ASCORBIC-ACID UPTAKE BY ISOLATED RAT HEPATOCYTES - STIMULATORY EFFECT OF DIQUAT, A REDOX CYCLING COMPOUND

The toxicity of redox cycling compounds which generate the formation of active oxygen species is commonly accepted to be associated with a decrease of cellular reductants involved in cellular defence. However, when hepatocytes were incubated with diquat, an established redox cycler, in the presence of ascorbic acid (AA) (1 mM), the intracellular level of AA was increased. The effects of diquat on AA uptake were investigated in isolated rat hepatocytes. Incubation of hepatocytes with diquat plus AA (1 mM) resulted in about a 2-fold increased accumulation which occurred in a time-dependent manner reaching a steady state after 15 min at 37-degrees. The initial AA uptake rate was dependent on the AA concentration added. This process is described by Michaelis-Menten kinetics (apparent K(m) = 953 +/- 59 muM and V(max) = 2.68 nmol/min/10(6) cells). Characterization of AA accumulation showed it to be inhibited: by incubation at 4-degrees; with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an inhibitor of intracellular ATP production; by decreasing the extracellular Na+ concentration or incubating with ouabain; with pfloretin, a glucose transport inhibitor; and with glucose, a competitive inhibitor of AA transport. Replacement of AA with its oxidized form, dehydroascorbic acid, in the absence of diquat enhanced AA accumulation by 2.5-fold and apparently prevented further accumulation by added diquat. In addition, maintaining AA reduced with dithiothreitol inhibited the diquat effect. Diquat-induced AA accumulation was inhibited (65%) by desferrioxamine, a free-iron chelator, but not by catalase and/or superoxide dismutase or different antioxidants. In contrast, incubation with other active oxygen species generating systems including bipyridilium structural analogues, paraquat and benzyl viologen, had no effect on AA accumulation in hepatocytes. These results suggest that diquat-induced AA accumulation by hepatocytes occurs by a specific mediated transport system rather than as a consequence of cytotoxicity and may involve the presence of free-iron.