Article Cite This: Environ. Sci. Technol. 2019, 53, 6804−6813
pubs.acs.org/est
Uptake, Elimination, and Biotransformation Potential of a Progestagen (Cyproterone Acetate) in Tilapia Exposed at an Environmental Concentration Shan Liu,† Haochang Su,§ Heng-Xiang Li,† Jin-Jun Liu,†,∥ Lang Lin,†,∥ Xiang-Rong Xu,*,† Lin-Zi Zuo,† and Jian-Liang Zhao*,‡
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†
Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, P. R. China ‡ The Environmental Research Institute, MOE Key Laboratory of Theoretical Chemistry of Environment, South China Normal University, Guangzhou 510006, P. R. China § Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, P. R. China ∥ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *
ABSTRACT: Although the distribution of progestagens in aquatic environments has been widely reported, details on their uptake, elimination, and biotransformation in fish have received little attention. This study investigated the uptake, elimination, and biotransformation potential of a progestagen, cyproterone acetate (CPTA), in Nile tilapia (Oreochromis niloticus) exposed to an environmentally relevant concentration under semistatic regimes. CPTA in tilapia tissues followed a similar pattern, reaching a concentration plateau within 4 days of exposure, and dropping to below limits of quantitation within 4 days of elimination. The calculated steady-state bioconcentration factors suggest a low bioconcentration potential of CPTA in juvenile tilapia. Results of enzymatic hydrolysis treatments revealed that no conjugates of CPTA were present in tissues, but conjugated biotransformation products of CPTA were found in bile, liver, and muscle. Most CPTA entered tissues and then was biotransformed into seven different products by phase I and phase II metabolism. The concentrations of endogenous cortisol were significantly influenced by CPTA in plasma and liver during the uptake period. These findings suggest that biotransformation products of CPTA should be considered for the assessment of the bioconcentration potential and ecological effects of progestagens.
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its concentrations up to 262 ng L−1, 2330 ng L−1, 10 ng L−1, and 76 ng L−1, respectively.9,15 Most studies to date have focused on the ecotoxicology of CPTA. It has been reported that high concentrations of CPTA (at μg L−1 level) could alter plasma sex steroid concentrations in mummichog (Fundulus heteroclitus),16 and inhibit gametogenesis in Japanese medaka (Oryzias latipes).17 Lower environmental concentrations (ng L−1) of CPTA appear to evoke somewhat less dramatic responses,18 but were still able to down-regulate transcription of key developmental proteins in zebrafish embryos.19 In addition to these adverse effects on aquatic organisms, CPTA is predicted to bioaccumulate in tissues due to its
INTRODUCTION
Progestagens have received considerable attention by scientists and the public in recent years, due to their potential endocrine disrupting effects on aquatic organisms.1,2 Natural and synthetic progestagens in the aquatic environment mainly originate from humans, livestock, and aquaculture.3−5 As an important representative of synthetic progestagens, cyproterone acetate (CPTA) is used in the treatment of androgenization in women,6 treatment of prostate cancer,7 chemical castration in men,8 contraception,6 and animal growth stimulation in livestock and aquaculture.9,10 CPTA is most widely applied in human use in France,11 Switzerland,1 the Czech Republic,12 and Germany.13 Synthetic progestagens are expected to be more resistant to biodegradation in the environments than natural versions.14 As a consequence, CPTA is frequently detected in wastewater from wastewater treatment plants, livestock, aquaculture, and surface water with © 2019 American Chemical Society
Received: May 14, 2019 Accepted: May 22, 2019 Published: May 22, 2019 6804
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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Environmental Science & Technology hydrophobicity (log Kow = 4.2) (Table S1 of the Supporting Information, SI). A few studies have focused on the bioconcentration of medroxyprogesterone acetate, norethindrone, and levonorgestrel in different tissues of fish under controlled laboratory conditions,20−22 and all three progestagens showed low potential to bioconcentrate in fish tissues.20−22 In contrast, CPTA has been observed to have a high detection frequency, bioaccumulative potential, and detectable concentrations in tissues of fish from aquaculture farms in China.10 However, no studies on the uptake, elimination, and biotransformation of CPTA in fish have been reported. The objective of the present study was to conduct a controlled laboratory experiment to evaluate the uptake, elimination, bioconcentration, and biotransformation potential of CPTA in different tissues of Nile tilapia (Oreochromis niloticus). Nile tilapia (Oreochromis niloticus), one of the most important species in commercial fisheries and widely distributed in freshwater environment around the world,23 was selected in this study. We examined the impact of CPTA on endogenous steroids and the potential indicators of CPTA presence in different tissues, in order to address knowledge gaps concerning the bioconcentration, biotransformation potential, and influence on endogenous steroids of CPTA in tilapia at an environmentally relevant concentration. These findings will be informative for the development of aquaculture management policies regarding CPTA.
CPTA in the tissues (Table S3), a nominal concentration of 100 ng L−1 was selected. The experimental setup consisted of one control (0.0001% [v/v] of dimethyl sulfoxide [DMSO], equivalent to 1.1 mg L−1) and one CPTA group (100 ng L−1) with the same DMSO concentration. For each group, there were four glass tanks (150 L capacity containing 114 L water), in quadruplicate (three tanks for tissue distribution, uptake, elimination, endogenous steroids analysis, and one tank for biotransformation product analysis), filled with dechlorinated tap water (Figure S1). At the initiation of exposure (t0: 0 h), 15 fish were randomly assigned to each glass tank. Before daily water replacements, water samples were collected to detect the temperature, pH, dissolved oxygen, and CPTA concentrations (Tables S4 and S5). On days 0, 1, 2, 4, 7, and 12 for uptake and days 12, 13, 14, 16, and 24 for elimination, three fish were randomly taken from each group and anesthetized with 0.01% tricaine methanesulfonate (Sigma-Aldrich). All experiments were performed in three replicates. Since CPTA was not detected in tissues on day 24, only days 12, 13, 14, and 16 were selected as elimination periods. Consequently, the experiment ran for 16 days including 12 days uptake and 4 days elimination. Blood, bile, liver, and muscle were immediately collected and weighed. The blood was collected using a prerinsed sodium heparin syringe and then stored in a 2 mL plastic vial for 8 h at 4 °C. The plasma was obtained from the supernatant in the vial after centrifugation at 10 000g for 10 min. The bile was obtained from the gall bladder by a syringe needle and stored in a 2 mL cryogenic vial. Approximately 1 g of muscle and 0.1 g of liver (both in wet weights) were dissected and stored in cryogenic vials separately. All tissues except the plasma were immediately stored at −20 °C until extraction. The experimental design for biotransformation products analysis only included 12 days of uptake. The tissue and water samples were collected on days 0 and 12 to identify the biotransformation products. All procedures were approved by the Animal Research Ethics Board of Chinese Academy of Sciences and were in accordance with the Guidelines of the Chinese Council on Laboratory Animal Care. Sample Pretreatment and Instrumental Analysis. Sample pretreatment and instrumental analysis followed previously established analytical methods with modifications.3,27,28 Briefly, water and plasma samples were extracted by solid-phase extraction using HLB cartridges. Bile samples were extracted by SAX/PSA-HLB tandem cartridges. Liver and muscle samples were homogenized, ultrasonicated, purified, and enriched using SAX/PSA-HLB tandem cartridges. Feed samples were freeze-dried, homogenized, and extracted by ultrasonication. The extracted tissue samples were divided into two 100-μL duplicates. One of the two 100-μL duplicates was used to measure the concentrations of target steroids (CPTA and endogenous steroids) in free form. Another 100-μL duplicate was subjected to enzymatic hydrolysis to release glucuronide and sulfate metabolites to free forms, followed by liquid−liquid extraction before analysis. The target steroid compounds and biotransformation products were analyzed using an Agilent 1200 LC-Agilent 6460 QQQ (RRLC-MS/ MS) and Agilent 1290 Infinity II LC-Agilent 6545 Q-TOF-MS, respectively. Detailed information is provided in the SI (Texts S2−S4 and Tables S1, S3, and S6). Quality Assurance, Quality Control, and Data Analysis. The limit of quantitation (LOQ) for each target compound was calculated based on the signal-to-noise ratio (SNR) near the target peak. LOQ is defined as ten times of the
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MATERIALS AND METHODS Chemicals. The synthetic progestagen cyproterone acetate (CPTA), along with seven endogenous steroids including three androgens [4-androstene-3,17-dione (AED), epi-androsterone (EADR), and testosterone (T)], two glucocorticoids [cortisol (CRL) and cortisone (CRN)], two progestagens [hydroxy progesterone (HP) and progesterone (P)], and three internal standards [testosterone-16,16,17-d3 (T-d3), cortisol-d2 (CRLd2) and progesterone-d9 (P-d9)] were used (Table S1). Oasis HLB cartridges (N-vinylpyrrolidone-m-divinylbenzene copolymer, 500 mg/6 mL or 200 mg/6 mL) and strong anion exchange/primary-secondary amine (SAX/PSA) cartridges (500 mg, 6 mL) were obtained from Waters Corporation (Milford, MA, U.S.A.) and CNW Technologies (Shanghai, China), respectively. Details regarding chemicals and materials are summarized in the SI (Text S1). Fish Exposure. In August 2017, juvenile tilapia (17 ± 3.3 g wet weight) were purchased from the Fish Germplasm Center of Guangdong Province (Guangzhou, China) and allowed to acclimatize to laboratory conditions for 2 weeks before use. The experiments generally followed the guidelines of the Organisation for Economic Co-operation and Development (OECD).24 The optimal temperature for growth of most of tilapiine species is reported to be between 25 and 28 °C.25 Tilapia mortality starts at 13.6 °C and total mortality occurs at 8.6 °C.26 Considering the optimal temperature of tilapia and the OECD recommended test temperature,24 we set 22 °C as the experimental exposure temperature. All fish were maintained under a 16:8-h (light/dark) photoperiod and fed with commercial pellets at a rate of 1% body weight per day. Uneaten food and feces were siphoned daily from the tested tanks after feeding (0.5−1 h). The experiment was conducted in semistatic regimes with the aerated water being replaced every day. On the basis of reported environmental concentrations of CPTA (Table S2) and the limits of quantification of 6805
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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Environmental Science & Technology
Figure 1. Uptake (0−12 days) and elimination (12−16 days) of CPTA in tissues of Nile tilapia exposed to 100 ng L−1 CPTA. The red and blue circles represent uptake and elimination data, respectively, and solid lines are kinetic fittings followed a pseudo-first-order model. The blue hollow circles represent data below limits of quantitation.
concentration (ng L−1) in the exposure water. The tissue burden of CPTA is calculated by the equation Burden = Ctissue × Mtissue, where Ctissue is the mean concentration of CPTA in fish tissue at certain time point (ng g−1 or μg L−1) and Mtissue is the mean mass of tissue per fish. Details on these coefficients are provided in the SI (Text S5). One-way analysis of variance (ANOVA) was used to assess differences in fish weight, length, and steroid concentrations among sampling days. Prior to ANOVA analysis, KolmogorovSimirnov and Levene’s tests were used to test the assumptions of normality and homogeneity of variances, respectively. Independent-samples t-test was used to compare endogenous steroid concentrations between exposure and control groups, and CPTA concentrations between enzymatic hydrolysis and nonenzymatic hydrolysis samples. Statistical significance was accepted at p < 0.05 level. A Pearson correlation analysis was conducted to identify correlation among different steroids in tissues. All data analyses were performed using software SPSS 17.0 and Sigma Plot 12.5.
SNR. The LOQs for the target analytes were in the ranges of 0.12 to 5.9 ng L−1 in water, 0.036 to 2.0 ng g−1 in muscle, 0.66 to 34 ng g−1 in liver, 0.37 to 7.6 μg L−1 in plasma, and 0.76 to 4.3 μg L−1 in bile, respectively (Table S3). The recoveries for all target steroids in matrix spiked samples of water, muscle, liver, bile, and plasma were generally observed in the range of 70 to 130% (Table S3). Both intra- and interday precisions of the RRLC-MS/MS instrument were examined. For the intraday precision, a standard solution (50 μg L−1 of each compound) was injected successively seven times. For the interday experiment, five standard solutions (50 μg L−1 of each compound) were analyzed on five different days over a onemonth interval. The relative standard deviation was less than 15%. For the detailed information, please refer to the SI (Text S3 and Table S3). Tissue-plasma partition coefficients (Kp) were calculated for the evaluation of the affinity between CPTA and macromolecules (i.e., albumin, globulins and/or lipoproteins) in plasma and other tissues.29 The values of Kp for different tissues were calculated by the ratio of CPTA levels (mean concentrations) in tissues (bile, liver, and muscle) and corresponding values (mean concentrations) in plasma at each time point during the uptake period. Steady-state bioconcentration factors (BCFss) and tissue burdens were calculated to assess the bioconcentration potential in different tissues. BCFss is calculated by the equation BCFss = 1000 × Cf(ss)/Cw, where Cf(ss) is CPTA concentration in fish tissue (ng g−1 or μg L−1) at the end of the uptake phase, and Cw is CPTA
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RESULTS AND DISCUSSION Levels of CPTA and Endogenous Steroids in Water. No steroids were detected in feed samples, indicating that CPTA in fish tissues only came from water exposure (Table S7). For the control group, no CPTA was detected in water samples (Table S5). For the exposure group, the concentrations of CPTA in water samples was 84 ± 11 ng L−1 during the uptake period, with relative standard deviation within 20% 6806
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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calculate the kinetic parameters,24 the valid sampling points were insufficient and may have led to uncertainty in the calculation of these parameters. Thus, we did not discuss the kinetic parameters. During the uptake period, concentrations of CPTA showed similar trends in fish tissues (except for bile), quickly increasing from day 0 to day 2 and remaining stable from day 4 onward (Figure 1). For bile samples, the concentrations of CPTA reached a steady state on day 7. In addition, tissue-plasma coefficients (Kp) were calculated.29 During the uptake period, the mean Kp values of CPTA ranged from 1.9 to 4.3 and 0.67 to 2.0 in liver and bile samples, respectively, which were much higher than those in muscle samples, indicating that CPTA showed a high affinity for macromolecules (i.e., albumin, globulins and/or lipoproteins) in liver and bile but not in muscle (Table S12). This difference was probably due to the detoxification and digestion activities of the liver and gall bladder for CPTA,36 as well as the low bioaccumulative potential of the muscle.10 During the elimination period, the concentrations of CPTA detected in all tissues decreased quickly and dropped below LOQs between days 12 and 16 (Figure 1), indicating that all tissues showed strong elimination abilities. Similar results have been found in previous studies. For example, the half-lives of 17α-ethinylestradiol in goldfish (Carassius auratus) and trenbolone in rainbow trout (Oncorhynchus mykiss) were 0.38 d37 and 0.99 d,38 respectively. These results indicate that CPTA, like estrogens and androgens, can be quickly eliminated from fish tissues. Bioconcentration Patterns of CPTA in Different Tissues. BCFss values for CPTA on day 12 were 76, 321, 115, and 20 in the plasma, bile, liver, and muscle, respectively (Table S13). These were higher than those of fish exposed to 50−118 μg L−1 of progestagens under laboratory conditions (4.3−37.8),20,21 but markedly lower than fish exposed to < LOQ-1 ng L−1 of progestagens in aquaculture farms, treated sewage effluents, or rivers (42−65 000).10,39,40 These differences in BCF values are probably due to differences in exposure concentrations and time.41 Higher exposure concentrations could increase the absolute mass in tissues, but BCF values in fish tissues showed an inverse bell pattern with the increased exposure concentration.39,41 For example, when fish were exposed to the low concentrations (ng L−1) of estrogens, the BCF values increased with the increased exposure concentration.39,41 In contrast, when fish were exposed to the relatively high concentrations (μg L−1), the BCF values decreased with the increased exposure concentrations, probably due to the saturation of uptake/elimination processes or the functional impairment of metabolism and excretion of toxicants from the fish body.39,41 Exposure time also likely plays an important role. Several studies have confirmed that the bioconcentration potential of xenobiotic pollutants in tissues can increase with exposure time.41,42 In short-term exposure, the tissues of common carp (Cyprinus carpio) and channel catfish (Ictalurus punctatus) showed a strong bioconcentration potential for medroxyprogesterone acetate and norethindrone in the initial several days.20,21 In long-term exposure, the concentrations of estrogens in muscle of crucian carp (Carassius carassius) increased with exposure time over the first 12 months.41 A target chemical with BCF value less than 1000 is not considered to be bioaccumulative.43 The maximum BCF value of CPTA in this study was below 1000, demonstrating a low bioconcentration potential of CPTA in juvenile tilapia,
of the nominal concentration (Table S5), consistent with the guidelines in the OECD.24 The stability of CPTA in the exposure tanks during 24 h with a nominal concentration of 100 ng L−1 was tested. The concentrations of CPTA at the beginning of exposure (t0: 0 h) and prior to water renewal (t24: 24 h) were 80 ± 3.4 ng L−1 and 87 ± 3.9 ng L−1, respectively (Table S8). These results indicate that CPTA was stable during the uptake experiment and could satisfy the requirements of toxicokinetic models. Notably, there were detectable levels of CPTA ( plasma, bile > muscle (ANOVA, p < 0.05), consistent with the bioaccumulation patterns of medroxyprogesterone acetate and norethindrone for several species.20,21 During the uptake period, the mean concentrations of CPTA in plasma, bile, liver, and muscle samples from the exposure group were in the ranges of 6.3−13 μg L−1, 4.8−21 μg L−1, 19−27 ng g−1, and 1.7−2.5 ng g−1, respectively, close to synthetic progestagens detected in wild environments, marketed fish and aquaculture farms, but lower than those detected in laboratory conditions with high exposure concentrations (μg L−1) (Tables S10 and S11). It is worth noting that the maximum concentration of CPTA (13 μg L−1) in plasma samples was close to the human therapeutic CPTA plasma level (15 μg L−1), suggesting that CPTA might elicit a pharmacological effect on fish.2,35 To test whether conjugates of CPTA were an important repository, enzymatic hydrolysis was also conducted for all tissue samples from the exposure group. No statistically significant difference (independent-samples t-test, p > 0.05) was found between enzymatic hydrolysis and nonenzymatic hydrolysis treatments at the same sampling points (Figure S2), suggesting that no glucuronidated or sulfated conjugates of CPTA were present in tilapia tissues. The reason that no conjugate was detected was probably due to the absence of a hydroxyl group in CPTA which could be directly conjugated with glucuronic acid or sulfate. Uptake and Elimination of CPTA by Tilapia. The uptake and elimination processes of CPTA followed a pseudofirst-order model (Figure 1). Although our sampling points could basically meet the OECD minimum sampling method to 6807
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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Figure 2. 1) Relative tissue burdens of CPTA at different time points; 2) contribution of each fraction to the overall CPTA and biotransformation products (free forms and conjugated forms of products) in different tissues on day 12.
Figure 3. Proposed pathways (①−⑦) for the biotransformation of CPTA in fish tissues and water samples. Products (D1−D3) and products (P1− P4) were identified by LC-Q-TOF MS with enzymatic hydrolysis and nonenzymatic hydrolysis, respectively. Intermediates (T1 and T2) were inferred by proposed pathway.
a maximum on day 2 and then fluctuated with exposure time. Muscle contributed most of the total body burden of CPTA, although the residual concentration of CPTA in muscle was the lowest (Figure 2 and Table S14). This was owing to the mass fraction of muscle relative to the total body being at the maximum with a percentage of 67%,44 indicating that muscle was the most important storage tissue in tilapia. Biotransformation Products. The list of identified biotransformation products, the corresponding total ion
apparently different from adult tilapia (BCF, 3400−16 000).10 These results also suggest that a short-term exposure under laboratory conditions might underestimate the bioconcentration ability of the target pollutant. The tissue burdens of CPTA were also calculated to assess the bioconcentration potential. During the uptake period, the tissue burdens of CPTA were up to 9.0 ng, 2.6 ng, 2.2 ng, and 29 ng per fish in the plasma, bile, liver, and muscle, respectively (Table S14). The total tissue burden of CPTA quickly reached 6808
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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Figure 4. Concentrations of endogenous steroids in different tissues during the uptake periods (0−12 d) and the elimination periods (12−16 d). *p < 0.05, **p < 0.01 indicate significant differences between the exposure group and control group.
proterone acetate in these species.46 In this study, 15βhydroxycyproterone acetate was searched for in water and tissue samples by LC-Q-TOF MS using the purpose-built database, but it was not detected. This differences may reflect metabolic differences between species or different instrument sensitivities. Furthermore, two transformation products (P1 and P4) were found in water samples. Besides liver, other tissues including kidney, gill, and intestine are also important metabolic organs in fish.45 Thus, biotransformation products detected in the water samples may have originated from excretion via kidney, gills, and feces. It is worth noting that three new products, 6-dechlorinated3-dihydro-15-hydroxy-CPTA (D1), 6-dechlorinated-3,6-tetrahydro-17α-deacetylated-CPTA (D2), and 6-dechlorinated-3dihydro-17α-deacetylated-CPTA (D3), were found after enzymatic hydrolysis treatment, indicating that these products should exist as conjugated forms (glycosylation/sulfation) in bile, liver, and muscle following phase II metabolism (Figures S14−S17). The conjugation reaction is predicted to happen at position C-3 of Ring-A, because there is a hydroxyl group at position C-3 and the conjugated products of steroids at the same position have been reported in previous studies.45,47 Proposed Biotransformation Pathways for CPTA. Two intermediates (T1 and T2) were added in Figure 3 for speculating the proposed biotransformation pathways. On the basis of the identified biotransformation products with
chromatograms, and extracted ion chromatograms in different media are shown in Table S15 and Figures S3 and S4. Four free forms of biotransformation products, 6-dechlorinated-15hydroxy-17α-deacetylated-CPTA (P1), 6-dechlorinated-11,15dihydroxy-CPTA (P2), 6-dechlorinated-6-dihydro-17α-deacetylated-CPTA (P3), and 6-dechlorinated-22-dihydro-CPTA (P4), were tentatively identified based on the mass/mass spectra from LC-Q-TOF MS analyses of fish tissues and water samples following nonenzymatic hydrolysis treatment (Figures S5−S13). Conjugated products were not identified by LC-QTOF MS in any sample following nonenzymatic hydrolysis treatment due to the low matching degree. Only one free form of biotransformation product was found in plasma, bile, and liver samples, and two free forms of biotransformation products were found in muscle (Figure 3). To our knowledge, these four biotransformation products are reported here for the first time. Dechlorination, hydrolysis, hydrogenation, and hydroxylation reactions could explain the biotransformation mechanism, indicating that phase I metabolism played an important role during the biotransformation of CPTA in tilapia. Phase I metabolism would increase the polarity and water solubility of the biotransformation products.45 These results differed from the biotransformation products of CPTA identified in dogs, monkeys, and humans.46 It has been reported that CPTA is metabolized primarily by hydroxylation via CYP3A4, forming the major metabolite 15β-hydroxycy6809
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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progestins may alter glucocorticoid receptors-mediated transcription52 or steroidogenesis enzymes,53 which could influence the production of glucocorticoids and their concentrations in fish. Besides glucocorticoid activities, CPTA also has antiandrogenic activity.1 The concentrations of plasma T in adult mummichog (Fundulus heteroclitus) and zebrafish (Danio rerio) decreased even under exposure to CPTA in water at 10 ng L−116 and in food at 0.8 mg g−1,54 respectively. However, there were no significant differences in the plasma samples of the two androgens AED and T at different time points between the control and exposure groups (ANOVA, p > 0.05). These results demonstrate that the different sensitivities of androgens in fish plasma might be influenced by species or life-stage.16,54,55 Interspecies differences have been confirmed in the binding affinity between androgen receptors and CPTA, which competes for binding sites in the brain and gonads of goldfish but not in rainbow trout.55 Only one endogenous cortisol was detected in bile samples. During the first 7 days, no apparent changes in cortisol levels in bile were detected in either the exposure (168 ± 63 μg L−1) or the control group (192 ± 91 μg L−1) (Figure 4). In the exposure group, the concentrations of cortisol detected in the bile samples increased on the last day of uptake and during the elimination period. However, there were no significant differences in cortisol levels detected in the bile samples at different time points between the exposure and control groups (ANOVA, p > 0.05). These results demonstrate that the level of cortisol in the bile adapted readily under a relatively low CPTA exposure concentration and short exposure time. Similarly to bile samples, the only endogenous steroid detected in the liver was cortisol (Figure 4). In contrast to bile samples, the concentrations of cortisol in the liver samples during the uptake period were significantly influenced by CPTA (ANOVA, p < 0.05). This was probably owing to different binding affinities of CPTA for the same steroid receptor in different tissues.55 Compared with the control group, the levels of cortisol in the exposure group showed an initial steady state during the first 4 days, then gradually increased over time during the uptake period (Figure 4). The concentrations of cortisol on day 12 were still significantly higher (p < 0.05) than at previous time points, indicating that the balance of cortisol did not appear to reach a steady-state plateau by the end of the uptake period (day 12). During the elimination period, the levels of cortisol quickly decreased to the control levels within 2 days, demonstrating that the effects of CPTA on cortisol in liver could be rapidly restored after a short time and a low concentration exposure. The three endogenous steroids, cortisol, AED, and T, were detected in muscle (Figure 4). Cortisol was detected more frequently (100%) than AED (46−63%) or T (29−58%) in either the exposure or control group. During the uptake period, the concentrations of cortisol detected in muscle samples were stable during the first 7 days, then showed an approximately 4fold increase on day 12 compared with the control group (ANOVA, p < 0.05). This indicates that CPTA did not have an impact on the levels of cortisol in the muscle for the first 7 days. During the elimination period, there were no significant differences among different sampling points. On day 16, the concentration of cortisol was still about 2-fold higher than detected in the control group at the same time point (ANOVA, p < 0.05), indicating that the levels of cortisol did not easily return to normal levels during the 4-days elimination period.
different pretreatments, seven biotransformation pathways were tentatively proposed for CPTA (Figure 3). The first two pathways (① and ②) occurred at positions C-6 (Ring-B), C-15 (Ring-D), and C-17α (Ring-D). CPTA was initially transformed into intermediate T1 by hydrolysis at position C17α and dechlorination at position C-6. As we only detected the products on day 12, we could not deduce the orders of hydrolysis and dechlorination. For chlorinated compounds, dechlorination is a common metabolic pathway of phase I metabolism in fish.48,49 Hydrolysis at position C-17 can increase the metabolic capacity of the parent.11 Subsequently, the intermediate T1 was transformed into products P1 and P3 by hydroxylation at position C-15 and hydrogenation at position C-6, respectively. Introduction of hydroxyl groups into the steroid rings somewhat increases the water solubility of the molecule.45 Besides hydroxylation, reduction (hydrogenation) is a general pathway of progestagens in tissue.11 In the third pathway, a dihydroxylated product (P2) was found. Products with similar hydroxylation positions have been reported in progestagen metabolites.11,50 After dechlorination reaction at position C-6, CPTA was converted into P4 by hydrogenation at position C-22 in the fourth pathway. Three new products (D1−D3) were found after enzymatic hydrolysis treatment. Similar to the first two pathways, CPTA was initially transformed into intermediate T1 by hydrolysis and dechlorination. Then the hydrogenated products D2 (at positions C-3 and C-6) and D3 (at position C-3) were formed in the fifth and sixth pathways, respectively. In the seventh pathway, CPTA was transformed into intermediate T2 by dechlorination and subsequently into D1 by hydroxylation at position C-15 and hydrogenation at position C-3. In comparison with products obtained by nonenzymatic hydrolysis treatment, the biggest difference with these new products was the reduction on position C-3. Hydrogenation at position C-3 can decrease the progestagenic activity and further facilitates conjugation and elimination.45,51 On the basis of the peak area ratios of biotransformation products to the overall CPTA and biotransformation products (free and conjugated forms of products), the yields for the products were estimated (Figure 2 and Table S16). After 12 days of exposure, residual levels of biotransformation products (both in free and conjugated forms) remained high (99% in plasma, 94% in bile, 96% in liver, and 97% in muscle) (Figure 2), demonstrating that the bulk of the CPTA entering tissues was biotransformed. No conjugated product was detected in plasma. On the contrary, a high proportion of conjugated products were identified in bile (50%), liver (86%), and muscle (49%). However, the absolute peak area and the proportion of the specific conjugated form (glycosylation or sulfation) were unavailable due to lack of corresponding standards to quantify. Impact of CPTA on Endogenous Steroids in Different Tissues. Four endogenous steroids were detected in plasma samples (Figure 4), with cortisol being the most dominant. The other endogenous steroids, cortisone (detection frequency, 100%), AED (96−100%), and T (41−56%), had high detection frequencies in the plasma samples for different groups. In the control group, the levels of these four steroids were stable throughout the exposure period, indicating that the addition of DMSO had no significant effect on endogenous steroids in tilapia. In the exposure group, the concentrations of the glucocorticoid cortisol in the plasma samples during the uptake period were influenced by CPTA and showed an increasing trend (Figure 4). Recent studies have noted that 6810
DOI: 10.1021/acs.est.9b02891 Environ. Sci. Technol. 2019, 53, 6804−6813
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For AED and T, there were some increases on day 2, but no significant differences were found in levels of these two androgens in the muscle samples between the exposure and control groups (ANOVA, p > 0.05), indicating that CPTA exposure did not influence these two androgens in the muscle samples within the exposure time. Chemical Indicators of Steroids in Fish Tissues. We found positive correlations between cortisol and CPTA in plasma and liver samples during the uptake experiment (p < 0.05) (Figure S18). These results differed from those in a previous study of wild fish, in which the androgen AED was found to be a reliable indicator of levels of most steroids (including CPTA) with relatively high detection rates in plasma.10 The difference in indicator accuracy between the laboratory experiment and the field investigation may be related to the differences in species, life-stages, exposure time, and types of steroids. For instance, higher levels of progestagens were detected in tissues of tilapia than those of spotted scat under the same culture condition and at a similar stage of growth,10 indicating a species-specific influence on the distribution of steroids. Life-stage is another important factor. In a previous study, adult tilapia showed stronger bioaccumulation of CPTA than the juveniles in the current study.10 Longterm and short-term exposures have been observed to cause similar patterns of change in exogenous steroids in fish tissues (i.e., increasing at first then remaining stable).20,21,41 Besides biological factors, the physicochemical property of a steroid, for example log Kow, is also an important influence factor. Log Kow had positive correlation with Log BAF (the bioaccumulation factor) for the distribution of steroids in fish tissues.10 In contrast, fish gender had little or no influence on bioconcentration of steroids in fish tissues. It has been reported that the levels of exogenous estrogens were similar among males and females of wild fish collected downstream of municipal effluents.56 In teleosts, cortisol is indispensable for homeostasis and mediation of disease resistance.52 A lifelong exposure to chemical pollutants may lead to an exhaustion of the cortisolproducing endocrine system in fish,57 possibly as a result of reduced sensitivity of cortisol to environmental pollutants. Therefore, cortisol is better suited as a chemical indicator to predict levels of CPTA in plasma and liver of Nile tilapia under short-term exposure to CPTA. Environmental Implications. This study provides a comprehensive assessment of the uptake, elimination, and biotransformation potential of CPTA in Nile tilapia exposed to an environmentally relevant concentration. On the basis of the results of uptake and elimination, CPTA is quickly eliminated from fish tissues. Therefore, cultured fish should be moved to unpolluted water for several days’ elimination prior to sale, which will be effective in reducing human health risks via ingestion of progestagen-contaminated fish. Alternatively, a reduced daily intake of tilapia may be recommended for sensitive populations such as children and pregnant women. We also observed that the bulk of the CPTA in tissues was transformed, but information regarding these biotransformation products is generally lacking. Further studies on the biotransformation of CPTA are needed for further understanding of the fate, bioaccumulation behavior, and toxicity of these byproducts in aquatic organisms.
Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.9b02891. Detailed sample preparation, instrumental analysis, data analysis, and additional tables and figures (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Phone/fax: +86 20 89100753; e-mail:
[email protected] (X.R.X.). *Phone/fax: +86 20 39311065; e-mail:
[email protected]. edu.cn (J.-L.Z.). ORCID
Shan Liu: 0000-0001-7432-185X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge the support from the National Natural Science Foundation of China (Nos. 41501534 and 41876129); the National Key Research and Development Program of China (Nos. 2017YFC0506302 and 2016YFC0502805); Natural Science Foundation of Guangdong Province, China (Nos. 2014A030310223 and 2016A030313156); and Science and Technology Planning Project of Guangdong Province, China (No. 2017B030314052).
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REFERENCES
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