In vitro toxicity of hydrogen peroxide against normal vs. tumor rat hepatocytes: Role of catalase and of the glutathione redox cycle

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Abstract
Hydrogen peroxide produced by stimulated phagocytic cells or during the metabolism of drugs, is toxic to various cell types. The aim of this study was to investigate its toxicity against normal vs. tumor rat hepatocytes. Isolated normal hepatocytes and tumor hepatocytes from three hepatocarcinoma cell lines, Fao, C2 (Faof1 C2) and HTC, were incubated in the presence of a H2O2-generating system consisting of glucose and varied concentrations of glucose oxidase. The toxicity of H2O2 was quantified by measuring the percentage of lactate dehydrogenase activity released in the culture medium after various times of incubation. By comparison to normal hepatocytes, tumor hepatocytes exhibited an increased susceptibility to lysis by H2O2. At a concentration of 100 mU per ml, glucose oxidase induced a lactate dehydrogenase activity release of only 6.1 ± 2.2% (mean ± S.E.) from normal hepatocytes and of 71.0 ± 2.9, 45.5 ± 2.5 and 34.7 ± 3.4% from Fao, C2 and HTC cells, respectively, after an 18-hr incubation. At a concentration of 10 mU per ml, glucose oxidase had no toxic effect to normal hepatocytes or HTC cells, whereas it induced a lactate dehydrogenase activity release of 58.7 ± 7.6 and 51.2 ± 5.6% from Fao and C2 cells, respectively. In addition, the time courses of lactate dehydrogenase activity release, studied with 500 mU per ml glucose oxidase, demonstrated that Fao cells, C2 cells and, to a lesser degree, HTC cells were lysed more rapidly than normal hepatocytes. The toxicity of glucose oxidase was suppressed by the addition of catalase, indicating that it was actually mediated by H2O2. Catalase activity expressed per cell number and the components of the glutathione redox cycle were decreased in tumor cells by comparison to normal hepatocytes. In addition, treatment of normal hepatocytes with aminotriazole, an inhibitor of catalase, and with 1,3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of glutathione reductase, rendered these cells as susceptible as tumor hepatocytes to the effects of H2O2. In conclusion, tumor hepatocytes were more susceptible to the toxic effect of H2O2 than normal hepatocytes. This difference is probably due to a decrease in the H2O2 detoxication systems that we studied, i.e. catalase and glutathione redox cycle, in tumor cells.