Effects of ascorbic acid on 3H-thymidine incorporation by isolated mouse thymocytes

The 15-min incorporation of 3H-thymidine (Tdr) and 3H-uridine (Ur) into nucleic acids of freshly isolated mouse thymocytes decreased steadily with time of preincubation at 37 degrees C. Sodium ascorbate at 5 mM prevented the decline of 3H-Tdr incorporation by preventing the decrease in its uptake. No such effect was noted on the incorporation of 3H-Ur, suggesting that ascorbate might be more specific for cells in or near the S phase of the cell cycle. The ene-diol group on the ascorbate molecule was required for this function, as ascorbyl-2-sulfate was ineffective and dehydroascorbate (DHA) reduced 3H-Tdr incorporation even further. Ascorbate was also inhibitory at a lower concentration (0.1 mM) or lower cell density. Thiols such as dithiothreitol or reduced glutathione seemed to act like 0.1 mM rather than 5 mM ascorbate. The inhibition by 0.1 mM ascorbate was presented by 0.1% bovine serum albumin (BSA), catalase or anaerobiosis. BSA had its own protective effects on the cells, since at 0.1% it increased the uptake of both 3H-Tdr and 3H-Ur. The combined effects of 5 mM ascorbate and 0.1% BSA on 3H-Tdr uptake were additive, but some synergism was noted at the lower BSA concentrations. These results suggest that with low concentrations of ascorbate (0.1 mM) oxidative reactions occur in vitro, resulting in the accumulation of the toxic hydroxyl radical (. OH). High concentrations apparently override this inhibition by a mechanism possibly involving an increase in critical cellular thiol groups.