Monocrotophos pesticide affects synthesis and conversion of sex steroids through multiple targets in male goldfish (Carassius auratus)

Monocrotophos (MCP) is an organophosphorus pesticide that is median-toxic to fish. MCP pesticide resulted in an increase of 17 beta estradiol following a decrease in testosterone in male goldfish (Carassius auratus). To fully understand the mechanism of MCP pesticide that causes the imbalance between male and female hormones, we determined the levels of plasma cholesterol, spermatic steroidogenic acute regulatory protein mRNA, steroidogenesis enzyme mRNA, plasma sex hormone synthesis intermediates, and effectual hormones in male goldfish exposed to MCP pesticide at nominal concentrations of 0.01, 0.10, and 1.00 mg/L for 21 days in a semi-static exposure system. The results indicated that MCP pesticide (a) led to decreased steroidogenic acute regulatory protein mRNA levels; (b) decreased mRNA levels of cholesterol side chain cleavage enzyme and cytochrome P450 17 alpha hydroxylase, which are steroidogenesis enzymes involved in androgen synthesis; and (c) increased cytochrome P450 aromatase mRNA levels, a steroidogenesis enzyme involved in the synthesis of effectual estrogen. The present study provides evidence that MCP pesticide affects synthesis and conversion of sex steroids through multiple targets in male goldfish.

(CYP19/P450arom), 20 beta hydroxysteroid dehydrogenase (20 beta HSD), cytochrome P450 11beta (CYP11beta/ P450 11beta ), and 11 beta hydroxysteroid dehydrogenase (11 beta HSD), all of which are potential target sites of xenobiotics. Govoroun et al. demonstrated that E 2 inhibited spermatic P450c17, 3 beta HSD, and P450 11beta gene expression in rainbow trout (Oncorhynchus mykiss) 10 . MCP pesticide might affect synthesis and conversion of sex hormones via a number of pathways, such as changing the contents of synthesis substrates or influencing gene expression and activities of steroidogenesis enzymes.
This study was conducted to fully understand the mechanism by which MCP pesticide causes an imbalance between male and female hormones in male goldfish. First, effects on plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were examined to determine whether a lack of substrates for sex hormone synthesis is responsible for inhibited T levels. Second, spermatic StAR mRNA levels were quantified, which could indicate the influence of MCP on the transport of cholesterol from the outer to the inner mitochondrial membrane. Third, mRNA levels of eight kinds of steroidogenesis enzymes and plasma levels of six kinds of sex hormone synthesis intermediates and four kinds of effectual hormones were determined, to elucidate the effects of MCP pesticide on steroidogenesis in male goldfish.

Effects of MCP pesticide on gonadosomatic index (GSI).
The fishes were sampled in summer with degraded gonads. GSI of the control male goldfish was 0.14 ± 0.06%, which was not different from that in any of the MCP pesticide treatments studied (Fig. 1).

Effects of MCP pesticide on cholesterol levels.
In the control group, the content of plasma HDL-C was higher than that of LDL-C. TC, HDL-C, and LDL-C levels were not influenced by MCP pesticide exposure (Fig. 2).

Discussion
In this study, mechanisms by which MCP pesticide affects sex hormone synthesis and conversion of male goldfish were discussed from four aspects: (a) cholesterol levels, (b) gene expression of StAR and steroidogenesis enzymes; (c) intermediate products of the process of sex hormone synthesis; and (d) levels of effectual hormones (Fig. 6).
In bony fish, the substrates of steroid synthesis are mainly provided by exogenous cholesterol 11 . For example, climbing perch (Anabas testudineus) used plasma cholesterol as the raw material of sex hormone synthesis 15 . Low-density lipoproteins (LDL) play a leading role in mammals, but most cholesterol was transported by high-density lipoproteins (HDL) in fish 7,16,17 . In this study, plasma levels of LDL-C and HDL-C were both maintained following MCP pesticide exposure, suggesting that the content of raw material for sex hormone synthesis was not affected.
StAR can transfer cholesterol to the inner mitochondrial membrane, providing a reaction substrate for synthesis of pregnenolone, which is catalyzed by P450scc. In rainbow trout, StAR transcriptional levels were closely

76% (Clarias gariepinus)
20-beta-HSD KC193327 97% (Cyprinus carpio) Adenine ring binding domain: X-Asp-X-X-Asp; Sequence for a β sheet: Gly-Gly-X-Asp-X-X-X-Asn-Asn-Ala-Gly-X; Active site: Thr-Asn-X-X-Gly-Thr-X-X-X-X-X; Substrate binding site: X-X-Asn-X-Ser-Ser-X; Enzyme active site: Tyr-X-X-X-Lys 12, 13 ; Sequence regulating hydrogen bonds in nicotinamide ring: X-X-X-Asn-X-X-X-Pro-Gly-X-X-X- Coenzyme I binding domain: Gly-Phe-Gly; Conserved sequence of 11-beta-HSDs: Cys-Met-Glu-Val-Asn-Phe-Phe-Gly; Enzyme active site: Tyr-Gly-X-Ser-Lys-Ala-Ala Table 1. Alignment of the deduced amino acid sequences of goldfish steroidogenesis enzyme genes with those of other animal species. related to changes in plasma steroid hormones 18 . P450scc catalyzes the first step of steroidogenesis, making it a key enzyme during this process. MCP pesticide inhibited gene expression levels of StAR in male goldfish testes, leading to the reduction of cholesterol that was transported to the inner mitochondrial membrane, and the decrease in plasma levels of pregnenolone caused by MCP pesticide exposure is consistent with the inhibition of P450scc gene expression.
Pregnenolone, 17α-hydroxypregnenolone, and dehydroepiandrosterone can be converted to progesterone, 17α-hydroxyprogesterone, and androstenedione, respectively. These reactions were all catalyzed by 3-beta-HSD. P450c17 is one of the key enzymes of the steroidogenesis pathway in teleostean gonads. There are two kinds of P450c17 in bony fish: P450c17-I and P450c17-II. P450c17-I shows both 17α-hydroxylase and 17,20-lyase activity, whereas P450c17-II has only 17α-hydroxylase activity. Pregnenolone and progesterone can be converted to 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, respectively, by 17α-hydroxylase activity; 17,20-lyase activity can break the chemical bonds between C17 and C20 and transform 17α-hydroxypregnenolone into dehydroepiandrosterone or 17α-hydroxyprogesterone into androstenedione. We found a significant reduction of plasma T in male goldfish after MCP pesticide exposure in our previous study 5 , and we observed that MCP pesticide exposure resulted in decreased plasma androstenedione in this study, which is the substrate of T. The down-regulation of androgens by MCP pesticide corresponded to decreases in P450c17 mRNA levels.
Estradiol is formed stepwise, starting with aromatization of androstenedione to estrone, followed by conversion of estrone to E 2 via 17-beta-HSD activity, or conversion of androstenedione to T via 17-beta-HSD, followed by aromatization of T to E 2 . The intermediates androstenedione and estrone have low bioactivity, whereas T and Scientific RepoRts | 7: 2306 | DOI:10.1038/s41598-017-01935-6 E 2 are effectual hormones with high bioactivity. MCP pesticide increased P450arom A gene expression in male goldfish gonads, and this is consistent with our previous study 5 .
P450 11beta catalyzes T into 11-beta-hydroxytestosterone 19 , and 11-beta-HSD2 further converts 11-beta-hydroxytestosterone into 11-KT 20 . The reduction of P450 11beta gene expression led to inhibition of 11-KT synthesis 10, 21 , whereas Kusakabe et al. suggested that plasma 11-KT levels had less relevance to 11-beta-HSD2 gene expression in the testes 22 . In this study, we observed significant declines of 11-beta-HSD2 and P45011beta mRNA levels, but there was no significant change in 11-KT.
Androgen is responsible for development of the testes. Based on the appearance and histologic features of the testes, the development process in goldfish can be classified into six periods 23 . Individuals were sampled in August in this study, when testes had degraded to period III after spermiation, and GSI of the control male goldfish was as low as 0.14 ± 0.06%. Although an abnormally low plasma concentration of T was observed concomitant with an increase in MCP pesticide concentration in our previous study 5 , GSI was not affected in this study. This may be explained by the unchanged 11-KT levels, because in general 11-KT, rather than T, is the dominant androgen in most fish species, including goldfish.
Studies on endocrine disruption of exogenous compounds, particularly regarding steroidogenesis, have focused mainly on gene expression and activity of steroidogenesis enzymes 24,25 , especially aromatase 26,27 , although multiple factors could be involved in steroidogenesis. In this research, we demonstrated that MCP pesticide acted on many target sites, affecting the transport of cholesterol and gene expression of steroidogenesis enzymes, leading to abnormal levels of sex hormone synthesis intermediates and effectual hormones, and thus resulting in a proportional imbalance of sex hormones in male goldfish.

Materials and Methods
Fish exposure and sample protocol. Goldfish (8.4 ± 0.6 cm standard length; 17.6 ± 3.6 g wet mass) were obtained from a local dealer in Qingdao, China. Following acclimation in the laboratory at 24-26 °C and under a 16 h light/8 h dark cycle for two weeks, fish were exposed to 0.01, 0.10, and 1.00 mg/L MCP pesticide (40% water-soluble preparation), the concentrations of which were 1/10000, 1/1000, and 1/100 of the 96-h LC 50 (about 100 mg/L, our unpublished results), respectively 5 . Experiments were conducted in 70 L aquaria containing 50 L dechlorinated tap water using a semi-static toxicity test. Twenty liters of water was renewed daily to keep the MCP concentrations constant. Fish were fed a non-estrogenic pelletized diet daily. Additionally, the fish were handled according to the National Institute of Health Guidelines for the handling and care of experimental animals. The animal utilization protocol was approved by the Institutional Animal Care and Use Committee of the Ocean University of China.
After 21 days of exposure [28][29][30][31][32][33] , goldfish were anesthetized in 75 mg/L MS-222 (Sigma-Aldrich, St. Louis, MO, USA). Blood was taken from the caudal vein using chilled heparinized syringes. After centrifugation (700 g, 15 min, 4 °C or 2810 g, 10 min, 4 °C), plasma was frozen at −80 °C for cholesterol or hormone quantification. Individuals were identified as male or female during dissection. The testes were weighed and related to body weight to determine  GSI (GSI = gonad weight/body weight × 100%), and then gonad samples were frozen in liquid nitrogen and stored at −80 °C for steroidogenesis enzyme gene quantification. In addition, ovaries and testes collected from unexposed fish were frozen in liquid nitrogen and stored at −80 °C for steroidogenesis enzyme gene cloning.
Cholesterol quantification. After twice dilution with 0.7% saline, plasma was analyzed using a fully automatic biochemistry analyzer to determine the content of TC, HDL-C, and LDL-C, with commercial kits obtained from Shanghai Fenghui Medical Science and Technology Co., LTD, Shanghai Rongsheng Biological Pharmaceutical Co., LTD, Beijing Beihua Kangtai Clinical Reagent Co., LTD, and Shenzhen-Mindray Biological Medical Electronics Co., LTD, China, respectively. The assay detection ranges were 0-20 mmol/L for TC, 0.05-6.0 mmol/L for HDL-C, and 0-9 mmol/L for LDL-C, respectively. The inter-and intra-assay coefficients of variation for TC and LDL-C were ≤5%. The inter-and intra-assay coefficients of variation for HDL-C were ≤2.5% and ≤4.0%, respectively.    10 mM each dNTP, and 4.5 μL diethypyrocarbonate (DEPC) water, incubated at 65 °C for 5 min, and then rapidly frozen. Reverse transcription reaction II was built with 4.0 μL 5× first strand synthesis buffer, 2.0 μL 0.1 mol/L DTT, 1.0 μL cloned ribonuclease inhibitor (40 U/μL, Takara Bio Inc., Shiga, Japan), and 12.0 μL reverse transcription reaction I, hatched at 37 °C for 2 min, and then hatched at 37 °C for 50 min after 1 μL M-MLV reverse transcriptase (200 U/μL) was added. The reaction was terminated by heating the reaction mixture at 70 °C for 15 min.
To clone cDNA fragments of steroidogenesis enzyme genes, including P450scc, 3-beta-HSD, 20-beta-HSD, 17-beta-HSD1, P450 11beta , and 11-beta-HSD2, from goldfish gonads, primers were designed for conserved amino acid sequence regions based on alignments of related gene sequences in other fish species that are closely related to goldfish ( Table 2). Primers designed by Primer Premier 5.0 were compounded by Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China. The polymerase chain reaction (PCR) was conducted with an initial denaturation step at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30s, annealing temperature (Table 2) for 30s, 72 °C for 1 min, and a final extension step at 72 °C for 5 min. Amplified PCR products were cloned in pGM-T easy vector (Takara Bio Inc., Shiga, Japan) and sequenced (Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). MegAlign and GeneDoc programs in DNAStar were used to analyze the homology of the deduced amino acid sequences of goldfish steroidogenesis enzymes with those of other vertebrates.
Quantitative real-time PCR assays were developed to examine the expression patterns of steroidogenesis enzyme genes and StAR in response to MCP pesticide based on the cloned goldfish steroidogenesis enzyme cDNA fragments and StAR, P450c17, P450arom A, beta actin (internal control 1), and 18S rRNA (internal control 2) sequences for goldfish published in GenBank. Primers designed by Primer Premier 5.0 were compounded by Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China (Table 3). Real-time PCR was performed in 20 μL reaction mixtures consisting of SYBR ® Premix Ex Taq TM II (Takara Bio Inc., Dalian, China), 0.4 μL ROX Reference Dye II (Takara Bio Inc., Shiga, Japan), 0.8 μL sense primer (10 μM), 0.8 μL anti-sense primer (10 μM), 4.0 μL cDNA, and 4.0 μL ultrapure water (sterile). The real-time PCR reactions were incubated at 95 °C for 30s, followed by 40 cycles of 95 °C for 5s, and 60 °C for 30s. Melting curve analyses were conducted to differentiate between the desired PCR products and primer-dimers or DNA contaminants. In addition, 2% agarose gel electrophoresis of the PCR products was performed to confirm the presence of single amplicons of the correct predicted size (data not shown). Gene expression was measured in duplicate. Neither beta actin nor 18S rRNA levels were affected by any of the experimental conditions in the study. The relative target gene mRNA levels were normalized using the geometric mean of the beta actin and 18S rRNA gene expression levels following the formula 2 −ΔΔCt and plotted on a logarithmic scale 34 .
Hormone quantification. Fish hormone ELISA Kits (R&D Systems, Inc., Minneapolis, MN, USA) were used to quantify plasma pregnenolone, progesterone, 17 alpha hydroxypregnenolone, 17 alpha hydroxyprogesterone, 17 alpha, 20 beta double hydroxyprogesterone, dehydroepiandrosterone, androstenedione, estrone, 11 beta hydroxytestosterone, and 11-KT levels according to the manufacturer's instructions. The concentration of each hormone was calculated from the linear part of the standard curve, and plasma was diluted when the hormone concentration exceeded the linear range.
Statistics. All data were tested for normality and homoscedasticity. Kruskal-Wallis H test followed by Dunnett's correction was used to analyze GSI, TC, HDL-C, LDL-C, dehydroepiandrosterone, 17 alpha hydroxyprogesterone, androstenedione, estrone, 3-beta-HSD, 11-beta-HSD2, 20-beta-HSD, P450scc, P450c17, P45011beta, and StAR data for an abnormal distribution or heteroscedasticity. A one-way analysis of variance (ANOVA) followed by Tukey's test was adopted for statistical analyses of effectual hormones and the other two intermediates, as well as steroidogenesis enzymes 17-beta-HSD1 and P450arom, because all assumption were met for parametric analyses. Effect size was also considered during statistical analysis. η 2 was calculated as η 2 = SS effect / SS total for parametric analysis and η 2 = H/(N−1) for Kruskal-Wallis tests. By convention, 0.01, 0.06, and 0.15 are considered "small", "medium", and "large"-effect sizes, respectively.