| Nano Zinc Oxide(n-ZnO)is one kind of metal oxide nanomaterials which is widely used in cosmetics and other daily commodities.Through various ways,it has free access to human beings.In real scenario,n-ZnO may have the risk of co-exposure with nanomaterials of different dimensions.Graphene oxide,a promising two dimentional nanomaterials,is likely to be exposed with n-ZnO in the future.Different shapes of n-ZnO and graphene oxide lead to huge differences in biological effects,which may produce unique combined effects in the compound exposure and finally generate complicated human health risks.In this study,A549 cell model were used to evaluate the joint cytotoxicity between n-ZnO and graphene oxide,explore the influence of the combined effects on cell gene expression and metabolic process,finally analyze toxicity contribution derived from interaction between two nanomaterials.The detail results are listed below:(1)Combined toxicity effects:n-ZnO exposure caused cell viability decline,intracellular reactive oxygen species(ROS)accumulation,overexpression of antioxidant enzymes,decrease of mitochondrial membrane potential,damage of cell membrane integrity,showing a significant dose effect toxicity in A549 cells.Graphene oxide under concentrations of 1,5,10 mg/L did not show significant toxic effects but decreased the cytotoxicity of n-ZnO,resulted in increased EC50 value of cell viability inhibition test.More over,graphene oxide could reduce intracellular ROS accumulation,decrease catalase expression,weaken mitochondrial membrane potential change and lessen lactate dehydrogenase leakage,showing obvious antagonistic effect.(2)The effect of co-exposure on cell transcriptomes and metabolomics:the transcriptomes study of A549 cells showed that 10 mg/L graphene oxide,20 mg/L nZnO and their combined exposure caused different expression of genes.The DEG numbers were 304,772 and 1145 respectively.Among them,n-ZnO alone and combined exposure group caused significant difference in gene function and pathway enrichment,however,graphene oxide did not induce similar effects.Gene Ontology functional annotation showed that co-exposure of n-ZnO and graphene oxide had a significant impact on the additional 40 types of functional categories,of which 11 were associated with cell membrane function.Through KEGG pathway analysis we also found that co-exposure of n-ZnO and graphene oxide caused more pathway abnormal.In addition,the metabolism analysis of A549 cells revealed that single and combined exposure of 20 mg/L n-ZnO had a greater impact on the expression of metabolites which lead to disturbance in the process of amino acid metabolism,carbohydrate metabolism and so on.Moreover,the addition of graphene oxide could weaken the effects of n-ZnO on the expression of metabolites,presenting antagonistic effect.(3)The contribution of nanomaterial interaction to joint cytotoxicity:We found that the main toxic mechanism of n-ZnO was to enter into cells through endocytosis,and gradually released zinc ions in the endosome,showing a Troy Trojan effect.Graphene oxide could prevent the endocytosis of n-ZnO through covering the cell membrane surface and lead to decreased bio-availability of n-ZnO,exhibiting the antagonistic effect.In addition,graphene oxide did not show significant effects on the deposition of n-ZnO and dissolution of zinc ions,meanwhile,its adsorption capacity of zinc ions was also very weak under the experimental condition,which was not the main mechanism of the joint effects.In summary,this study explored the combined cytotoxicity and its underlying mechanism of graphene oxide and n-ZnO,finding the antagonistic effect of graphene oxide on n-ZnO.Our study provides a new way of thinking for the joint effects of different kind nanomaterials and provides basic informations on potential co-exposure risk of the two nanomaterials. |
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