| As a common organophosphorus flame retardant,tris(2-butoxyethyl)phosphate(TBOEP)is detected in nature environment and aquatic animals extensively.Despite previous researches have reported the developmental toxicity of TBOEP in zebrafish(Danio rerio)larvae,its underlying mechanisms are not clear until now.Therefore,our research was aimed to investigate the potential mechanisms of toxicity.In this study,zebrafish embryos were used as reasearch models and exposed to 0,20?g/L,200 ?g/L,1000 ?g/L and 2000 ?g/L TBOEP from 2 hours post fertilization(2 hpf)to120 hpf.The survival rate,hatching rate,malformation rate,heart rate and body length of zebrafish embryos were observed and counted to evaluate the acute developmental toxicity of TBOEP.Samples were colleated at 12 hpf,24 hpf,72 hpf and 120 hpf to measure the expression of genes related with development and growth.Apoptosis staining analysis was performed at 72 hpf and 120 hpf and biochemical analysis was performed at 120 hpf at the end of exposure to evaluate the damage mechanism and investigate the potential toxicity mechanism.The results are shown below:1.TBOEP can cause acute developmental toxicity of zebrafish embryos,change the basic growth parameters of zebrafish embryos and inhibit their growth and development.Survival rate and hatching rate(81.5 ± 0.8% and 75.8 ± 1.2%,respectively)of zebrafish embryos in 2000 ?g/L groups were significantly lower than that in control group(91.1 ±0.3% and 83.2 ± 1.3%,respectively),and the malformation rate(8.1 ± 0.4%)were significantly higher than that in control group(2.3 ± 0.6%).At 12 hpf,the length of zebrafish embryos in 1000 ?g/L and 2000 ?g/L concentration groups(1.64 ± 0.05 mm and1.59 ± 0.06 mm,respectively)decreased significantly than control group(1.72 ± 0.06 mm).At 72 hpf,the length of zebrafish embryos in 200 ?g/L,1000 ?g/L and 2000 ?g/L concentration groups(3.32 ± 0.03 mm,3.25 ± 0.05 mm and 3.15 ± 0.04 mm,respectively)decreased significantly than control group(3.44 ± 0.07 mm).At 120 hpf,the length of zebrafish embryos in 200 ?g/L,1000 ?g/L and 2000 ?g/L concentration groups(3.87 ± 0.13 mm,3.79 ± 0.13 mm and 3.70 ± 0.09 mm,respectively)decreased significantly than control group(4.04 ± 0.11 mm).2.TBOEP can cause cardiac development toxicity and damage heart development.At 72 hpf heart rate of zebrafish larvae in 1000 ?g/L and 2000 ?g/L concentration groups decreased significantly,which were 148 ± 4.3 beats / min and 142 ± 5.1 beats / min respectively while in control group was 163.4 ± 6.2 beats / min.At 72 hpf and 120 hpf,AO staining indicated that there was apoptosis around heart area in zebrafish larvae.Similarly,TUNEL assay also showed that there was apoptosis in the heart region at 120 hpf in zebrafish larvae.3.TBOEP could down-regulate Wnt signal pathway and change the expression of related genes.The expression of axin1,axin2 and dvl3 was decreased while fzd,?-catenin,pkc,wnt11,sox9 b and nkx2.5 was increased.Correlation analysis indicated that expression of these genes was significantly correlated with body length.BIO,which was a specific activator of Wnt signaling pathway,could recover the body length and expression of genes which were changed before.4.TBOEP can also lead to oxidative stress.The content of reactive oxygen species(ROS)increased by 27.3% and 58.3% while the activity of superoxide dismutase(SOD)decreased by 17.9% and 23.1% in 1000 ?g/L and 2000 ?g/L concentration groups.Meanwhile the activity of catalase(CAT)decreased by 19.4% in 1000 ?g/L and 2000 ?g/L concentration group.In conclusion,our research demonstrated that TBOEP could cause acute developmental toxicity and cardiac developmental toxicity,and investigated underlying mechanisms of toxicity.TBOEP could down-regulate Wnt signal pathway to inhibit development and growth in the early developmental stage of zebrafish. |
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