Aquatic Toxicology 160 (2015) 69–75
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Dose-dependent compensation responses of the hypothalamic-pituitary-gonadal-liver axis of zebrafish exposed to the fungicide prochloraz Yao Dang a , John P. Giesy b,c,d,e , Jianghua Wang a,∗ , Chunsheng Liu a,∗ a
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada c Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China d School of Biological Sciences, University of Hong Kong, SAR, Hong Kong, China e State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China b
a r t i c l e
i n f o
Article history: Received 8 November 2014 Received in revised form 5 January 2015 Accepted 7 January 2015 Available online 8 January 2015 Keywords: HPG axis Compensation responses Zebrafish Prochloraz Aromatase inhibitor
a b s t r a c t Compensation responses and adaptability of hypothalamic-pituitary-gonadal (HPG) axis have been reported in fish exposed to model chemicals, however due to its importance in predictive toxicology further study was needed to elucidate details of the integrated responses to model chemicals. Transcriptional profiles of the hypothalamic-pituitary-gonadal (HPG) axis and concentrations of 17-estradiol (E2) in plasma were measured in male and female zebrafish that had been exposed to one of seven concentrations of the fungicide, prochloraz: low (1, 3 or 10 g/L), medium (30 or 100 g/L) or high concentrations (300 or 1000 g/L) for 4 days. In zebrafish exposed to the low and medium concentrations of prochloraz, compensation responses of the HPG axis through transcription, occurred in brain (up-regulation of gnrh, gnrhr and lhˇ) and both brain and gonad (up-regulation of steroidogenic genes), respectively. Concentrations of E2 in plasma and expression of estrogen receptor 1 (er1) and vitellogenins (vtgs) in liver did not change. This result suggested that compensatory responses were successful in maintaining homeostasis. In zebrafish exposed to the two greatest concentrations, compensatory responses occurred in brain, gonad and liver through up-regulation of er2ˇ, but it failed to maintain concentration of E2 in blood plasma and expression of er1 and vtgs in liver. Collectedly, the results observed in this study allowed characterization of dose-dependent compensatory responses along the HPG axis and liver and identified key linkages between compensatory responses occurring in brain, gonad and liver after exposure to prochloraz. © 2015 Elsevier B.V. All rights reserved.
1. Introduction As an imidazole fungicide, prochloraz is registered for various agricultural uses throughout the world (http://pubchem.ncbi. nlm.nih.gov/summary/summary.cgi?cid=73665). This chemical can act as an inhibitor of cytochrome P450 (CYP) 14␣-demethylase (CYP51), which is a key enzyme in the synthesis of ergosterol, and therefore inhibits growth of fungi (van den Bossche et al., 1987, 1982). However, prochloraz can also inhibit activities of other CYP enzymes, including cytochrome P450 c17␣-hydroxylase/17,20lyase (CYP17) and aromatase (CYP19) (Ankley et al., 2009). In
∗ Corresponding authors. Tel.: +86 27 87282113; fax: +86 27 87282114. E-mail addresses: [email protected] (J. Wang), [email protected] (C. Liu). http://dx.doi.org/10.1016/j.aquatox.2015.01.003 0166-445X/© 2015 Elsevier B.V. All rights reserved.
vertebrates, the gene product of CYP17 is responsible for synthesis of testosterone (T) and CYP19 catalyzes conversion of T to 17-estradiol (E2). Therefore, inhibition of activities of the two enzymes would decrease production of both T and E2. These effects have been previously confirmed experimentally, both in vitro and in vivo, in mammals, where exposure to prochloraz significantly inhibited activities of both CYP17 and CYP19 enzymes and decreased concentrations of T and E2 in blood plasma (Blystone et al., 2007; Mason et al., 1987; Noriega et al., 2005; Sanderson et al., 2002; Vinggaard et al., 2000). In fish, treatment with prochloraz decreased concentrations of T and E2 in blood plasma, and affected reproductive function (Ankley et al., 2005, 2009; Marca Pereira et al., 2011a, b; Liu et al., 2011; Skolness et al., 2011; Villeneuve et al., 2007; Zhang et al., 2008a,). Due to its effectiveness as an inhibitor of CYP17 and CYP19 enzymes, prochloraz has been used as a model chemical for studying the responses of
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hypothalamic-pituitary-gonadal (HPG) axes of vertebrates (Ankley et al., 2005, 2009; Marca Pereira et al., 2011a, b; Liu et al., 2011; Villeneuve et al., 2007; Zhang et al., 2008a). The HPG axis is a dynamic endocrine system, that maintains physiological conditions of reproduction by various homeostatic feedback mechanisms during exposure to stressors including chemicals (Ankley et al., 2009). Compensatory responses of the HPG axis have been documented in fish exposed to model chemicals (Ankley et al., 2009; Liu et al., 2011; Skolness et al., 2011; Villeneuve et al., 2007; Zhang et al., 2008a, b, c). For example, results of several studies have demonstrated that exposure to fadrazol, which inhibits the enzymatic activity of CYP19, resulted in decreased concentrations of E2 in blood plasma and caused a time- and dose-dependent up-regulation of genes of HPG axis in small fish. Up-regulation of those genes was considered to be a compensatory mechanism to return concentrations of E2 in blood plasma to pre-exposure values (Villeneuve et al., 2009; Zhang et al., 2008c). Similarly, treatment with prochloraz significantly decreased concentrations of T and E2 in blood plasma and resulted in a comprehensive up-regulation of genes along the HPG axes of fishes (Ankley et al., 2009; Liu et al., 2011; Skolness et al., 2011; Zhang et al., 2008a). Furthermore, in two time-course studies, inhibitory effects of prochloraz or fadrozole on production of E2 in fathead minnow were transitory and did not persist during the 8-day exposure phase, which demonstrated the effectiveness of compensatory responses. Termination of the exposure resulted in recovery of expression of genes and concentrations of steroid hormones, including a brief period of “overcompensation” immediately after cessation of exposure (Ankley et al., 2009; Villeneuve et al., 2009). These results suggest key compensatory responses of the HPG axis after exposure to chemical stressors, and highlight the need to consider these compensatory responses when developing approaches to assess potential risks of chemicals (Ankley et al., 2009; Villeneuve et al., 2009). To better understand compensatory mechanisms in fishes and more accurately predict effects of chemicals based on modes of action a more comprehensive characterization of these dosedependent compensatory responses was still needed. Objectives of the present study were to: (1) examine dose-dependent expression behaviors of HPG axis and genes expressed in liver; (2) compare sensitivities of genes; and (3) identify linkages between responses occurring at different organs, including brain, gonad and liver of zebrafish. 2. Materials and methods 2.1. Chemicals and reagents Prochloraz and TRIzol regent were obtained from Sigma (St. Louis, MO, USA) and Invitrogen (New Jersey, NJ, USA), respectively. Reverse transcription and SYBR Green kits were purchased from Takara (Dalian, Liaoning, China). 17-estrogen (E2) enzyme immunoassay (EIA) kits were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). All the other reagents used in this study were of analytical grade. 2.2. Fish and chemical exposure Zebrafish were maintained in flow-through tanks at 28 ± 0.5 ◦ C with a 12:12 light/dark cycle, and water pH, hardness and dissolved oxygen were routinely monitored. Before exposure, 5-month old males and females (sexual maturity) were acclimated in 15-L tanks filled with 10 L of carbon-filtered water for 1 week. After acclimation, fish were exposed to 0, 1, 3, 10, 30, 100, 300, 1000, 3000 or 10,000 g/L (0, 0.0027, 0.0080, 0.027, 0.080, 0.27, 0.80, 2.7, 8.0
or 27 M) prochloraz for 4 days. Concentrations were selected based on previous studies, where comprehensive compensation responses of HPG axis genes would be expected to occur (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a). Five females and five males were exposed in each of 2 replicated tanks for each concentration. One half of the water in each tank was replaced daily with fresh carbon-filtered water containing corresponding concentration of prochloraz. Both control and exposure groups received 0.01% DMSO since previous study demonstrated that such DMSO concentration did not affect reproductive function (Han et al., 2013). During the exposure period, survival was recorded. After exposure, fish were euthanized and blood was collected for plasma hormone analysis as described before (Liu et al., 2009). Tissues from brain (including hypothalamus and pituitary), gonad and liver were sampled and preserved in TRIzol reagent for subsequent RNA isolation. In this study, experimental procedures were carried out following the approved protocol by Institutional Animal Care and Use Committee (IACUC) of Huazhong Agricultural University. 2.3. Quantification of hormones Briefly, plasma was obtained by centrifugation (5000 × g for 5 min at 4 ◦ C) of whole blood. Plasma from 2 fish was pooled for quantification by use of a commercial EIA kit as described previously (Liu et al., 2009). Briefly, plasma (3 L) from each pooled sample was diluted with 500 L ultrapure water and extracted thrice with 2 mL of ethyl ether, and the ether phase was collected and evaporated. After that, residues were redissolved with EIA buffer provided in the kit and E2 was quantified following manufacturer’s instructions. The limit of quantification was 6 pg/L, and the intra- and inter-assay coefficients of variance (CV) were in ovary > in liver (Table 1). The rank order of OECPs were genes in brain > in ovary > in liver (Table 1).
in brain, testis and liver (Fig. 3 and S4–S6, see Supporting Information). In brain, expressions of gnrh2, gnrhr1, gnrhr2, gnrhr3, gnrhr4, lhˇ, er1 and er2˛ were up-regulated, compared with their levels of expression control. In testis, expression of star, cyp17 and cyp19a were significantly up-regulated, while expressions of er1 and er2˛ were down-regulated. In liver, exposure to 1000 g/L prochloraz down-regulated expression of vtg1, but no statistically significant effects were observed when fish were exposed to 300 g/L. Treatment with 300 g/L prochoraz significantly up-regulated expression of er2˛, while exposure to the greater concentration (1000 g/L) did not change expression of that gene. Similar to females, the rank order of gene sensitivities in males was genes in brain > in testis > in liver (Table 1). The rank order of OECPs were genes in brain > in testis > in liver (Table 1).
3.4. Transcriptional responses in males
4. Discussion
Exposure to low concentrations (1, 3 or 10 g/L) of prochloraz significantly changed expression of some genes in brain, while expression of all genes measured in testis and liver were not changed (Fig. 3 and S4–S6, see Supporting Information). Genes which were changed in brain were gnrh2, gnrhr1, gnrhr2, gnrhr4, lhˇ, er1 and er2˛. Treatment with medium concentrations of prochloraz (30 or 100 g/L) caused significant changes in expression of some genes monitored in brain and testis, while no significant effects on expression of genes monitored in liver were observed (Fig. 3 and S4–S6, see Supporting Information). In brain, exposure to 30 or 100 g/L significantly up-regulated expression of gnrh2, gnrhr1, gnrhr2, gnrhr3, gnrhr4, lhˇ, er1 and er2˛. In testis, expression of cyp17 and cyp19a was up-regulated, while expression of er1 and er2˛ were unchanged. No significant effects on expression of other genes monitored in testis compared with the control were observed. Exposure to the greatest concentrations of prochloraz (300 or 1000 g/L) significantly changed expression some genes monitored
Recently, compensatory responses and adaptability of the HPGaxis, represented as up-regulation of genes along this axis, were reported in fishes exposed to model chemicals, such as fadrozole, prochloraz, kethconazole and highlighted the need to consider compensatory responses when developing approaches to assess potential risks of chemicals (Ankley et al., 2007, 2009, 2012; Villeneuve et al., 2009a, 2009b, 2013; Breen et al., 2013). For the first time, the results observed in this study further demonstrated that in female and male zebrafish exposed to low concentration prochloraz, transcriptionally compensatory responses of HPG axis and liver only occurred in brain; in medium concentration groups the compensatory responses occurred in both brain and gonad; in high concentration groups, the compensatory responses seemed to occur in brain, gonad and liver. Direct effects of prochloraz on concentrations of E2 in blood plasma and expression of er1 and vtgs in liver of female and male zebrafish were consistent with prochloraz’s anticipated mode of action. Prochloraz is an inhibitor of various CYP enzymes. Results of
Table 1 Endpoints determined for gene expression in HPG axis and liver of female and male zebrafish exposed to different concentration of prochloraz. Genes
Females
Males
LOEC
EC50
OECP(s)
OMRV
LOEC
EC50
OECP(s)
OMRV
Brain
gnrh2 gnrh3 gnrhr1 gnrhr2 gnrhr3 gnrhr4 fshˇ lhˇ er1 er2˛
NE 3 NE ≤1 ≤1 ≤1 NE 3 ≤1 3
NE 2.8 (2.3, 3.2) NE 0.7 (0.7, 0.7) 0.9 (0.8, 1.0) 0.7 (0.7, 0.7) NE 23.7 (19.3, 28.1) 0.9 (0.8, 1.0) 2.5 (2.4, 2.6)
NE 3,10 NE 1, 3, 10, 30, 100, 300, 1000 1, 3, 10, 30, 100, 300, 1000 1, 3, 10, 30, 100, 300, 1000 NE 3, 10, 30, 100, 300, 1000 1, 3, 10, 30, 100, 300 3, 10, 30, 100, 300
NE 3.0 (2.2, 3.9) NE 1.6 (1.5, 1.7) 2.3 (2.2, 2.3) 1.7 (1.6, 1.7) NE 28.7 (25.2, 32.2) 1.0 (0.9, 1.1) 0.8 (0.7, 0.9)
10 NE ≤1 1 30 ≤1 NE 10 ≤1 ≤1
83.4 (75.5, 91.3) NE 72.3 (67.0, 77.6) 88.3 (80.4, 96.2) 60.8 (56.4, 65.2) 134.2 (123.7, 144.7) NE 438.3 (389.2, 487.4) 175.1 (162.0, 188.2) 439.8 (399.5, 480.1)
10, 30, 1000 NE 1, 30, 100, 300, 1000 1, 30, 100, 300, 1000 30, 100, 300, 1000 1, 10, 30, 100, 300, 1000 NE 100, 300, 1000 1, 30, 100, 300, 1000 1, 30, 100, 300, 1000
1.2 (1.2, 1.3) NE 0.7 (0.7, 0.7) 1.2 (1.1, 1.3) 1.7 (1.5, 1.9) 2.9 (2.5, 3.2) NE 3.2 (2.7, 3.7) 2.0 (1.8, 2.2) 2.2 (2.0, 2.4)
Gonad
fshr lhr hmgr star cyp11a 3ˇhsd cyp17 17ˇhsd cyp19a er1 er2˛
300 30 NE 30 300 NE 100 100 30 300 100
NE 31.5 (29.2, 33.8) NE NE 98.4 (91.7, 105.7) NE 105.4 (98.4, 112.4) 130.5 (120.0, 141.0) 125.4 (114.9, 135.9) NE 85.8 (80.5, 91.1)
300 30, 100, 300, 1000 NE 30, 100, 300, 1000 300, 1000 NE 100, 300, 1000 100, 300 30, 100, 300, 1000 300 100, 300, 1000
2.3 (2.0, 2.6) 3.0 (2.8, 3.2) NE 4.3 (3.6, 4.9) 1.0 (1.0, 1.0) NE 2.0 (1.7, 2.3) 2.1 (1.8, 2.3) 7.6 (7.3, 7.9) 1.8 (1.7, 2.0) 0.9 (0.8, 1.0)
NE NE NE 300 NE NE 30 NE 30 100 30
NE NE NE 150.2 (137.9, 9162.5) NE NE 463.5 (431.9, 495.1) NE 187.6 (172.7, 202.5) 170.8 (155.9, 185.7) 75.4 (70.1, 80.7)
NE NE NE 300, 1000 NE NE 30, 100, 300, 1000 NE 30, 100, 300, 1000 100, 300, 1000 30, 100, 300, 1000
NE NE NE −2.7 (−3.0, −2.4) NE NE 4.9 (4.5, 5.3) NE 4.4 (3.7, 5.1) −1.1 (−1.1, −1.1) −1.1 (−1.2, −1.0)
Liver
er1 vtg1 vtg2 er2˛
300 300 300 300
NE NE NE NE
300, 1000 300, 1000 300, 1000 300, 1000
−3.4 (−3.9, −3.0) −29.7 (−32.6, −26.8) −20.8 (−23.4, −18.2) 1.8 (1.6, 2.0)
NE 1000 NE 300
NE NE NE NE
NE 1000 NE 300
NE −4.9 (−5.6, −4.2) NE 0.9 (0.9, 1.0)
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Organs
LOEC: the lowest observed effect concentration, g/L; EC50 : the concentration to induce half of the maximal response, g/L; OMRV: observed maximal response value, fold change relative control; OECPs: observed effect concentration points, g/L; NE: not estimated because the number of genes with statistically significant response was ≤1 or the maximal response was not reached.
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previous studies suggested that CYP19A activity was a more sensitive target of prochloraz than other steroidogenic CYPs (e.g., CYP17) and plasma volume was limited and could only be used for detection of a single hormone (Ankley et al., 2009). Therefore, only E2 was quantified in plasma. Consequently, we expected exposure to prochloraz to impair the synthesis, decreasing concentration of E2 in blood plasma. Results observed in this study are consistent with this expectation, with a significant decrease in concentrations of E2 in blood plasma in females and males exposed to 300 or 1000 g/L prochloraz. In fish, synthesis of vtg in the liver is under the control of er, and is induced by estrogens, such as E2. Therefore, decreased concentrations of E2 in blood plasma would result in down-regulated expression of er1 and vtgs in liver. The results observed in this study were consistent with these hypotheses. Previous studies also reported that treatment with 300 or 1000 g/L prochloraz significantly decreased concentrations of E2 in blood plasma and down-regulated the expression of er1 and vtgs in liver of small fish, including zebrafish, medaka and fathead minnow (Ankley et al., 2005, 2009; Liu et al., 2011; Skolness et al., 2011; Zhang et al., 2008a). Results of this study were consistent with those of previous observations that small fish (e.g., fathead minnow, medaka and zebrafish) have the capacity to mount a compensatory response to the direct inhibitory effects of prochloraz on blood plasma E2 concentration by up-regulating expression of genes included in brain and gonad (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a). Similar to previous studies, where time- or dose-dependent upregulation of genes included in brain and gonad were observed in small fish exposed to prochloraz (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a), the expression of some genes was also induced in the present study. However, in this study, we provided more details and insights on dose-dependent compensatory response along HPG-axis and liver by using more exposure doses compared with previous studies. In female and male zebrafish exposed to low and medium concentration prochloraz, compensatory responses occurred in brain and both brain and gonad, respectively in this study. In fish, gnrh can induce the synthesis of fsh and lh by gnrhr in brain, and then fsh and lh are secreted by the pituitary and bind to their receptors (fshr and lhr) in the gonad to induce steroidogenesis (Liu et al., 2011; Zhang et al., 2008a). In addition, sex hormones (e.g., E2) in blood can enter into brain and work by their receptors (e.g., er) for negative feedback regulation (Liu et al., 2011; Zhang et al., 2008a). In this study, up-regulation of gnrhrs, lhˇ and ers in brain was observed after exposure to low and medium concentration prochloraz for 4 days. Direct effects of prochloraz on the expression of these genes were excluded in previous study using in vitro brain explant exposure (Liu et al., 2011), therefore, up-regulation of these genes was considered as a compensation response for decreased concentration of E2. However, in this study, exposure to low concentrations prochloraz did not decrease concentrations of E2 in blood plasma and vtgs expression in liver. A possible explanation was that the inhibitory effect of prochloraz on E2 might be transitory and occurred in initial stage of exposure, but concentration of E2 recovered due to compensation responses and returned to control levels by the end of 4-day exposure. This has been confirmed by designed experiments in fathead minnows (Ankley et al., 2009; Villeneuve et al., 2009), where the inhibitory effects of low concentration prochloraz or fadrozole on concentrations of E2 in blood plasma in fathead minnow only occurred within 1 day of exposure but returned to the control level by the end of the 8day exposure phase. In previous studied using fathead minnow or Japanese medaka medaka (Oryzias latipes) as animal models, expression of gnrhrs, lhˇ and ers kept unchanged after exposure to prochloraz (Ankley et al., 2009; Skolness et al., 2011; Zhang et al., 2008a), although expression of chicken-II-type gnrh (cgnrh
II) was significantly up-regulated in female medaka after 3 g/L prochloraz exposure for 7 days (Zhang et al., 2008a). However, consistent with the results of this study, our previous study found that exposure to 300 g/L prochloraz for 12 or 48 h caused a strong upregulation of several gnrhrs in brain of female zebrafish (Liu et al., 2011). Therefore, these differences might be explained by species differences. For low concentration exposure, our results demonstrated that compensatory up-regulation of gene expression only occurred in brain, not in gonad and liver, but the compensation is successful since no significant effect on concentrations of E2 were observed by the end of the 4-day exposure. These results suggested that other feedback mechanisms might be included. For example, in a previous study it was reported that treatment with LH in immature rat leydig cells significantly increased the activity of steroidogenic enzyme (CYP11A) (van Haren et al., 1995). Therefore, the feedback mechanisms might have also included responses at other levels than transcription, such as increased enzyme activity or protein synthesis. Further studies are needed to explore the details of other compensation responses. For medium concentration exposure, compensatory responses included up-regulation of steroidogenic genes except for genes included in brain above. Similar compensatory up-regulations of these genes were also reported in fathead minnow and medaka exposed to 30-g/L prochloraz for 8 days (Ankley et al., 2009; Liu et al., 2011). Similarly, exposure to 30 or 100 g/L prochlorza did not change plasma E2 concentrations and vtgs expression of liver in female and male zebrafish in the present study, suggesting the effectiveness of compensation responses. Transcriptional compensation seems to involve up-regulation of genes in brain, gonad and liver in female and male zebrafish after exposure to greatest concentration prochloraz. In this study, up-regulation of gnrhrs, lhˇ and ers was also observed in fish exposed to the greatest concentrations, which suggests consistency in responses. In gonad, abundances of several genes were increased and cyp17 and cyp19a was the most up-regulated gene. In a previous study the most up-regulated gene in female fathead minnow exposed to 300-g/L prochloraz codes was cyp19a (Ankley et al., 2009). In medaka, a significant up-regulation of cyp17 and cyp19a was also observed in females exposed to 300-g/L prochloraz for 7 days (Zhang et al., 2008a). Consistent with the results of the present study and previous published papers (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a), a study reported that compensatory responses of female fathead minnow to cyp19a inhibitor (fadrozole)-induced decrease in concentrations of E2 in blood plasma were associated with up-regulation of steroidogenic genes in ovaries (Villeneuve et al., 2009). In liver, besides down-regulation of er1 and vtgs observed, exposure to the greatest concentration prochloraz up-regulated expression of er2˛. Down-regulation of vtgs and er1 was considered to be a direct effect of prochlorazinduced decreases in production of E2, and up-regulation of er2˛ might be a mechanism of compensation to decreased production of vtgs. In a recent study it was found that er2 was also involved in E2-induced vtg synthesis in fish (Yost et al., 2014). Therefore, further study is needed to explore the possible roles of er2␣ in fish exposed to prochloraz in future. In summary, in this study, dose-dependent responsive behaviors of genes involved in HPG axis and liver were investigated and linkages between compensatory responses occurred in brain, gonad and liver in terms of degrees of prochloraz stresses. Two points are especially notable in this regard. First, in low-dose exposure groups transcriptional compensation responses of HPG axis occurred only in brain, suggesting that other down-stream compensatory mechanisms might be involved, therefore, further studies are needed to explore those possibilities. Second, upregulation of er2˛ in liver was considered as a compensatory mechanism for decreased vtgs production due to exposure to
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prochloraz. This result suggests that compensatory responses also occur in liver. However, these responses, although critical to understanding the mechanisms of chemicals, need to be examined and integrated in a broader system to support more reliable predictions of toxicity of chemicals.[1] Additionally, it should be noted that in low and medium concentration groups, E2 concentrations kept unchanged and we speculated that transcriptionally compensatory responses (e.g., up-regulation of genes involved in HPG axis and liver) were successful in maintaining homeostasis. However, we did not measure protein contents or activities of enzymes to further support our hypothesis. Therefore, further studies are needed in future to explore these possibilities.
Acknowledgements This work was supported by Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (Program No. 2014RC001) to Dr. Chunsheng Liu. This work was also supported by the National Natural Science Foundation of China (31370525) and the Fundamental Research Funds for the Central Universities (2014PY027) to Dr. Jianghua Wang. Prof. Giesy was supported by the program of 2012 “High Level Foreign Experts” (#GDW20123200120) funded by the State Administration of Foreign Experts Affairs, the P.R. China to Nanjing University and the Einstein Professor Program of the Chinese Academy of Sciences. He was also supported by the Canada Research Chair program, a Visiting Distinguished Professorship in the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox. 2015.01.003.
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Dose-dependent compensation responses of the hypothalamic-pituitary-gonadal-liver axis of zebrafish exposed to the fungicide prochloraz Yao Dang a , John P. Giesy b,c,d,e , Jianghua Wang a,∗ , Chunsheng Liu a,∗ a
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada c Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China d School of Biological Sciences, University of Hong Kong, SAR, Hong Kong, China e State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China b
a r t i c l e
i n f o
Article history: Received 8 November 2014 Received in revised form 5 January 2015 Accepted 7 January 2015 Available online 8 January 2015 Keywords: HPG axis Compensation responses Zebrafish Prochloraz Aromatase inhibitor
a b s t r a c t Compensation responses and adaptability of hypothalamic-pituitary-gonadal (HPG) axis have been reported in fish exposed to model chemicals, however due to its importance in predictive toxicology further study was needed to elucidate details of the integrated responses to model chemicals. Transcriptional profiles of the hypothalamic-pituitary-gonadal (HPG) axis and concentrations of 17-estradiol (E2) in plasma were measured in male and female zebrafish that had been exposed to one of seven concentrations of the fungicide, prochloraz: low (1, 3 or 10 g/L), medium (30 or 100 g/L) or high concentrations (300 or 1000 g/L) for 4 days. In zebrafish exposed to the low and medium concentrations of prochloraz, compensation responses of the HPG axis through transcription, occurred in brain (up-regulation of gnrh, gnrhr and lhˇ) and both brain and gonad (up-regulation of steroidogenic genes), respectively. Concentrations of E2 in plasma and expression of estrogen receptor 1 (er1) and vitellogenins (vtgs) in liver did not change. This result suggested that compensatory responses were successful in maintaining homeostasis. In zebrafish exposed to the two greatest concentrations, compensatory responses occurred in brain, gonad and liver through up-regulation of er2ˇ, but it failed to maintain concentration of E2 in blood plasma and expression of er1 and vtgs in liver. Collectedly, the results observed in this study allowed characterization of dose-dependent compensatory responses along the HPG axis and liver and identified key linkages between compensatory responses occurring in brain, gonad and liver after exposure to prochloraz. © 2015 Elsevier B.V. All rights reserved.
1. Introduction As an imidazole fungicide, prochloraz is registered for various agricultural uses throughout the world (http://pubchem.ncbi. nlm.nih.gov/summary/summary.cgi?cid=73665). This chemical can act as an inhibitor of cytochrome P450 (CYP) 14␣-demethylase (CYP51), which is a key enzyme in the synthesis of ergosterol, and therefore inhibits growth of fungi (van den Bossche et al., 1987, 1982). However, prochloraz can also inhibit activities of other CYP enzymes, including cytochrome P450 c17␣-hydroxylase/17,20lyase (CYP17) and aromatase (CYP19) (Ankley et al., 2009). In
∗ Corresponding authors. Tel.: +86 27 87282113; fax: +86 27 87282114. E-mail addresses: [email protected] (J. Wang), [email protected] (C. Liu). http://dx.doi.org/10.1016/j.aquatox.2015.01.003 0166-445X/© 2015 Elsevier B.V. All rights reserved.
vertebrates, the gene product of CYP17 is responsible for synthesis of testosterone (T) and CYP19 catalyzes conversion of T to 17-estradiol (E2). Therefore, inhibition of activities of the two enzymes would decrease production of both T and E2. These effects have been previously confirmed experimentally, both in vitro and in vivo, in mammals, where exposure to prochloraz significantly inhibited activities of both CYP17 and CYP19 enzymes and decreased concentrations of T and E2 in blood plasma (Blystone et al., 2007; Mason et al., 1987; Noriega et al., 2005; Sanderson et al., 2002; Vinggaard et al., 2000). In fish, treatment with prochloraz decreased concentrations of T and E2 in blood plasma, and affected reproductive function (Ankley et al., 2005, 2009; Marca Pereira et al., 2011a, b; Liu et al., 2011; Skolness et al., 2011; Villeneuve et al., 2007; Zhang et al., 2008a,). Due to its effectiveness as an inhibitor of CYP17 and CYP19 enzymes, prochloraz has been used as a model chemical for studying the responses of
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hypothalamic-pituitary-gonadal (HPG) axes of vertebrates (Ankley et al., 2005, 2009; Marca Pereira et al., 2011a, b; Liu et al., 2011; Villeneuve et al., 2007; Zhang et al., 2008a). The HPG axis is a dynamic endocrine system, that maintains physiological conditions of reproduction by various homeostatic feedback mechanisms during exposure to stressors including chemicals (Ankley et al., 2009). Compensatory responses of the HPG axis have been documented in fish exposed to model chemicals (Ankley et al., 2009; Liu et al., 2011; Skolness et al., 2011; Villeneuve et al., 2007; Zhang et al., 2008a, b, c). For example, results of several studies have demonstrated that exposure to fadrazol, which inhibits the enzymatic activity of CYP19, resulted in decreased concentrations of E2 in blood plasma and caused a time- and dose-dependent up-regulation of genes of HPG axis in small fish. Up-regulation of those genes was considered to be a compensatory mechanism to return concentrations of E2 in blood plasma to pre-exposure values (Villeneuve et al., 2009; Zhang et al., 2008c). Similarly, treatment with prochloraz significantly decreased concentrations of T and E2 in blood plasma and resulted in a comprehensive up-regulation of genes along the HPG axes of fishes (Ankley et al., 2009; Liu et al., 2011; Skolness et al., 2011; Zhang et al., 2008a). Furthermore, in two time-course studies, inhibitory effects of prochloraz or fadrozole on production of E2 in fathead minnow were transitory and did not persist during the 8-day exposure phase, which demonstrated the effectiveness of compensatory responses. Termination of the exposure resulted in recovery of expression of genes and concentrations of steroid hormones, including a brief period of “overcompensation” immediately after cessation of exposure (Ankley et al., 2009; Villeneuve et al., 2009). These results suggest key compensatory responses of the HPG axis after exposure to chemical stressors, and highlight the need to consider these compensatory responses when developing approaches to assess potential risks of chemicals (Ankley et al., 2009; Villeneuve et al., 2009). To better understand compensatory mechanisms in fishes and more accurately predict effects of chemicals based on modes of action a more comprehensive characterization of these dosedependent compensatory responses was still needed. Objectives of the present study were to: (1) examine dose-dependent expression behaviors of HPG axis and genes expressed in liver; (2) compare sensitivities of genes; and (3) identify linkages between responses occurring at different organs, including brain, gonad and liver of zebrafish. 2. Materials and methods 2.1. Chemicals and reagents Prochloraz and TRIzol regent were obtained from Sigma (St. Louis, MO, USA) and Invitrogen (New Jersey, NJ, USA), respectively. Reverse transcription and SYBR Green kits were purchased from Takara (Dalian, Liaoning, China). 17-estrogen (E2) enzyme immunoassay (EIA) kits were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). All the other reagents used in this study were of analytical grade. 2.2. Fish and chemical exposure Zebrafish were maintained in flow-through tanks at 28 ± 0.5 ◦ C with a 12:12 light/dark cycle, and water pH, hardness and dissolved oxygen were routinely monitored. Before exposure, 5-month old males and females (sexual maturity) were acclimated in 15-L tanks filled with 10 L of carbon-filtered water for 1 week. After acclimation, fish were exposed to 0, 1, 3, 10, 30, 100, 300, 1000, 3000 or 10,000 g/L (0, 0.0027, 0.0080, 0.027, 0.080, 0.27, 0.80, 2.7, 8.0
or 27 M) prochloraz for 4 days. Concentrations were selected based on previous studies, where comprehensive compensation responses of HPG axis genes would be expected to occur (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a). Five females and five males were exposed in each of 2 replicated tanks for each concentration. One half of the water in each tank was replaced daily with fresh carbon-filtered water containing corresponding concentration of prochloraz. Both control and exposure groups received 0.01% DMSO since previous study demonstrated that such DMSO concentration did not affect reproductive function (Han et al., 2013). During the exposure period, survival was recorded. After exposure, fish were euthanized and blood was collected for plasma hormone analysis as described before (Liu et al., 2009). Tissues from brain (including hypothalamus and pituitary), gonad and liver were sampled and preserved in TRIzol reagent for subsequent RNA isolation. In this study, experimental procedures were carried out following the approved protocol by Institutional Animal Care and Use Committee (IACUC) of Huazhong Agricultural University. 2.3. Quantification of hormones Briefly, plasma was obtained by centrifugation (5000 × g for 5 min at 4 ◦ C) of whole blood. Plasma from 2 fish was pooled for quantification by use of a commercial EIA kit as described previously (Liu et al., 2009). Briefly, plasma (3 L) from each pooled sample was diluted with 500 L ultrapure water and extracted thrice with 2 mL of ethyl ether, and the ether phase was collected and evaporated. After that, residues were redissolved with EIA buffer provided in the kit and E2 was quantified following manufacturer’s instructions. The limit of quantification was 6 pg/L, and the intra- and inter-assay coefficients of variance (CV) were in ovary > in liver (Table 1). The rank order of OECPs were genes in brain > in ovary > in liver (Table 1).
in brain, testis and liver (Fig. 3 and S4–S6, see Supporting Information). In brain, expressions of gnrh2, gnrhr1, gnrhr2, gnrhr3, gnrhr4, lhˇ, er1 and er2˛ were up-regulated, compared with their levels of expression control. In testis, expression of star, cyp17 and cyp19a were significantly up-regulated, while expressions of er1 and er2˛ were down-regulated. In liver, exposure to 1000 g/L prochloraz down-regulated expression of vtg1, but no statistically significant effects were observed when fish were exposed to 300 g/L. Treatment with 300 g/L prochoraz significantly up-regulated expression of er2˛, while exposure to the greater concentration (1000 g/L) did not change expression of that gene. Similar to females, the rank order of gene sensitivities in males was genes in brain > in testis > in liver (Table 1). The rank order of OECPs were genes in brain > in testis > in liver (Table 1).
3.4. Transcriptional responses in males
4. Discussion
Exposure to low concentrations (1, 3 or 10 g/L) of prochloraz significantly changed expression of some genes in brain, while expression of all genes measured in testis and liver were not changed (Fig. 3 and S4–S6, see Supporting Information). Genes which were changed in brain were gnrh2, gnrhr1, gnrhr2, gnrhr4, lhˇ, er1 and er2˛. Treatment with medium concentrations of prochloraz (30 or 100 g/L) caused significant changes in expression of some genes monitored in brain and testis, while no significant effects on expression of genes monitored in liver were observed (Fig. 3 and S4–S6, see Supporting Information). In brain, exposure to 30 or 100 g/L significantly up-regulated expression of gnrh2, gnrhr1, gnrhr2, gnrhr3, gnrhr4, lhˇ, er1 and er2˛. In testis, expression of cyp17 and cyp19a was up-regulated, while expression of er1 and er2˛ were unchanged. No significant effects on expression of other genes monitored in testis compared with the control were observed. Exposure to the greatest concentrations of prochloraz (300 or 1000 g/L) significantly changed expression some genes monitored
Recently, compensatory responses and adaptability of the HPGaxis, represented as up-regulation of genes along this axis, were reported in fishes exposed to model chemicals, such as fadrozole, prochloraz, kethconazole and highlighted the need to consider compensatory responses when developing approaches to assess potential risks of chemicals (Ankley et al., 2007, 2009, 2012; Villeneuve et al., 2009a, 2009b, 2013; Breen et al., 2013). For the first time, the results observed in this study further demonstrated that in female and male zebrafish exposed to low concentration prochloraz, transcriptionally compensatory responses of HPG axis and liver only occurred in brain; in medium concentration groups the compensatory responses occurred in both brain and gonad; in high concentration groups, the compensatory responses seemed to occur in brain, gonad and liver. Direct effects of prochloraz on concentrations of E2 in blood plasma and expression of er1 and vtgs in liver of female and male zebrafish were consistent with prochloraz’s anticipated mode of action. Prochloraz is an inhibitor of various CYP enzymes. Results of
Table 1 Endpoints determined for gene expression in HPG axis and liver of female and male zebrafish exposed to different concentration of prochloraz. Genes
Females
Males
LOEC
EC50
OECP(s)
OMRV
LOEC
EC50
OECP(s)
OMRV
Brain
gnrh2 gnrh3 gnrhr1 gnrhr2 gnrhr3 gnrhr4 fshˇ lhˇ er1 er2˛
NE 3 NE ≤1 ≤1 ≤1 NE 3 ≤1 3
NE 2.8 (2.3, 3.2) NE 0.7 (0.7, 0.7) 0.9 (0.8, 1.0) 0.7 (0.7, 0.7) NE 23.7 (19.3, 28.1) 0.9 (0.8, 1.0) 2.5 (2.4, 2.6)
NE 3,10 NE 1, 3, 10, 30, 100, 300, 1000 1, 3, 10, 30, 100, 300, 1000 1, 3, 10, 30, 100, 300, 1000 NE 3, 10, 30, 100, 300, 1000 1, 3, 10, 30, 100, 300 3, 10, 30, 100, 300
NE 3.0 (2.2, 3.9) NE 1.6 (1.5, 1.7) 2.3 (2.2, 2.3) 1.7 (1.6, 1.7) NE 28.7 (25.2, 32.2) 1.0 (0.9, 1.1) 0.8 (0.7, 0.9)
10 NE ≤1 1 30 ≤1 NE 10 ≤1 ≤1
83.4 (75.5, 91.3) NE 72.3 (67.0, 77.6) 88.3 (80.4, 96.2) 60.8 (56.4, 65.2) 134.2 (123.7, 144.7) NE 438.3 (389.2, 487.4) 175.1 (162.0, 188.2) 439.8 (399.5, 480.1)
10, 30, 1000 NE 1, 30, 100, 300, 1000 1, 30, 100, 300, 1000 30, 100, 300, 1000 1, 10, 30, 100, 300, 1000 NE 100, 300, 1000 1, 30, 100, 300, 1000 1, 30, 100, 300, 1000
1.2 (1.2, 1.3) NE 0.7 (0.7, 0.7) 1.2 (1.1, 1.3) 1.7 (1.5, 1.9) 2.9 (2.5, 3.2) NE 3.2 (2.7, 3.7) 2.0 (1.8, 2.2) 2.2 (2.0, 2.4)
Gonad
fshr lhr hmgr star cyp11a 3ˇhsd cyp17 17ˇhsd cyp19a er1 er2˛
300 30 NE 30 300 NE 100 100 30 300 100
NE 31.5 (29.2, 33.8) NE NE 98.4 (91.7, 105.7) NE 105.4 (98.4, 112.4) 130.5 (120.0, 141.0) 125.4 (114.9, 135.9) NE 85.8 (80.5, 91.1)
300 30, 100, 300, 1000 NE 30, 100, 300, 1000 300, 1000 NE 100, 300, 1000 100, 300 30, 100, 300, 1000 300 100, 300, 1000
2.3 (2.0, 2.6) 3.0 (2.8, 3.2) NE 4.3 (3.6, 4.9) 1.0 (1.0, 1.0) NE 2.0 (1.7, 2.3) 2.1 (1.8, 2.3) 7.6 (7.3, 7.9) 1.8 (1.7, 2.0) 0.9 (0.8, 1.0)
NE NE NE 300 NE NE 30 NE 30 100 30
NE NE NE 150.2 (137.9, 9162.5) NE NE 463.5 (431.9, 495.1) NE 187.6 (172.7, 202.5) 170.8 (155.9, 185.7) 75.4 (70.1, 80.7)
NE NE NE 300, 1000 NE NE 30, 100, 300, 1000 NE 30, 100, 300, 1000 100, 300, 1000 30, 100, 300, 1000
NE NE NE −2.7 (−3.0, −2.4) NE NE 4.9 (4.5, 5.3) NE 4.4 (3.7, 5.1) −1.1 (−1.1, −1.1) −1.1 (−1.2, −1.0)
Liver
er1 vtg1 vtg2 er2˛
300 300 300 300
NE NE NE NE
300, 1000 300, 1000 300, 1000 300, 1000
−3.4 (−3.9, −3.0) −29.7 (−32.6, −26.8) −20.8 (−23.4, −18.2) 1.8 (1.6, 2.0)
NE 1000 NE 300
NE NE NE NE
NE 1000 NE 300
NE −4.9 (−5.6, −4.2) NE 0.9 (0.9, 1.0)
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Organs
LOEC: the lowest observed effect concentration, g/L; EC50 : the concentration to induce half of the maximal response, g/L; OMRV: observed maximal response value, fold change relative control; OECPs: observed effect concentration points, g/L; NE: not estimated because the number of genes with statistically significant response was ≤1 or the maximal response was not reached.
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previous studies suggested that CYP19A activity was a more sensitive target of prochloraz than other steroidogenic CYPs (e.g., CYP17) and plasma volume was limited and could only be used for detection of a single hormone (Ankley et al., 2009). Therefore, only E2 was quantified in plasma. Consequently, we expected exposure to prochloraz to impair the synthesis, decreasing concentration of E2 in blood plasma. Results observed in this study are consistent with this expectation, with a significant decrease in concentrations of E2 in blood plasma in females and males exposed to 300 or 1000 g/L prochloraz. In fish, synthesis of vtg in the liver is under the control of er, and is induced by estrogens, such as E2. Therefore, decreased concentrations of E2 in blood plasma would result in down-regulated expression of er1 and vtgs in liver. The results observed in this study were consistent with these hypotheses. Previous studies also reported that treatment with 300 or 1000 g/L prochloraz significantly decreased concentrations of E2 in blood plasma and down-regulated the expression of er1 and vtgs in liver of small fish, including zebrafish, medaka and fathead minnow (Ankley et al., 2005, 2009; Liu et al., 2011; Skolness et al., 2011; Zhang et al., 2008a). Results of this study were consistent with those of previous observations that small fish (e.g., fathead minnow, medaka and zebrafish) have the capacity to mount a compensatory response to the direct inhibitory effects of prochloraz on blood plasma E2 concentration by up-regulating expression of genes included in brain and gonad (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a). Similar to previous studies, where time- or dose-dependent upregulation of genes included in brain and gonad were observed in small fish exposed to prochloraz (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a), the expression of some genes was also induced in the present study. However, in this study, we provided more details and insights on dose-dependent compensatory response along HPG-axis and liver by using more exposure doses compared with previous studies. In female and male zebrafish exposed to low and medium concentration prochloraz, compensatory responses occurred in brain and both brain and gonad, respectively in this study. In fish, gnrh can induce the synthesis of fsh and lh by gnrhr in brain, and then fsh and lh are secreted by the pituitary and bind to their receptors (fshr and lhr) in the gonad to induce steroidogenesis (Liu et al., 2011; Zhang et al., 2008a). In addition, sex hormones (e.g., E2) in blood can enter into brain and work by their receptors (e.g., er) for negative feedback regulation (Liu et al., 2011; Zhang et al., 2008a). In this study, up-regulation of gnrhrs, lhˇ and ers in brain was observed after exposure to low and medium concentration prochloraz for 4 days. Direct effects of prochloraz on the expression of these genes were excluded in previous study using in vitro brain explant exposure (Liu et al., 2011), therefore, up-regulation of these genes was considered as a compensation response for decreased concentration of E2. However, in this study, exposure to low concentrations prochloraz did not decrease concentrations of E2 in blood plasma and vtgs expression in liver. A possible explanation was that the inhibitory effect of prochloraz on E2 might be transitory and occurred in initial stage of exposure, but concentration of E2 recovered due to compensation responses and returned to control levels by the end of 4-day exposure. This has been confirmed by designed experiments in fathead minnows (Ankley et al., 2009; Villeneuve et al., 2009), where the inhibitory effects of low concentration prochloraz or fadrozole on concentrations of E2 in blood plasma in fathead minnow only occurred within 1 day of exposure but returned to the control level by the end of the 8day exposure phase. In previous studied using fathead minnow or Japanese medaka medaka (Oryzias latipes) as animal models, expression of gnrhrs, lhˇ and ers kept unchanged after exposure to prochloraz (Ankley et al., 2009; Skolness et al., 2011; Zhang et al., 2008a), although expression of chicken-II-type gnrh (cgnrh
II) was significantly up-regulated in female medaka after 3 g/L prochloraz exposure for 7 days (Zhang et al., 2008a). However, consistent with the results of this study, our previous study found that exposure to 300 g/L prochloraz for 12 or 48 h caused a strong upregulation of several gnrhrs in brain of female zebrafish (Liu et al., 2011). Therefore, these differences might be explained by species differences. For low concentration exposure, our results demonstrated that compensatory up-regulation of gene expression only occurred in brain, not in gonad and liver, but the compensation is successful since no significant effect on concentrations of E2 were observed by the end of the 4-day exposure. These results suggested that other feedback mechanisms might be included. For example, in a previous study it was reported that treatment with LH in immature rat leydig cells significantly increased the activity of steroidogenic enzyme (CYP11A) (van Haren et al., 1995). Therefore, the feedback mechanisms might have also included responses at other levels than transcription, such as increased enzyme activity or protein synthesis. Further studies are needed to explore the details of other compensation responses. For medium concentration exposure, compensatory responses included up-regulation of steroidogenic genes except for genes included in brain above. Similar compensatory up-regulations of these genes were also reported in fathead minnow and medaka exposed to 30-g/L prochloraz for 8 days (Ankley et al., 2009; Liu et al., 2011). Similarly, exposure to 30 or 100 g/L prochlorza did not change plasma E2 concentrations and vtgs expression of liver in female and male zebrafish in the present study, suggesting the effectiveness of compensation responses. Transcriptional compensation seems to involve up-regulation of genes in brain, gonad and liver in female and male zebrafish after exposure to greatest concentration prochloraz. In this study, up-regulation of gnrhrs, lhˇ and ers was also observed in fish exposed to the greatest concentrations, which suggests consistency in responses. In gonad, abundances of several genes were increased and cyp17 and cyp19a was the most up-regulated gene. In a previous study the most up-regulated gene in female fathead minnow exposed to 300-g/L prochloraz codes was cyp19a (Ankley et al., 2009). In medaka, a significant up-regulation of cyp17 and cyp19a was also observed in females exposed to 300-g/L prochloraz for 7 days (Zhang et al., 2008a). Consistent with the results of the present study and previous published papers (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a), a study reported that compensatory responses of female fathead minnow to cyp19a inhibitor (fadrozole)-induced decrease in concentrations of E2 in blood plasma were associated with up-regulation of steroidogenic genes in ovaries (Villeneuve et al., 2009). In liver, besides down-regulation of er1 and vtgs observed, exposure to the greatest concentration prochloraz up-regulated expression of er2˛. Down-regulation of vtgs and er1 was considered to be a direct effect of prochlorazinduced decreases in production of E2, and up-regulation of er2˛ might be a mechanism of compensation to decreased production of vtgs. In a recent study it was found that er2 was also involved in E2-induced vtg synthesis in fish (Yost et al., 2014). Therefore, further study is needed to explore the possible roles of er2␣ in fish exposed to prochloraz in future. In summary, in this study, dose-dependent responsive behaviors of genes involved in HPG axis and liver were investigated and linkages between compensatory responses occurred in brain, gonad and liver in terms of degrees of prochloraz stresses. Two points are especially notable in this regard. First, in low-dose exposure groups transcriptional compensation responses of HPG axis occurred only in brain, suggesting that other down-stream compensatory mechanisms might be involved, therefore, further studies are needed to explore those possibilities. Second, upregulation of er2˛ in liver was considered as a compensatory mechanism for decreased vtgs production due to exposure to
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prochloraz. This result suggests that compensatory responses also occur in liver. However, these responses, although critical to understanding the mechanisms of chemicals, need to be examined and integrated in a broader system to support more reliable predictions of toxicity of chemicals.[1] Additionally, it should be noted that in low and medium concentration groups, E2 concentrations kept unchanged and we speculated that transcriptionally compensatory responses (e.g., up-regulation of genes involved in HPG axis and liver) were successful in maintaining homeostasis. However, we did not measure protein contents or activities of enzymes to further support our hypothesis. Therefore, further studies are needed in future to explore these possibilities.
Acknowledgements This work was supported by Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (Program No. 2014RC001) to Dr. Chunsheng Liu. This work was also supported by the National Natural Science Foundation of China (31370525) and the Fundamental Research Funds for the Central Universities (2014PY027) to Dr. Jianghua Wang. Prof. Giesy was supported by the program of 2012 “High Level Foreign Experts” (#GDW20123200120) funded by the State Administration of Foreign Experts Affairs, the P.R. China to Nanjing University and the Einstein Professor Program of the Chinese Academy of Sciences. He was also supported by the Canada Research Chair program, a Visiting Distinguished Professorship in the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox. 2015.01.003.
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