in order to assess the gonadosomatic index (GSI = gonad weight (g)/body weight (g) × 100). For RNA analysis, ovaries were collected individually, whereas brains and livers of females were pooled for each replicate (n = 4). Pooling was necessary due to the small sample sizes and varying extraction efficiencies and to control for interindividual variability. Blood was taken and pooled from four of the dissected fish in each replicate using heparinized needles. After centrifugation, the blood plasma was stored at −80 °C for hormone analysis. One female fish and one male fish per replicate (total of four fish per treatment) were fixed in Bouin’s after opening of the abdominal area for histological examination. Embryo Exposure. At 2−4 h post fertilization (hpf), 120 blastula-stage embryos per replicate were randomly placed in 150 mL covered glass beakers containing 100 mL of reconstituted water at 27 ± 1 °C with the appropriate concentration of MPA and DDG. Exposure experiments were performed using a semistatic procedure as previously described for several progestins and RU486.10 The present experiment included five replicates for MPA and DDG at nominal concentration of 5, 50, and 500 ng/L each, as well as their mixtures (5 + 5, 50 + 50, and 500 + 500 ng/L). Included were also five replicates of water control and solvent control containing 0.01% DMSO. The concentrations of MPA and DDG were selected based on the transcriptional responses of progestins (2−200 ng/L) in zebrafish eleuthero-embryos in our previous study10 and the relative steroid hormone receptor binding activities in vitro.17,21 The lowest concentration was environmentally realistic. Every 24 h, lethal and sublethal effects were evaluated, and dead embryos removed. Water was completely exchanged every 24 h by transferring embryos to new beakers containing appropriate compound concentrations. At 48, 96, and 144 hpf, 25 embryos or eleuthero-embryos, respectively, were pooled and stored in RNA later for further molecular analysis. Chemical Analysis. The analytical methods described previously10,11 were adapted for the determination of MPA and DDG concentrations in exposure waters. The analysis was performed using solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (HPLC-MS-MS), and the recoveries for MPA and DDG were 101.5 ± 5.1% and 96.7 ± 4.9%, respectively. The limits of quantification of the method were calculated directly from samples as the values giving a signal-to-noise ratio (S/N) of 10 and were 0.28 and 1.3 ng/L for MPA and DDG, respectively. Medroxyprogesterone (MP) was used as internal standard (IS). Detailed information about the analytical procedures used for chemical analysis is provided in the SI. Histology. After anesthesia, one male and one female fish per replicate tank (total of n = 4 per treatment) were randomly taken, opened at the abdominal site and fixed in Bouin’s solution for about 24 h. Slices were prepared and histological analysis were performed as previously described.22 Three cross sections were taken from different tissue levels along the gonadaxis from each fish. Subsequently, two sections (out of three) from different regions of the ovary and testis, respectively, were examined for each individual fish. The staging and histological alterations were evaluated according to guidelines (OECD, 2010; U.S. EPA, 2007) and our previous studies.12,13 4638
DOI: 10.1021/es505575v Environ. Sci. Technol. 2015, 49, 4636−4645
Article
Environmental Science & Technology
Figure 1. Reproductive and histological effects in zebrafish after exposure to MPA, DDG and their binary mixtures. (A) Cumulative mean number of eggs per female per day after 2 weeks pre-exposure (left) followed by 3 weeks exposure (right) to solvent control (0.01% DMSO), and different concentrations of MPA, DDG and their mixtures. Each curve represents the mean values of four replicate tanks per day and treatment. (B) Average number of eggs per female per day during the last week of exposure (day 15−21). Each bar represents the mean value ± SD of four replicates per treatment. *p-value < 0.05, **p-value < 0.01, and ***p-value
