Article pubs.acs.org/est
p,p′‑DDE Induces Gonadal Intersex in Japanese Medaka (Oryzias latipes) at Environmentally Relevant Concentrations: Comparison with o,p′‑DDT Jianxian Sun, Chen Wang, Hui Peng, Guomao Zheng, Shiyi Zhang, and Jianying Hu* MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, People’s Republic of China S Supporting Information *
ABSTRACT: Previous studies have reported high body burdens of dichlorodiphenyltrichloroethane (DDT) and its metabolites in wild fishes worldwide. This study evaluated the adverse effects of 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene (p,p′-DDE) and o,p′-DDT on gonadal development and reproduction by exposing transgenic Japanese medaka (Oryzias latipes) from hatch for 100 days. While both p,p′-DDE and o,p′-DDT induced intersex in male medaka, the lowest observable effective concentration (LOEC) of o,p′-DDT was 57.7 ng/g ww, about 5-fold lower than that (272 ng/g ww) of p,p′-DDE. Since LOECs of both chemicals were comparable to the body concentrations in wild fish, DDT contamination would likely contribute to the occurrence of intersex observed in wild fish. Exposure to o,p′-DDT resulted in much higher expression of vitellogenin in liver of males than p,p′-DDE, accordant with the higher potency of o,p′-DDT than p,p′-DDE to induce intersex. This phenomenon could be partly explained by the significantly elevated levels of 17β-estradiol in plasma of males exposed to o,p′-DDT, in addition to its estrogenic activity via the estrogen receptor. Significantly lower fertilization (p = 0.006) and hatchability (p = 0.019) were observed in the 13 intersex males. This study for the first time demonstrated the induction of intersex and reproductive effects of p,p′-DDE and o,p′-DDT at environmentally relevant concentrations.
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INTRODUCTION A wide variety of chemicals released into the aquatic environment have been shown to interfere with hormonal control of sexual differentiation and reproduction in fish.1 There is laboratory evidence that substances mimicking estrogens are able to impair gonadal development including gonadal growth retardation, testicular degeneration and incidences of intersex in males.2−5 Gonadal intersex in male is related to the disturbance of spermatogenesis and impairment of reproductive success caused by estrogenic chemicals, such as the reduction of milt volume, sperm density, and fertility,6−8 and intersex in testis has been widely observed in freshwater and marine fish species.8−11 The pollutants, which have been reported to induce intersex in fish, mainly include classic phenolic estrogenic compounds, such as natural and synthetic estrogens and nonylphenol.12 In addition to estrogenic compounds, antiandrogenic contaminants have been associated with the widespread feminization of wild fish as observed in U.K. rivers.13 However, the intersex in male fish was induced by typical antiandrogenic chemicals, such as flutamide, vinclozolin, and cyproterone acetate only at high concentrations far from environmental levels.14,15 Dichlorodiphenyltrichloroethane (DDT) has been widely used for agricultural and public health purposes since the 1940s,16 and relatively high concentrations of DDTs dominated by 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p′-DDE, © XXXX American Chemical Society
the metabolite of p,p′-DDT) have still been detected in wild organisms in recent years.17−19 Due to biomagnification,20 p,p′DDE usually has relatively high internal exposure concentrations in wild fish compared with chemicals with lower bioaccumulation or without biomagnification. p,p′-DDE is an androgen receptor antagonist,21,22 and p,p′-DDE in Chinese sturgeon (Acipenser sinensis)18 has been detected at higher concentrations (349 ng/g ww in liver) than those required to inhibit androgen receptor transcriptional activity in in vitro assay.21 p,p′-DDE has also been identified as the major causative chemical that contributed to antiandrogenic activity in wild seals.23 As an androgen receptor antagonist, p,p′-DDE is able to prevent the transcription of androgen associated genes and reduce the sperm count in male fish.24 In lab exposure experiments, p,p′-DDE greatly induced vitellogenin (VTG) in male Japanese medaka (Oryzias latipes)25 and estrogen related genes were significantly up-regulated in male zebrafish,26 exerting obvious estrogenic activity of which mechanism has been explained by its antiandrogenic activity. Thus, as an antiandrogenic, bioaccumulative, and environmentally ubiquitous chemical, it would be interesting to determine whether Received: October 13, 2015 Revised: November 24, 2015 Accepted: November 25, 2015
A
DOI: 10.1021/acs.est.5b05042 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology
0.1 mg/L CaCO3, pH 7.7 ± 0.2, dissolved oxygen 7.8 ± 0.3 mg/L, and temperature 25 ± 1 °C. Exposure Design. Medaka larvae collected on the first day after hatch were exposed to solvent (acetone, 0.01‰ in water), or 0.02, 0.2, 2, and 20 μg/L p,p′-DDE, or 0.005, 0.05, 0.5, and 5 μg/L o,p′-DDT, in a flow-through system with four full water renewals every 24 h. The exposure was performed in glass tanks filled with 4 L carbon-filtered water for the first month, and fish were transferred to 12 L glass tanks as to accommodate growth. After 100 days of exposure, liver, and testis from six male medaka of each group were used for gene expression analysis. Six males from each exposure group were chosen for reproductive tests, and the remaining fish were anesthetized with tricaine methanesulfonate (MS-222). The gonad of male fish in each treatment group was isolated for intersex observation by GFP fluorescence, and three were used for tissue sections for histopathological observation. Two additional sets of exposures were completed with 0.5 and 12.5 μg/L of p,p′-DDE and 0.25 and 6.25 μg/L of o,p′-DDT for intersex incidence investigation only. Numbers of fish in each group were similar to the first set, and details were listed in Supporting Information Table S2. Observation of Intersex in Male Medaka. Testis of male medaka were isolated for observation of florescence under a fluorescence stereoscopic microscope (Leica M165 FC, Germany), with a 10× objective lens and GFP filters set. Fluorescent images were recorded by a color digital cooled charge-coupled device (CCD) camera (Leica DFC310 FX, Germany). The intersex incidence was calculated as the sum of male fish with any level of observed severity of intersex relative to that of males in the exposure group. The testis from each fish was then stored in 10% formaldehyde solution until histological intersex determination. Histological Examination. Fixed gonad samples were dehydrated and embedded in paraffin blocks, sectioned at 5−10 μm, stained with hematoxylin and eosin (H&E), and examined by light microscopy for routine histological and morphometrical analyses. Histological measurements were taken using an ocular micrometer. Evaluation of Reproductive Success. After 100 days of exposure, male and female medaka of each group were judged for sex by the presence of distinct secondary sex characteristics. Six males from each treatment group were mated to control female in clean water, and eggs were collected for 3 days. Three parameters, spawning (number of eggs per female per day), percentage of fertilization, and hatching rate were used to assess reproductive success. Presence of intersex was also observed in males following the reproductive study and reproductive data from these intersex males were selected to investigate the effects of intersexuality on male reproduction. Spawned eggs were carefully collected daily at 2−3 h postfertilization during the final week of exposure and gently separated. Mean numbers of spawned eggs per female were calculated for each exposure group. A Leica M165 FC microscope was used to observe fertilization and eleutheroembryo development. Fertilized eggs were identified under a microscope before the late morula stage (Stage 9) and fertilization success was calculated by dividing the number of spawned eggs by that of fertilized eggs. All fertilized eggs were collected and cultured in glass plates using carbon-treated water without chemical exposure until hatched. Embryos were observed each day until hatch. Hatching success was calculated by hatched fries/fertilized eggs.
p,p′-DDE could induce intersex and cause reproductive abnormalities in fish at environmentally relevant levels. In addition, although the use of DDT has been banned in most countries, DDT containing 15−21% o,p′-DDT is still used in several countries for malaria vector control, dicofol production, and antifouling paint for fishing ships.27,28 o,p′DDT and its metabolites (o,p′-DDD and o,p′-DDE) could disrupt the endocrine system by directly activating estrogen receptors.29 The relative binding affinity (RBA) of o,p′-DDT to 17β-estradiol (100) with the estrogen receptor in rainbow trout was 0.43, which was comparable to bisphenol A (0.21) and 4-toctylphenol (3.2).30 In vivo studies have shown that o,p′-DDT can induce intersex in Japanese medaka at much higher exposure concentrations (5−50 μg/L) than environmentally relevant concentrations.3,4 However, it remains unclear whether o,p′-DDT could induce intersex and cause reproductive abnormalities in fish at environmentally relevant levels. Investigations of potential intersex incidences and reproductive effects by pollutants at environmentally relevant concentrations is challenging due to the facts that (1) large sample sizes are required to achieve results with statistical power at environmental concentration, and (2) conventional histopathological observations of intersex can be easily overlooked due to the limited observation area of the testes and time required in this process. In this study, we investigated the intersex incidence and the related adverse reproductive effects of o,p′DDT, a typical estrogenic chemical and p,p′-DDE, a typical antiandrogenic chemical at environmentally relevant concentrations using pMOSP1-EGFP transgenic Japanese medaka, which was developed to specifically indicate the occurrence of gonadal intersex in male Japanese medaka by use of fluorescence measurement of expressed green fluorescence protein (GFP).31 Such a comprehensive comparison study provides a chance to better understand the estrogenic potency to induce intersex of fish by an antiandrogenic chemical.
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MATERIALS AND METHODS Chemicals and Reagents. p,p′-DDE and o,p′-DDT were obtained from Wellington Laboratories Inc. (Ontario, Canada). PCB121 was purchased from AccuStandard (Connecticut). 17β-Estradiol (17β-E2) and d3-E2 were purchased as powders from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Dansyl chloride (DNS) was obtained from Sigma-Aldrich. Dichloromethane (DCM), n-hexane, and acetone were pesticide residue grade and from OmniSolv (EM Science, KS). Sodium sulfate, silica gel (100−200 mesh size), and aluminumoxide (neutral, 150 mesh size) were purchased from Beijing Chemical Reagent (Beijing, China). Fish Maintenance. Transgenic Japanese medaka, which is a specific and sensitive biosensor for indicating intersex occurrence in male medaka fish by green fluorescence protein (GFP),31,32 were used in this study. In this transgenic fish, the green fluorescence protein (GFP) reporter gene was derived by the regulatory elements of the OSP1 gene, which is a specific and sensitive molecular biomarker for indicating intersex occurrence in male medaka fish exposed to estrogenic chemicals.31 The transgenic GFP was faithfully expressed in ovaries and in testes with intersex, perfectly mimicking the expression pattern of endogenous OSP1.31,32 Fish were cultured in flow-through tanks under conditions that facilitated breeding (16:8 light/dark cycle) and were fed live brine shrimp (Artemia nauplii) twice daily. Water used in the experiment was filtered through activated carbon and had a hardness of 8.1 ± B
DOI: 10.1021/acs.est.5b05042 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Table 1. Number of Fish in p,p′-DDE and o,p′-DDT Exposure Groups and Concentrations of Chemicals number of fish groups (μg/L)
female
male
intersex number (%)
0
38
40
NDb
23 25 18 19
26 22 22 25
2 3 5 6
57.7 ± 22.6a
20
28
2 (7.1%)
474 ± 219
27
29
4 (13.8%)
2092 ± 806
30
36
9 (25.0%)
29184 ± 14994
20
16
11 (68.8%)
solvent
blank
p,p′-DDE
0.02 0.2 2 20
272 ± 135 1194 ± 83.4 19613 ± 9627 257252 ± 61921
o,p′-DDT
0.005
o,p′-DDE 1.1 ± 0.2 o,p′-DDD 12.0 ± 2.3 o,p′-DDT 44.5 ± 23.6 o,p′-DDE 11.2 ± 1.7 o,p′-DDD 96.1 ± 15.3 o,p′-DDT 367 ± 205 o,p′-DDE 31.6 ± 10.6 o,p′-DDD 529 ± 159 o,p′-DDT 1530 ± 638 o,p′-DDE 350 ± 167 o,p′-DDD 3109 ± 1056 o,p′-DDT 25725 ± 14340
0.05
0.5
5
a
conc. in fish (ng/g ww)
(7.7%) (13.6%) (22.7%) (24.0%)
Total concentration of o,p′-DDTs (o,p′-DDE+o,p′-DDD+o,p′-DDT). bBelow detection limit.
Gene Expression Analysis. Liver and testis of male medaka fish were stored in liquid nitrogen until gene expression analysis. Total RNA was extracted from the liver and gonad samples using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocols. To prevent genomic DNA contamination, total RNA was digested by DNase I (TaKaRa Biotechnology, China) then purified. First-strand cDNA synthesis and relative quantitation in real time RT-PCR were performed according to methods described previously.33 Primers for quantification of mRNA of each gene were based on previously published paper.25 All primer sequences are shown in Table S1. To minimize DNA contamination, most primer pairs span at least one intron of the genomic sequence. Ribosomal protein L7 (RPL-7), an appropriate endogenous control for gene expression profiling in EDCs studies,33 was used as the internal control. Relative gene expression was evaluated by comparative cycle threshold (Ct) as described by Applied Biosystems (Foster City, CA). Analysis of DDTs in Medaka. Whole bodies of medaka fish were cut into pieces and spiked with a surrogate standard (PCB 121), and extracted with accelerated solvent extraction (Dionex ASE-200, Sunnyvale, CA) by a DCM/n-hexane (1:1 v/v) mixture solution at 100 °C and 1500 psi, which were performed with two 10 min cycles. The extraction of samples was concentrated to approximately 2 mL and passed through a glass column packed with 1 g of Na2SO4 and 8 g of acidified silica (48% H2SO4). After application of the sample, the column was eluted with 15 mL of n-hexane and 10 mL of DCM. The eluate was further cleaned on a neutral alumina column (4 g of sodium sulfate, 4 g of neutral alumina, and 4 g of sodium sulfate). The column was eluted with 20 mL of n-hexane and 25 mL of 60% DCM in n-hexane. The extraction solution was evaporated to dryness under a gentle stream of nitrogen and redissolved with n-hexane. Identification and quantification of DDTs were performed by a gas chromatography−mass spectrometer (GC-MS) (Agilent 6890N) equipped with a 5975C mass spectrometer and an Equity 1701 fused silica capillary column (30 cm × 0.25 mm × 0.25 μm film thickness;
J&W Scientific). A splitless injector was used, and the injector was held at 250 °C. The temperature program was from 70 °C (2 min) to 220 °C at a rate of 20 °C/min (keeping this temperature for 10 min), and to 280 °C at 20 °C/min, which was held for 6 min. The ion source temperature was maintained at 280 °C. The carrier gas was helium at a constant flow rate of 2 mL/min. The recoveries of p,p′-DDE, o,p′-DDE, o,p′-DDD, and o,p′-DDT ranged from 88.9% to 116.4%. The recovery of PCB 121 was 93.9 ± 14.2%. The method detection limits (MDLs) were set to the instrumental minimum detectable amounts with a signal-to-noise ratio of 3. The MDLs of p,p′DDE, o,p′-DDE, o,p′-DDD, and o,p′-DDT were 9.9, 7.2, 2.8, 19.3 ng/g ww, respectively. Detection of Plasma 17β-E2. Blood samples from eight male medaka were collected by a centrifuge method, and blood from two males were pooled.34 Following centrifugation, plasma were collected for analysis of 17β-E2. 17β-E2 in plasma from each group (n = 4) were analyzed using a method previously described.35 Aliquots (2−5 μL) of plasma samples were spiked with 0.02 ng surrogate (d2-βE2) and diluted with 100 μL ultrapure water prepared by the Milli-Q RC apparatus (Millipore, Bedford, MA). Samples were extracted twice with 300 μL MTBE, vortex for 5 min, and centrifuged at 4000g for 5 min. Following extraction, organic fractions were dried under a gentle stream of nitrogen. Extracts were redissolved in 100 μL DNS (1 mg/mL in acetone), vortex-mixed for 1 min, and incubated at 60 °C for 10 min. The derivatizations were diluted by 100 μL nanopure water and extracted with 2 mL hexane twice by vortex for 5 min and centrifuged at 4000g for 5 min. The final extracts were dried under a gentle stream of nitrogen and redissolved in 20 μL methanol. The extracts were analyzed using a Waters ACQUITY UPLC system (Waters, MA) with a Waters Micromass Quattro Premier XE (triple-quadrapole) detector operated in electrospray positive mode (ESI+ mode). Detailed information on equipment conditions and quality assurance/quality control are provided in the Supporting Information. C
DOI: 10.1021/acs.est.5b05042 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 1. Presence of intersex in male medaka exposed to p,p′-DDE. (A) Dose−response relationship of intersex incidence (%) and intrabody concentrations (ng/g ww). (B) Testis of normal transgenic male medaka. (C,D) Testis with intersex severity at level 1 and level 2 observed in p,p′DDE exposure. Scale bar = 800 μm in B, C, and D. (E−I) Severity index and related incidence of intersex in transgenic male medaka in control (E) and in each p,p′-DDE treatment groups 0.02 (F), 0.2 (G), 2 (H), and 20 μg/L (I).
Statistical Analyses. Statistical analyses were carried out using SPSS 19.0 (SPSS Inc., IL). A nonlinear regression to investigate relationships between body concentrations and intersex incidences after exposure to p,p′-DDE or o,p′-DDT was produced using Matlab (version 2008b). The reproductive effects due to intersexuality of male fish were analyzed by t-test. Differences with p < 0.05 were considered statistically significant.
evaluated by the number of primary oocytes in the whole testis as described in our previous paper.31 After 100 days of exposure to p,p′-DDE, the ratios (0.88−1.3) between male and female (determined by gonad phenotype) in all exposure groups were similar to that (1.05) in control, showing no obvious change (Table 1), but intersex with different severities were observed in testis of males. Traditional histological examination was also performed to further verify the presence of oocytes in these male fish (Figure S1). Intersex severity indexes of male medaka fish was arbitrarily graded into five levels according to our previously paper:31 Level 0, no detectable primary oocytes with GFP expression in the testis; level 1, 1−10 primary oocytes observed in the testis; level 2, 11−100 primary oocytes observed in the testis; level 3, more than 100 primary oocytes observed in the testis; level 4, sex reversal. Intersex severities at level 1 and level 2 were observed in p,p′-DDE exposure groups (Figure 1C and D). The intersex severity index showed a dose dependent increase with p,p′DDE exposure concentrations. In the 0.02 μg/L and 0.2 μg/L exposure groups, all intersex male medaka were at level 1; in the 2 μg/L and 20 μg/L exposure groups, intersex at level 2 was observed (Figure 1E−I). The intersex incidences of male medaka fish exposed to p,p′DDE were 7.7% (2/26, p = 0.16), 13.6% (3/22, p = 0.053), 17.8% (8/40, p < 0.001), 22.7% (5/22, p = 0.008), 25% (7/28, p < 0.001), and 24.0% (6/25, p < 0.005) in the exposure groups with internal concentrations of 271.6, 1193.6, 6240, 19 612.8, 1 653 554, and 2 57 251.8 ng/g ww, respectively, showing a significant dose−response relationship (Figure 1A). Several papers have highlighted the estrogenic activity of antiandrogenic chemicals in fish, and intersex has also been observed in male medaka after exposure to some antiandrogenic chemicals such as flutamide, vinclozolin, and cyproterone acetate.14,15 Vinclozolin can induce intersex in males of medaka (7%) at relatively high concentrations (5000 μg/L) following exposure during a similar period to this study.15 Relative to Hdihydrotestosterone (DHT), the binding affinity to androgen receptor of vinclozolin (0.02) has been reported to be similar that of p,p′-DDE (0.027),37 thus, the intersex observed at a much lower concentration (0.02 μg/L) of p,p′-DDE than
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RESULTS AND DISCUSSION Accumulation of p,p′-DDE and o,p′-DDT. DDTs are bioaccumulative, and therefore body concentrations are very important to the outcome of exposure. In this study, body concentrations of p,p′-DDE in male medaka were determined. After 100 days of exposure, body concentrations were 272 ± 135, 1,194 ± 83.4, 19,613 ± 9,627, and 257,252 ± 61,921 ng/g ww corresponding with waterborne concentrations of 0.02, 0.2, 2, and 20 μg/L of p,p′-DDE, respectively (Table 1). During exposure, 12−29% of o,p′-DDT were metabolized into o,p′DDE and o,p′-DDD as shown in Table 1 and Table S2. Since o,p′-DDT, o,p′-DDE, and o,p′-DDD have similar binding activities in a yeast-based human estrogen receptor gene transcription assay with half-effective concentrations (EC50) of 1.81 × 10−3 M, 3.32 × 10−3 M, and 5.34 × 10−3 M, respectively,29 total concentrations of these three o,p′-isomers were calculated in this study. Total body concentrations of o,p′DDTs in medaka were 57.7 ± 23, 474 ± 219, 2,092 ± 806, and 29,184 ± 14,994 ng/g ww after 100-day exposure to 0.005, 0.05, 0.5, and 5 μg/L, respectively (Table 1). Thus, p,p′-DDE and o,p′-DDTs in male medaka fish showed approximately 0.5−1.35 × 104 fold accumulation following a 100-day exposure (Table 1). Accumulation of o,p′-DDT was comparable to a previous study in female medaka (4.4 × 104) exposed for 2 weeks.4 The fact that p,p′-DDE and o,p′-DDT are more accumulative than classic (xeno)estrogens like 17β-E2, estrone, and nonylphenol (NP) would lead to relatively great effects of p,p′-DDE and o,p′-DDT in vivo compared with in vitro as reported in a previous study.36 Gonadal Intersex in Male Medaka Exposed to p,p′DDE. Intersex severities in transgenic medaka fish were D
DOI: 10.1021/acs.est.5b05042 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 2. Presence of intersex in male medaka exposed to o,p′-DDT. (A) Dose−response relationship of intersex incidence (%) and intrabody concentrations (ng/g ww). (B-D) Testis with intersex severity at level 1 (B), level 2 (C), and level 4 (D) in o,p′-DDT exposure groups. Scale bar = 800 μm in B, C, and D. (E−I) Severity index and related incidence of intersex in transgenic male medaka in control (E) and in each o,p′-DDT treatment groups 0.005 (F), 0.05 (G), 0.5 (H), and 5 μg/L (I).
44 830 ng/g ww of o,p′-DDT, respectively, showing a dose− response relationship (Figure 2A, Table 1). The LOEC for o,p′DDT to induce 7.1% intersex incidence was 57.7 ng/g ww, the corresponding waterborne concentration of 0.005 μg/L, which was 400-fold lower than the concentration (1.92 μg/L) in a previous study at which intersex was observed in one of six male medaka exposed to o,p′-DDT from hatch to adult for 4 weeks.2 The sensitive, fast, convenient, and accurate transgenic medaka used in this study allowed us to increase the sample size to obtain a more reliable dose response curve (Figure 2A). It should be noted that the LOEC of o,p′-DDT for inducing intersex (57.7 ng/g ww, 7.1%) was about 5-fold lower than that of p,p′-DDE (271.6 ng/g ww, 7.7%), indicating that the feminization potency to induce intersex in male medaka fish by o,p′-DDT was much greater than p,p′-DDE. Based on the dose response relationship between body concentrations and intersex incidence of o,p′-DDT (Figure 2A), and concentrations reported in Oreochromis mossambicus from South Africa (110−4035 ng/g Lw),38 intersex incidence which could be caused by o,p′-DDT was quantitatively assessed. The intersex incidence was calculated to be 12.4 ± 10.4%, slightly higher than the risk posed by p,p′-DDE (8.05 ± 3.2%) assuming that medaka has similar sensitivity with fish from the Luvuvhu River, even though p,p′-DDE presented with 2−10 fold higher body concentrations than o,p′-DDT in wild fish.38 The calculated intersex incidences caused by these two DDT isomers were 2−3 fold lower than those reported in wild fish populations (48−66%), suggesting that other chemicals may also contribute to intersexuality of wild fish, or that these fish species may be more sensitive to DDTs compared to Japanese medaka. The results in this study provided evidence that both p,p′-DDE and o,p′-DDT would contribute to the intersex incidence observed in wild fish although interspecies variations may limit the precision to extrapolate our laboratory dose− response relationships to wild fish species. Gene Expression and Plasma Estradiol Levels. In vitro studies have reported that o,p′-DDT is an estrogen receptor agonist with a 50% inhibition concentration (IC50) of 5 μM, and p,p′-DDE is an antiandrogenic chemical with an IC50 of 5
vincozolin may be attributed to its high bioaccumulation and/ or the sensitivity of the method for intersex detection used in this study. The lowest observable effective concentration (LOEC) of p,p′-DDE to induce 7.7% intersex incidence was 0.02 μg/L, from which the corresponding body concentration was 272 ± 135 ng/g ww, which was roughly 100 fold higher than concentrations in fish from Bohai Bay (4.7 ng/g ww in white flower croaker, 2.8 ng/ww in mullet, and 2.8 ng/g ww bartail flathead),20 and slightly lower than concentrations (349 ng/g ww in liver) in Chinese sturgeon from the Yangtze River.19 This LOEC was much lower than concentrations detected in Oreochromis mossambicus (1764−7609 ng/g in fish fat samples) from a DDT-sprayed area, the Luvuvhu River in South Africa38 and concentrations (780 ng/g ww) in shovelnose sturgeon (Scaphirhynchus albus) from the Mississippi River.10 The intersex incidence of 48−63% has been reported in male Oreochromis mossambicus from the Luvuvhu River,38 and 29% in male shovelnose sturgeon from the Mississippi River.10 Based on the relationship between body concentrations and intersex incidences in medaka obtained in this study and the concentrations of p,p′-DDE reported in fish from the Luvuvhu River, the intersex incidence by p,p′-DDE was assessed to be 8.05 ± 3.2% assuming that medaka has similar sensitivity as fish from the Luvuvhu River. Intersex in Male Medaka Exposed to o,p′-DDT. Intersex with different severities at level 1, level 2, and level 4 (Figure 2B, C, and D) were observed in the testis of males exposed to o,p′-DDT at different concentrations. The intersex severity showed dose-dependent increases with increasing o,p′-DDT exposure concentrations. Intersex at level 1 and level 2 were observed at exposure concentrations of 0.005, 0.05, and 0.5 μg/ L of o,p′-DDT, and sex-reversal (level 4) was induced when exposure concentration increased to 5 μg/L (Figure 2E-I). The intersex incidences in male medaka fish exposed to o,p′DDT were 7.1% (2/28, p = 0.18), 13.8% (4/29, p = 0.038), 11.8% (6/51, p < 0.001), 25.0% (9/36, p = 0.003), 68.6% (11/ 16, p < 0.001), and 100% (40/40, p < 0.001) at body concentrations of 57.7, 474.2, 1394, 2091.6, 29 183.8, and E
DOI: 10.1021/acs.est.5b05042 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 3. Relative gene expression of VTG1, VTG2, CHG-H, and CHG-L in livers and CYP19a and CYP17 in gonads of male medaka fish after exposure to p,p′-DDE (A) and o,p′-DDT (B). * indicates p < 0.05.
μM.21 Therefore, the stronger potency to induce intersex of o,p′-DDT than p,p′-DDE in vivo could be attributed to differences in their mechanisms of endocrine disruption. Direct binding and activation of the ER may elicit stronger potency to induce intersex than indirectly induced estrogenic activity by inhibiting androgenic activity in male fish. To explore this hypothesis, expression of estrogen-related genes including VTG-1, VTG-2, CHG-H, and CHG-L in liver of male medaka were determined. The expression of VTG-1 and VTG-2 were significantly up-regulated in a dose-dependent manner, in fish exposed to 0.02, 0.2, 2, and 20 μg/L of p,p′-DDE, relative to control, and responses ranged from 15.5 ± 0.42 to 37.8 ± 16.2fold, and 8.2 ± 0.83 to 24.0 ± 42.7-fold, respectively (Figure 3A). The dose-dependent increase in CHG-H (174 ± 90.7 to 726 ± 360 fold) and CHG-L (16.3 ± 11.7 to 75.3 ± 35.7 fold) were also found in p,p′-DDE exposure groups. The upregulation of these genes were in agreement with a previous study on p,p′-DDE exposure.25 In o,p′-DDT exposure groups, the dose-dependent up-regulation of VTG-1 and VTG-2 (ranging from 3.1 ± 2.2 to 1060 ± 6,714-fold and 4.6 ± 5.8 to 919 ± 2383-fold) were 3−30 fold higher than that resulting from p,p′-DDE exposure at similar concentrations. The upregulation of CHG-H and CHG-L which ranged from 1216 ± 1821 to 43 189 ± 322 106-fold and 54.3 ± 88.4 to 1103 ± 2156-fold, respectively, were also observed in the liver of male medaka (Figure 3B), which were 8−250 fold higher than those in male medaka exposed to similar concentrations of p,p′-DDE. This finding further suggested a stronger estrogenic effect of o,p′-DDT than p,p′-DDE, which is accordant with the higher intersex incidence in o,p′-DDT exposure groups than p,p′-DDE. Stronger estrogenic activity of o,p′-DDT was also observed in the summer flounder (Paralichthys dentatus) where levels of vitellogenin protein were induced after exposure to o,p′-DDT, though there are no reported changes in fish exposed to similar concentrations of p,p′-DDE.39 The effects of p,p′-DDE and o,p′-DDT on the biosynthesis of endogenous 17β-E2 in male medaka were further investigated.
Expressions of CYP19a mRNA and CYP17 mRNA in testis were up-regulated by 5.5−7.6-fold and 1.8−3.6-fold in p,p′DDE exposed male fish, respectively. As for o,p′-DDT, whereas gene expression of CYP17 was up-regulated by 1.2−14.2-fold, which were slightly higher than that of p,p′-DDE, CYP19a were greatly up-regulated by 7.7−62.0-fold (Figure 3B). Since gonadal aromatase (CYP19a) is important to the synthesis of 17β-E2, the dose-dependent up-regulation of gonadal CYP19a in males would increase endogenous concentrations of 17β-E2 which would further contribute to estrogenic effects, and therefore plasma 17β-E2 concentrations were determined. While there were no significant variations in concentrations of 17β-E2 between males from p,p′-DDE exposure groups and the control group, the plasma 17β-E2 levels in males from the 0.005, 0.05, 0.5, and 5 μg/L o,p′-DDT exposure groups were 1.1 ± 0.2, 0.9 ± 0.2, 1.3 ± 0.3, and 2.6 ± 1.0 ng/mL, respectively, significantly higher than those in males from the control group (0.4 ± 0.3 ng/mL) (Figure 4). The increase in expression of CYP19a and elevated levels of plasma 17β-E2 were also observed in male medaka after exposure to another estrogenic compound (EE2).40 Thus, the endogenously induced 17β-E2 in male fish from o,p′-DDT groups could
Figure 4. Concentrations of 17β-estradiol (17β-E2) in plasma of male medaka from o,p′-DDT exposure groups. Blood of two male medaka of the same group were pooled together. Each group contained four replicates (n = 4). Significance was shown with (*), where * indicates p < 0.05; ** indicates p < 0.01. F
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both antiandrogenic and estrogenic chemicals especially with high accumulative ability should be of significant concern.
partly contribute to its relatively high estrogenic effects which induce VTG and CHG, and intersex in male medaka. Reproductive Effects of Gonadal Intersex in Male Medaka. The reproductive effects of male fish with intersex has been documented in wild fish such as wild roach (Rutilus rutilus), and adverse effects on fertilization success have also been observed,8 posing risk to the population.7,41 To investigate the reproductive effects of both p,p′-DDE and o,p′-DDT due to intersexuality in male fish, six male medaka randomly chosen from each exposure group were mix-mated with control female fish in clean water. Of the total 48 male fish in the reproductive study, 13 males elicited intersex (8 in p,p′-DDE treated, and 5 in o,p′-DDT treated groups). When reproductive success data obtained from these 13 male fish were compared to data for fish from control groups, it was found that no significant variation in fecundities occurred, but fertilization rates (p = 0.006) and hatching rates (p = 0.019) were significantly inhibited compared to those from control groups (Figure 5). Similar
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b05042. Text, figures and tables addressing (1) Detailed method for analysis of 17β-E2; (2) Primer sequences of genes used in real-time PCR; (3) Number of fish in p,p′-DDE and o,p′-DDT exposure groups and concentrations of chemicals; (4) Intersex testis of transgenic medaka and light micrograph of the same testis by paraffin section with H&E staining (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone/fax: 86-10-62765520; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China [41330637 and 41171385] is gratefully acknowledged.
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Figure 5. Reproductive toxicity of intersex male medaka exposed to p,p′-DDE and o,p′-DDT. These 13 intersex males were from p,p′-DDE (8 males) and o,p′-DDT (5 males) exposure groups. Reproductive data of the 13 intersex males paired with control females were pooled and compared to control (both male and female were from control). * indicates p < 0.05.
REFERENCES
(1) Kime, D. E. Endocrine Disruption in Fish; Springer Science & Business Media, 1998. (2) Cheek, A. O.; Brouwer, T. H.; Carroll, S.; Manning, S.; McLachlan, J. A.; Brouwer, M. Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption. Environ. Health Persp. 2001, 109, 681. (3) Edmunds, J. S.; McCarthy, R. A.; Ramsdell, J. S. Permanent and functional male-to-female sex reversal in d-rR strain medaka (Oryzias latipes) following egg microinjection of o,p′-DDT. Environ. Health Persp. 2000, 108, 219. (4) Metcalfe, T. L.; Metcalfe, C. D.; Kiparissis, Y.; Niimi, A. J.; Foran, C. M.; Benson, W. H. Gonadal development and endocrine responses in Japanese medaka (Oryzias latipes) exposed to o,p′-DDT in water or through maternal transfer. Environ. Toxicol. Chem. 2000, 19, 1893− 1900. (5) Leet, J. K.; Sassman, S.; Amberg, J. J.; Olmstead, A. W.; Lee, L. S.; Ankley, G. T.; Sepúlveda, M. S. Environmental hormones and their impacts on sex differentiation in fathead minnows. Aquat. Toxicol. 2015, 158, 98−107. (6) Jobling, S.; Beresford, N.; Nolan, M.; Rodgers-Gray, T.; Brighty, G.; Sumpter, J.; Tyler, C. Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents. Biol. Reprod. 2002, 66, 272−281. (7) An, W.; Hu, J. Y.; Giesy, J. P.; Yang, M. Extinction risk of exploited wild roach (Rutilus rutilus) populations due to chemical feminization. Environ. Sci. Technol. 2009, 43, 7895−7901. (8) Jobling, S.; Coey, S.; Whitmore, J.; Kime, D.; Van Look, K.; McAllister, B.; Beresford, N.; Henshaw, A.; Brighty, G.; Tyler, C. Wild intersex roach (Rutilus rutilus) have reduced fertility. Biol. Reprod. 2002, 67, 515−524. (9) Hinck, J. E.; Blazer, V. S.; Schmitt, C. J.; Papoulias, D. M.; Tillitt, D. E. Widespread occurrence of intersex in black basses (Micropterus spp.) from US rivers, 1995−2004. Aquat. Toxicol. 2009, 95, 60−70. (10) Harshbarger, J.; Coffey, M.; Young, M. Intersexes in Mississippi river shovelnose sturgeon sampled below Saint Louis, Missouri, USA. Mar. Environ. Res. 2000, 50, 247−250. (11) Urushitani, H.; Katsu, Y.; Kato, Y.; Tooi, O.; Santo, N.; Kawashima, Y.; Ohta, Y.; Kisaka, Y.; Lange, A.; Tyler, C. R.; Johnson,
decreasing fertilization and hatchability rates were observed in intersex males from p,p′-DDE groups and o,p′-DDT groups, but the significances were weak possibly due to the decreased sample size and statistical power, which were 0.024 and 0.076 for intersex males from p,p′-DDE groups (n = 8), and 0.081 and 0.085 for o,p′-DDT groups (n = 5). Of the 13 male medaka with gonadal intersex, one showed serious intersex at level 4, this fish was from the exposure group of o,p′-DDT at 5 μg/L. The female paired with this male still spawned eggs every day as normal, but none of the spawned eggs were fertilized, as such, no eggs hatched. This indicated that the sex-reversed male medaka continued to perform normal mating behavior but with no sperm or low quality sperm. Further study into the mechanism of this effect is warranted. Overall, this study for the first time clarified that both the antiandrogenic chemical, p,p′-DDE and the estrogenic chemical, o,p′-DDT could cause intersexuality in fish at environmentally relevant concentrations. Such a comprehensive study provided further information on the estrogenic potency to induce intersex of male fish via the antiandrogenic pathway. High concentrations of p,p′-DDE are widely reported in wild life, and therefore would likely contribute to the presence of intersex in wild fish. The risk of o,p′-DDT should also be of concern for wild fish, considering its stronger potency than p,p′DDE. Intersex in male fish significantly inhibited fertilization and hatchability, indicating that a reduced capacity to produce viable offspring would occur following exposure to estrogenic compounds and/or antiandrogenic compounds. Therefore, G
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(29) Gaido, K. W.; Leonard, L. S.; Lovell, S.; Gould, J. C.; Babaï, D.; Portier, C. J.; McDonnell, D. P. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 1997, 143, 205−212. (30) Matthews, J.; Celius, T.; Halgren, R.; Zacharewski, T. Differential estrogen receptor binding of estrogenic substances: a species comparison. J. Steroid Biochem. Mol. Biol. 2000, 74, 223−234. (31) Zhao, Y. B.; Wang, C.; Xia, S.; Jiang, J. Q.; Hu, R.; Yuan, G. X.; Hu, J. Y. Biosensor medaka for monitoring intersex caused by estrogenic chemicals. Environ. Sci. Technol. 2014, 48, 2413−2420. (32) Zhao, Y. B.; Hu, J. Y. Development of a molecular biomarker for detecting intersex after exposure of male medaka fish to synthetic estrogen. Environ. Toxicol. Chem. 2012, 31, 1765−1773. (33) Zhang, Z. B.; Hu, J. Y. Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR. Toxicol. Sci. 2006, 95, 356−368. (34) Babaei, F.; Ramalingam, R.; Tavendale, A.; Liang, Y.; Yan, L. S. K.; Ajuh, P.; Cheng, S. H.; Lam, Y. W. Novel blood collection method allows plasma proteome analysis from single zebrafish. J. Proteome Res. 2013, 12, 1580−1590. (35) Chang, H.; Wan, Y.; Naile, J.; Zhang, X. W.; Wiseman, S.; Hecker, M.; Lam, M. H.; Giesy, J. P.; Jones, P. D. Simultaneous quantification of multiple classes of phenolic compounds in blood plasma by liquid chromatography−electrospray tandem mass spectrometry. J. Chromatogr. A 2010, 1217, 506−513. (36) Legler, J.; Zeinstra, L. M.; Schuitemaker, F.; Lanser, P. H.; Bogerd, J.; Brouwer, A.; Vethaak, A. D.; de Voogt, P.; Murk, A. J.; van der Burg, B. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity. Environ. Sci. Technol. 2002, 36, 4410−4415. (37) Scippo, M.-L.; Argiris, C.; Van De Weerdt, C.; Muller, M.; Willemsen, P.; Martial, J.; Maghuin-Rogister, G. Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors. Anal. Bioanal. Chem. 2004, 378, 664− 669. (38) Barnhoorn, I. E.; Van Dyk, J. C.; Pieterse, G.; Bornman, M. Intersex in feral indigenous freshwater Oreochromis mossambicus, from various parts in the Luvuvhu River, Limpopo Province, South Africa. Ecotoxicol. Environ. Saf. 2010, 73, 1537−1542. (39) Mills, L. J.; Gutjahr-Gobell, R. E.; Haebler, R. A.; Horowitz, D. J. B.; Jayaraman, S.; Pruell, R. J.; McKinney, R. A.; Gardner, G. R.; Zaroogian, G. E. Effects of estrogenic (o,p′-DDT; octylphenol) and anti-androgenic (p,p′-DDE) chemicals on indicators of endocrine status in juvenile male summer flounder (Paralichthys dentatus). Aquat. Toxicol. 2001, 52, 157−176. (40) Kashiwada, S.; Kameshiro, M.; Tatsuta, H.; Sugaya, Y.; Kullman, S. W.; Hinton, D. E.; Goka, K. Estrogenic modulation of CYP3A38, CYP3A40, and CYP19 in mature male medaka (Oryzias latipes). Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2007, 145, 370−378. (41) Harris, C. A.; Hamilton, P. B.; Runnalls, T. J.; Vinciotti, V.; Henshaw, A.; Hodgson, D.; Coe, T. S.; Jobling, S.; Tyler, C. R.; Sumpter, J. P. The consequences of feminization in breeding groups of wild fish. Environ. Health Persp. 2010, 119, 306−311.
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p,p′‑DDE Induces Gonadal Intersex in Japanese Medaka (Oryzias latipes) at Environmentally Relevant Concentrations: Comparison with o,p′‑DDT Jianxian Sun, Chen Wang, Hui Peng, Guomao Zheng, Shiyi Zhang, and Jianying Hu* MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, People’s Republic of China S Supporting Information *
ABSTRACT: Previous studies have reported high body burdens of dichlorodiphenyltrichloroethane (DDT) and its metabolites in wild fishes worldwide. This study evaluated the adverse effects of 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene (p,p′-DDE) and o,p′-DDT on gonadal development and reproduction by exposing transgenic Japanese medaka (Oryzias latipes) from hatch for 100 days. While both p,p′-DDE and o,p′-DDT induced intersex in male medaka, the lowest observable effective concentration (LOEC) of o,p′-DDT was 57.7 ng/g ww, about 5-fold lower than that (272 ng/g ww) of p,p′-DDE. Since LOECs of both chemicals were comparable to the body concentrations in wild fish, DDT contamination would likely contribute to the occurrence of intersex observed in wild fish. Exposure to o,p′-DDT resulted in much higher expression of vitellogenin in liver of males than p,p′-DDE, accordant with the higher potency of o,p′-DDT than p,p′-DDE to induce intersex. This phenomenon could be partly explained by the significantly elevated levels of 17β-estradiol in plasma of males exposed to o,p′-DDT, in addition to its estrogenic activity via the estrogen receptor. Significantly lower fertilization (p = 0.006) and hatchability (p = 0.019) were observed in the 13 intersex males. This study for the first time demonstrated the induction of intersex and reproductive effects of p,p′-DDE and o,p′-DDT at environmentally relevant concentrations.
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INTRODUCTION A wide variety of chemicals released into the aquatic environment have been shown to interfere with hormonal control of sexual differentiation and reproduction in fish.1 There is laboratory evidence that substances mimicking estrogens are able to impair gonadal development including gonadal growth retardation, testicular degeneration and incidences of intersex in males.2−5 Gonadal intersex in male is related to the disturbance of spermatogenesis and impairment of reproductive success caused by estrogenic chemicals, such as the reduction of milt volume, sperm density, and fertility,6−8 and intersex in testis has been widely observed in freshwater and marine fish species.8−11 The pollutants, which have been reported to induce intersex in fish, mainly include classic phenolic estrogenic compounds, such as natural and synthetic estrogens and nonylphenol.12 In addition to estrogenic compounds, antiandrogenic contaminants have been associated with the widespread feminization of wild fish as observed in U.K. rivers.13 However, the intersex in male fish was induced by typical antiandrogenic chemicals, such as flutamide, vinclozolin, and cyproterone acetate only at high concentrations far from environmental levels.14,15 Dichlorodiphenyltrichloroethane (DDT) has been widely used for agricultural and public health purposes since the 1940s,16 and relatively high concentrations of DDTs dominated by 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p′-DDE, © XXXX American Chemical Society
the metabolite of p,p′-DDT) have still been detected in wild organisms in recent years.17−19 Due to biomagnification,20 p,p′DDE usually has relatively high internal exposure concentrations in wild fish compared with chemicals with lower bioaccumulation or without biomagnification. p,p′-DDE is an androgen receptor antagonist,21,22 and p,p′-DDE in Chinese sturgeon (Acipenser sinensis)18 has been detected at higher concentrations (349 ng/g ww in liver) than those required to inhibit androgen receptor transcriptional activity in in vitro assay.21 p,p′-DDE has also been identified as the major causative chemical that contributed to antiandrogenic activity in wild seals.23 As an androgen receptor antagonist, p,p′-DDE is able to prevent the transcription of androgen associated genes and reduce the sperm count in male fish.24 In lab exposure experiments, p,p′-DDE greatly induced vitellogenin (VTG) in male Japanese medaka (Oryzias latipes)25 and estrogen related genes were significantly up-regulated in male zebrafish,26 exerting obvious estrogenic activity of which mechanism has been explained by its antiandrogenic activity. Thus, as an antiandrogenic, bioaccumulative, and environmentally ubiquitous chemical, it would be interesting to determine whether Received: October 13, 2015 Revised: November 24, 2015 Accepted: November 25, 2015
A
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0.1 mg/L CaCO3, pH 7.7 ± 0.2, dissolved oxygen 7.8 ± 0.3 mg/L, and temperature 25 ± 1 °C. Exposure Design. Medaka larvae collected on the first day after hatch were exposed to solvent (acetone, 0.01‰ in water), or 0.02, 0.2, 2, and 20 μg/L p,p′-DDE, or 0.005, 0.05, 0.5, and 5 μg/L o,p′-DDT, in a flow-through system with four full water renewals every 24 h. The exposure was performed in glass tanks filled with 4 L carbon-filtered water for the first month, and fish were transferred to 12 L glass tanks as to accommodate growth. After 100 days of exposure, liver, and testis from six male medaka of each group were used for gene expression analysis. Six males from each exposure group were chosen for reproductive tests, and the remaining fish were anesthetized with tricaine methanesulfonate (MS-222). The gonad of male fish in each treatment group was isolated for intersex observation by GFP fluorescence, and three were used for tissue sections for histopathological observation. Two additional sets of exposures were completed with 0.5 and 12.5 μg/L of p,p′-DDE and 0.25 and 6.25 μg/L of o,p′-DDT for intersex incidence investigation only. Numbers of fish in each group were similar to the first set, and details were listed in Supporting Information Table S2. Observation of Intersex in Male Medaka. Testis of male medaka were isolated for observation of florescence under a fluorescence stereoscopic microscope (Leica M165 FC, Germany), with a 10× objective lens and GFP filters set. Fluorescent images were recorded by a color digital cooled charge-coupled device (CCD) camera (Leica DFC310 FX, Germany). The intersex incidence was calculated as the sum of male fish with any level of observed severity of intersex relative to that of males in the exposure group. The testis from each fish was then stored in 10% formaldehyde solution until histological intersex determination. Histological Examination. Fixed gonad samples were dehydrated and embedded in paraffin blocks, sectioned at 5−10 μm, stained with hematoxylin and eosin (H&E), and examined by light microscopy for routine histological and morphometrical analyses. Histological measurements were taken using an ocular micrometer. Evaluation of Reproductive Success. After 100 days of exposure, male and female medaka of each group were judged for sex by the presence of distinct secondary sex characteristics. Six males from each treatment group were mated to control female in clean water, and eggs were collected for 3 days. Three parameters, spawning (number of eggs per female per day), percentage of fertilization, and hatching rate were used to assess reproductive success. Presence of intersex was also observed in males following the reproductive study and reproductive data from these intersex males were selected to investigate the effects of intersexuality on male reproduction. Spawned eggs were carefully collected daily at 2−3 h postfertilization during the final week of exposure and gently separated. Mean numbers of spawned eggs per female were calculated for each exposure group. A Leica M165 FC microscope was used to observe fertilization and eleutheroembryo development. Fertilized eggs were identified under a microscope before the late morula stage (Stage 9) and fertilization success was calculated by dividing the number of spawned eggs by that of fertilized eggs. All fertilized eggs were collected and cultured in glass plates using carbon-treated water without chemical exposure until hatched. Embryos were observed each day until hatch. Hatching success was calculated by hatched fries/fertilized eggs.
p,p′-DDE could induce intersex and cause reproductive abnormalities in fish at environmentally relevant levels. In addition, although the use of DDT has been banned in most countries, DDT containing 15−21% o,p′-DDT is still used in several countries for malaria vector control, dicofol production, and antifouling paint for fishing ships.27,28 o,p′DDT and its metabolites (o,p′-DDD and o,p′-DDE) could disrupt the endocrine system by directly activating estrogen receptors.29 The relative binding affinity (RBA) of o,p′-DDT to 17β-estradiol (100) with the estrogen receptor in rainbow trout was 0.43, which was comparable to bisphenol A (0.21) and 4-toctylphenol (3.2).30 In vivo studies have shown that o,p′-DDT can induce intersex in Japanese medaka at much higher exposure concentrations (5−50 μg/L) than environmentally relevant concentrations.3,4 However, it remains unclear whether o,p′-DDT could induce intersex and cause reproductive abnormalities in fish at environmentally relevant levels. Investigations of potential intersex incidences and reproductive effects by pollutants at environmentally relevant concentrations is challenging due to the facts that (1) large sample sizes are required to achieve results with statistical power at environmental concentration, and (2) conventional histopathological observations of intersex can be easily overlooked due to the limited observation area of the testes and time required in this process. In this study, we investigated the intersex incidence and the related adverse reproductive effects of o,p′DDT, a typical estrogenic chemical and p,p′-DDE, a typical antiandrogenic chemical at environmentally relevant concentrations using pMOSP1-EGFP transgenic Japanese medaka, which was developed to specifically indicate the occurrence of gonadal intersex in male Japanese medaka by use of fluorescence measurement of expressed green fluorescence protein (GFP).31 Such a comprehensive comparison study provides a chance to better understand the estrogenic potency to induce intersex of fish by an antiandrogenic chemical.
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MATERIALS AND METHODS Chemicals and Reagents. p,p′-DDE and o,p′-DDT were obtained from Wellington Laboratories Inc. (Ontario, Canada). PCB121 was purchased from AccuStandard (Connecticut). 17β-Estradiol (17β-E2) and d3-E2 were purchased as powders from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Dansyl chloride (DNS) was obtained from Sigma-Aldrich. Dichloromethane (DCM), n-hexane, and acetone were pesticide residue grade and from OmniSolv (EM Science, KS). Sodium sulfate, silica gel (100−200 mesh size), and aluminumoxide (neutral, 150 mesh size) were purchased from Beijing Chemical Reagent (Beijing, China). Fish Maintenance. Transgenic Japanese medaka, which is a specific and sensitive biosensor for indicating intersex occurrence in male medaka fish by green fluorescence protein (GFP),31,32 were used in this study. In this transgenic fish, the green fluorescence protein (GFP) reporter gene was derived by the regulatory elements of the OSP1 gene, which is a specific and sensitive molecular biomarker for indicating intersex occurrence in male medaka fish exposed to estrogenic chemicals.31 The transgenic GFP was faithfully expressed in ovaries and in testes with intersex, perfectly mimicking the expression pattern of endogenous OSP1.31,32 Fish were cultured in flow-through tanks under conditions that facilitated breeding (16:8 light/dark cycle) and were fed live brine shrimp (Artemia nauplii) twice daily. Water used in the experiment was filtered through activated carbon and had a hardness of 8.1 ± B
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Environmental Science & Technology Table 1. Number of Fish in p,p′-DDE and o,p′-DDT Exposure Groups and Concentrations of Chemicals number of fish groups (μg/L)
female
male
intersex number (%)
0
38
40
NDb
23 25 18 19
26 22 22 25
2 3 5 6
57.7 ± 22.6a
20
28
2 (7.1%)
474 ± 219
27
29
4 (13.8%)
2092 ± 806
30
36
9 (25.0%)
29184 ± 14994
20
16
11 (68.8%)
solvent
blank
p,p′-DDE
0.02 0.2 2 20
272 ± 135 1194 ± 83.4 19613 ± 9627 257252 ± 61921
o,p′-DDT
0.005
o,p′-DDE 1.1 ± 0.2 o,p′-DDD 12.0 ± 2.3 o,p′-DDT 44.5 ± 23.6 o,p′-DDE 11.2 ± 1.7 o,p′-DDD 96.1 ± 15.3 o,p′-DDT 367 ± 205 o,p′-DDE 31.6 ± 10.6 o,p′-DDD 529 ± 159 o,p′-DDT 1530 ± 638 o,p′-DDE 350 ± 167 o,p′-DDD 3109 ± 1056 o,p′-DDT 25725 ± 14340
0.05
0.5
5
a
conc. in fish (ng/g ww)
(7.7%) (13.6%) (22.7%) (24.0%)
Total concentration of o,p′-DDTs (o,p′-DDE+o,p′-DDD+o,p′-DDT). bBelow detection limit.
Gene Expression Analysis. Liver and testis of male medaka fish were stored in liquid nitrogen until gene expression analysis. Total RNA was extracted from the liver and gonad samples using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocols. To prevent genomic DNA contamination, total RNA was digested by DNase I (TaKaRa Biotechnology, China) then purified. First-strand cDNA synthesis and relative quantitation in real time RT-PCR were performed according to methods described previously.33 Primers for quantification of mRNA of each gene were based on previously published paper.25 All primer sequences are shown in Table S1. To minimize DNA contamination, most primer pairs span at least one intron of the genomic sequence. Ribosomal protein L7 (RPL-7), an appropriate endogenous control for gene expression profiling in EDCs studies,33 was used as the internal control. Relative gene expression was evaluated by comparative cycle threshold (Ct) as described by Applied Biosystems (Foster City, CA). Analysis of DDTs in Medaka. Whole bodies of medaka fish were cut into pieces and spiked with a surrogate standard (PCB 121), and extracted with accelerated solvent extraction (Dionex ASE-200, Sunnyvale, CA) by a DCM/n-hexane (1:1 v/v) mixture solution at 100 °C and 1500 psi, which were performed with two 10 min cycles. The extraction of samples was concentrated to approximately 2 mL and passed through a glass column packed with 1 g of Na2SO4 and 8 g of acidified silica (48% H2SO4). After application of the sample, the column was eluted with 15 mL of n-hexane and 10 mL of DCM. The eluate was further cleaned on a neutral alumina column (4 g of sodium sulfate, 4 g of neutral alumina, and 4 g of sodium sulfate). The column was eluted with 20 mL of n-hexane and 25 mL of 60% DCM in n-hexane. The extraction solution was evaporated to dryness under a gentle stream of nitrogen and redissolved with n-hexane. Identification and quantification of DDTs were performed by a gas chromatography−mass spectrometer (GC-MS) (Agilent 6890N) equipped with a 5975C mass spectrometer and an Equity 1701 fused silica capillary column (30 cm × 0.25 mm × 0.25 μm film thickness;
J&W Scientific). A splitless injector was used, and the injector was held at 250 °C. The temperature program was from 70 °C (2 min) to 220 °C at a rate of 20 °C/min (keeping this temperature for 10 min), and to 280 °C at 20 °C/min, which was held for 6 min. The ion source temperature was maintained at 280 °C. The carrier gas was helium at a constant flow rate of 2 mL/min. The recoveries of p,p′-DDE, o,p′-DDE, o,p′-DDD, and o,p′-DDT ranged from 88.9% to 116.4%. The recovery of PCB 121 was 93.9 ± 14.2%. The method detection limits (MDLs) were set to the instrumental minimum detectable amounts with a signal-to-noise ratio of 3. The MDLs of p,p′DDE, o,p′-DDE, o,p′-DDD, and o,p′-DDT were 9.9, 7.2, 2.8, 19.3 ng/g ww, respectively. Detection of Plasma 17β-E2. Blood samples from eight male medaka were collected by a centrifuge method, and blood from two males were pooled.34 Following centrifugation, plasma were collected for analysis of 17β-E2. 17β-E2 in plasma from each group (n = 4) were analyzed using a method previously described.35 Aliquots (2−5 μL) of plasma samples were spiked with 0.02 ng surrogate (d2-βE2) and diluted with 100 μL ultrapure water prepared by the Milli-Q RC apparatus (Millipore, Bedford, MA). Samples were extracted twice with 300 μL MTBE, vortex for 5 min, and centrifuged at 4000g for 5 min. Following extraction, organic fractions were dried under a gentle stream of nitrogen. Extracts were redissolved in 100 μL DNS (1 mg/mL in acetone), vortex-mixed for 1 min, and incubated at 60 °C for 10 min. The derivatizations were diluted by 100 μL nanopure water and extracted with 2 mL hexane twice by vortex for 5 min and centrifuged at 4000g for 5 min. The final extracts were dried under a gentle stream of nitrogen and redissolved in 20 μL methanol. The extracts were analyzed using a Waters ACQUITY UPLC system (Waters, MA) with a Waters Micromass Quattro Premier XE (triple-quadrapole) detector operated in electrospray positive mode (ESI+ mode). Detailed information on equipment conditions and quality assurance/quality control are provided in the Supporting Information. C
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Figure 1. Presence of intersex in male medaka exposed to p,p′-DDE. (A) Dose−response relationship of intersex incidence (%) and intrabody concentrations (ng/g ww). (B) Testis of normal transgenic male medaka. (C,D) Testis with intersex severity at level 1 and level 2 observed in p,p′DDE exposure. Scale bar = 800 μm in B, C, and D. (E−I) Severity index and related incidence of intersex in transgenic male medaka in control (E) and in each p,p′-DDE treatment groups 0.02 (F), 0.2 (G), 2 (H), and 20 μg/L (I).
Statistical Analyses. Statistical analyses were carried out using SPSS 19.0 (SPSS Inc., IL). A nonlinear regression to investigate relationships between body concentrations and intersex incidences after exposure to p,p′-DDE or o,p′-DDT was produced using Matlab (version 2008b). The reproductive effects due to intersexuality of male fish were analyzed by t-test. Differences with p < 0.05 were considered statistically significant.
evaluated by the number of primary oocytes in the whole testis as described in our previous paper.31 After 100 days of exposure to p,p′-DDE, the ratios (0.88−1.3) between male and female (determined by gonad phenotype) in all exposure groups were similar to that (1.05) in control, showing no obvious change (Table 1), but intersex with different severities were observed in testis of males. Traditional histological examination was also performed to further verify the presence of oocytes in these male fish (Figure S1). Intersex severity indexes of male medaka fish was arbitrarily graded into five levels according to our previously paper:31 Level 0, no detectable primary oocytes with GFP expression in the testis; level 1, 1−10 primary oocytes observed in the testis; level 2, 11−100 primary oocytes observed in the testis; level 3, more than 100 primary oocytes observed in the testis; level 4, sex reversal. Intersex severities at level 1 and level 2 were observed in p,p′-DDE exposure groups (Figure 1C and D). The intersex severity index showed a dose dependent increase with p,p′DDE exposure concentrations. In the 0.02 μg/L and 0.2 μg/L exposure groups, all intersex male medaka were at level 1; in the 2 μg/L and 20 μg/L exposure groups, intersex at level 2 was observed (Figure 1E−I). The intersex incidences of male medaka fish exposed to p,p′DDE were 7.7% (2/26, p = 0.16), 13.6% (3/22, p = 0.053), 17.8% (8/40, p < 0.001), 22.7% (5/22, p = 0.008), 25% (7/28, p < 0.001), and 24.0% (6/25, p < 0.005) in the exposure groups with internal concentrations of 271.6, 1193.6, 6240, 19 612.8, 1 653 554, and 2 57 251.8 ng/g ww, respectively, showing a significant dose−response relationship (Figure 1A). Several papers have highlighted the estrogenic activity of antiandrogenic chemicals in fish, and intersex has also been observed in male medaka after exposure to some antiandrogenic chemicals such as flutamide, vinclozolin, and cyproterone acetate.14,15 Vinclozolin can induce intersex in males of medaka (7%) at relatively high concentrations (5000 μg/L) following exposure during a similar period to this study.15 Relative to Hdihydrotestosterone (DHT), the binding affinity to androgen receptor of vinclozolin (0.02) has been reported to be similar that of p,p′-DDE (0.027),37 thus, the intersex observed at a much lower concentration (0.02 μg/L) of p,p′-DDE than
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RESULTS AND DISCUSSION Accumulation of p,p′-DDE and o,p′-DDT. DDTs are bioaccumulative, and therefore body concentrations are very important to the outcome of exposure. In this study, body concentrations of p,p′-DDE in male medaka were determined. After 100 days of exposure, body concentrations were 272 ± 135, 1,194 ± 83.4, 19,613 ± 9,627, and 257,252 ± 61,921 ng/g ww corresponding with waterborne concentrations of 0.02, 0.2, 2, and 20 μg/L of p,p′-DDE, respectively (Table 1). During exposure, 12−29% of o,p′-DDT were metabolized into o,p′DDE and o,p′-DDD as shown in Table 1 and Table S2. Since o,p′-DDT, o,p′-DDE, and o,p′-DDD have similar binding activities in a yeast-based human estrogen receptor gene transcription assay with half-effective concentrations (EC50) of 1.81 × 10−3 M, 3.32 × 10−3 M, and 5.34 × 10−3 M, respectively,29 total concentrations of these three o,p′-isomers were calculated in this study. Total body concentrations of o,p′DDTs in medaka were 57.7 ± 23, 474 ± 219, 2,092 ± 806, and 29,184 ± 14,994 ng/g ww after 100-day exposure to 0.005, 0.05, 0.5, and 5 μg/L, respectively (Table 1). Thus, p,p′-DDE and o,p′-DDTs in male medaka fish showed approximately 0.5−1.35 × 104 fold accumulation following a 100-day exposure (Table 1). Accumulation of o,p′-DDT was comparable to a previous study in female medaka (4.4 × 104) exposed for 2 weeks.4 The fact that p,p′-DDE and o,p′-DDT are more accumulative than classic (xeno)estrogens like 17β-E2, estrone, and nonylphenol (NP) would lead to relatively great effects of p,p′-DDE and o,p′-DDT in vivo compared with in vitro as reported in a previous study.36 Gonadal Intersex in Male Medaka Exposed to p,p′DDE. Intersex severities in transgenic medaka fish were D
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Figure 2. Presence of intersex in male medaka exposed to o,p′-DDT. (A) Dose−response relationship of intersex incidence (%) and intrabody concentrations (ng/g ww). (B-D) Testis with intersex severity at level 1 (B), level 2 (C), and level 4 (D) in o,p′-DDT exposure groups. Scale bar = 800 μm in B, C, and D. (E−I) Severity index and related incidence of intersex in transgenic male medaka in control (E) and in each o,p′-DDT treatment groups 0.005 (F), 0.05 (G), 0.5 (H), and 5 μg/L (I).
44 830 ng/g ww of o,p′-DDT, respectively, showing a dose− response relationship (Figure 2A, Table 1). The LOEC for o,p′DDT to induce 7.1% intersex incidence was 57.7 ng/g ww, the corresponding waterborne concentration of 0.005 μg/L, which was 400-fold lower than the concentration (1.92 μg/L) in a previous study at which intersex was observed in one of six male medaka exposed to o,p′-DDT from hatch to adult for 4 weeks.2 The sensitive, fast, convenient, and accurate transgenic medaka used in this study allowed us to increase the sample size to obtain a more reliable dose response curve (Figure 2A). It should be noted that the LOEC of o,p′-DDT for inducing intersex (57.7 ng/g ww, 7.1%) was about 5-fold lower than that of p,p′-DDE (271.6 ng/g ww, 7.7%), indicating that the feminization potency to induce intersex in male medaka fish by o,p′-DDT was much greater than p,p′-DDE. Based on the dose response relationship between body concentrations and intersex incidence of o,p′-DDT (Figure 2A), and concentrations reported in Oreochromis mossambicus from South Africa (110−4035 ng/g Lw),38 intersex incidence which could be caused by o,p′-DDT was quantitatively assessed. The intersex incidence was calculated to be 12.4 ± 10.4%, slightly higher than the risk posed by p,p′-DDE (8.05 ± 3.2%) assuming that medaka has similar sensitivity with fish from the Luvuvhu River, even though p,p′-DDE presented with 2−10 fold higher body concentrations than o,p′-DDT in wild fish.38 The calculated intersex incidences caused by these two DDT isomers were 2−3 fold lower than those reported in wild fish populations (48−66%), suggesting that other chemicals may also contribute to intersexuality of wild fish, or that these fish species may be more sensitive to DDTs compared to Japanese medaka. The results in this study provided evidence that both p,p′-DDE and o,p′-DDT would contribute to the intersex incidence observed in wild fish although interspecies variations may limit the precision to extrapolate our laboratory dose− response relationships to wild fish species. Gene Expression and Plasma Estradiol Levels. In vitro studies have reported that o,p′-DDT is an estrogen receptor agonist with a 50% inhibition concentration (IC50) of 5 μM, and p,p′-DDE is an antiandrogenic chemical with an IC50 of 5
vincozolin may be attributed to its high bioaccumulation and/ or the sensitivity of the method for intersex detection used in this study. The lowest observable effective concentration (LOEC) of p,p′-DDE to induce 7.7% intersex incidence was 0.02 μg/L, from which the corresponding body concentration was 272 ± 135 ng/g ww, which was roughly 100 fold higher than concentrations in fish from Bohai Bay (4.7 ng/g ww in white flower croaker, 2.8 ng/ww in mullet, and 2.8 ng/g ww bartail flathead),20 and slightly lower than concentrations (349 ng/g ww in liver) in Chinese sturgeon from the Yangtze River.19 This LOEC was much lower than concentrations detected in Oreochromis mossambicus (1764−7609 ng/g in fish fat samples) from a DDT-sprayed area, the Luvuvhu River in South Africa38 and concentrations (780 ng/g ww) in shovelnose sturgeon (Scaphirhynchus albus) from the Mississippi River.10 The intersex incidence of 48−63% has been reported in male Oreochromis mossambicus from the Luvuvhu River,38 and 29% in male shovelnose sturgeon from the Mississippi River.10 Based on the relationship between body concentrations and intersex incidences in medaka obtained in this study and the concentrations of p,p′-DDE reported in fish from the Luvuvhu River, the intersex incidence by p,p′-DDE was assessed to be 8.05 ± 3.2% assuming that medaka has similar sensitivity as fish from the Luvuvhu River. Intersex in Male Medaka Exposed to o,p′-DDT. Intersex with different severities at level 1, level 2, and level 4 (Figure 2B, C, and D) were observed in the testis of males exposed to o,p′-DDT at different concentrations. The intersex severity showed dose-dependent increases with increasing o,p′-DDT exposure concentrations. Intersex at level 1 and level 2 were observed at exposure concentrations of 0.005, 0.05, and 0.5 μg/ L of o,p′-DDT, and sex-reversal (level 4) was induced when exposure concentration increased to 5 μg/L (Figure 2E-I). The intersex incidences in male medaka fish exposed to o,p′DDT were 7.1% (2/28, p = 0.18), 13.8% (4/29, p = 0.038), 11.8% (6/51, p < 0.001), 25.0% (9/36, p = 0.003), 68.6% (11/ 16, p < 0.001), and 100% (40/40, p < 0.001) at body concentrations of 57.7, 474.2, 1394, 2091.6, 29 183.8, and E
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Figure 3. Relative gene expression of VTG1, VTG2, CHG-H, and CHG-L in livers and CYP19a and CYP17 in gonads of male medaka fish after exposure to p,p′-DDE (A) and o,p′-DDT (B). * indicates p < 0.05.
μM.21 Therefore, the stronger potency to induce intersex of o,p′-DDT than p,p′-DDE in vivo could be attributed to differences in their mechanisms of endocrine disruption. Direct binding and activation of the ER may elicit stronger potency to induce intersex than indirectly induced estrogenic activity by inhibiting androgenic activity in male fish. To explore this hypothesis, expression of estrogen-related genes including VTG-1, VTG-2, CHG-H, and CHG-L in liver of male medaka were determined. The expression of VTG-1 and VTG-2 were significantly up-regulated in a dose-dependent manner, in fish exposed to 0.02, 0.2, 2, and 20 μg/L of p,p′-DDE, relative to control, and responses ranged from 15.5 ± 0.42 to 37.8 ± 16.2fold, and 8.2 ± 0.83 to 24.0 ± 42.7-fold, respectively (Figure 3A). The dose-dependent increase in CHG-H (174 ± 90.7 to 726 ± 360 fold) and CHG-L (16.3 ± 11.7 to 75.3 ± 35.7 fold) were also found in p,p′-DDE exposure groups. The upregulation of these genes were in agreement with a previous study on p,p′-DDE exposure.25 In o,p′-DDT exposure groups, the dose-dependent up-regulation of VTG-1 and VTG-2 (ranging from 3.1 ± 2.2 to 1060 ± 6,714-fold and 4.6 ± 5.8 to 919 ± 2383-fold) were 3−30 fold higher than that resulting from p,p′-DDE exposure at similar concentrations. The upregulation of CHG-H and CHG-L which ranged from 1216 ± 1821 to 43 189 ± 322 106-fold and 54.3 ± 88.4 to 1103 ± 2156-fold, respectively, were also observed in the liver of male medaka (Figure 3B), which were 8−250 fold higher than those in male medaka exposed to similar concentrations of p,p′-DDE. This finding further suggested a stronger estrogenic effect of o,p′-DDT than p,p′-DDE, which is accordant with the higher intersex incidence in o,p′-DDT exposure groups than p,p′-DDE. Stronger estrogenic activity of o,p′-DDT was also observed in the summer flounder (Paralichthys dentatus) where levels of vitellogenin protein were induced after exposure to o,p′-DDT, though there are no reported changes in fish exposed to similar concentrations of p,p′-DDE.39 The effects of p,p′-DDE and o,p′-DDT on the biosynthesis of endogenous 17β-E2 in male medaka were further investigated.
Expressions of CYP19a mRNA and CYP17 mRNA in testis were up-regulated by 5.5−7.6-fold and 1.8−3.6-fold in p,p′DDE exposed male fish, respectively. As for o,p′-DDT, whereas gene expression of CYP17 was up-regulated by 1.2−14.2-fold, which were slightly higher than that of p,p′-DDE, CYP19a were greatly up-regulated by 7.7−62.0-fold (Figure 3B). Since gonadal aromatase (CYP19a) is important to the synthesis of 17β-E2, the dose-dependent up-regulation of gonadal CYP19a in males would increase endogenous concentrations of 17β-E2 which would further contribute to estrogenic effects, and therefore plasma 17β-E2 concentrations were determined. While there were no significant variations in concentrations of 17β-E2 between males from p,p′-DDE exposure groups and the control group, the plasma 17β-E2 levels in males from the 0.005, 0.05, 0.5, and 5 μg/L o,p′-DDT exposure groups were 1.1 ± 0.2, 0.9 ± 0.2, 1.3 ± 0.3, and 2.6 ± 1.0 ng/mL, respectively, significantly higher than those in males from the control group (0.4 ± 0.3 ng/mL) (Figure 4). The increase in expression of CYP19a and elevated levels of plasma 17β-E2 were also observed in male medaka after exposure to another estrogenic compound (EE2).40 Thus, the endogenously induced 17β-E2 in male fish from o,p′-DDT groups could
Figure 4. Concentrations of 17β-estradiol (17β-E2) in plasma of male medaka from o,p′-DDT exposure groups. Blood of two male medaka of the same group were pooled together. Each group contained four replicates (n = 4). Significance was shown with (*), where * indicates p < 0.05; ** indicates p < 0.01. F
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both antiandrogenic and estrogenic chemicals especially with high accumulative ability should be of significant concern.
partly contribute to its relatively high estrogenic effects which induce VTG and CHG, and intersex in male medaka. Reproductive Effects of Gonadal Intersex in Male Medaka. The reproductive effects of male fish with intersex has been documented in wild fish such as wild roach (Rutilus rutilus), and adverse effects on fertilization success have also been observed,8 posing risk to the population.7,41 To investigate the reproductive effects of both p,p′-DDE and o,p′-DDT due to intersexuality in male fish, six male medaka randomly chosen from each exposure group were mix-mated with control female fish in clean water. Of the total 48 male fish in the reproductive study, 13 males elicited intersex (8 in p,p′-DDE treated, and 5 in o,p′-DDT treated groups). When reproductive success data obtained from these 13 male fish were compared to data for fish from control groups, it was found that no significant variation in fecundities occurred, but fertilization rates (p = 0.006) and hatching rates (p = 0.019) were significantly inhibited compared to those from control groups (Figure 5). Similar
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b05042. Text, figures and tables addressing (1) Detailed method for analysis of 17β-E2; (2) Primer sequences of genes used in real-time PCR; (3) Number of fish in p,p′-DDE and o,p′-DDT exposure groups and concentrations of chemicals; (4) Intersex testis of transgenic medaka and light micrograph of the same testis by paraffin section with H&E staining (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone/fax: 86-10-62765520; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China [41330637 and 41171385] is gratefully acknowledged.
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Figure 5. Reproductive toxicity of intersex male medaka exposed to p,p′-DDE and o,p′-DDT. These 13 intersex males were from p,p′-DDE (8 males) and o,p′-DDT (5 males) exposure groups. Reproductive data of the 13 intersex males paired with control females were pooled and compared to control (both male and female were from control). * indicates p < 0.05.
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decreasing fertilization and hatchability rates were observed in intersex males from p,p′-DDE groups and o,p′-DDT groups, but the significances were weak possibly due to the decreased sample size and statistical power, which were 0.024 and 0.076 for intersex males from p,p′-DDE groups (n = 8), and 0.081 and 0.085 for o,p′-DDT groups (n = 5). Of the 13 male medaka with gonadal intersex, one showed serious intersex at level 4, this fish was from the exposure group of o,p′-DDT at 5 μg/L. The female paired with this male still spawned eggs every day as normal, but none of the spawned eggs were fertilized, as such, no eggs hatched. This indicated that the sex-reversed male medaka continued to perform normal mating behavior but with no sperm or low quality sperm. Further study into the mechanism of this effect is warranted. Overall, this study for the first time clarified that both the antiandrogenic chemical, p,p′-DDE and the estrogenic chemical, o,p′-DDT could cause intersexuality in fish at environmentally relevant concentrations. Such a comprehensive study provided further information on the estrogenic potency to induce intersex of male fish via the antiandrogenic pathway. High concentrations of p,p′-DDE are widely reported in wild life, and therefore would likely contribute to the presence of intersex in wild fish. The risk of o,p′-DDT should also be of concern for wild fish, considering its stronger potency than p,p′DDE. Intersex in male fish significantly inhibited fertilization and hatchability, indicating that a reduced capacity to produce viable offspring would occur following exposure to estrogenic compounds and/or antiandrogenic compounds. Therefore, G
DOI: 10.1021/acs.est.5b05042 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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