The Toxicity Effect And Molecular Mechanism Of Silver Nanoparticles On Euplotes Vannus And Tetrahymena Thermophila

AgNP is one of the most widely used metal nanomaterials,and its potential biological and environmental risks have raised widespread concern.To date,the biological toxicity of AgNP and the corresponding mechanisms have been reported,but the molecular mechanisms about how AgNP affects organisms and how organisms direct the cell to a detoxifying reaction and damage repair under AgNP stress are poorly understood.This study used integrated analysis of transcriptomic,microRNAomic,lncRNAomic and biochemical approaches to investigate how ciliates(Euplotes vannus and Tetrahymena thermophila)cope with AgNP-induced stress,with multiple lines of evidence indicating novel mechanisms in these ciliates.The main results are as follow:(1)The 12h LC50 of Ag+and AgNP to E.vannus were 2.18 ± 0.06 and 30.01 ±1.98 mg/L,respectively.The 24h IC50 of Ag+,Ag-Cit10,Ag-Cit80 and Ag-PVP80 to T.thermophila were 0.86 ± 0.06,1.64± 0.14,2.68 ± 0.13 and 2.93 ± 0.08 mg/L,respectively.(2)AgNP exposure caused toxicity in the ciliates by eliciting oxidative stress.After AgNP exposure for 24h,as high as 1.38 ± 0.15 mg/g of silver was detected in E.vannus,while AgNP-rich as black spots were observed on the cell membrane of T.thermophila.The KEGG and GO enrichment analysis suggested that endocytic pathways may be involved in the uptake of AgNP in E.vannus,while the Ag+transport by both ciliates might be mediated via the ion channels on the cell membrane.The ROS and MDA contents in both ciliates were significantly increased,while the ATP content was significantly reduced,suggesting the lipid peroxidation and mitochondrial damage.Transcriptomic data showed that several genes that relevant to energy metabolism,electron transfer and DNA damage repair were significantly up regulated,suggesting that the AgNP lead to the mitochondrial dysfunction and cell cycle arrest.(3)The ciliates responded to oxidative stress by changing the expression levels of antioxidant enzymes,directing the cell to a detoxifying reaction.After AgNP exposure,the content of GPx was significantly increased,while the content of Gr was significantly reduced in both ciliates,suggesting that the cells elicited an antioxidant response.Transcriptomic data showed that several antioxidant genes,such as GPx,Gr,glutathione synthetase and superoxide dismutase were differentially expressed.KEGG and GO enrichment analysis revealed that multiple antioxidant-related biological pathways were significantly enriched,such as glutathione metabolism,kinase activity,and oxidoreductase activity,etc.,supporting that the cells elicited an antioxidant response.(4)The post-transcriptional regulation by microRNA and lncRNA in ciliates play important roles in responding to the toxicity of AgNP.A total of 16 differentially expressed microRNAs were identified in E.vannus after AgNP exposure,and 4640 target genes were predicted.Data suggest that by inhibiting gene expression,these microRNAs inhibited many important biological functions of E.vannus,including TCA cycle,oxidative phosphorylation,and cell cycle,which contributed to E.vannus to cope with the toxicity of AgNP at different exposure times.On the other hand,a total of 1250 differentially expressed lncRNAs were identified in T.thermophila after Ag+,Ag-Cit10,Ag-Cit80 and Ag-PVP80 exposure.These lncRNAs exhibited a dynamic expression pattern,and based on the lncRNA-mRNA co-expression networks,they seemed to play a key role in inhibiting cell growth,activating cell membrane ion channels and regulating redox enzyme activity,which enabled T.thermophila to cope with the stress of Ag+and AgNP.