Toxicities And Mechanism Of Nano-ZnO On Swin Bladder Of Zebrafish Embryos

In recent years,nano-ZnO is one of the most widely used artificial nanomaterials in the world,However,the widely use of nano-ZnO inevitably increases the ecological risk in aquatic environment.With the cumulate of nano-ZnO in aquatic environment,it is necessary to study the biological safety and toxicity mechanism of nano-ZnO.In this study,zebrafish embryos as the test organisms and nano-ZnO as the research object,the toxic effects of of nano-ZnO with different exposure concentrations on the development of swim bladder were studied.According to the dissolution of nano-ZnO,the corresponding concentration of Zn²⁺treatment groups were set up.We start the test from 0 hours post fertilization?0 hpf?,based on 5 different developmental stages of swim bladder,which included 0 hpf?before hatching,0?120 hpf,0?168 hpf,after hatching?120 hpf,and 120?168hpf.In addition,we also investigate the toxic mechanism of nano-ZnO through the QPCR and molecular biotechnology.The main conclusions of this study are as follows:?1?The suspension of nano-ZnO has extremely poor stability,with the concentration increases,its sedimentation rate will increase and the absolute value of Zeta potential will decrease.?2?Nano-ZnO can cause swim bladder defect and spinal curvature in 168 hpf zebrafish,and also cause zebrafish has a high mortality rate at high concentration.However,the dissolution of Zn²⁺from nano-ZnO suspension was not the only or primary reason for the mortality of zebrafish and damage to the swim bladder.?3?A dose-response relationship was observed that damage rates of swim bladder and spinal or mortalities of zebrafish increased with the increasing nano-ZnO concentrations.Exposure to 15 mg/L nano-ZnO caused the mortality of zebrafish and damage to swim bladder and spinal curvature?90%of control?,while exposure to 10 mg/L led to a reduction in the area of the seim bladder?70%of control?.?4?The result shows that the sensitivity of early developmental stage?0?168 hpf?of zebrafish to nano-ZnO had the following order:budding stage?0 hpf-before hatching?>inflating stage?after hatching-120 hpf?>inflation complete stage?120?168 hpf?.The developmental area and time of swim bladder and the length of zebrafish were also affected by nano-ZnO exposure.?5?QPCR result shows that exposure to nano-ZnO,the 72 hpf zebrafish m RNA transcription levels of Bone Morphogenetic protein-2?bmp-2?,Bone Morphogenetic protein-4?bmp-4?and the bladder developing-related genes Sonic Hedgehog?shha?and Indian Hedgehog?ihha?were all decreased.Nano-ZnO can suppres the expression of bmp-2 and bmp-4,which led to the damage to bone development in zebrafish.And nano-ZnO also interrupts the Hh signaling,suppres the expression of ihha and shha,and leads to the damage of the epithelial layer,mesenchyina and the outer mesenchymal tissue of swim baldder.?6?There was a significant correlation between the damage rate to swim bladder and the mortality of zebrafish?r=0.978,p<0.01?,indicating that the damage to swim bladder was one of the main reason for the death of zebrafish after exposure to nano-ZnO.However,the toxicological mechanism of nano-ZnO on the swim bladder of zebrafish,especially for the budding stage and inflating stages,is unknown and should be further investigated.