Mechanisms of the killing of cultured hepatocytes by hydrogen peroxide

Abstract

Mechanisms of H 2O 2-induced cell injury were explored in primary cultures of rat hepatocytes. Cells prepared from male rats and cultured for 1 day prior to treatment were killed by H 2O 2 either added directly to the medium at 0.25–2 m m or generated in situ by glucose oxidase (0.25–2 U/ml) or xanthine oxidase (20–120 m m/ml) and 2 m m xanthine. Catalase protected the cells in each case. Lipid peroxidation as measured by the accumulation of malondialdehyde (MDA) preceded the cell death due to H 2O 2 added directly to the cultures or generated in the medium. The antioxidants N,N′-diphenyl p-phenylenediamine (DPPD) and promethazine prevented the accumulation of MDA in both cases and protected the cells treated with H 2O 2 directly. DPPD and promethazine did not react directly with H 2O 2. Other antioxidants including butylated hydroxytoluene, vitamin E, and N-propylgallate had varied protective activity against the addition of H 2O 2 in proportion to their ability to reduce MDA accumulation. In glucose oxidasetreated cultures, DPPD and promethazine prevented the cell killing during the first hour but failed to protect between 1 and 3 h despite prevention of lipid peroxidation. The cell killing between 1 and 3 h in the presence of DPPD was prevented by catalase indicating its dependence upon continued generation of H 2O 2. Further addition of H 2O 2 in the presence of DPPD also increased the number of dead cells without lipid peroxidation. The data are consistent with at least two mechanisms of hepatocyte killing by H 2O 2. The first pathway is prevented by the antioxidants DPPD and promethazine and is very likely related to the peroxidation of membrane phospholipids. The second is independent of lipid peroxidation yet dependent upon the continued presence of H 2O 2.