The Synthetic Progestin Levonorgestrel Is a Potent Androgen in the

Article pubs.acs.org/est

The Synthetic Progestin Levonorgestrel Is a Potent Androgen in the Three-Spined Stickleback (Gasterosteus aculeatus) Johan Svensson,†,* Jerker Fick,‡ Ingvar Brandt,† and Björn Brunström† †

Department of Environmental Toxicology, Uppsala University, Norbyvägen 18A, SE-75 236 Uppsala, Sweden Department of Chemistry, Umeå University, Linneaus väg 6, SE-90 187, Umeå, Sweden

‡

S Supporting Information *

ABSTRACT: The use of progestins has resulted in contamination of aquatic environments and some progestins have in experimental studies been shown to impair reproduction in fish and amphibians at low ng L−1 concentrations. The mechanisms underlying their reproductive toxicity are largely unknown. Some progestins, such as levonorgestrel (LNG), exert androgenic effects in mammals by activating the androgen receptor (AR). Male three-spined stickleback (Gasterosteus aculeatus) kidneys produce spiggin, a gluelike glycoprotein used in nest building, and its production is directly governed by androgens. Spiggin is normally absent in females but its production in female kidneys can be induced by AR agonists. Spiggin serves as the best known biomarker for androgens in fish. We exposed adult female sticklebacks to LNG at 5.5, 40, and 358 ng L−1 for 21 days. Androgenic effects were found at LNG concentrations ≥40 ng L−1 including induction of spiggin transcription, kidney hypertrophy, and suppressed liver vitellogenin transcription. These are the first in vivo quantitative data showing that LNG is a potent androgen in fish supporting the contention that androgenic effects of certain progestins contribute to their reproductive toxicity.

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INTRODUCTION Active pharmaceutical ingredients (APIs) have become worldwide aquatic contaminants. Extensive use and poor removal by sewage treatment plants (STPs) have led to heavy input of APIs into the aquatic environments. Compounds from human and veterinary medicine have been detected in STP effluents, surface and ground waters, sediments, and biota.1−9 Although APIs are detected in relatively low concentrations (frequently ng L−1 in surface water up to μg L−1 in STP effluent), their generally high potency and specificity for biological targets, many of which are highly conserved between species, have raised concern about potential adverse effects on aquatic organisms.10,11 Research on the effects of APIs in the environment has hitherto focused chiefly on synthetic steroidal estrogens like 17α-ethinylestradiol (EE2), which has been shown in numerous studies to impair reproduction in aquatic organisms, even at subng L−1 levels.12 Far less attention has been paid to progestins; steroidal hormones analogous to progesterone (P4), used mainly in contraception and hormone replacement therapy.13 In contraception, progestins are used singly or in combination with an estrogen. The progestins are however chiefly responsible for the contraceptive effect (inhibition of ovulation and production of a cervical mucus plug), whereas the estrogen mainly secures bleeding regularity.14 All progestins activate progesterone receptors (PRs) but, depending on the © 2013 American Chemical Society

parent compound from which they are derived (P4, testosterone or spironolactone), many also interact with other hormone receptors and exert different combinations of progestagenic, antigonadotropic, (anti)androgenic, (anti)estrogenic, glucocorticoidogenic, and antimineralocorticoidogenic effects.13,15,16 Progestins have been detected in surface waters at concentrations ranging from 3 to 199 ng L−1.3,4,7,8,17,18 It was recently shown that the commonly used progestin levonorgestrel (LNG) has a high potential for bioaccumulation in fish to the extent that a water concentration of 1 ng L−1 gave rise to a fish plasma concentration exceeding the human therapeutic plasma concentration.9 Recent studies have shown that low ng L−1 concentrations of progestins can inhibit reproduction in fish and amphibians,19−25 but the underlying mechanisms are not fully understood. It appears from fish studies that the androgenic progestins LNG and norethindrone (NET) affect reproduction at lower concentrations (0.8 and 1.2 ng L−1, respectively) than the nonandrogenic P4 and drospirenone (DRSP) (100 and 6500 ng L−1, respectively). As could be expected, LNG and NET also caused masculinization of Received: Revised: Accepted: Published: 2043

October 22, 2012 January 29, 2013 January 30, 2013 January 30, 2013 dx.doi.org/10.1021/es304305k | Environ. Sci. Technol. 2013, 47, 2043−2051

Environmental Science & Technology

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females.19,20,25 Consequently, there are indications that the reproductive toxicity of progestins may partly be mediated by activation of the androgen receptor (AR). To determine the androgenicity of LNG in fish, we studied effects on the three-spined stickleback (Gasterosteus aculeatus), a species uniquely responsive to androgens. Three-spined sticklebacks produce spiggin, and spiggin serves as the only known molecular biomarker for androgen exposure. This 203 kDa glycoprotein is produced in the kidney of males during spawning season and is used as an adhesive in nest-building.26 Spiggin production is accompanied by pronounced kidney hypertrophy and is directly governed by androgens, mainly 11ketotestosterone (11-KT), which shows a peak in plasma levels during the spawning season.26−29 Spiggin production is absent in female sticklebacks under normal conditions but is readily induced by exposure to exogenous androgens, serving as the androgenic equivalent to the estrogenic biomarker vitellogenin. The spiggin protein can be quantified with ELISA,30−34 and the transcript with quantitative real-time PCR (qPCR).35 LNG is both a strong PR agonist and a moderate AR agonist having binding affinities for human PR and AR of 323 and 58% of those of P4 and testosterone, respectively.36 We chose LNG as test compound due to its widespread use, frequent environmental detection,4,6−9 strong bioconcentration,9 and reprotoxic effects.19 Utilizing the unique androgen-specific biomarkers of the three-spined stickleback, the present study reports the first in vivo quantitative determination of the androgenic potency of LNG in fish.

be due to the chemical exposure only and not cues from the physical environment. Six female fish were randomly allocated to each aquarium. Female sex identification was based on the absence of erythrophores in the mouth. The fish were fed frozen bloodworms every other day, and feces and other debris were removed daily. Exposure renewal was conducted each day, concurrently with the exchange of 6 L of water. The new water was a priori dosed with 60 μL of LNG solution or for the vehicle control aquaria with 60 μL of acetone. The acetone concentration in the aquaria therefore never exceeded 0.001%, which is below the maximum level (0.002%) recommended for long-term reproductive studies.38 Water quality was monitored in all aquaria at least twice each week. Mean ± SD values measured were: temperature, 14.7 ± 0.4 °C; pH, 7.8 ± 0.2; dissolved oxygen, 87.5 ± 4.5% of air saturation value (ASV); conductivity, 509.2 ± 9.6 μS. Triplicate water samples for chemical analysis were taken in each aquarium approximately 1.5 h after water exchange on day 1, 7, 14, and 21 of exposure. No mortalities or signs of abnormal appearance or behavior were recorded throughout the exposure period. The study was approved by the Uppsala Local Ethics Committee for Research on Animals. At the end of the exposure period, all fish were killed by decapitation and weighed. Ovaries, liver, and kidney were excised and weighed to the nearest milligram. Relative organ weights were expressed as ovary somatic, liver somatic, and kidney somatic indices (OSI, LSI and KSI, respectively) calculated as follows: (the organ weight/body weight) × 100. Female sex was confirmed by visual inspection of the gonads. One fish in the 100 ng L−1 group was identified as a male and was excluded from subsequent analyses. Kidney samples from 8 fish and liver samples from all 12 fish from each exposure group were snap frozen in liquid nitrogen and stored at −80 °C until further processing. The remaining 4 kidneys from each group were fixed in phosphate buffered formaldehyde (4%, HistoLab). Kidney Histology and Image Analysis. After fixation, whole kidneys were dehydrated in increasing concentrations of ethanol and embedded in hydroxyethyl methacrylate (Leica Historesin, Heidelberg, Germany). Nine transverse sections (2 μm) were cut from the middle part of each kidney, with 40 μm between sections. Six of these were stained with hematoxylineosin. Histological measurement of the height of the epithelium lining the secondary proximal tubules (KEH) was made under a light microscope with an attached image analyzer. The height was measured only in circularly shaped tubule sections with a well-developed epithelium and a small but distinct lumen. The measurement was carried out using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Three sections were examined for each fish, and three tubules were measured in each section. The KEH was measured four times in each tubule, once in each quarter. Three sections from each fish were also stained with periodic acidSchiff (PAS), which highlights carbohydrate-rich macromolecules such as the glycoprotein spiggin. All histological evaluation was made using coded slides. RNA Isolation and Reverse Transcription. Total RNA was isolated from kidney and liver samples using the Aurum Total RNA Fatty and Fibrous Tissue kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as described by the manufacturer. RNA purity and quantity were determined spectrophotometrically (260/280 and 260/230 nm ratios 2 or above for all samples) using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). RNA quality was verified by

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MATERIALS AND METHODS Animals and Test Chemical. Three-spined sticklebacks were caught in Ö resund on the west coast of Sweden in December 2009 and were brought to the aquarium facility at the Evolutionary Biology Centre, Uppsala University. The fish were held in Uppsala tap water in flow-through holding tanks and were fed a mixture of frozen bloodworms (Chironomus; Ruto B.V., Zevenhuizen, The Netherlands) and dried flake food (Aller Aqua, Christiansfeld, Denmark) every two days. To maintain reproductive quiescence, the water temperature was 10 °C and the photoperiod 8:16 h light/dark.37 The fish were maintained under these conditions until the start of the experiment in May 2011. LNG (CAS: 797−63−7, HPLC grade, >98% purity) was obtained from Sigma-Aldrich (Steinheim, Germany). Stock solutions were prepared in acetone. LNG Exposure and Tissue Sampling. A total of 48 adult female three-spined sticklebacks were exposed to either acetone (vehicle control) or to LNG at nominal concentrations of 10, 100, or 1000 ng L−1 for 21 days in a semistatic system. All treatments were performed in duplicates. The exposure concentrations were chosen based on the results from a pilot study and measured environmental concentrations. Acetone was chosen as vehicle since no adverse effects have been shown using water concentrations up to 0.2% in long-term studies in fish.38 Eight 10 L plastic aquaria (Ferplast, Vicenza, Italy) were used as test vessels. The aquaria were filled with 8 L of Uppsala tap water, which was constantly aerated using air stones. Each aquarium was randomly assigned one of the four exposure concentrations. Water temperature was kept at 15 °C and the light regimen was 8:16 h light/dark. Keeping the photoperiod and temperature at levels which inhibit the reproductive system was to make sure that any change in reproductive status would 2044

dx.doi.org/10.1021/es304305k | Environ. Sci. Technol. 2013, 47, 2043−2051


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including chemicals used, chromatographic details and selected reaction monitoring (SRM) transitions are available in the Supporting Information. Data Analysis and Statistics. All graphical illustrations and statistical analyses were made using GraphPad Prism version 5.01 (GraphPad Software Inc., CA, USA). Normality and homogeneity of variance were tested for using ShapiroWilk and Bartlett’s tests, respectively. Data were log transformed if needed to meet the assumptions of parametric statistics. Differences in organosomatic indices, KEH and relative mRNA transcription were analyzed using one-way ANOVA followed by post hoc step-down t tests with Bonferroni-Holm correction to compare exposure group means to the vehicle control.43 Results are presented as mean ± SEM.

agarose gel electrophoresis. The RNA isolates were stored at −80 °C until further processing. Synthesis of cDNA was performed from 900 ng RNA of each sample using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Prior to qPCR each cDNA sample was diluted 10-fold with nucleasefree water. Quantitative Real-Time PCR. The qPCR analysis was conducted on a Rotor-Gene 6000 DNA amplification system (Qiagen, Hilden, Germany) using the iQ SYBR Green Supermix kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) as described by the manufacturer. The transcript levels of the genes for spiggin, androgen receptor β (ARβ) and CYP1A were determined in the kidney, and for vitellogenin in the liver. Gene-specific primer sequences and cycling profiles for spiggin, vitellogenin, CYP1A, and ubiquitin were found in the published literature.35,39,40 Primers for ARβ were constructed from a GenBank (http://www.ncbi.nlm.nih.gov/genbank/) ARβ sequence using ClustalW (http://www.ebi.ac.uk/Tools/msa/ clustalw2) and Primer3 (http://frodo.wi.mit.edu/) programs. All primers were synthesized by Sigma-Aldrich (St. Louis, MO, USA). Detailed information about the qPCR reactions is provided in Tables S1 and S2 of the Supporting Information. Three potential reference genes were employed in both tissues; β-actin, elongation factor α (EF1-α), and ubiquitin. The transcriptions of β-actin and EF1-α were found to differ significantly between groups in the kidney, whereas ubiquitin transcription was stable in both tissues. Ubiquitin was therefore employed as the reference gene. All samples were amplified in triplicate. Negative controls in each PCR assay were a no template control (NTC), where nuclease-free water was added to the reaction instead of cDNA template, and a no reverse transcriptase control (RTC), where nuclease-free water had been added to the cDNA synthesis reaction instead of the reverse transcriptase enzyme. Melt curve analysis from 55° to 95 °C was performed after each PCR run, ensuring the amplification of single PCR products. To determine the reaction efficiency of each primer pair, a standard curve was produced from serial (1:4) dilutions of pooled cDNA. To verify that the correct genes had been amplified, the nucleotide sequence of the PCR products was determined by sequence analysis. In brief, PCR was conducted using FideliTaq PCR Master Mix (2X) (Affymetrics, Inc., Cleveland, OH, USA) and PCR products were purified using GenElutePCR Clean-Up Kit (Sigma-Aldrich, St. Louis, MO, USA). Sequencing reactions were performed by Uppsala Genome Center (Uppsala University). The generated nucleotide sequences were submitted to a BLAST search in GenBank’s and Ensembl’s G. aculeatus nucleotide databases, and showed 100% identity with their intended targets. Calculation of Relative Gene Transcription. Ratios of mRNA transcription were calculated using the Pfaffl method.41,42 Triplicate amplification data from the genes of interest were averaged and normalized to ubiquitin to make up for differences in mRNA input and reverse transcription efficiency. The mean transcription ratios in the exposed groups were normalized to the mean transcription ratio in the vehicle control group and reported as a fold change relative to the control. Chemical Analysis. Water samples were extracted and analyzed using an in-line SPE-column coupled to liquid chromatography-tandem mass spectrometry. Detailed description of the chemical analysis and the in-line extraction,

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RESULTS Measured Concentrations of LNG. Measured concentrations in the aquaria were lower than the nominal, with average recoveries of 55 (5.5 ng L−1), 40 (40 ng L−1) and 36% (358 ng L−1) in the lowest, mid, and highest concentration, respectively. The lower than nominal concentrations were probably due to accumulation into the fish and adsorption of the compound to the aquaria and silicone tubings. Concentrations were however stable during the course of the experiment and the measured concentrations are henceforth used in the presentation and interpretation of the data. LNG was not detected in the vehicle control aquaria. Gross Morphology. Mean body weight was 3.04 ± 0.80 g, with no difference between groups (data not shown). Neither OSI nor LSI was affected by LNG treatment (data not shown). KSI was significantly increased (p < 0.0001) in both the 40 ng L−1 and the 358 ng L−1 group showing an approximate doubling of relative kidney size (Table 1). Table 1. Kidney Somatic Index (KSI) and Kidney Epithelium Height (KEH) in Female Three-Spined Sticklebacks (G. aculeatus) after 21 Days of Aqueous Exposure to Levonorgestrel or Vehicle (Acetone) Onlya group vehicle 5.5 ng L−1 40 ng L−1 358 ng L−1 a b

KSI (%) 0.84 0.86 1.78 1.68

± ± ± ±

0.17, n = 12 0.22, n = 12 0.41b, n = 11 0.30b, n = 12

KEH (μm) 7.8 ± 0.9, n = 4 8.4 ± 0.8, n = 3 20.3 ± 0.4b, n = 3 24.5 ± 1.4b, n = 4

Mean ± SEM, n refers to the number of kidneys measured. Significantly different from vehicle control (p < 0.0001).

Kidney Histology. Histological evaluation of the kidneys showed that exposure to LNG at 40 and 358 ng L−1 caused a significant increase (p < 0.0001) of the height of the proximal tubule epithelium (Table 1). KEH was approximately 2.5 and 3 times higher in the 40 ng L−1 and the 358 ng L−1 group respectively compared with the KEH in the control group. Kidney sections stained with PAS also showed intense staining of the proximal tubule epithelia in the groups exposed to LNG at 40 and 358 ng L−1 and weak staining in the control and 5.5 ng L−1 groups (Figure.1). Gene Transcription. Exposure to LNG significantly induced (p < 0.0001) kidney spiggin transcription at 40 and 358 ng L−1 (fold change approximately 150 000 and 250 000, respectively; part A of Figure 2). There was a small but significant decrease (p = 0.0421) in spiggin transcription at 5.5 2045



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Figure 1. Kidney sections of female three-spined sticklebacks (G. aculeatus) after 21 days of aqueous exposure to vehicle (acetone) only (A) or levonorgestrel (B−D). Sections were stained with periodic acid-Schiff (PAS). All microphotographs are taken under the same magnification. ((A) Vehicle control; kidney epithelium height (KEH) = 7.8 μm. (B) 5.5 ng L−1; KEH = 8.4 μm. (C) 40 ng L−1; KEH = 20.3 μm. (D) 358 ng L−1; KEH = 24.5 μm). Note strong PAS staining in C and D.

Figure 2. Relative mRNA transcription of spiggin (A), CYP1A (C), and ARβ (D) in kidney and vitellogenin (B) in liver of female three-spined stickleback (G. aculeatus) after 21 days of aqueous exposure to levonorgestrel or vehicle (acetone) only. Eight kidneys were analyzed in each group. Number of livers analyzed in each group was 11 in the 40 ng L−1 group and 12 in all other groups. Data are presented as fold change (mean + SEM) relative to vehicle control and significant differences are marked with asterisks (*p = 0.0421 (A); *p = 0.0191 (B); ***p < 0.0001 (A−D)).

ng L−1 (fold change −2.8), implying a U-shaped dose− response curve. A dose-dependent down-regulation of liver

vitellogenin transcription at 40 ng L−1 (p = 0.0191) and 358 ng L−1 (p < 0.0001) was observed (fold change −9.0 and −142.6, 2046



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respectively; part B of Figure 2 ). Kidney transcription of CYP1A was decreased (p < 0.0001) at 40 and 358 ng L−1 (fold change −17.7 and −21.5, respectively; part C of Figure 2), whereas ARβ transcription appeared to be unaffected by LNG exposure (part D of Figure 2).

Furthermore, they show that the androgenic potency of waterborne LNG in three-spined stickleback is remarkably high, being similar to that of MT and even surpassing those of DHT and 17β-trenbolone (TB). Spiggin induction by MT has been reported at 10 ng L−1, but the LOECs for DHT and TB are 2000 and 3041 ng L−1, respectively.31,35,55 It should be noted however that DHT and TB LOECs were determined for induction of spiggin protein and not transcripts, so different assay sensitivities might contribute to the observed differences in potency.31,55 The LOEC of 40 ng L−1 for both KSI and KEH is the lowest LOEC reported for these end points thus far. The LOEC of MT was 100 ng L−1 for both KSI and KEH.31,33 The response at 40 ng L−1 seems to correspond to maximal spiggin production by the kidneys. There are probably LNG concentrations between 5.5 and 40 ng L−1, which would cause partial spiggin induction like the barely 10-fold increase caused by MT at 10 ng L−1.35 The binding affinity of LNG to recombinant rat AR has been shown to be 18% of that of DHT.56 Higher affinity to rat AR than LNG was also shown for MT (33% of that of DHT) and TB (126% of that of DHT).56 It should be noted, however, that the binding affinities of these compounds for the stickleback AR are unknown. Furthermore, teleosts possess at least two AR isoforms, which differ in tissue distribution and ligand binding specificities, further aggravating predictions of androgenic potency.57 The high androgenic potency of LNG in sticklebacks exposed via the water is not only related to its AR affinity but is also a reflection of its strong uptake from water into fish. The measured bioconcentration factor (BCF) of LNG in rainbow trout (Oncorhynchus mykiss) was 12 000, much higher than the predicted BCF of 46 based on its octanol−water partition coefficient.9 This was attributed to sequestration of LNG entering the gills by binding to SHBG.46 Water-borne sex steroids are very efficiently taken up by fish and uptake rates appear to reflect the steroids’ binding affinity for SHBG.46,58,59 A difference in bioconcentration would seemingly account for the difference in potency between LNG and TB, which has a measured BCF of 13 in fathead minnow (Pimephales promelas).48 By contrast, LNG is more potent than DHT in terms of spiggin induction, but DHT has higher affinity to both rat AR and fish SHBG.46,56 However, reports show that 11-KT, despite having lower binding affinity than DHT to fish AR, can be a more potent inducer of AR-mediated transcription.60−62 We found that LNG exposure caused a dose-dependent decrease in vitellogenin transcription. Exogenous androgens can modulate plasma vitellogenin concentrations and liver transcription.47−49,63−67 Vitellogenin is inducible by aromatizable androgens such as MT, which are converted by CYP19A into E2.49,66 The nonaromatizable androgens DHT and TB on the other hand suppress vitellogenin levels.47,48,63,64,67 This suppression appears to be caused not by AR activation but rather by lowered E2 plasma concentrations.48,64 Decreased plasma sex steroid levels is a common effect of exogenous androgen exposure,35,48−50,65 and the androgenic progestin NET has been found to cause decreased sex steroid levels in fathead minnows.20 These effects could be due to negative feedback on the HPG axis or increased clearance rate of endogenous steroids because of displacement from SHBG.49 Involvement of the HPG axis is supported by the finding that LNG caused lowered expression of luteinizing hormone (LH) in brains of African clawed frogs (Xenopus laevis). 22 Consequently, LNG probably inhibits vitellogenin transcription by causing decreased plasma concentrations of E2. It should be

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DISCUSSION The results of the present study show that LNG has strong androgenic and antiestrogenic effects in female three-spined sticklebacks at water concentrations ≥40 ng L−1. Spiggin induction is considered to be mediated via the AR, as androgen-induced spiggin production is blocked by the model AR antagonist flutamide.31,32,34 Recently, a qPCR assay was developed for measuring tissue-specific spiggin gene transcription.35 This assay was able to detect spiggin mRNA induction at a 10-fold lower concentration of 17α-methyltestosterone (MT) (10 ng L−1) than in studies on expression at the protein level.31 Previous studies32,35 have demonstrated that levels of spiggin mRNA seem to correspond to protein levels, as both basal mRNA and basal protein levels are 5 to 6 orders of magnitude higher in males than in females. We therefore chose to analyze transcript concentrations of spiggin as a marker for AR activation in the present study. LNG massively induced spiggin transcription and caused kidney hypertrophy at 40 and 358 ng L−1. The relatively small difference in spiggin transcription between the 40 and 358 ng L−1 groups suggests that these values represent the maximal spiggin production, and conforms to the highest levels of induction in previous studies.31,32,35,44,45 There was an apparent decrease in spiggin transcription in fish exposed to an LNG concentration of 5.5 ng L−1. Possible explanations could be that LNG lowered plasma 11-KT levels via negative feedback on the hypothalamus-pituitary-gonad (HPG) axis and/or that 11-KT was cleared from the plasma due to displacement from sex hormone binding globulin (SHBG) by LNG. The binding affinity of LNG to SHBG (52% of that of 5α-dihydrotestosterone (DHT)) is higher than that of 11-KT (28% of that of DHT).46 Several studies report lowered endogenous androgen levels in fish exposed to exogenous androgens and androgenic progestins.20,35,47−50 Possibly, LNG at 5.5 ng L−1 was able to lower 11-KT levels but not induce spiggin transcription, whereas higher LNG concentrations caused induction. However, this study and many previous ones have shown that spiggin production is very low in unexposed females and to determine a further reduction in spiggin transcription involves methodological uncertainty.28,31,33,51 Even though mRNA alterations are not always indicative of a functional response, the morphological data corroborate the qPCR analysis as a true measurement of spiggin production. The increase in relative kidney size reflects the increased thickness of the proximal tubule epithelium occurring as the function of this tissue changes from metabolism and excretion to secretion of spiggin.52 The observed strong PAS staining in the affected fish indicates high intracellular concentrations of glycoproteins, that is spiggin. Even though increased KSI and KEH are supposedly less sensitive responses than induction of spiggin protein expression determined with ELISA or spiggin mRNA transcription determined using qPCR,29,31,35,53 these morphological effects were detected at the same LNG concentration as spiggin induction in the present study. Our results on spiggin induction probably reflect the androgenic properties of LNG separately from its progestagenic properties, as P4 does not induce spiggin production.28,34,54 2047



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affected male mate-calling behavior.24 Molecular data suggest that, even though synthetic progestins are strong PR agonists in mammals, they interact very poorly with teleost PRs,74 which supports that the androgenicity of progestins is of major importance for their effects in fish. Moreover, in progestinexposed zebrafish (Danio rerio) embryos, the altered pattern of gene transcription caused by P4 was dissimilar to changes in transcription caused by LNG and NET,75 further supporting that the mode of action of androgenic and nonandrogenic progestins differs. Taken together, the present and previous reports indicate that the adverse effects on fish reproduction by synthetic progestins are to a considerable extent caused by their androgenic properties. LNG has been detected in environmental samples in several countries,4,6−9 with a highest concentration of 30 ng L−1 in STP effluent and 7 ng L−1 in surface water.7,8 This means that environmental levels of LNG are lower than those having effects in the present study. However, other progestins are also frequently detected in the aquatic environment and additive effects are likely. That certain progestins seem to cause effects via their androgenicity implies that they also may have additive effects with other androgenic pollutants in aquatic environments. In conclusion, the results of the present study show that LNG has androgenic and antiestrogenic effects in female threespined stickleback at concentrations ≥40 ng L−1, manifested as altered gene transcription and morphological changes. Further studies are warranted to determine to what extent the reproductive impairment by pharmaceutical progestins in aquatic vertebrates is due to their androgenic properties and how mixtures of progestins may act together to affect reproduction.

noted however that the vitellogenin-depressing effect of LNG might not be entirely due to its androgenic properties, as it was recently shown that P4 caused a U-shaped decrease in vitellogenin expression in female fathead minnows.23 The mechanism behind this is however unknown. Kidney CYP1A expression was analyzed to determine the effect of LNG on an enzyme expressed in the proximal tubule epithelium,68−70 the site of spiggin synthesis, but supposedly not directly regulated by the AR. There is no conclusive evidence in the literature supporting the observed CYP1A suppression as a direct effect of AR or PR activation. There are some reports71 of aryl hydrocarbon receptor (AhR)−AR crosstalk in mammals but results are inconclusive. A plausible explanation for the decrease in CYP1A expression could be that a reduced level of this enzyme reflects a general down regulation of normal kidney function in response to AR activation when the kidney transforms from an excretory organ to a secretory gland. Androgen-induced transformation of the proximal tubule epithelium into its glandular spiggin-secreting state leads to a marked reduction of ion reuptake and excess water excretion.52 Presence of ion and water transporting membrane systems in enterocytes in mature males and androgen-treated females suggests that the intestine takes over osmoregulation when the kidney produces spiggin,52 a process also directly governed by androgens.72 LNG exposure did not significantly alter the expression of ARβ in the kidney. This conforms to previous studies on sticklebacks that have shown little effect of androgens on kidney ARβ expression,35,45,62,72 even when the same treatment strongly induced spiggin synthesis.35,45 This lack of autoregulation seems to apply generally to stickleback AR,72 and no androgen response element (ARE) has been found in the promoter region of cloned stickleback AR.62 Adverse effects consequent to continuous spiggin induction have not been shown. However, juvenile sticklebacks exposed to MT for 2 months had heavily inflamed kidneys.51 MT exposure caused 17−31% mortality without affecting growth or condition parameters indicating that the kidney inflammation might have been a contributing factor to mortality.51 As previously stated the kidney at least partially ceases to function as an excretory and osmoregulatory organ when it starts producing spiggin,52 but any long-term effects of this are unknown. It is however plausible that a chronic spiggin production, normally occurring only in males for about three months per year,27 is energetically costly. The suppression of vitellogenesis by LNG exposure is more likely than the induced spiggin production to be a direct adverse effect. A correlation between suppressed vitellogenin levels in females and decreased fecundity has been shown.73 The correlation was highly similar even though the tested chemicals inhibited vitellogenesis by different mechanisms of action.73 This indicates that female vitellogenin suppression in itself can adversely affect reproduction,73 and may account for some of the previously reported reprotoxic effects of LNG and NET. The present study clearly establishes LNG as a potent and efficient androgen in fish attesting to the previous reports of its masculinization of female fish and amphibians.19,20,25 Furthermore, the model androgens DHT and TB have adverse effects in fish resembling those caused by LNG and NET.47,48,50 It is plain from previous reports that androgenic progestins cause reproductive impairment in fish at lower concentrations than nonandrogenic ones.19,20,23 An androgenic effect of LNG was also isolated in African clawed frogs where LNG but not P4

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ASSOCIATED CONTENT

S Supporting Information *

Detailed descriptions of the qPCR reactions and chemical analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +46 18 471 2600, fax: +46 18 471 64 25, e-mail: johan. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Margareta Mattsson and Stefan Gunnarsson for expert work and guidance on the histological analysis, and Erika Jansson and Gaëtan Philippot for excellent work on the qPCR. This work was supported by the Swedish Foundation for Strategic Environmental Research (Mistra), through the MistraPharma research programme.

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REFERENCES

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