Toxicity Of GO On Zebrafish Embryos In Vitro And In Vivo

Graphene oxide(GO)is widely used in the fields of electronics,energy,environmental protection,biomedicine due to the excellent physical properties and chemical stability.It may be discharged into ecosystems,especially aquatic ecology system,which poses a threat to aquatic life and human health.Previous studies have found that GO can induce oxidative stress and apoptosis in fish tissues,organs or embryos.However,the research on the research on the GO induced fish cell apoptosis is limited.The mechanism of apoptosis is unclear.In this study,zebrafish(Danio rerio)embryos and zebrafish embryo fibroblasts(ZF4)were used for in vivo and in vitro experiments.The uptake of GO into zebrafish embryos and the effects on embryo development,oxidative stress,and apoptosis.The internalization of GO,intracellular ROS generation,organelle damage and apoptosis,and the pathways of GO induced apoptosis were further analyzed.In vivo experiments show that GO can adhere to the surface of embryo chorion and can cross the chorion,enter the zebrafish embryo,mainly accumulate in the yolk.With the increase of exposure time and embryo development,GO mainly accumulates in the head,abdomen and yolk sac.GO in embryo can cause developmental toxicity in zebrafish embryos,including the delayed hatching and development,increased heartbeat,deformity rate and increased mortality.In addition,GO enhance the ROS production in embryos,activates CAT and SOD enzymes,and reduces the GSH content at same time,causing oxidative stress,and triggers apoptosis.Through RT-qPCR experiments,it was found that GO can down-regulate the expression of the anti-apoptotic bcl-2,up-regulate the expression of the pro-apoptotic gene bax,and finally activate the expression of caspase-3,thereby triggers the caspase-dependent apoptosis pathway and cause apoptosis.Therefore,the development of zebrafish embryos are negatively affect.In vitro experiments show that GO can be endocytosed into ZF4 cells and mainly accumulate in lysosomes.The internalized GO increases the ROS level in ZF4 cells significantly,cause cellular damage and dysfunction,increase lysosomal membrane permeability,decrease mitochondrial membrane potential,disturbs normal cellular physiological activilies,and ultimately leads to apoptosis.The expression levels of apoptosis-related genes in ZF4 cells found that GO alters mitochondrial permeability by affecting the balance between pro-apoptotic bax and anti-apoptotic bcl-2 on the mitochondrial membrane,resulting in a decrease of mitochondrial membrane potential,activates caspase-3 and finally trigger cell apoptosis.In summary,GO shows toxic effects on zebrafish embryos and ZF4 cells.The experiments in vivo and in vitro achieve consistent results.In vivo experiments shows that GO can cause developmental toxicity of embyos through oxidative stress and apoptosis.In vitro experiments confirmed that GO can induce apoptosis of zebrafish embryo through the mitochondrial pathway from the subcellular and molecular levels.Tthe mechanism of GO-induced zebrafish embryo developmental toxicity and cell apoptosis analyzed systematically both in vivo and in vitro.This study supplements the information on the pathways of GO-induced fish cell apoptosis and provides better understanding on the ecotoxicity of GO.