Di-(2-Ethylhexyl)-Phthalate(DEHP) Alters Locomotor Behavior Function Through Affecting Dopaminergic Signaling Transmission In Zebrafish(Danio Rerio)

Background and objective:Di-(2-ethylhexyl)phthalate(DEHP)was existed widely in the natural environment and organism with its toxicity getting great concern.The epidemiological investigation showed that DEHP exposure was not only associated with reduced neural reflex in infants,but also with behavioral characteristics of preschool children,especially attention deficit and hyperactivity disorder-related symptoms.Animal study found that DEHP has certain neurotoxic effects,which could affect the development of nervous system and cause altered neurobehavior in animals.However,few studies have deeply evaluated the effect of DEHP on locomotor behavior and the possible mechanisms.The normal functioning of dopaminergic signaling is very important for the regulation of animal neurobehavior and the cognitive function of the adult animal.In this study,the effect of DEHP on locomotor behavior of zebrafish larvae and the underlying mechanisms were investigated using zebrafish at early developmental stage as an animal model.Materials and methods:Zebrafish embryos at 6 hours post-fertilization(hpf)were exposed to 0,1,2.5,5,10 mg/L DEHP solution until 7 days post-fertilization(dpf).Survival rate,hatching rate,malformation rate,body length at 96 hpf and heart rate at 72 hpf in each concentration group were observed and recorded.Embryo spontaneous activity was assessed at 24,26,28,30 and 32 hpf,and larval locomotor behavior was analyzed under light-to-dark transition stimulus at 5,6 and 7 dpf using zebrafish behavioral analysis system(Noldus,Netherlands).After behavior analysis,larvae in each group were collected for following analysis.Gene expression analysis was performed for genes related to dopamine system and apoptosis by real-time quantitative PCR(RT-q PCR).The dopamine(DA)level in zebrafish was measured using DA ELISA Kit.Acridine orange(AO)staining was used to detect apoptotic cells in larval brain.MDA content and SOD activity of zebrafish was determined with the MDA assay kit and SOD assay kit.Results:1.The body length of larvae was reduced with DEHP exposure.Embryo spontaneous movement frequency was prominently inhibited in 2.5 mg/L DEHP exposure group at 32 hpf and in 5,10 mg/L exposure groups at 28,30 and 32 hpf.2.At 5 dpf,DEHP exposure did not result in any significant effects on larval locomotor behavior during the light period,while during the dark period larvae from 2.5,5 and 10 exposure groups show shorter diatance moved and a significant downward trend in velocity compared to the control.Additionally,5 dpf larvae exposed to 5 and 10 mg/L DEHP also exhibited lower activity during the dark period than control fish.At 6 and 7 dpf,compared with the control,2.5,5 and 10 mg/L DEHP resulted in a significant decrease in distance moved and activity and an obvious downward trend in velocity during both the light and dark period.3.Among the genes related to dopamine system,tyrosine hydroxylase(th),dopamine active transporter(dat)and monoamine oxidase(mao)genes were downregulated in 2.5,5 and 10mg/L exposure groups;dopamaine receptor D1(drd1)and dopamaine receptor D2a(drd2a)genes were upregulated in 2.5 mg/L exposure group;dopamaine receptor D2a(drd2a),dopamaine receptor D2b(drd2b),dopamaine receptor D4a(drd4a)and dopamaine receptor D4c(drd4c)genes were downregulated in 5 and 10 mg/L exposure groups.4.The level of dopamine in larvae was significantly reduced in a concentration-dependent manner and showed significant differences in the larvae exposed to 2.5,5 and 10 mg/L DEHP compared with the control.5.AO staining showed that no obvious apoptotic cells were observed in the brain of larvae in the control group,whereas obvious apoptotic cells appeared in the brain of larvae in 2.5,5and 10 mg/L exposure groups.Among the genes related to apoptosis,p53 and caspase-3genes were significantly upregulated in 2.5,5 and 10 mg/L exposure groups;bax and caspase-9 genes were significantly upregulated in 5 and 10 mg/L exposure groups;bcl2gene was significantly upregulated in 1 mg/L exposure group but was significantly downregulated in 5 and 10 mg/L exposure group.The bcl-2/bax ratio was increased in 1mg/L exposure group but was decreased in 5 and 10 mg/L exposure group.6.Significant increases in MDA level were observed in 5 and 10 mg/L exposure groups.The activity of SOD was markedly increased in 1 mg/L exposure group but was significantly reduced in 2.5,5 and 10 mg/L exposure groups.Conclusions:1.DEHP resulted in the inhibition of the embryonic spontaneous movement and altered larval locomotor behavior.2.DEHP disrupted the gene expression of dopamine system and reduced the dopamine level in larvae,affecting the dopaminergic signaling transmission in zebrafish.3.DEHP caused apoptosis and oxidative stress that promoted the dysregulation of dopaminergic signaling transmission in part,resulting in the altered locomotor behavior in zebrafish.