Inhibition of Etoposide-induced DNA Damage and Cytotoxicity in L1210 Cells by Dehydrogenase Inhibitors and Other Agents

The mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-0-ethylidene-β-D-glucopyranoside) (VP-16), an important antitumor agent, is unclear. There is evidence that DNA may be the target of action because VP-16 causes single-strand and double-strand breaks in DNA and produces cytotoxicity over a similar dose range. We have hypothesized that an enzyme system, such as dehydrogenase, catalyzes an oxidation-reduction reaction involving the pendant phenolic group which forms an active metabolite that causes the DNA damage and cytotoxicity. To test our hypothesis, we investigated the effect of disulfiram, an aldehyde dehydrogenase inhibitor, and its metabolite, diethyldithiocarbamate, on VP-16-induced DNA damage in L1210 cells. Using the alkaline elution technique to assay DNA damage, we found that disulfiram and diethykjithiocerbamate inhibited VP-16-induced single-strand breaks. Both compounds were also capable of significantly reducing VP-16-induced cytotoxicity. Oxalic add, pyrophosphate, and malonic acid, competitive inhibitors of succinate dehydrogenase, and the naturally occurring dehydrogenase substrates, succinic acid, β-glycerophosphate, and isocitric add, also blocked the effects of VP-16. Free-radical scavengers were also studied. While sodium benzoate was particularly effective in preventing drug-induced DNA damage and cytotoxicity, a number of other scavengers were not. Our data are consistent with the hypothesis that VP-16 is activated by an enzyme such as a dehydrogenase which transforms it into an active intermediate resulting in DNA damage and, consequently, cell death.