Research On The Nuclear Receptors Mediated Endocrine Disrupting Effects Of Pesticides And Their Metabolites

The annual production and consumption of pesticides in China is the largest worldwide,so studies about the ecological and health risks of pesticides had always been the research hotspot.Present studies have reported that pesticides could enter the soil,air or water system via spray-drift,volatilization and runoff events,and truned into numerous metabolites.However,studies about management and risks assessment of metabolites of pesticides and the remains scare.Herein,we use zebrafish embryo toxicity assay and MTS assay to comparatively evaluate the toxicity of pesticides and their metabolites.On the other hand,we use nuclear receptors mediated dual-luciferase reporter gene assays,PCR assay and molecular docking to comprehensively explore the nuclear receptors mediated endocrine disrupting effects of pesticides and their metabolites.The major work and achievements of this study can be divided into two parts:(1)The identification of the metabolites of chlorothalonil in zebrafish(Danio rerio)and their embryo toxicity and endocrine effects at environmentally relevant levels.Chlorothalonil is a broad spectrum fungicide with high annual production and environmental contamination.Despite its high consumption,studies regarding the potential ecological risks of chlorothalonil,especially its metabolites,to aquatic organisms are still limited.In this study,we selected the zebrafish(Danio rerio)as the in vivo model and first identified the metabolite(4-hydroxychlorothalonil)of chlorothalonil in zebrafish by tandem quadrupole/orthogonal-acceleration time-of-flight(Q-TOF).Besides,PCR assay was also used to explore the mechanism of the biotransformation of chlorothalonil mediated by CYP450.Then,the in vivo and in vitro models were applied to comprehensively estimate the embryo toxicity and potential endocrine effect of chlorothalonil and 4-hydroxychlorothalonil.The data from zebrafish embryo toxicity showed that the lowest observed effect concentrations of both chlorothalonil and 4-hydroxychlorothalonil were 50 ?g/L after 96 h of exposure.The mortality rate of the 4-hydroxychlorothalonil was 2.6-fold higher than that of the parent compound at the concentration of 50 ?g/L.Dual-luciferase reporter gene assays indicated that both chlorothalonil and 4-hydroxychlorothalonil exerted estrogen receptor ?(ER?)agonist activity with REC20 values of 2.4×10-8 M and 3.6×10-8 M,respectively.However,only 4-hydroxychlorothalonil exhibited both thyroid receptor ?(TR?)agonistic and antagonistic activities.Lastly,we employed molecular docking to predict the binding affinity of chlorothalonil and 4-hydroxychlorothalonil with ER? or TR?.The results revealed that the potential endocrine effect of chlorothalonil and 4-hydroxychlorothaloni might be attributed to the different binding affinities with the receptors.In conclusion,our studies revealed that 4-hydroxychlorothalonil exhibited potent endocrine-disrupting effects compared to its parent compound,chlorothalonil.The results provided here remind us that the assessment of the potential ecological and health risks of the metabolites of fungicides in addition to their parent compounds should arouse great concerns.(2)Researches on the GR and MR mediated endocrine disrupting effects of 4 pesticides and 21 metabolites.Pyriproxyfen,Malathion,Benalaxyl and Fenoxaprop-ethyl are four widely used pesticides.For their high consumption and numerous metabolites,the ecological and health risk assessment of these pesticides and their metabolites is of great importance.In this study,CHO-K1 cells was used as a model and combined with MTS assay to contrast the toxicity of 4 pesticides and 21 metabolites;dual-luciferase reporter gene assays was also used to investigate their potential endocrine disrupting effects via the GR and the MR.As a powerful tool,molecular docking had been successfully applied to show detailed information about the possible interactions between these chemicals and the ligand binding pocket of GR and MR.In addition,PCR was used to comprehensively reveal the variation of gene expression in H295 R cells under the exposure of these chemicals.Results of MTS assay showed that Pry-A,Pry-C,Pry-D,Pry-H,ML-A and BX-B would exhibit cytotoxicity,and ML-A showed the most potent toxicity which would even significantly inhibited the cell viability at the concentration of 10-8 M.Dual-luciferase reporter gene assays showed that only Pry-F,Pry-G,ML,ML-A showed GR mediated antagonistic activity with the order of ML-A > Pry-F > Pry-G > ML,and their RIC20 values were 10-10 M,3.1×10-8 M,1.6×10-6 M and 7.2×10-6 M,respectively.Results of molecular docking is in accordance with that of dual-luciferase reporter gene assays.PCR assay showed that Pry-F,Pry-G,ML,ML-A would significantly change the expression level of genes involved in synthesis and transport of steroid hormone.The expression of 3?HSD2,CYP11B1,CYP11B2 showed a significant up-regulation under low concentration exposure.These data will be helpful in understanding the potential ecological and health risks of pesticides and their metabolites.