Toxic Effect And Mechanism Of Aryl Hydrocarbon Receptor Agonistic Pesticide Mepanipyrim In Zebrafish

With the rapid development of modern society and the continuous improvement of people’s material living standards,the demand for pesticides is gradually increasing.More than 1000 kinds of pesticides are commonly used in the whole world,and the annual use of pesticides in the world is about 1-3.5 million tons.Different from persistent organic pollutants,pesticides are easier to be degraded and metabolized,but their residues are often detected in the ecological environment and animals due to the huge amount of use.Pesticide pollution has seriously affected people’s health and the dynamic balance of ecological environment.Among these commonly used pesticides,some belong to aryl hydrocarbon receptor(AhR)agonists.AhR agonists have strong toxicity to cardiovascular,nervous and visual systems of animals.Currently,the commonly used methods for the detection of AhR agonists are EROD enzyme activity assay and luciferase reporter gene assay in vitro,but the detection sensitivity is limited and not convenient.Subsequently,transgenic zebrafish for the detection of AhR agonists have come out one after another,but the traditional transgenic zebrafish based on the unmodified cyp1a gene promoter is also limited by the detection sensitivity.Therefore,a highly sensitive transgenic zebrafish Tg(cyp1a-12DRE-egfp)for screening of AhR agonists was constructed based on the recombinant zebrafish cyp1a gene promoter.The sensitivity of the transgenic zebrafish to TCDD was 1 ng/L.In this study,combined with the previous results of in silico simulation screening in our laboratory,we verified the AhR agonistic activity of 14 potential AhR agonists in vivo and in vitro assays.In vitro,11 kinds of pesticides showed AhR agonistic activity.In vivo,14 kinds of pesticides showed AhR agonistic activity.In this study,zebrafish was used as the research model to study the toxic effect and mechanism of mepanipyrim,a typical AhR agonist.It mainly include the accumulation of mepanipyrim in zebrafish,the toxicity of mepanipyrim to zebrafish’s cardiac development,visual development and neural development and its involved mechanism.Our results showed that:1)Mepanipyrim has stronger binding ability to AhR.2)Mepanipyrim was easy to be degraded and metabolized in zebrafish embryos and larvae.3)Mepanipyrim exposure could lead to the pericardial edema,accelerated heart rate and arrhythmia in 72 hpf larvae.The cardiomyocyte size of larval heart increased significantly,and the expression of genes related to myocardial contraction and calcium signaling pathway changed significantly.Cytochrome c oxidase family genes were found to be potential AhR target genes in vitro reporter gene experiments.AhR antagonist CH223191 can effectively attenuate mepanipyrim induced cardiac developmental toxicity,which is mainly manifested by the significant reduction of pericardial edema,the improvement of heart rate acceleration and arrhythmia,the recovery of normal cardiomyocyte size,and the recovery of normal expression of genes related to myocardial contraction and calcium signaling pathway.4)Mepanipyrim exposure could change the eye size of 72 hpf zebrafish larvae.The length of long axis and short axis of eyes decreased significantly,and the thickness of retinal cell layers(RPE,ONL,INL,GCL)decreased significantly.There were significant changes in the genes of phototransduction signal pathway in larvae.In vitro reporter gene experiment,grklb was found to be a potential AhR target gene.5)Mepanipyrim exposure significantly enhanced the light sensitivity of 5 dpf larvae,and the larvae became more active under 2500 lx light intensity.Larvae were more sensitive to long wavelength(red light)and medium long wavelength(green and blue light)light stimulation,and the mRNA expression of opn1mwl,opn1mw2,opn1lw1,and opn1lw2,which were sensitive to long wavelength and medium long wavelength light,were significantly changed.6)Mepanipyrim exposure could lead to hyperactivity of 7 dpf larvae,which was similar to Huntington’s disease and epilepsy.The contents of GABA and the activities of Na+/K+-Ca2+-ATPase were significantly increased.There were significant changes in the expression of genes related to skeletal muscle cell contraction,synaptic transmission of brain neurons and calcium signaling pathway.