Enantioselective Endocrine Disrupting Effects Of Phenylpyrazole Chiral Insecticides

Pesticides plays a very important role in agricultural production.However,the harm of pesticides to animals,plants and even human beings frequently occurs due to the irrational use of pesticides.Fipronil is a phenylpyrazole chiral pesticide,which was restricted due to its high toxicity,toxic metabolism,endocrine disrupting effects,and unfriendly environment.It was reported that there were significant differences between the enantiomers.Phenylpyrazole chiral insecticides ethiprole and flufiprole were developed and popularized as alternatives to fipronil.Ethiprole and flufiprole had the similar structures to fipronil.It were still not clear whether they had the endocrine disrupting effect and whether there are differences between enantiomers.In this study,the endocrine-disrupting effects(EDEs)of phenylpyrazole chiral pesticides and their enantiomers were studied through the in vitro difluoromycin reporter gene assay,zebrafish exposure test and molecular dynamics simulation.The main research conclusions are as follows:(1)In the aspect of hormone agonistic effect,none of phenylpyrazoles shown estrogen effect or thyroid hormone effect.In terms of estrogen antagonism,R-(+)-flufiprole,R-(-)-fipronil and R-(+)-ethiprole showed stronger estrogen antagonism.The corresponding 20%relative inhibitory concentration(RIC20)values were values were 1.37,1.35 and 1.34 times of the respective enantiomers,with certain stereoselectivity.The tendency of the antagonistic activity was:R-(-)-fipronil>R-(+)-flufiprole>rac-fipronil>R-(+)-ethiprole>rac-ethiprole>rac-flufiprole>S-(+)-fipronil>S-(-)-flufiprole>S-(-)-ethiprole.In terms of TRβ-mediated EDEs of thyroid hormones,only S-(-)-flufiprole and S-(-)-ethiprole exhibited enantioselective anti-thyroid hormone effects and calculated RIC20,showing certain stereoselectivity.In addition,fipronil and its enantiomers had no thyroid hormone antagonistic effects.(2)The acute toxicity of the enantiomers of flufiprole and ethiprole except R-ethiprole to zebrafish embryos were both low toxicity.After treatment with S-(-)-flufiprole and S-(-)-ethiprole,the expression of the thyroid-stimulating hormone gene(TSHβ)was significantly induced;The expression of the deiodinase(Diol)was significantly suppressed,and the deiodinase(Dio2)was unchanged compared to the control;The transcription of the TRa gene was not changed and the TRβ was significantly decreased.It is speculated that the mechanism of the thyroid hormone interference effect may be the inhibition of the expression of the deiodinase Diol and the decrease of T3 concentration in the body.Due to the existence of a negative feedback mechanism,the expression of TSHβ is up-regulated,or the pesticide may have caused the expression of TSHβ because of the T3 concentration.The decline also led to anti-thyroid hormone effects(downregulation of TRβ).(3)The order of acute toxicity of the ethiprole and flufiprole to juveniles fish was same as that to embryo.After exposure to R-(+)-flufiprole and R-(+)-ethiprole,the expression of HPG axis related genes was significantly affected.The expression of 17β-HSD3,Cyp19a and Cyp19b were significantly down-regulated.And,the expression of cyp19b was down more,meaning that Cyp19b showed more sensitive.ERa and Vtg gene expression were inhibited.A possible mechanism for the anti-estrogenic effect was that the pesticides inhibited the activity of hydroxysteroid dehydrogenase(17β-HSD3)and aromatase(Cyp17,Cyp19a and Cyp19b).Then.the conversion from cholesterol to androstenedione to estradiol were inhibited.Finally,the level of estrogen E2 in the body was down,which leading to anti-estrogenic effects(down-regulated of Vtgl,Vtg2 and ERα).(4)R-(+)-flufiprole,R-(+)-ethiprole and R-(-)-fipronil formed more interaction forces with the ERa receptor protein(hydrogen bond,hydrophobic interaction and Pi-Pi bond).And,they had lower total binding free energy(ΔGcalc),and ΔEvdw.For TRβ receptors.S-(+)-fipronil,S-(-)-flufiprole and S-(-)-ethiprole were better than S-(+)-fipronil.In the simulation R-(+)-flufiprole and R-(+)-ethiprole form more hydrogen bonds and hydrophobic interactions.and the total binding free energy also shows the same trend.Although S-(+)-fipronil shows a stronger affinity for TRβ receptor protein than R-(-)-fipronil.However,the ΔGcalc and ΔEsurf of S-(+)-fipronil were lower than those of flufiprole and ethiprole.The above results validated the conclusion of the reporter gene assay that the R-(-)-fipronil.R-(+)-flufiprole and R-(+)-ethiprole had anti-estrogenic effects,while S-(-)-flufiprole.and S-(-)-ethiprole had anti-thyroid hormone effects.