Xiaona Zhang

Monocrotophos pesticide modulates the expression of sexual differentiation genes and causes phenotypic feminization in zebrafish (Danio rerio)

Monocrotophos (MCP) is an organophosphorus pesticide moderately toxic to fish, and it has significant estrogenic properties in vivo. In this study, zebrafish (Danio rerio) were exposed to 0.001, 0.010, and 0.100 mg/L 40% MCP pesticide in a semi-static manner from fertilization to 40 days post-hatching. Histological analyses were performed to determine whether sex differentiation in zebrafish was affected by MCP, and the mRNA expression levels of genes involved in sexual differentiation were quantified by real-time PCR to clarify the possible mechanism(s) of action. The results revealed a prominent increase in the proportion of females (71%) in the 0.100 mg/L MCP pesticide treatment as well as the presence of one intersex individual in each of the groups exposed to 0.001 and 0.100 mg/L MCP. MCP exposure stimulated forkhead transcription factor gene L2 (foxl2) expression and suppressed doublesex/mab-3 related transcription factor 1 (dmrt1) expression, indirectly leading to elevated gonadal aromatase (cyp19a1a) gene expression, which should promote phenotypic feminization. In addition, MCP treatment increased the transcription of brain aromatase (cyp19a1b), resulting in an indirect impact on sexual differentiation. The results from this investigation can be used for risk and hazard assessment of MCP pesticide.

Disruption of the Thyroid System by the Thyroid-Disrupting Compound Aroclor 1254 in Juvenile Japanese Flounder (Paralichthys olivaceus)

Polychlorinated biphenyls (PCBs) are a group of persistent organochlorine compounds that have the potential to disrupt the homeostasis of thyroid hormones (THs) in fish, particularly juveniles. In this study, thyroid histology, plasma TH levels, and iodothyronine deiodinase (IDs, including ID1, ID2, and ID3) gene expression patterns were examined in juvenile Japanese flounder (Paralichthys olivaceus) following 25- and 50- day waterborne exposure to environmentally relevant concentrations of a commercial PCB mixture, Aroclor 1254 (10, 100, and 1000 ng/L) with two-thirds of the test solutions renewed daily. The results showed that exposure to Aroclor 1254 for 50 d increased follicular cell height, colloid depletion, and hyperplasia. In particular, hypothyroidism, which was induced by the administration of 1000 ng/L Aroclor 1254, significantly decreased plasma TT4, TT3, and FT3 levels. Profiles of the changes in mRNA expression levels of IDs were observed in the liver and kidney after 25 and 50 d PCB exposure, which might be associated with a reduction in plasma THs levels. The expression level of ID2 mRNA in the liver exhibited a dose-dependent increase, indicating that this ID isotype might serve as sensitive and stable indicator for thyroid-disrupting chemical (TDC) exposure. Overall, our study confirmed that environmentally relevant concentrations of Aroclor 1254 cause significant thyroid disruption, with juvenile Japanese flounder being suitable candidates for use in TDC studies.

Impairment of the cortisol stress response mediated by the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish (Danio rerio) exposed to monocrotophos pesticide.

In teleosts, an important component of the stress response is coordinated by the hypothalamic-pituitary-interrenal (HPI) axis. Environmental contaminants might disrupt the stress axis and consequently affect the stress response in fish. To investigate the effect of monocrotophos (MCP) pesticide on the stress response of fish and its potential mechanisms, adult zebrafish (Danio rerio) were exposed to 0, 1, 10, and 100μg/L of a 40% MCP-based pesticide for 21d, after which time fish were subjected to a 3-min air-exposure stressor. Concentrations of the whole-body cortisol were measured by radioimmunoassay and abundances of transcripts of proteins involved in the HPI axis were determined using quantitative real-time PCR. Results showed that 100μg/L of MCP pesticide decreased whole-body cortisol levels of female zebrafish in response to an acute stressor, but without any effect on the cortisol response in males. 100μg/L MCP pesticide reduced POMC and GR expression in the brain, MC2R and P45011β expression in the head kidney, but enhanced 20β-HSD2 expression in the head kidney, suggesting that MCP damaged the HPI axis involving acting at pituitary regulatory levels, inhibiting cortisol synthesis and stimulating cortisol catabolism, or disturbing the negative feedback regulation. Additionally, MCP depressed liver GR transcription but did not affect phosphoenolpyruvate carboxykinase and tyrosine aminotransferase expression in zebrafish, suggesting a role for this pesticide in reducing target tissue responsiveness to cortisol. Considered together, the reduced ability to elevate cortisol levels in response to an acute stress may be an endocrine dysfunction occurring in zebrafish subchronically exposed to MCP pesticide.

Anti-estrogenic effect of semicarbazide in female zebrafish (Danio rerio) and its potential mechanisms.

Semicarbazide (SMC), a member of the hydrazine family, has various toxic effects and has been detected in organisms, aquatic environments, and food. SMC exposure inhibited the transcription of hepatic vitellogenin and estrogen receptors in female zebrafish (Danio rerio), suggesting that it had anti-estrogenic properties. In order to elucidate the mechanisms underlying these, we exposed female zebrafish to SMC and used enzyme-linked immunosorbent assays to examine plasma 17β-estradiol (E2) and testosterone (T) levels. Gonad histology was analyzed and the mRNA expression of genes involved in the reproductive axis, the gamma-aminobutyric acid (GABA) shunt, and leptin was quantified by real-time PCR. Zebrafish were exposed to 1, 10, 100, or 1000μg/L SMC in a semi-static system for 96hours or 28 days. Plasma E2 levels were significantly decreased and ovarian maturation was inhibited by SMC, suggesting that its anti-estrogenic effect was exerted by reducing endogenous E2 levels. This was likely due to the SMC-mediated inhibition of cytochrome P450 (CYP) 19A mRNA levels, because this enzyme catalyzes the conversion of T to E2 in the gonads. In addition, down-regulation of the mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, steroidogenic acute regulatory protein, CYP17, and 17beta-hydroxysteroid dehydrogenase was observed; this was predicted to reduce T concentrations and further contribute to the reduced E2 levels. SMC-induced changes in the expression of these steroidogenic genes correlated with decreased transcription of gonadotropic hormones (follicle-stimulating hormone and luteinizing hormone) and significantly elevated leptin expression. Furthermore, SMC also altered expression of the key enzyme in gamma-aminobutyric acid (GABA) synthesis, GABA receptors, and salmon gonadotropin-releasing hormone, thus affecting gonadotropin expression. Overall, SMC acted at multiple sites related to reproduction to reduce plasma E2 levels, consequently exerting an anti-estrogenic effect in female zebrafish. These effects were observed at environmentally relevant concentrations and highlight the importance of controlling SMC contamination.

Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus).

Goldfish (Carassius auratus) vitellogenin (Vtg) is an efficient biomarker for estrogen contamination in aquatic environments. In this study, Vtg and lipovitellin (Lv) were purified from the plasma of 17β-estradiol (E2)-induced male goldfish and unfertilized eggs of females, and were used to generate polyclonal antibodies against Vtg (anti-Vtg) and Lv (anti-Lv), respectively. SDS-PAGE and Western blot were performed to confirm the specificity of the two antibodies and the immunological similarity between Vtg and Lv. As anti-Lv recognized more antigen epitopes than anti-Vtg, it was used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for goldfish Vtg with purified Lv as the standard. The detection limit of the assay was 1.82ng/mL, and the working range was 3.9-250ng/mL. The use of Lv instead of Vtg as the standard provided greater precision and strengthened the robustness of the sandwich ELISA. Western blot and the Lv-based ELISA were used to detect Vtg inductions in surface mucus and plasma of E2-induced goldfish. The surface mucus Vtg level in E2-induced males was significantly higher than that in the control males and E2-induced females, and was much closer to the plasma Vtg level in E2-induced males than that in E2-induced females. Therefore, the surface mucus Vtg level of male goldfish may be a reliable indicator of estrogenic activity in the aquatic environment.

Monocrotophos Pesticide Decreases the Plasma Levels of Total 3,3′,5-Triiodo-l-Thyronine and Alters the Expression of Genes Associated with the Thyroidal Axis in Female Goldfish (Carassius auratus)

Our recent study showed that monocrotophos (MCP) pesticide disrupted the hypothalamic-pituitary-thyroid (HPT) axis in male goldfish (Carassius auratus); however, the effects of MCP on the thyroid system in female goldfish are remain unclear. In the present study, plasma thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels were evaluated in female goldfish exposed to 0.01, 0.10, and 1.00 mg/L of 40% MCP-based pesticide for 21 days in a semi-static exposure system. Expression profiles of HPT axis-responsive genes, including transthyretin (ttr), deiodinases (d1, d2, and d3), tshβ, thyrotropin-releasing hormone (trh), and corticotrophin-releasing hormone (crh), were determined. The results indicated that MCP decreased the plasma levels of total 3,3′,5-triiodo-l-thyronine (TT3) and the ratio of TT3 to total 3,3′,5,5′-l-thyroxine (TT4), and induced alternative expression of TH-related genes. Exposure to 0.01 and 0.10 mg/L MCP pesticide resulted in the up-regulation of ttr mRNA. The reduction of plasma TT3 levels was partly attributed to an increase in the metabolism of T3 in the liver, as revealed by the highly elevated hepatic d1 and d3 mRNA levels in the MCP treatment groups, and the expression of hepatic d3 showed a negative correlation with the plasma TT3/TT4 levels in females. Moreover, the plasma TSH levels were lower in females exposed to 0.01 and 0.10 mg/L MCP pesticide, whereas the up-regulation of tshβ mRNA levels was compensated by the decreased plasma TT3 levels. These results indicated that MCP had the potential to influence several pathways of HPT axis homeostasis in female goldfish.

Lipovitellin as an antigen to improve the precision of sandwich ELISA for quantifying zebrafish (Danio rerio) vitellogenin

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a core biomarker for screening environmental estrogens in test guidelines of the Organization for Economic Cooperation and Development. To accurately quantify zebrafish Vtg, lipovitellin (Lv), the main Vtg-derived yolk protein, was used as the antigen to establish a sandwich enzyme-linked immunosorbent assay (ELISA). The purified Lv was a phospholipoglycoprotein with apparent molecular weight of ~445kDa, and separated into three polypeptides corresponding to ~117, ~102, and ~23.8kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunological analysis confirmed the specificity of the anti-Lv antibody for Vtg and the immunological similarity between Vtg and Lv. Using the purified Lv and anti-Lv antibody, a sandwich ELISA with a detection limit of 4.3ng/mL and a detection range from 7.8 to 250ng/mL was developed. The intra- and inter-assay coefficients of variation were both below 10%. Moreover, the Lv standard curve was nearly identical to the Vtg standard curve, and paralleled serial whole-body homogenate dilutions of male zebrafish exposed to 17β-estradiol, demonstrating that the Lv-based ELISA could be used for quantification of zebrafish Vtg. Zebrafish Lv showed high stability during purification process, heat treatment, -80°C storage, and repeated freeze/thaw cycles. Additionally, the standard curve of Lv stored at -80°C for 3months exhibited higher robustness than that of Vtg stored under the same conditions. Finally, the usefulness of the ELISA for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to monocrotophos.

Development of a lipovitellin-based goldfish (Carassius auratus) vitellogenin ELISA for detection of environmental estrogens

The susceptibility of vitellogenin (Vtg) to degradation is a major problem affecting the robustness of enzyme-linked immunosorbent assay (ELISA) for goldfish (Carassius auratus) Vtg. In this study, a phospholipoglycoprotein with molecular mass of ∼420kDa was purified from goldfish egg extracts and it produced a single band corresponding to ∼112kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the amino acid composition of the purified protein was comparable to that of lipovitellin (Lv) from other fish species. Thus, the purified protein was identified as goldfish Lv. Purified Lv and anti-Lv polyclonal antiserum were used to develop an ELISA with a detection range between 31.25 and 1000ngmL(-)(1). The intra- and inter-assay coefficients of variation were 6.45% and 7.08%, respectively. The immunological similarity between goldfish Vtg and Lv was confirmed by immunoelectrophoresis and Western blot. Goldfish Lv showed higher stability than Vtg after -80°C storage, multiple freeze/thaw cycles, and heat treatment. Moreover, the use of treated Lv in the ELISA did not change the slopes of standard curves. Parallelism between the Lv standard curve and plasma dilution curves of vitellogenic females confirmed the validity of the assay for quantifying plasma Vtg. The Lv-based Vtg ELISA was further applied to evaluate the estrogenic activity of monocrotophos pesticide.

Effects of monocrotophos pesticide on steroidogenesis and transcription of steroidogenic enzymes in rainbow trout RTG-2 cells involving the protein kinase A signal pathway

Monocrotophos (MCP) pesticide, listed as a UNEP Prior Informed Consent chemical, has been proved to exert toxic effects on the reproductive system of teleost fishes by changing the balance of sex steroid hormones. To investigate the effects of MCP on steroidogenesis in vitro, the rainbow trout (Oncorhynchus mykiss) gonadal cell line RTG-2 was exposed to different MCP concentrations for 48 h. The levels of 17 β-estradiol (E(2)) and testosterone in the medium were measured by radioimmunoassay and the expression of steroidogenic acute regulatory protein and cytochrome P450 enzymes CYP11A1, CYP17, and CYP19A was detected by quantitative real-time PCR. The results showed that 1.0 and 10.0 μg/L MCP pesticide induced E(2) levels and promoted steroidogenic enzyme expression. The possible mechanisms of MCP steroidogenic activity were investigated using inhibitors of protein kinase A (PKA) and protein kinase C. The PKA inhibitor H-89 abrogated the 10.0 μg/L MCP-induced transcriptional up-regulation of steroidogenic enzymes, suggesting an involvement of PKA-dependent mechanism in the disruption of steroidogenesis by the MCP pesticide in rainbow trout RTG-2 cells.

Estrogenic effects associated with bisphenol a exposure in male zebrafish (Danio rerio) is associated with changes of endogenous 17β-estradiol and gene specific DNA methylation levels

The binding affinity of bisphenol A (BPA) to estrogen receptors (ERs) is much lower than that of 17β-estradiol (E2), and whether there are other molecular mechanisms responsible for the estrogenic action of BPA in vivo currently remains unknown. The objective of this study was to explore the potential association between the estrogenic effect induced by bisphenol A in vivo and changes of endogenous E2 and gene specific DNA methylation levels. After a waterborne exposure of male zebrafish to 500, 1000, or 1500μg/L of BPA for 21d, vitellogenin (VTG) concentration in whole body homogenate, plasma E2 and testosterone levels, hepatic ERs mRNA expressions, gonadal cyp19a1a and cyp17a1 mRNA expressions, and methylation levels of hepatic esr1 and gonadal cyp19a1as promoters were determined. Our results indicated that for the 500 and 1500μg/L treatment groups, VTG might be induced mainly by the elevated E2 levels; increases of E2 levels could be partly explained by the up-regulated expression of gonadal aromatase, mRNA levels of which were found to be negatively related to the methylation levels of both its promoter and one CpG site. In addition, upon BPA exposure, hepatic esr1 mRNA levels were also negatively related to the methylation levels of both its promoter and one CpG site. These observations provide evidence for the non-ERs mediated mechanisms underlying the estrogenic action of BPA on male zebrafish.