Shaoguo Ru

Estrogenic effects of monocrotophos evaluated by vitellogenin mRNA and protein induction in male goldfish (Carassius auratus).

Monocrotophos (MCP) is listed as a Prior Informed Consent chemical, and it is still used extensively in developing countries. It has been seen that MCP shows high toxicity in fish and aquatic ecosystems. To confirm whether MCP shows estrogenic effects on fish, the induction of vitellogenin (VTG) in male goldfish (Carassius auratus) following a 21 day exposure to 0.01, 0.10 and 1.00 mg x L(-1) 40% MCP pesticide in a semi-static exposure system was evaluated by hepatic mRNA and plasma protein. Real-time reverse transcriptase polymerase chain reaction assay showed that MCP significantly increased hepatic mRNA expression of VTG in male goldfish in a dose-dependent manner. Western blot analysis revealed the presence of two VTG subunits with molecular masses of 134 kDa and 110 kDa in the plasma of the exposure groups, which were not observed in that of the control males, indicating the induction of VTG protein synthesis and secretion by MCP in male goldfish. Furthermore, the levels of plasma VTG protein in the control group and the 0.01, 0.10 and 1.00 mg x L(-1) 40% MCP pesticide exposure groups were quantified by enzyme-linked immunoassay, and revealed concentrations of 53.8+/-5.6 ng x mL(-1), 285.5+/-5.0 ng x mL(-1), 972.9+/-7.4 ng x mL(-1) and 678.4+/-38.0 ng x mL(-1), respectively. The overlapping results of VTG mRNA and protein indicated that MCP had significant estrogenic properties and was thus a potential endocrine disruptor.

Effects of monocrotophos on the reproductive axis in the male goldfish (Carassius auratus): potential mechanisms underlying vitellogenin induction.

Monocrotophos (MCP) is a highly toxic organophosphorus pesticide that has been banned in many countries. Both vitellogenin mRNA expression and secretion were significantly induced in male goldfish by exposure to an MCP-based pesticide, suggesting that MCP has significant estrogenic properties. To elucidate the mechanisms of action of MCP on vitellogenin induction, we used radioimmunoassay to examine the effect of MCP treatment on plasma 17beta-estradiol and testosterone levels in male goldfish (Carassius auratus). We also investigated the potential impacts of MCP treatment on aromatase expression, on the synthesis and secretion of pituitary gonadotropins and on the regulation of hypothalamic gonadotropin-releasing hormones by real-time PCR and radioimmunoassay. Experiments were carried out during the period of gonadal late recrudescence following a 21-day exposure to 0.01, 0.10 and 1.00 mg L(-1) of a pesticide containing 40% MCP in a semi-static exposure system. The results indicated that males in each MCP treatment group had much higher plasma levels of 17beta-estradiol, suggesting that the induction of VTG production by MCP was indirectly caused by elevated levels of endogenous 17beta-estradiol. MCP-induced plasma 17beta-estradiol levels via interference with the reproductive axis at multiple potential sites in male goldfish: (a) MCP exposure enhanced the mRNA expression of gonadal aromatase, the enzyme that converts androgens into estrogens, consequently reducing plasma levels of testosterone and increasing plasma concentrations of 17beta-estradiol; (b) MCP treatment increased follicle-stimulating hormone beta subunit mRNA expression and protein secretion and decreased luteinizing hormone beta subunit mRNA expression and protein secretion, thus interfering with gonadotropin synthesis and secretion at the pituitary level and leading to the disruption of reproductive endocrine control and androgen and estrogen balance.

Exposure to monocrotophos pesticide during sexual development causes the feminization/demasculinization of the reproductive traits and a reduction in the reproductive success of male guppies (Poecilia reticulata).

Monocrotophos is a highly toxic organophosphorus pesticide that has been confirmed to be an endocrine-disrupting chemical. To evaluate the influence of this pollutant on the reproductive system of male fish, we studied the sex steroid levels, reproductive traits, sex ratio, and reproductive success in male guppies (Poecilia reticulata) exposed to 40% monocrotophos pesticide at the nominal concentrations of 0.01, 0.10, and 1.00 mg/L for 90 days from birth to adulthood in a semi-static exposure system. Radioimmunoassay and western blot analyses demonstrated that the long-term exposure to monocrotophos pesticide during the sexual development of male guppies caused a significant increase in 17β-estradiol levels and consequently induced vitellogenin synthesis, suggesting the feminization of the males. Monocrotophos pesticide also caused a significant decrease in testosterone levels, which consequently inhibited testis growth and reduced the sperm count and the area and intensity of their sexually attractive orange spots, which collectively indicated the significant demasculinization of the male sexual characteristics. Furthermore, these changes in the sexual characteristics at the cellular and organ levels translated into ecologically important effects on the reproductive success at the individual level, as measured by a decrease in offspring production and survival rate. The present study provides the first evidence that monocrotophos pesticide can cause severe reproductive abnormalities in fish due to its endocrine-disrupting action.

Disruptions in aromatase expression in the brain, reproductive behavior, and secondary sexual characteristics in male guppies (Poecilia reticulata) induced by tributyltin.

Although bioaccumulation of tributyltin (TBT) in fish has been confirmed, information on possible effects of TBT on reproductive system of fish is still relatively scarce, particularly at environmentally relevant levels. To evaluate the adverse effects and intrinsic toxicological properties of TBT in male fish, we studied aromatase gene expression in the brain, sex steroid contents, primary and secondary sexual characteristics, and reproductive behavior in male guppies (Poecilia reticulata) exposed to tributyltin chloride at the nominal concentrations of 5, 50, and 500 ng/L for 28 days in a semi-static exposure system. Radioimmunoassay demonstrated that treatment with 50 ng/L TBT caused an increase in systemic levels of testosterone of male guppies. Gonopodial index, which showed a positive correlation with testosterone levels, was elevated in the 5 ng/L and 50 ng/L TBT treated groups. Real-time PCR revealed that TBT exposure had inhibiting effects on expression of two isoforms of guppy aromatase in the brain, and these changes at the molecular levels were associated with a disturbance of reproductive behavior of the individuals, as measured by decreases in frequencies of posturing, sigmoid display, and chase activities when males were paired with females. This study provides the first evidence that TBT can cause abnormalities of secondary sexual characteristics in teleosts and that suppression of reproductive behavior in teleosts by TBT is due to its endocrine-disrupting action as an aromatase inhibitor targeting the nervous system.

An integrated approach combining chemical analysis and an in vivo bioassay to assess the estrogenic potency of a municipal solid waste landfill leachate in Qingdao.

Various adverse effects related to landfill leachate have made leachates an important issue in past decades, and it has been demonstrated that landfill leachate is an important source of environmental estrogens. In this study, we employed chemical analysis of some already evaluated estrogenic substances, in combination with a bioassay using several specific biomarkers (e.g., plasma vitellogenin and sex steroids, enzyme activity of gonad gamma-glutamyl transpeptidase, and gonadosomatic index) to evaluate the estrogenic activities in outlets from different stages of the leachate treatment process. The results indicated that 5 environmental estrogens (4-t-octylphenol, bisphenol A, di-ethyl phthalate, di-n-butyl phthalate, and diethylhexyl phthalate) were detected by a gas chromatography-mass spectrometry, and the concentrations in leachate samples were 6153 ng/L, 3642 ng/L, 2139 ng/L, 5900 ng/L, and 9422 ng/L, respectively. Leachate (1∶200 diluted) induced the synthesis of plasma vitellogenin and led to decreased enzyme activity of gonad gamma-glutamyl transpeptidase and gonadosomatic index in male goldfish (Carassius auratus) after a 28-day exposure, while increased circulating 17β-estradiol level was also observed in males exposed to treated effluent. Although the target EEs were partially removed with removal rates varying from 87.2% to 99.77% by the “membrane bioreactor+reverse osmosis+aeration zeolite biofilter” treatment process, the treated effluent is still estrogenic to fish. The method combined chemical techniques with the responses of test organisms allowing us to identify the group of estrogen-like chemicals so that we were able to evaluate the overall estrogenic effects of a complex mixture, avoiding false negative assessments.

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.

Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide

Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17β-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460 kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6 ng/mL and a detection range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were 2.4-6.8% and 6.7-10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00 mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.

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.

Effects of monocrotophos on the reproductive axis in the female goldfish (Carassius auratus)

Monocrotophos (MCP) is a highly toxic organophosphorus pesticide. To elucidate the influence of MCP on female fish reproduction, plasma 17beta-estradiol, testosterone and gonadotropin levels and aromatase and gonadotropin beta subunit gene expression levels were examined in female goldfish (Carassius auratus) following a 21-day exposure to 0.01, 0.10 and 1.00 mg L-(1) 40% MCP-based pesticide in a semi-static exposure system. The results indicated that MCP induced increases in plasma 17beta-estradiol levels and the 17beta-estradiol/testosterone ratio via interference with the reproductive axis at multiple potential sites through two mechanisms: (a) MCP exposure enhanced the mRNA expression of gonadal aromatase, the enzyme that converts androgens into estrogens, consequently reducing plasma testosterone levels and increasing plasma concentrations of 17beta-estradiol; and (b) MCP treatment increased follicle-stimulating hormone beta subunit mRNA expression and secretion and decreased luteinizing hormone beta subunit mRNA expression and secretion, leading to the disruption of reproductive endocrine control and androgen and estrogen balance. This study provided convincing evidence for reproductive toxicology of MCP by disrupting of the HPG axis at multiple sites in female goldfish.