Activation of poly(ADP-ribose)polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly iso lated rat hepatocytes caused DNA damage and loss of membrane integrity. Suc h DNA lesions are likely to be single strand breaks since neither caryolysi s nor chromatine condensation was seen in electron micrographs from tBOOH-t reated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, in dicating the absence of internucleosomal DNA cleavage, a classical reflecti on of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a d ramatic drop in both NAD(+) and ATP, The inhibition of PARP by 3-aminobenza mide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH, The lack of th e protective effect of PARP inhibitor, therefore, does not implicate PARP i n the mechanism of tBOOH-induced cytotoxicity, Electron micrographs also sh ow no mitochondrial swelling in cells under oxidative stress, but such orga nelles were mainly located around the nucleus, a picture already observed i n autoschizis, a new suggested kind of cell death which shows both apoptoti c and necrotic morphological characteristics. (C) 2000 Academic Press.