The Mechanical Study Of Nanocoppers' Toxicity Using Genomics And Proteomics Technologies

Nanoscale materials are already becoming commercially available for industrial applications and consumer use. Despite the widespread use of nanomaterials, knowledge of the toxicity and potential health risks associated with nanomaterial use is extremely limited. Recent in vivo and in vitro studies have suggested that inhalation and dermal absorption of some nanomaterials may have adverse health effects and that the use of medical products containing nanomaterials may lead to chronic health risks. Furthermore, the unique and diverse physicochemical properties of nanoscale materials suggest that their toxicological properties may differ from those of the corresponding bulk materials.Our study design is based on the framework of systems toxicology. On one hand, the the'traditional'toxicology approach were used to study the body weight, physiology, clinical chemistry, histopathology of the rats exposed to nanocoppers. On the other hand, the'-omics'technologies including genomics, proteomics and metabonomics, were used to obtain the mechanical information about toxicity of nanocoppers. At last, both data will be integrate to lead to a systems toxicology understanding of nanocoppers.In the subacute toxicity study, male Wistar rats orally gavaged the 200 mg/kg nanocoppers for 5 days showed significant changes of the serum clinical biochemistry parameters, including the aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, alanine aminotransferase, triglyceride and total bile acid increased significantly, and alkaline phosphatase and total cholesterol decreased slightly. All the results indicated that the effects produced by nano-copper at a dose of 100 or 50 mg/kg/d were less than those induced at a higher dose of 200 mg/kg/d. Nano-copper induced overt hepatotoxicity and nephrotoxicity at 200 mg/kg/d for 5 d, which mainly involved scattered dot hepatocytic necrosis and widespread renal proximal tubule necrosis. For the rats treated with micro-copper, only slightly swelling of the proximal tubule epithelia were found.In the study of the gene expression profile of the rat liver and kidney exposed to the nanocoppers for 5 days, we found that, in 200mg/kg nanocoppers group rat liver, the dramatically changed genes can be classified to a few categories, including response to stress, metabolic process, electron transport, regulation of cell proliferation, etc. The related KEGG pathways includes Cell adhesion molecules (CAMs), signaling pathways, Antigen processing and presentation, Cytokine-cytokine receptor interaction, Metabolism of xenobiotics by cytochrome P450, and metabolic process, etc. While in 200mg/kg microcoppers group rat liver, only 9 genes were found significantly changed, and no more than 2 genes can be classified to a category or pathway. In 200mg/kg nanocoppers group rat kidney, the dramatically changed genes can be classified to a few categories, including metabolic process, biological regulation, transport, response to stress, developmental process, cell proliferation, cell differentiation and cell death, etc. The related KEGG pathways includes, valine, leucine and isoleucine degradation, Cell cycle, Oxidative phosphorylation, Fatty acid metabolism, Metabolism of xenobiotics by cytochrome P450, Complement and coagulation cascades and metabolic process etc.In the following proteomic study we found that, there are 19 proteins dramatically expressed in 200mg/kg nanocoppers group rat liver can be identified, including Carbonic anhydrase 3, Catalase, Glutathione S-transferase, etc. The 19 proteins can be classified to Response to oxidative stress, Nitrogen metabolism and Glutathione metabolic process. We also identified 7 rat liver proteins both dramatically expressed in nanocoppers 100mg/kg and 200mg/kg group, including Argininosuccinate synthase, Isocitrate dehydrogenase, Hydroxymethylglutaryl-CoA synthase, etc. They were related to Generation of precursor metabolites and energy, and Response to extracellular stimulus functional categories. Only 3 proteins dramatically expressed in 200mg/kg microcoppers group rat liver can be identified, and they are Keratin 8, Keratin 18 and Calreticulin. In rat kidney, we identified 54 proteins dramatically changed in 200mg/kg nanocoppers group, including Glutathione peroxidase 3, ATP synthase subunit beta, Regucalcin, etc. The 54 proteins can be classified to oxidative hosphorylation, Response to oxidative stress, tricarboxylic acid cycle, Urea cycle and metabolism of amino groups, cellular homeostasis, cytoskeleton organization, etc. There are 19 rat kidney proteins both dramatically expressed in nanocoppers 100mg/kg and 200mg/kg group were identified, including Isocitric dehydrogenase, Ubiquinol-cytochrome-c reductase complex core protein 1, Electron transfer flavoprotein subunit alpha, etc. They are related to oxidative phosphorylation, response to metal ion and tricarboxylic acid cycle. While in 200mg/kg microcoppers group, no kidney protein were found dramatically changed.We also detect the content of protein thiol and MDA in nanocoppers exposed rat liver and kidney, the results were consistent to the genomics and proteomics findings and verifyed the oxidative stress play a important role in the hepatotoxicity and nephrotoxicity induce by nanocoppers.At last, we integrated the genomics and proteomics results to the metabonomics findings obtained in the previous study in our lab. We found the changes induced by nanocoppers at gene, protein and metabolite level were consistent. In rat liver, a lot of genes, proteins and metabolites related to the tricarboxylic acid cycle, Urea cycle and metabolism of amino groups pathways were identified dramatically changed at same time. Furthermore, the genes and proteins related to Response to oxidative stress, Glutathione metabolic process, electron transport, Synthesis and degradation of ketone bodies also dramatically changed together. In rat kidney, we found a lot of genes, proteins and metabolites related to the tricarboxylic acid cycle, Glycolysis / Gluconeogenesis and Urea cycle and metabolism of amino groups pathways significantly changed together. And the genes and proteins related to oxidative phosphorylation, Cellar calcium ion homeostasis Glutathione metabolic process also dramatically changed at same time. Based on these findings, we speculated that these pathways maybe play important roles in in the hepatotoxicity and nephrotoxicity induce by nanocoppers. And the–omics technologies used in nanotoxicoloy together were proved to be feasible.In summary, liver and kidneys were the main target organs of the rats repeatedly treated with nano-copper for 5 days. Response to oxidative stress, energy metabolism dysfunction, especially the inhibition of tricarboxylic acid cycle and oxidative phosphorylation process maybe the mechanisms of hepatotoxicity and nephrotoxicity induce by nanocoppers. And the disturbing of Cellar calcium ion homeostasis maybe another reason for nephrotoxicity induce by nanocoppers. The–omics technologies used in nanotoxicoloy study together were proved to be valuable.