Peripheral serum iTRAQ-based proteomic characteristics of carbon tetrachloride-induced acute liver injury in Macaca fascicularis

Abstract

Carbon tetrachloride (CCl4) is a potent chemical compound that can induce liver cells necrosis. The purpose of this study was to evaluate the hepatic toxicity of CCl4 exposure in Macaca fascicularis to explore the liver toxicity mechanism using a proteomic approach. One animal (no.F6) was intoxicated by oral gavage with 15 % CCl4 solution (10 mL/kg, dissolved in edible peanut oil), and was sacrificed at 48 h after CCl4 administration. Another blank control animal (no.F4) was sacrificed at the same time. The liver cells of the blank control animal showed normal hepatocyte morphology. However, the hepatocytes at 48 h time point after CCl4 administration showed necrosis and vacuolation histopathologically. The animal No.F7∼F12 and no.M7∼M12 were administrated by gavage with 15 % CCl4 solution (10 mL/kg, dissolved in edible peanut oil). Blood samples were collected before gavage administration, and served as the 0 h blank control samples. Then, blood samples were collected at 2 h, 48 h, 72 h and 168 h after CCl4 exposure, and served as the test samples. Routine biochemistry and immunical parameters were performed using biochemistry analyzer for all serum. Then the serum from male and female animals at 0 h, 2 h, 48 h, and 72 h was mixed, respectively. The peripheral serum proteins at 0 h, 2 h, 48 h, and 72 h were extracted, then the proteins were enzymatically hydrolyzed and the peptides were isotopic labeled by isobaric tags for relative and absolute quantification (iTRAQ). Finally, the UniProt Protein Sequence Library of Macaca fascicularis was queried to identify and compare the differential proteins between different time points. The results showed that, as traditional biomarkers of liver injury, alanine aminotransferases (ALT) and aspartate aminotransferases (AST) showed a typical time-effect curve. Compared with 0 h, there were totally 55, 323, and 158 differential proteins (P value <0.05, Ratio fold >1.5, FDR<0.05) at 2 h, 48 h and 72 h, respectively. GO enrichment analysis of differentially expressed proteins only at 48 h involved 3 cellular components (P adjust value <0.05), and differential proteins at other time points had no significant enrichment. Furthermore, KEGG enrichment analysis showed that the toxicity effect of CCl4 at different time points after administration was mediated through 22 pathways such as biosynthesis of antibiotics, carbon metabolism, biosynthesis of amino acids, peroxisome, cysteine and methionine metabolism, arginine biosynthesis, and complement and coagulation cascades (P adjust value <0.05). Among them, the counts of signaling pathway involved biosynthesis of antibiotics, carbon metabolism and biosynthesis of amino acids were more than 10 and the three pathways may play a greater role in toxicity progress after administration of CCl4. PPI network analysis showed that there were 3, 52, and 13 nodes in the interaction of differential proteins at 2 h, 48 h, and 72 h, respectively. In conclusion, many differential proteins in peripheral blood were detected after CCl4 administration, and the GO and KEGG enrichment analysis showed the toxicological mechanisms of CCl4-induced liver injury and potential protection reaction mechanism for CCl4 detoxication may be related with multi biological processes, signaling pathway and targets.