Toxic Mechanism Of 3nm Ferric Oxide Nanoparticles On Zebrafish Embryo Development

Ferric oxide nanoparticles(FeNPs)have broad application prospects in the field of biomedicine.They can be used as drug carriers,molecular probes and contrast agents to participate in disease treatment and clinical diagnosis.Sub-5 nm nanoparticles are characterized by ultra-small size,narrow distribution,good biocompatibility and superparamagnetism,which can be used as T1 contrast agent to provide high sensitivity tumor imaging.Under the effect of external magnetic field,tumor tissues can be targeted by FeNPs for magnetocaloric therapy.FeNPs can also be used for cell magnetic labeling and multimodal molecular imaging probe construction.Moreover,due to the ultra-small size effect,it is superior to other nanoparticles and can be quickly cleared by kidney and liver.In order to promote the practical application of sub-5 nm FeNPs,we took zebrafish embryos as object,studied the developmental toxicity of zebrafish embryos,and evaluated their biological safety.Zebrafish is a model organism for vertebrate research.The transparent embryo and in vitro fertilization of zebrafish provide great convenience for research of developmental biology.In recent years,construction of transgenic zebrafish strains with fluorescent protein specific target cells has further enhanced the advantages of zebrafish as a model organism.In order to explore the effect of Fe₂O₃ nanoparticles on vertebrates,we used 3nm FeNPs,with 6 nm and 12 nm FeNPs as control,to study the toxicity and molecular mechanism of FeNPs on zebrafish embryonic development.The main conclusions are as follows:1.The effects of FeNPs with different sizes and concentrations on zebrafish embryo viability:in this study,zebrafish embryos were exposed to 3 nm,6 nm and 12 nm FeNPs.The results showed as follows:(1)The embryonic mortality of zebrafish increased with the increase of FeNPs concentration and exposure time;(2)The mortality of zebrafish embryo was inversely proportional to the size of FeNPs,that is,the smaller the particle size,the stronger the toxicity;(3)The total malformation rate of zebrafish embryo increased with the increase of FeNPs concentration and the decrease of particle size.The malformations were microcephaly,microphthalmos,pericardial edema,yolk cyst,vertebral curvature,tail dysplasia,developmental retardation and even failure;(4)3 nm FeNPs had strong oxidative damage to zebrafish embryos;(5)3 nm FeNPs mediated embryo apoptosis,and the main action site was digestive system.2.The effects of FeNPs on embryonic organ development of zebrafish:at the concentration of 40 mg/L,3 nm FeNPs was used to detect the developmental toxicity of FeNPs to zebrafish embryonic organs.The results as follows:(1)The effect of FeNPs on cardiact development:using heart transgenic fluorescent zebrafish Tg(cmlc2:EGFP),the toxic effects of 3 nm FeNPs on the heart included vascular hypoplasia,cerebral vasoconstriction,cardiac regional retardation,abnormal distribution of caudal vein vessels,trunk internodal vascular occlusion,and general contraction of aorta;(2)The effect of FeNPs on vascular development:using vascular transgenic fluorescent zebrafish Tg(flk1:EGFP),the toxic effects of 3 nm FeNPs onon blood vessels were as follows:Vascular hypoplasia,cerebral vasoconstriction,heart regional development retardation,abnormal distribution of tail vein blood vessels,trunk interganglion vascular occlusion,general contraction of aorta and other blood circulation system abnormalities;(3)The effect of FeNPs on liver development:zebrafish liver transgenic line Tg(lfabp:m Cherry)was used.After exposure to FeNPs,the liver volume was significantly reduced.The smaller the FeNPs were,the more restricted the liver development was,and the stronger the developmental toxicity was to the liver;(4)The effect of FeNPs on kidney development:using kidney transgenic line Tg(wt1b:EGFP),FeNPs with smaller size had more severe developmental toxicity to kidney,which showed that the glomeruli of anterior kidney were reduced,the neck of renal tubules was obviously absent,and the curvature of proximal convoluted tubules disappeared;(5)The effect of FeNPs on cartilage development:It was found that the head bones of the larve exposed to 3 nm FeNPs became smaller,the angle of hyoid bone was curved,the Merkel bone became shorter,the morphology of angle parotid bone was disordered,the development of pharyngeal scale was not obvious,and the development of pectoral fin was defective,while 6 nm and 12 nm had no significant effect on the development of cartilage.3.The effects of FeNPs on neuromuscular and motor system development in zebrafish:At a concentration of 40 mg/L,3 nm FeNPs were used to detect the neuromuscular developmental toxicity of FeNPs to zebrafish embryos.The results as follows:(1)The effects of FeNPs on nerve development:using transgenic line Tg(eef1a1l1:EGFP),it was found that the central nervous system and peripheral nervous system were significantly atrophied in 48 hpf embryos treated with 3 nm FeNPs,and the development of nervous system was abnormal in 3 nm FeNPs treated group;(2)The effect of FeNPs on skeletal muscle development:using polarized light to detect 48 hpf zebrafish embryos treated with FeNPs,it was found that the average birefringence intensity of muscle tissue in 3 nm treatment group was only 42%of that of control group,skeletal muscle development was not complete and muscle fiber bundle was disordered;(3)Analysis of zebrafish embryo movement trajectory:in the free swimming experiment of zebrafish exposed to 3 nm FeNPs,the swimming distance and moving speed of zebrafish were decreased by 65%and82%,respectively,compared with control group.4.In order to study the molecular mechanism of the developmental toxicity of 3 nm FeNPs to zebrafish embryos at different stages,48 hpf,72 hpf,96 hpf and 120 hpf embryos treated with 3 nm FeNPs were collected and high-throughput sequenced.The following results were obtained:(1)In the 48 hpf group,the developmental signals of the eye and sensory system were significantly affected by nanoparticles,as well as the development of heart and blood vessels;(2)The differentially expressed genes in 72 hpf group focused on eye development and light perception,including synapses,light stimulation perception,synaptic signal,light transduction,retinol metabolism,etc;(3)At 96 hpf,the differentially expressed genes in zebrafish embryos were concentrated in sensory perception,light stimulation perception,apoptosis,lysosome,phagosome,iron apoptosis,etc;(4)At 120 hpf,the differentially expressed genes were concentrated in iron homeostasis,trivalent iron binding,iron apoptosis,cell necrosis and valley cystine metabolism,etc.5.The transcriptome data of 48 hpf,72 hpf,96 hpf and 120 hpf were analyzed by the weighted gene co-expression network analysis(WGCNA).It was found that the molecular mechanism of 3 nm FeNPs on embryonic development was probably caused by inflammatory reaction and cell death.The key hub genes plcd1a and prkcdb were screened by weighted network analysis.Plcd1a is related to nerve signal transduction,neurotransmitter hormone and neuromuscular signal,indicating that 3 nm FeNPs mainly acts on the signal transmission of neuromuscular system in zebrafish embryos,which is consistent with the phenotype found by using transgenic fluorescent fish by affecting GTPase protein coupled Wnt/Ca2+signal transduction,namely 3 nm FeNPs affect the development of neuromuscular system and motor ability of embryo;the increase of prkcdb expression will lead to the increase of downstream ERK and ROS,which is consistent with the previous conclusion.The effect of 3 nm FeNPs on embryo activity is manifested in oxidative stress on cells.In summary,the developmental toxicity and molecular mechanism of 3 nm FeNPs were systematically studied.It was found that 3 nm FeNPs had effects on embryonic activity,heart development,vascular development,liver development,kidney development,cartilage development,neuromuscular system development and motor ability of zebrafish embryos.The molecular mechanism of 3 nm FeNPs at development of zebrafish embryos was discussed at transcriptome level.This study lays a foundation for the study of biological safety of iron oxide nanoparticles.