Neurodevelopmental Mechanism Of Cypermethrin Exposure In Early Zebrafish Embryogenesis

The nervous system during embryogenesis is highly sensitive to exogenous chemicals,and it is extremely susceptible to the interference from environmental pollutants such as pesticides.Largely and widely used pyrethroids can enter the human body through a variety of ways.At present,pyrethroids and their metabolites have been commonly detected in the urine of pregnant women and breast milk around the world.Pyrethroids can cross the placental barrier or be delivered through breastfeeding or other ways to developing fetuses and infants,which may cause adverse neurodevelopmental outcomes.The developmental toxicity caused by pyrethroids has been paid great attention to,but the study on the effects of pyrethroid in early embryogenesis is insufficient,and the molecular mechanism of early neurodevelopmental toxicity is still unclear.In present study,cypermethrin,the most commonly detected in the environmental medium and human bodies,was used as a representative of pyrethroids.Test concentrations of 20,100 and 500μg/L were set by reference to the levels detected in the environment.Zebrafish was used as an experimental model.Exposure was carried out during early embryogenesis from fertilized egg to the somite stage（0-24hpf）.By evaluating the continuous effect of cypermethrin on embryonic development,detecting the accumulation and elimination of compounds in the embryo,combining RNA-Seq and molecular biology methods to explore the effects on embryonic transcriptomics,neurotransmitters,voltage-gated ion channels to comprehensively analyze the mechanism of cypermethrin on the early neural development of zebrafish embryos.The main findings are as follows:（1）By analyzing the embryonic developmental and behavioral levels after exposure of cypermethrin during early embryogenesis,it was found that cypermethrin did not affect the survival,growth or development of zebrafish,but significantly enhanced the activity of embryos in the embryonic membrane,leading to earlier hatching.After the exposure,the locomotor distance,maximum speed and activity of zebrafish larvae in the exposed group were still significantly higher than control,showing the continuous effect of cypermethrin on the embryo’s neurodevelopment.（2）By detecting the accumulation and elimination of cypermethrin in zebrafish embryos and larvae via UPLC-MS,it was found that cypermethrin could pass through the embryonic membrane and be rapidly accumulated by the developing embryos with a dose-dependant manner.Both zebrafish embryos and larvae had a certain metabolic capacity for cypermethrin with a higher elimination rate at the initial stage of elimination when under higher body load.（3）Through RNA-Seq,it was found that cypermethrin exposure during early embryogenesis would cause differentially expression of genes and change the expression time of cell composition and molecular biological function-related genes.Differentially expressed genes were significantly enriched in PI3K-Akt,estrogen receptor and extracellular matrix receptor interaction signaling pathways,showing alteration in cell growth,cell differentiation,cell cycle regulation and signal transduction processes.（4）Real-time quantitative PCR and enzyme-linked immunoassay were used to detect the expression and content of acetylcholine and Ca²⁺signaling pathway-related genes and substances.It was found that exposure of cypermethrin would significantly up-regulate the expression of choline transporter and nicotinic acetylcholine receptor,together with calmodulin and its downstream targets,calmodulin kinase,calcineurin,and nitric oxide synthase related genes during embryonic period.In addition,by adding Na⁺channel blockers,it was found that cypermethrin could cause abnormal opening of Na⁺channels.After the exposure,the molecular changes in Ca²⁺channels and acetylcholine returned to normal faster than Na⁺channels,which meant more sensitive effect on Na⁺channels.Present study explored the effects of cypermethrin exposure during early embryogenesis on zebrafish neurodevelopment and noted the activity of embryos in the embryonic membrane,the vital endpoint of developmental neurotoxicity.Combining chemical and molecular biology methods,it was found that cypermethrin could be rapidly accumulated by embryos during early exposure.By opening Na⁺channels,activating CALM-Ca N/Ca MKII/NOS signaling pathways,and up-regulating the expression of neurotransmitter receptors and solute carriers,it affected the transmission of nerve excitement,which in turn leaded to individual neurodevelopmental effects.Present study will provide a scientific reference for the neurodevelopmental toxicity assessment of pyrethroids.