Inhibition of lipid peroxidation without prevention of cellular injury in isolated rat hepatocytes

Abstract

The role of lipid peroxidation in tissue injury has been the subject of many investigations. Recently evidence has suggested that lipid peroxidation due to various chemicals is directly responsible for loss of hepatocyte viability. In the present study hepatocytes, isolated by a collagenase perfusion technique, were incubated in either a Tris buffer or Medium 199 with and without the antioxidant N,N′-diphenyl- p-phenylenediamine (DPPD) (2.5 μ m). In order to determine if there is a relationship between lipid peroxidation and cell injury, samples were taken over a 6-hr incubation period for estimation of concentration of thiobarbituric acid (TBA) reactants, diene conjugates of lipids, and loss of intracellular potassium ion (K +) and aspartate aminotransferase (AST). The content of reduced glutathione (GSH) was also assessed. The same parameters were also investigated in cells, which were suspended in Medium 199, incubated with either sodium iodoacetamide (100 μ m), diethyl maleate (4 m m), or sodium vanadate (400 μ m). The effects of DPPD (2.5 μ m) on the responses induced by these chemicals were also studied. Medium 199 was found to be a superior incubation buffer with respect to the cellular viability characteristics, leakage of intracellular K + and AST. Furthermore, GSH was maintained at much higher concentrations and there was less evidence of lipid peroxidation when Medium 199 was used rather than Tris. However, in the presence of DPPD the increase in concentration of TBA reactants was inhibited but DPPD had no protective effect on the release of K + and AST or the decrease in GSH concentration. The three chemical treatments, sodium iodoacetamide, diethyl maleate, and sodium vanadate, each caused loss of cell viability, marked and rapid decrease in GSH, and increases in the estimates of lipid peroxidation. DPPD completely inhibited the lipid peroxidation due to sodium iodoacetamide, but only delayed and did not prevent the loss of K + and had no effect on the loss of GSH. Similarly, DPPD inhibited the lipid peroxidation due to diethyl maleate but did not cause a significant change in the decline of cell viability or alteration in the loss of GSH. The decrease in cell viability produced by vanadium was prevented by DPPD, although at 6 hr of incubation intracellular K + was significantly lower in hepatocytes incubated with DPPD plus vanadium than those with DPPD alone. Again DPPD inhibited the increase in lipid peroxidation but did not alter the loss of GSH with vanadium treatment. Overall the results of this study suggest that lipid peroxidation is not totally responsible for the loss of cellular viability associated with incubation in different mediums or in response to chemicals such as sodium iodoacetamide and diethyl maleate.