| Monocrotophos (MCP) pesticide, listed as a UNEP Prior Informed Consentchemical, has been proved to exert toxic effects on the reproductive system of teleostfishes by changing the balance of sex steroid hormones. The biosynthesis of sexsteroid hormones in teleosts is regulated by the transcriptions and/or activities ofsteroidogenic enzyme and also under the control of pituitary-secreted gonadotropins.In view of avoiding the potential cross-talk among different tissues, we used RTG-2cells to investigate the mechanisms of MCP effects on steroidogenesis in fish. RTG-2cultures were exposed to40%MCP-containing pesticide, and the levels of E2and T inthe culture medium and the expression of the steroidogenic genes were subsequentlydetermined by radioimmunoassay and quantitative real-time PCR, respectively.Inhibition of PKAand PKC was performed to identify signaling mechanisms involvedin the MCP-induced disruption of steroidogenic gene transcription. The effects ofMCP pesticide on cAMP/PKA pathway, the certain signaling pathway involving inthe steroidogenesis, were also measured. In this study the effects of MCP pesticide onsteroidogenesis was investigated by evaluating the sex homornes levels, steroidogenicenzyme transcription and the modulated action of signaling pathway. The resultsacquired in present study provided a potential mechanism of modulatingsteroidogenesis by MCP pesticide.The results indicated that:(1)48-h IC50of MCP in RTG-2cells was calculated as243.80μg/L, and theviability of RTG-2cells was not affected by the pesticide at concentrations lower than160μg/L. The activities of certain enzymes were evaluated as biochemical factors tocharacterize the cell growth and physiological function, and the chosen exposuredoses of MCP pesticide did not affect the cell status.(2) Sex steroid levels in the culture medium were measured byradioimmunoassay following a48-h exposure to0.1,1.0,10.0, and100.0μg/L MCPpesticide. The E2levels in untreated control and cells treated with0.1μg/L MCPpesticide were both significantly lower than the initial concentration. The increase ofthe pesticide concentration to1.0and10.0μg/L correlated with a significantup-regulation of E2production by RTG-2cells compared with either control group or the initial concentration. All MCP groups showed a highly significant reduction in Tlevels after48h compared to T levels in initial medium. This suggested that MCPpesticide disrupted the biosynthesis of sex steroid hormones in RTG-2cells, andespecially promoted the E2production by the cells.(3) The expression levels of steroidogenic acute regulatory protein (StAR) andseveral steroidogenic key cytochrome P450enzymes (CYPs), including P450sidechain cleavage enzyme (CYP11A1), cytochrome P45017alpha-hydroxylase (CYP17),and gonadal aromatase (P450aromatase, CYP19A) in cells were detected with thereal-time PCR assay after48-h exposure to0.1,1.0,10.0, and100.0μg/L MCPpesticide. StAR mRNA levels were not affected by any of the MCP concentrationstested. CYP11A1and CYP17expression were enhanced after the treatment with1.0and10.0μg/L MCP pesticide, and CYP19A mRNA expression levels wereup-regulated on exposure to0.1,1.0, and10.0μg/L MCP pesticide. The datasuggested that MCP pesticide promoted the transcription of steroidogenic CYPsmRNA.(4) To determine the involvement of the PKA and PKC pathways in theMCP-induced transcriptional changes, RTG-2cells were treated with a combinationof10.0μg/L MCP pesticide and inhibitors of PKA and PKC for48-h. StAR,CYP11A1, CYP17, and CYP19A mRNA expression in cells were evaluated by real-time PCR again. StAR expression was not affected by10.0μg/L MCP pesticide andits combination with the inhibitors. However, CYP11A1, CYP17, and CYP19AmRNA expression induced by10.0μg/L MCP pesticide was markedlydown-regulated by the PKAinhibitor but was not affected by the PKC inhibitor. Thus,we hypothesized that the modulation of MCP on steroidogenic enzyme expressioninvolved the PKAsignaling pathway rathen than PKC cascade.(5) The concentration of cAMP in cell was analyzed by enzyme linkedimmunosorbent assay following a exposure to0.1,1.0,10.0, and100.0μg/L MCPpesticide for0.5,2,4,8,24, and48h. The data showed that MCP pesticide exposureincrease the concentration of cAMP within4h. Therefore, the activities of PKA wereestimated following a4-h exposure to0.1,1.0,10.0, and100.0μg/LMCP pesticide bya non-radioactive detection assay, and the result revealed an enhancement of PKAactivity induced by1.0,10.0, and100.0μg/L MCP pesticide exposure. These resultssupposed that MCP stimulated the cAMP/PKA signal transduction pathway, whichinvolved in the MCP modulated-CYPs expression. In conclusion, MCP promoted the expression of steroidogenic enzymesCYP11A1. CYP17, and CYP19A and caused significant changes in the production ofsteroid hormones E2by RTG-2fish cells. The MCP-induced transcriptional activationwas abrogated by the PKA inhibitor H-89, and MCP increase the concentration ofcAMP and activity of PKA, suggesting that MCP modulated the biosynthesis of sexsteroids hormones by interfering with the PKA signaling pathway. This was the firsttime to provide a convincing evidence for mechanism of MCP effects onsteroidogenesis. Further more our results provide a better understanding ofmechanisms underlying estrogenic effects of toxic chemicals similar to MCP, whichare unrelated to estrogen receptor activation, and also help to identify the mechanismof multiple toxic effects exerted by MCP pesticide. |
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