RELATIONSHIP OF THE METABOLISM OF VITAMIN-C AND VITAMIN-E IN CULTURED-HEPATOCYTES TREATED WITH TERT-BUTYL HYDROPEROXIDE

The relationship between the metabolism of alpha-tocopherol (alpha-T) (vitamin E) and that of ascorbic acid (vitamin C) was examined in cult ured hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Unl ike vitamin E, the cellular content of vitamin C did not decline after overnight culturing of freshly prepared hepatocytes. In addition, thi s basal vitamin C content was not affected by the presence of alpha-T phosphate in the overnight culture medium. Supplementation of the over night culture medium with vitamin C (10 mu M to 10 mM) increased the c ellular content of vitamin C by >1 order of magnitude. Increasing the cellular content of ascorbate increased the protection against TBHP to xicity, with or without the presence of a physiological content of vit amin E. In vitamin E-supplemented cells, a loss of alpha-T occurred wi thin 15 min of exposure to TBHP and before the decrease in cellular as corbate content. The vitamin C content declined in parallel with the l oss 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 w ith the ferric iron chelator deferoxamine or the antioxidant N,N'-diph enyl-1,4-phenylenediamine prevented the loss of ascorbate and the cell killing by TBHP in hepatocytes either sufficient or deficient in alph a-T. However, neither alpha-T nor ascorbate prevented the accumulation of DNA single-strand breaks caused by TBHP, indicating that these vit amins do not effectively scavenge the TBHP-derived radicals responsibl e for DNA damage. The data in the present study indicate that vitamins E and C act as independent antioxidants and that ascorbate does not r egenerate alpha-T in cultured rat hepatocytes.