Stress Mechanism Of Trichloropropyl Phosphate Exposure Induced Neurotoxicity In Zebrafish

Because of the adverse health effects of PBDEs,many developed countries have banned the use of these compounds as flame retardants.Therefore,alternative chemicals such as organophosphate flame retardant(OPFR)have become popular substitutes.What's more,with their usage rising(year by year)and frequently being detected in the environment,the OPFRs has been of great concern.In this study,tris(1-chloro-2-propyl)phosphate(TCPP),a typical organophosphorus flame retardant,was selected to treat zebrafish(Danio rerio)as a model organism.We systematically studied whether TCPP could cause embryonic developmental toxicity and affect the normal nerve development and behavior movement of zebrafish,and further explore the possible mechanism of inducing abnormal behavior.Through the research,the following conclusions are drawn:1.The LC50 of TCPP acute exposure to zebrafish was 49.71 mg / L,according to which the acute exposure concentration gradient of TCPP(TCPP concentration gradient was 0,5 mg/L,15 mg/L and 25 mg/L)was determined,and the apparent deformities of zebrafish embryos under TCPP stress were observed.TCPP exposure can inhibit the development of zebrafish embryos,prolong the incubation time of embryos,and lead to a series of morphological malformations such as yolk cyst,spinal curvature,swimming sac closure,pericardial cyst,venous sinus hemorrhage,eye dysplasia and so on.Oil red O staining showed that TCPP exposure could cause lipid metabolism disorder and fatty acid siltation in zebrafish.The increase of granulocytes in the early stage of zebrafish embryos exposed to TCPP was detected by neutral red staining technique,which confirmed that the early immune system of zebrafish was damaged.This series of experiments proved that TCPP exposure has a wide range of effects on the embryonic development of zebrafish.2.The behavioral results of embryo and juvenile fish showed that TCPP led to abnormal behavior,including the decrease of frequency of spontaneous movement,the decrease of average speed and distance,the decrease of activity ability of juvenile fish in the dark and the slow / insensitive response to acousto-optic stimulation.3.The detection of acetylcholinesterase(ACh E),a key enzyme in nerve conduction,showed that the activity of Ach E was inhibited and the normal nerve signal transmission was damaged after TCPP exposure.Compared with the control group,the accumulation of ROS was significantly increased in the TCPP exposure group,which caused oxidative stress in zebrafish.AO apoptotic staining and vascular transgenic fish Tg(flk 1:m Cherry)observation showed the ablation of head blood vessels and the occurrence of apoptosis of a large number of brain cells exposed to TCPP,while the staining of hair cells of lateral line colliculus(FM 1-43 dye)showed that the number of caudal nerve thalamic hair cells decreased and arranged disorderly.4.The results of pax6 a and nova1,WISH genes related to neurodevelopment showed that in the control group,pax6 a was mainly expressed in the eyes,but lower in the forebrain.After TCPP exposure,the expression of pax6 a in eyes and forebrain of juvenile fish were decreased significantly.At the same time,nova1 was mainly expressed in brain and was almost invisible in spine and eyes of juvenile fish but was decreased significantly in TCPP treatment.The differential expression of the two genes is consistent with the results of q-PCR,which may be the main reason for the abnormal behavior of zebrafish.5.The results of a series of behavioral analysis of TCPP chronic exposure from juvenile to adult fish showed that chronic TCPP exposure led to anxiety-like behavior,excessive panic in adult fish and the decrease of the ability to avoid risk;social interaction tests showed that long-term TCPP exposure led to abnormal communication,eccentricity and loneliness in adult fish;and T-maze test showed that zebrafish memory and cognitive function were seriously impaired.6.HE staining showed that the number of neurons decreased and glial cells proliferated to form glial scar in the brain of adult zebrafish exposed to TCPP,which suggested that there was neuronal apoptosis in the periventricular layer.Immunofluorescence detection showed that under normal conditions,the expressions of nova1 and pax6 a proteins were almost all located in the nucleus,while some of the TCPP exposure groups were expressed in the cytoplasm,and the levels of nova1 and pax6 a proteins were decreased significantly.This study provides an important reference for risk assessment and early warning of TCPP exposure,and provides a basis for early prevention and gene therapy of neurological diseases caused by pollutants.