Oleanolic acid (OA) is a triterpenoid compound, which exists in foods and some medicinal herbs. We have shown that among the ten triterpenoids, OA is very effective in decreasing the hepatotoxicity of carbon tetrachloride, acetaminophen, bromobenzene, thioacetamide, furosemide, cadmium, phalloidin, colchicine, and D-galactosamine plus endotoxin. OA has also been used for human liver diseases in China for the last decade. However, the mechanisms of hepatoprotection by OA are not clear. The overall purpose of this grant is to determine the mechanisms by which OA protects against some of these hepatotoxicants. The initial work will test the hypothesis that OA suppresses toxicant bioactivation as a means of protection. Total cytochrome P-450, b5, and NADPH-cytochrome-c reductase will be assayed spectrophotometrically; P-450 enzyme activities will be assessed by 7-alkoxyresorufins, testosterone oxidation and coumarin hydroxylation. P-450 protein (1A, 2A, 2B, 2E and 3A) levels will be determined by immunoblotting with specific polycolonal antibodies for P- 450 isozymes. Acetaminophen metabolites in vivo will be analyzed by HPLC to determine whether OA decreases acetaminophen toxicity by modifying its metabolism. Covalent binding of 14C-AA to liver proteins, and the formation of AA-GSH adducts by liver microsomes in vitro will also be determined. In addition, the activity of UDP-glucuronosyltransferase and sulfotransferase towards acetaminophen will be assayed to determine if OA increases these detoxication enzymes. Studies will also be performed to test the hypothesis that OA increases cellular defense mechanisms against toxicant insult, such as nonenzymatic components [glutathione (GSH), metallothionein (MT), ascorbic acid, alpha-tocopherol, Zn and Cu] and enzymatic components (GSH-peroxidase, GSH-reductase, GSH-S-transferases, superoxide dismutase, catalase and DT-diaphorase]. Whether OA decreases Cd toxicity by inducing MT will be examined in detail. The effect of OA on MT induction, MT isoforms and MT mRNA will be characterized. These studies will increase our understanding of how necrotic liver injury produced by hepatotoxicants can be prevented by OA, will increase our knowledge about liver injury and cell death via the protective studies, and will provide data that might be instrumental in developing a therapy for liver disorders.
