Oxidative mitochondrial DNA damage and deletion in hepatocytes of rejecting liver allografts in rats: Role of TNF-α

Abstract

An orthotopic liver transplant model in the rat was used to evaluate the role of tumor necrosis factor alpha (TNF‐α) in liver transplant rejection. There were significantly increased levels of TNF‐α mRNA and parallel increases in 8‐hydroxy‐2′ deoxyguanosine (8‐OHdG) indicative of oxidative DNA damage present 7 to 12 days after transplantation. Cells staining positively for 8‐OHdG were localized to the cytoplasm of hepatocytes adjacent to the TNF‐α expressing inflammatory cells in the portal areas or in patches surrounded by inflammatory cells in the hepatic sinusoids. Significantly more cells staining for 8‐OHdG were found in the allogeneic grafts that were strongly rejected than in the syngeneic controls or in the grafts placed in species that accepted the allograft permanently after a rejection episode. TUNEL reactivity lagged 2 days behind peak reactivity for 8‐OHdG. On day 12 after transplantation, many cells stained for both 8‐OHdG and TUNEL, indicating that the cells suffering oxidative DNA injury were undergoing apoptosis or death. Oxidative injury resulted in mtDNA deletion consisting of 4,834 base‐pairs. Studies of hepatocytes cultured from normal rats displayed dose‐dependent relationships between TNF‐α concentration and 8‐OHdG and mtDNA mutation. Repetitive intraperitoneal injection of Enbrel, a TNF receptor blocker, significantly decreased hepatocyte 8‐OHdG levels and the frequency of deleted mtDNA while greatly extending graft survival time. In conclusion, the data presented implicate TNF‐α as being capable of causing oxidative DNA damage and mtDNA mutation in hepatocytes. (HEPATOLOGY 2005;42:208–215.)