Effect Of Trichlorfon On The Embryonic Development Of Zebrafish

Trichlorfon(TCF)is an organophosphorus pesticide widely used in aquaculture,plantation and processing industries in China.Because of its high efficiency and low toxicity,it is often used as a water disinfectant and fish insecticides to control plankton in water.However,long-term repeated use of trichlorfon can cause residues and enrichment in water bodies and silt,which will be a great harm to the ecological environment and human health.As an extremely important aquatic population in the aquatic ecosystem,fish are also the main food source for human daily life.Therefore,the toxicity evaluation of organophosphorus pesticides on fish and other aquatic organisms and the study of toxicity mechanism have always been the focus of aquatic toxicology and it is also useful for ecological risk assessment.The present study aimed to investigate the toxicity and mechanism of trichlorfon on zebrafish embryonic development via the observation of classic toxicological phenotypes,gene levels,biochemical enzyme activities,and protein expression levels using zebrafish embryos as experimental model.Changes in morphological and phenotypic characteristics of zebrafish embryos after trichlorfon exposure were observed under a microscope.Alterations in gene transcription,enzyme activities,and proteins related to zebrafish embryonic development were analyzed by PCR,q PCR,spectrophotometer,and western blot.The main results were obtained as following:1.Trichlorfon acute toxicity on zebrafish embryosZebrafish embryos were grouped in parallel experiments according to the OECD chemical testing guidelines and exposed to 0,10,20,30,40,50,60,70,80,90,and 100 mg/L trichlorfon solutions,respectively.The results of the acute toxicity experiment showed that trichlorfon at high concentration had a lethal effect on zebrafish embryos.Then,the effect of half-lethal concentrations of trichlorfon on zebrafish embryos at 48,72,and 96 hpf were calculated according to the probability unit method,which were 50.225,41.360,and 25.133 mg/L,respectively.According to the grading standard of pesticide toxicity,trichlorfon is a moderately toxic substance to zebrafish.2.Developmental toxicity of trichlorfon on zebrafish embryosAccording to the 96-hpf half-lethal concentration of trichlorfon to zebrafish embryos,6 groups of non-lethal concentrations of trichlorfon solution were finally prepared.Zebrafish embryos were exposed to0,1.5,3,6,12,24 mg/L trichlorfon aqueous solution,respectively.Through 4-days of toxicity exposure and microscopic observations,the experimental results showed that trichlorfon causeed a series of adverse effects on the zebrafish embryonic development,such as abnormal autonomic movement,slowed heart rate,spinal curvature,body length inhibition,pericardial edema,reduced melanin deposition,and other a variety of significant teratogenic effects.3.Effect of trichlorfon on the expression of genes related to embryonic development of zebrafishIn order to further explore the toxicity mechanism of trichlorfon-induced abnormal development of zebrafish embryos,the embryos were exposed to 0,6 and 12 mg/L trichlorfm aqueous solutions for 4days.The expression of genes related to the development of zebrafish embryos was detected by fluorescent quantitative PCR.The results showed that the expressions of the genes TYR,Tbx2,LOX,BMP2 in the treated zebrafish embryos were significantly down-regulated,while Mef2 c and GH genes were firstly up-regulated but then down-regulated.4.Effect of trichlorfon on the antioxidant system of the embryos or larvae of zebrafishIn order to study the effect of trichlorfon on the antioxidant system of zebrafish embryos,the activities of three antioxidant enzymes and their gene expressions were determined.In addition,we also determined the oxidative damage of trichlorfon to zebrafish by using the kit to detect the content of MDA.The results of three enzyme activities determination and q PCR showed that the activities of SOD and GPX in embryos of zebrafish treated with trichlorfon increased first and then decreased with the increase of trichlorfon concentration,while the activity of cat increased,suggesting the oxidative damage to zebrafish embryos caused by trichlorfon.At the same time,the results of MDA detection showed that there was obvious lipid peroxidation in zebrafish embryos after trichlorfon exposure.5.Effect of trichlorfon on the levels of embryonic development related proteins in the embryos or larvae of zebrafishThe results of microscopic observation showed that trichlorfon caused the decrease of melanin deposition,pericardial edema,and heart rate slowing in the embryos or larvae of zebrafish.Meanwhile,the expression of TYR and Tbx2 genes was significantly down-regulated detected by fluorescent quantitative PCR.Therefore,the effects of trichlorfon on the melanin related Tyrp-1 protein and the heart related Tbx2 protein of zebrafish were studied in order to further explore the toxic mechanism of trichlorfon onzebrafish..The changes of Tyrp-1 protein and Tbx2 protein were detected by Western blot.The results showed that the levels of Tyrp-1 and Tbx2 proteins in the embryos or larvae of zebrafish exposed to trichlorfon decreased significantly compared with the control group,which was basically consistent with the results of phenotypic observation and q PCR.It can be concluded that trichlorfon can affect the Tyrp-1and Tbx2 proteins of zebrafish to inhibit the production of melanin and the development of heart.The results of this study indicate that trichlorfon has toxic effect on the embryonic heart,bone,nerve development and melanin formation of zebrafish,which is concentration and time dependent.Moreover,trichlorfon exposure caused teratogenic effects on zebrafish embryos,down-regulation of key genes for development,oxidative damage,and decreased expressions of embryonic development related proteins,affecting the fish embryonic development.The results of this study can provide a basis for aquatic ecological risk assessment and water pollution control of trichlorfon.