Investigation Of Z24 Hepatotoxic Mechanism In Rats By Proteomics

Angiogenesis is one of the keys to tumor growth and metastasis, so it is an effective strategy to block or restrain angiogenesis in oncotherapy. The birth of new anti-angiogenic drugs has provided new opportunity to tumor cure. Although many anti-angiogenic drugs have been used in clinic, the majority of chemical angiogenesis inhibitors were restricted because of toxicity. At present toxicity is the main obstruction in the development and clinical application of this kind of drugs, therefore it is important to systematically study the toxic effects and toxicological mechanisms of rejected compounds for more achievements. Z24 is a synthetic chemical angiogenesis inhibitor, and it acts on basic fibroblast growth factor and Bcl-2 protein.Investigations, both in vivo and in vitro, showed that Z24 could inhibit the growth of many types of tumor. However, its hepatotoxicity to rodents was discovered during the preclinical safety evaluation. By experience it is necessary to understand the toxic effect and mechanism of drug for successful development, therefore the mechanism of Z24-induced hepatotoxicity would be researched for further development. In this thesis, first Z24-induced hepatic injury rat models were made, then the proteomics of liver and plasma from Z24-treated and untreated rats were compared, and on the basis of function analysis of the differentially expressed proteins, Z24 hepatotoxicity mechanism were supposed, finally corresponding experiments were done to confirm the hypothesis. The main methods and results as follows:Duplication of Z24-induced injury of liver in ratsFemale wistar rats were intragastric administrated Z24 at dose of 0, 50, 100 and 200mg/kg per day for 5 days, respectively, then plasma biochemistry and liver histopathology were detected. It was found that the levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBI), direct bilirubin (DBI), triglyceride and total cholesterol increased and blood urea nitrogen decreased in plasma from 100 and 200mg/kg groups. By light microscope, hepatocyte vacuolar degeneration and fibroplasia was observed in all Z24-treated groups, just at different extent. By electron microscope, changes of ultramicrostructur could be found in 200mg/kg group, such as hepatocyte karyon crimpling, transparent mitochondrion, mitochondrial cristae breakage, and so on. These results confirmed that Z24 had damaged liver of the treated rats.Analysis of differentially expressed proteins in Z24-treated rats liver The whole protein extract was separated by two-dimensional gel electrophoresis (2-DE), and the profiles of protein expression were compared between Z24-treated and untreated groups, then differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight tandem mass spectrum (MALDI-TOF-TOF-MS). 22 proteins were identified, including dihydrolipoamide dehydrogenase, propionyl-CoA carboxylase alpha chain, enoyl-CoA hydratase, electron transfer flavoprotein (ETF) alpha polypeptide, ETF dehydrogenase and ATP synthase D chain, glycerol-3-phosphate dehydrogenase 1, ornithine aminotransferase precursor, branched chain ketoacid dehydrogenase E1 (alpha polypeptide), fumarylacetoacetase, glycine N-methyltrasferase, aldehyde dehydrogenase 7A1 and 1B1, 3-alpha-hydroxysteroid dehydrogenase (3α-HSD), Glutathione S-transferase alpha-5 (GST-α5), Glutathione S-transferase P (GST-P) and selenium binding protein 2, PDZ and LIM domain protein 1, and so on. All these proteins decreased, but GST-α5, GST-P and PDZ and LIM domain protein 1 increased.Analysis of differentially expressed proteins in Z24-treated rats plasma The method was the same as the previous method of liver proteomic analysis, and 11 differentially expressed proteins were identified, including fetub protein (Fetuin-B), argininosuccinate synthase (ASS), haptoglobin, clusterin precursor, apolipoprotein E precursor, serum albumin precursor, and so on. In these proteins, fetub protein and serum albumin precursor decreased, all the others increased. Function analysis of differentially expressed proteins and verification of some proteinsFunction and subcellular location of the identified differentially expressed proteins were look up in Swiss-Prot database and the proteins were classified by function based on Gene Ontology. In the other hand the expression of 3α-HSD, GST-P and Fetuin-B was checked by western blot. It was found that in differentially expressed liver proteins 12 of 22 located in mitochondrion and nearly one third, such as dihydrolipoamide dehydrogenase, enoyl-CoA hydratase, ETF dehydrogenase and ATP synthase D chain involved in carbohydrate, lipide and energy metabolism. Plasma differentially expressed proteins ASS and Fetuin-B were potential hepatotoxicity biomarker, and clusterin was related to apoptosis. Based on function analysis of those proteins, we supposed: 1) Z24 mainly inhibited carbohydrate aerobic oxidation, fatty acidβ-oxidation and oxidative phosphorylation pathway, which resulted in ATP production decreased and not enough to maintain the normal cell function, then promoted cell death, and apoptosis maybe the major mode of Z24-induced hepatocyte death. 2) Inhibition of fatty acidβ-oxidation resulted in free fatty acid (FFA) accumulation in cell and caused liver steatosis. 3) Fetub protein and ASS in plasma could be regarded as potential biomarkers of Z24-induced hepatotoxicity. The results of western blot were coincident with the proteomic results.Effect of Z24 on hepatocyte apoptosis and mitochondria functionZ24-induced hepatotoxicity rat models were made as previous method. Then mitochondria from Z24-treated and untreated rats liver were prepared, and indexes such as mitochondria swellen, transmembrane potential (ΔΨm), reactive oxygen species (ROS), NAD(P)H redox state and free calcium level were detected. On the other hand, hepatocytes apoptosis were detected in situ by TUNEL. It was found that free calcium and ROS increased, meanwhile NAD(P)H decreased in the mitochondria of Z24-treated groups. The results also indicated thatΔΨm decreased and mitochondria swelled in Z24-treated groups. Furthermore, more apoptosis cells were seen in Z24-treated groups than the untreated group. These results suggested that Z24 induced hepatocyte apoptosis maybe by mitochondria calcium overload which stimulated mitochondrial permeability transition pores (PTP) open and induced mitochondrial permeability transition (MPT), further causedΔΨm decrease, ROS increase, and mitochondria swellen, finally promoted hepatocyte apoptosis.Effect of Z24 on liver antioxidation systemZ24-induced hepatotoxicity rat models were made as previous method. Malonaldehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX), reduced glutathione (GSH) and glutathione-S-transferase (GST) in liver homogenate was detected by colorimetry. Results showed that in Z24-treated groups the GPX activity decreased, and the total SOD activity increased, which mainly duing to increased CuZn-SOD isoenzyme activity, Mn-SOD isoenzyme activity didn't obviously change, and GST activity increased, GSH level didn't decrease, but increased, and MDA level didn't increase obviously. These results suggested that antioxygen ability wasn't decreased and lipid peroxidation wasn't increased in Z24-treated rats liver, therefore we supposed that oxidative stress injury shouldn't play the main role in Z24-induced hepatotoxicity.Effect of Z24 on liver microsome cytochrome P450 (CYP450) enzyme systemZ24-induced hepatotoxicity rat models were made as previous method. Liver microsome was prepared by differential centrifugation, then some indexes including total CYP450, cytochrome b5 (Cyt-b5), NADPH-CYP450 reducase, CYP450 subtypes 1A2, 1B1, 2E1 and 3A4 were detected. It was found that the levels of total CYP450 and Cyt-b5 increased in the Z24-treated groups, and the activity of NADPH-CYP450 reducase increased, too. As for the CYP450 subtypes, the change was different, that was CYP2E1 and 1B1 obviously increased in all three Z24-treated groups, but CYP1A2 and 3A just increased in 200mg/kg groups. These results suggested that Z24 could alter the expression of CYP450 enzyme system and the induction to CYP2E1 and CYP1B1 maybe involved in Z24-induced hepatotoxicity.On the basis of above results, we made conclusions as follows:1) Z24 could damage rats liver, major changes in plasma were increased AST, ALP and TBI, and toxicity characters in pathology were liver steatosis and fibroplasia, and damaged target cellular organ was mitochondrion. Inhibition of fatty acidβ-oxidation resulted in FFA accumulation in cell and caused liver steatosis.2) Z24-induced rats hepatotoxicity mainly by inhibiting carbohydrate aerobic oxidation, fatty acidβ-oxidation and oxidative phosphorylation pathway, resulting in decreased ATP production and cell dysfunction and promoting apoptosis finally. Calcium metabolic disturbance stimulated PTP open and switched on hepatocyte apoptosis.3) Z24 could alter the expression of CYP450 enzyme system and the induction to CYP2E1 and CYP1B1 probably involved in Z24-induced hepatotoxicity.4) Oxidative stress injury wasn't the main role in Z24-induced hepatotoxicity.5) Fetuin-B and ASS in plasma could be regarded as potential biomarkers of Z24-induced hepatotoxicity.