Effects Of Acute Exposure To TBOEP (Tris(2-Butoxyethyl) Phosphate) On Motor Behavior And Axonal Growth In Zebrafish(Danio Rerio) Larvae

Tris(2-butoxyethyl)phosphate(TBOEP)is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem.Recently,the reproduction and development toxicology of TBOEP has been reported.However,fewer studies have assessed the neurotoxic effects in zebrafish(Danio rerio)larvae.In this study,zebrafish embryos were subjected to waterborne exposure of TBOEP at 0,50,500,1500 and 2500 ?g/L from 2 to 144-h post-fertilization(hpf).Behavioral measurements showed that TBOEP exposure reduced embryonic spontaneous movement and decreased swimming speed of larvae in response to dark stimulation.In accordance with these motor effects,TBOEP treatment reduced neuron-specific GFP expression in transgenic Tg(HuC-GFP)zebrafish larvae and inhibited the growth of secondary motoneurons,as well as decreased expression of marker genes related to central nervous system development in TBOEP treated group.Furthermore,increased contents of reactive oxygen species(ROS)and malondialdehyde(MDA),as well as reduction of superoxide dismutase(SOD)activity were detected in TBOEP exposure group.The present results showed that the alteration in motor neuron and oxidative stress could together lead to the motor behavior alterations induced by TBOEP.The results of this experiment are as follows:1.Results showed that the hatching rate at 96 hpf and the survival rate at 144 hpf decreased only in the highest exposure group.Meanwhile,exposure to 500,1500 and 2500 ?g/L TBOEP resulted in a significant decrease in heart rate of larvae at 96 hpf.Furthermore,significantly lower body length was found in 144 hpf larvae which had been exposed to 500,1500 and 2500 ?g/L TBOEP compared to the control.However,TBOEP did not significantly affect malformation rates compared with the control samples.2.In control embryos,side-to-side alternating contractions initiated at 20 hpf,and gradually reached a peak of 16 bends/min at 22 hpf,and then decreased slowly to 9 bend/min at 28 hpf.Embryos exposed to TBOEP at 500,1500 and 2500 ?g/L did not change the onset time of spontaneous movement(~20 hpf),but exhibited decreased bending frequency at 22 hpf.Significant decreases in spontaneous movement were observed every two hours in 2500 ?g/L TBOEP treated larvae from 20 to 28 hpf,being less than that of the control group.Locomotion activities in larvae at 144 hpf were further evaluated under alternating light-dark photoperiod stimulation.The swimming speed usually decreased rapidly when photoperiod was switched from dark to light and reversed vice versa in all groups.The average swimming speed of larvae exposed to TBOEP of 1500 and 2500 ?g/L were significantly lower than that of controls during 5-min dark period.3.Morphological analysis revealed that 1500 and 2500 ?g/L TBOEP exposure significantly reduced the length of the dorsal and ventral axon from secondary motor neuron.At the transcription level,we further assessed the expression of genes related to the axonal development by qRT-PCR.Expression of ?1-tublin was significantly decreased only in 2500 ?g/L TBOEP treatment group.Relative to controls,the mRNA levels of shha and syn2 a were significantly down-regulated at 144 hpf in larvae exposed to 500,1500 and 2500 ?g/L TBOEP.The transcript level of gap43 was significantly down-regulated after exposure to various concentrations of TBOEP as compared to that in the control.4.Exposure of transgenic Tg(HuC-GFP)zebrafish to TBOEP at 1500 and 2500 ?g/L showed a significant reduction in GFP expression in the brain and spinal cord at 144 hpf,being less than that of the control group.However,the lower concentrations of TBOEP(50 and 500 ?g/L)caused no significant difference.5.The expression of mbp and gfap was significantly down-regulated after exposure to various concentrations of TBOEP.Meanwhile,the transcript levels of elavl3 and nkx2.2a had a significant down-regulation in 500,1500 and 2500 ?g/L TBOEP treated groups.In addition,expression of nestin and nr4a2 b was significantly decreased after exposure to 2500 ?g/L TBOEP.However,transcription of neurogenin and ache was not significantly changed compared with the control group.6.A significant increase in ROS content was seen in 500,1500 and 2500 ?g/L TBOEP treated larvae relative to the control group.Meanwhile,a significant increase in MDA level was observed only in 2500 ?g/L TBOEP treatment group.The down-regulation of activity levels of SOD was observed in larvae exposed to TBOEP at 1500 and 2500 ?g/L.Taken together,these results indicated that TBOEP exposure affected the formation and growth of axons in secondary motor neurons associated with the neurobehavior of zebrafish larvae.Therefore,we should not ignore the toxic effects of TBOEP in the aquatic environment and we need to further study the toxic mechanism of TBOEP.