Neurotoxic Effects Of Silver Nanoparticles On Early Life-stage Development Of Zebrafish(Danio Rerio)

Silver nanoparticles(Ag NPs)have good photoelectric,catalytic,superconducting properties and bactericidal and disinfection activities,and have become the most widely used metal nanomaterials in the market.The commercial products containing Ag NPs are almost everywhere.The mass production and wide application of Ag NPs increase the opportunities for its release into the environment,as well as the risk of human exposure.Therefore,effects of Ag NPs on organisms and ecosystems have aroused wide concern.To study the neurotoxic effects of Ag NPs exposure on the early-life stages of zebrafish(Danio rerio),a series of experiments was carried out as following: Firstly,the embryonic zebrafish model was exposed to Ag NPs(0 μg/L,200 μg/L,400 μg/L,600 μg/L,800 μg/L and 1000 μg/L)for 7 days and the mortality rate,hatching rate,heart rate and abnormality rate were calculated.Meanwhile,changes in morphology of zebrafish larvae were observed by stereomicroscope to explore the developmental toxic effect of Ag NPs.Then,m RNA expression of five common internal reference genes(gapdh,ef1α,β-actin,rpl7 and rnf7)of zebrafish exposed to Ag NPs(0 μg/L,400 μg/L,600 μg/L,800 μg/L and 1000 μg/L)was measured by Real-time quantitive PCR in order to select the credible internal reference genes for expression analysis of objective genes.Finally,the m RNA expression levels of GABA receptor pathway related genes(gabra1,gat 1,abat and gad1b)in the zebrafish exposed to Ag NPs were detected by Real-time quantitative PCR to study the neurotoxic effects of Ag NPs from the perspective of neurotransmitters and their receptors.The results are as follows:1.According to the mortality rate,the 96 h-sublethal concentration(LC50)of the zebrafish exposed to Ag NPs was 1380.94 μg/L,and 168 h-LC50 was 1216.13 μg/L.Meanwhile,after Ag NPs exposure,survival rate,hatching rate,heart rate,abnormality rate and other indicators of zebrafish were significantly changed in a concentration-dependent manner.During different stages of embryonic development,melanin retardation,pericardial edema,yolk sac edema,spinal curvature and other malformations were also observed in Ag NP-exposed zebrafish.2.In this experiment,Best Keepe,ge Norm and Norm Finder softwares were selected to evaluate the stability of five candidate internal reference genes of zebrafish.The comprehensive evaluation method was applied to analyze the stability of the internal reference genes and the sequence was: β-actin > gapdh > rnf7 > rpl7 > ef1α.The most stable internal reference gene was β-actin.3.The m RNA expression of GABA receptor pathway related genes(gabra1,gat 1,abat and gad1b)in zebrafish larvae were changed by Ag NPs exposure.The transcriptional level of abat were significantly up-regulated in the low-concentration group,and gat1 m RNA expression was notably up-regulated in all the Ag NPs treatment groups(p < 0.05).The transcriptional profile of gabra1 was remarkably decreased in the high-concentration group(p < 0.01).There was no significant change in gad1 b m RNA expression.In summary,Ag NPs can affect the development of zebrafish embryos.The gene expression of GABA receptor pathway related genes(gabra1,gat 1,abat and gad1b)in zebrafish larvae were regulated by Ag NPs exposure.