The synthesis of vitamin C is substantially reduced in Osteogenic Disorder Shionogi (ODS) rats. Hepatocytes prepared from these rats contained approximately 12% of the wild-type content of this vitamin. In culture, the ascorbate content remained low in the absence of supplementation of the medium. Independent of their vitamin C status, cultured hepatocytes become depleted of vitamin E. Supplementation of the culture medium with 10C microM ascorbate and 1.2 microM alpha-tocopherol phosphate maintained the physiological content of both vitamins C and E in ODS hepatocytes. Thus, the antioxidant function of vitamins C and E could be evaluated in the presence of both or either vitamin or in the absence of both vitamins. Hepatocytes deficient in both vitamins were the most susceptible to lipid peroxidation (as measured by thiobarbituric acid) and to cell kllling within a 90-min exposure to 125-500 microM tert-butyl hydroperoxide (TBHP). Supplementation to achieve a physiological content of both vitamins C and E reduced the evidence of lipid peroxidation and abolished the cell killing. Supplementation with either vitamin alone resulted in an intermediate degree of both lipid peroxidation and cell killing. In ODS hepatocytes treated with TBHP, the decline in vitamin E preceded the decline in vitamin C. In ODS hepatocytes depleted of vitamin C, the loss of vitamin E after exposure to TBHP was greater than that in the presence of physiological levels of ascorbate. This greater loss of vitamin E in the face of a depletion of vitamin C was readily attributable to the increased peroxidation of lipids. Thus, the physiological level of vitamin C in cells does not seem to regenerate vitamin E. In contrast, the rate and extent of the depletion of vitamin C correlate with the degree of cell killing. These data document the antioxidant function of the physiological level of cellular vitamin C and relate this function to protection against peroxidative cell injury.
The disposition of vitamin E was examined in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Culturing of the cells overnight (18-20 hr) with approximately 60 nM alpha-tocopherol (alpha-T) equivalents [Williams' E medium, 18 nM tocopherol phosphate (alpha-TP), 9% fetal calf serum, 43 nM alpha-T] resulted in a content of alpha-T that was 16% of the concentration of vitamin E measured in freshly isolated hepatocytes. Supplementation of the medium with 1 microM alpha-TP maintained the alpha-T concentration of the cultured cells at the level of freshly isolated hepatocytes. Supplemented hepatocytes exposed to TBHP showed decreased lipid peroxidation and delayed cell killing, compared with hepatocytes not cultured overnight with alpha-TP. Killing of the supplemented cells by TBHP was accompanied by a loss of alpha-T. Pretreatment of supplemented hepatocytes with the iron chelator deferoxamine prevented much of the loss of alpha-T. At the same time, deferoxamine inhibited both the lipid peroxidation and cell killing. The antioxidant N,N'-diphenyl-1,4-phenylenediamine reduced the loss of alpha-T and significantly decreased lipid peroxidation. In the presence of N,N'-diphenyl-1,4-phenylenediamine, cell killing was delayed by 15 min and reduced in extent. Overnight supplementation of hepatocytes with nonesterified alpha-T, or vitamin E esters other than alpha-TP, similarly rendered the cells less sensitive to TBHP. The nonesterified alpha-T produced a higher cell-associated vitamin E concentration than did the esters; however, nonesterified alpha-T did not result in greater protection against TBHP. These data indicate that the mechanisms of the cell killing by TBHP are the same in cultured hepatocytes that contain low or physiological concentrations of vitamin E.
The relationship between the metabolism of alpha-tocopherol (alpha-T) (vitamin E) and that of ascorbic acid (vitamin C) was examined in cultured hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Unlike vitamin E, the cellular content of vitamin C did not decline after overnight culturing of freshly prepared hepatocytes. In addition, this basal vitamin C content was not affected by the presence of alpha-T phosphate in the overnight culture medium. Supplementation of the overnight culture medium with vitamin C (10 microM to 10 mM) increased the cellular content of vitamin C by > 1 order of magnitude. Increasing the cellular content of ascorbate increased the protection against TBHP toxicity, with or without the presence of a physiological content of vitamin E. In vitamin E-supplemented cells, a loss of alpha-T occurred within 15 min of exposure to TBHP and before the decrease in cellular ascorbate content. The vitamin C content declined in parallel with the loss of cell viability. Supplementation of the overnight culture medium with increasing concentrations of ascorbate progressively spared the depletion of alpha-T while preventing the cell killing. Pretreatment with the ferric iron chelator deferoxamine or the antioxidant N,N'-diphenyl-1,4-phenylenediamine prevented the loss of ascorbate and the cell killing by TBHP in hepatocytes either sufficient or deficient in alpha-T. However, neither alpha-T nor ascorbate prevented the accumulation of DNA single-strand breaks caused by TBHP, indicating that these vitamins do not effectively scavenge the TBHP-derived radicals responsible for DNA damage. The data in the present study indicate that vitamins E and C act as independent antioxidants and that ascorbate does not regenerate alpha-T in cultured rat hepatocytes.
The peripheral benzodiazepine receptor (PBzR) is associated with the outer mitochondrial membrane. Protoporphyrin IX (PPIX), an endogenous substance with high affinity for the PBzR, induced the inner membrane permeability transition (MPT) in respiring liver mitochondria de-energized by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Cyclosporin A (CyA), an inhibitor of the permeability transition, prevented this effect. In cultured hepatocytes, the MPT was measured as an increased [3H]sucrose-accessible space sensitive to CyA. Nanomolar concentrations of PPIX potentiated the induction of the MPT and the extent of cell killing in hepatocyte cultures de-energized by rotenone. CyA prevented the enhanced cell killing by PPIX. PPIX did not increase the rate or extent of ATP depletion, the loss of the mitochondrial membrane potential, or the accumulation of long chain acyl-CoA thioesters. The association of the PBzR with the voltage-dependent anion channel of the outer mitochondrial membrane and with the adenine nucleotide carrier of the inner membrane suggests that this complex mediates the transport of PPIX across the mitochondrial membranes. In turn, this same complex participates in the MPT. Thus, the same structural complex (PBzR, voltage-dependent anion channel, and adenine nucleotide carrier) can interact with the endogenous substrate PPIX to result in different functional consequences depending on the state of mitochondrial energization.
