Study On The Combined Effects Of Chlorpyrifos And Chlorothalonil On The Endocrine Disturbance Of Zebrafish

Chlorpyrifos is an organophosphorus insecticide,which acts as an insecticide by inhibiting the activity of acetylcholinesterase.Chlorothalonil is a kind of substituted benzene-type fungicide,which acts as a bactericidal action by inhibiting the cellular respiration enzymes of the pathogenic bacteria cells.Due to the slow degradation rate of chlorpyrifos and chlorothalonil in water,the relatively low soil adsorption,and the holding period is longer in the water body.Therefore,the toxicological study of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil is of great significance to ecological risk assessment.In this study,zebrafish were used as model organisms.The present study evaluated the effects of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil on endocrine system of zebrafish as well as the potential mechanisms,including hypothalamus-pituitary-thyroid axis(HPT),hypothalamic-pituitary-gonadal axis(HPG)and endocrine enzyme.The acute toxicity of zebrafish showed that the LC50 value of chlorpyrifos was 2.14 mg·L-1,and the LC50 value of chlorothalonil 96 h was 0.353 mg·L-1.Chlorpyrifos and chlorothalonil were respectively medium and highly toxic to zebrafish embryos.The LC50 value of the chlorpyrifos was 2.12 mg·L-1 and the LC50 value of the chlorothalonil was 0.079 mg·L-1 for 96h of zebrafish larvae.The chlorpyrifos and chlorothalonil were respectively medium and highly toxic to the zebrafish larvae.Additive index method is used to determine chlorpyrifos + chlorothalonil binary combination for the joint effect of zebrafish embryos.During the exposure time of 24 h,48 h and 72 h,the AI value respectively was-0.193,-0.123 and-0.093,and showed an antagonistic effect on the embryos of zebrafish.At 96 h,the AI value was 1.82,which showed synergistic effect.In this study,we explored the hypothalamic-pituitary-gonad axis(HPG)of zebrafish larvae in two aspects.On the one hand,real-time fluorescence quantitative PCR was used to detect the effects of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil on the expression of genes related to the gonadal axis of zebrafish larvae.On the other hand,the effects of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil were detected by enzyme-linked immunosorbent assay(ELISA)on the level of the yolk protein(VTG)of zebrafish larvae.The results of the experiment showed that the expression of the genes of ER??ER?1,VTG1?VTG2 and ER?2 was increased in chlorpyrifos,and there was no significant change in gene expression of CYP19a.Chlorothalonil promoted the up-regulation of the expression of ER?1?VTG1?VTG2 and ER?2,and the expression of CYP19a and ERa was not significantly changed compared with the control group.The combination of chlorpyrifos + chlorothalonil promoted the up-regulation of the expression of ER??ER?1?VTG2 and ER?2,and inhibits the expression of VTG1 gene,and there was no significant change in gene expression of CYP19a.Chlorpyrifos,chlorothalonil and chlorpyrifos+ chlorothalonil had no significant effect on the VTG content in zebrafish.The results showed that chlorpyrifos and chlorothalonil had certain estrogenic interference effect on the zebrafish.There was a significant decrease in the degree of interference with the gonadal axis of chlorpyrifos + chlorothalonil compared with the exposure to single agent.From the hypothalamus-pituitary-thyroid axis(HPT)regulation perspective,using real-time fluorescent quantitative PCR detection of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil dual combination of zebrafish larvae thyroid axis of the influence of related gene expression.The effects of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil on the level of the thyroid hormone of zebrafish were detected by enzyme-linked immunosorbent assay(ELISA).The results showed that chlorpyrifos and chlorothalonil had a certain thyroid endocrine disrupting effect on zebrafish larvae.There was a significant decrease in the degree of interference with the thyroid axis of chlorpyrifos + chlorothalonil compared with the exposure to single agent.Compared with control group,chlorpyrifos and chlorothalonil can promote the up-regulation of gene expression of CRH,TSH,D1,D2 and TRa,inhibit the expression of TPO gene,and there is no significant change in TG gene expression.The binary combination of chlorpyrifos + chlorothalonil can inhibit the expression of TRa and TPO genes,and there is no significant change in gene expression of CRH,TSH and TG.The content of T3 were increased and T4 were no significant change in the larvae of chlorpyrifos,chlorothalonil and chlorpyrifos + chlorothalonil.