Reduction of phenoxyl radicals of the antitumour agent etoposide (VP-16) by glutathione and protein sulfhydryls in human leukaemia cells: Implications for cytotoxicity.

Phenoxyl radicals are inevitable intermediates in the oxidative enzymatic metabolism of a phenolic antitumour drug, etoposide (VP-16), by peroxidases, cytochrome P-450, prostaglandin synthetase and tyrosinase, as well as in its interactions with oxygen and peroxyl radicals. It has been shown that one-electron reduction of the VP-16 phenoxyl radical by ascorbate and thiols prevents/delays its oxidative metabolism by tyrosinase both in model systems and in cell homogenates. To elucidate the role of endogenous thiols in the reduction of VP-16 phenoxyl radicals, K562 human leukaemia cells grown in Dulbecco's modified Eagle's medium which does not contain vitamin C (ascorbate) were used, thus excluding the ascorbate-dependent reduction of VP-16 phenoxyl radicals. VP-16 phenoxyl radicals were reduced by endogenous reductants in K562 cell homogenates, intracellular thiols mainly being responsible. Depletion of endogenous thiols by mersalyl acid resulted in almost complete inhibition of the ability of cell homogenates to reduce VP-16 phenoxyl radicals. Three systems were used to evaluate the contribution of thiol-dependent reduction of VP-16 phenoxyl radicals: (1) K562 cell homogenates depleted or supplemented with glutathione (GSH) in vitro; (2) homogenates derived from K562 cells with a decreased level of endogenous thiols and GSH (using a specific inhibitor of gamma-glutamyl cysteine synthetase, buthionine-S,R-sulfoximine; BSO) and (3) homogenates derived from K562 cells with increased content of endogenous thiols as a result of treatment with cadmium chloride. Depletion of thiols in K562 cells or cell homogenates proportionally decreased the ability of homogenates to reduce VP-16 phenoxyl radicals. Similarly, depletion or supplementation of K562 cells or cell homogenates with GSH proportionally decreased or increased the ability to reduce VP-16 phenoxyl radicals. Reduction of VP-16 phenoxyl radicals by K562 cell homogenates was similar to that obtained from cell homogenates isolated from K/VP.5 cells, a VP-16 resistant cell line derived from K562 cells. Elevation of endogenous thiols by cadmium chloride increased the ability of homogenates to reduce VP-16 phenoxyl radicals but did not reveal any significant difference in the ability of the two types of cells to interact with VP-16 radicals. Finally, BSO treatment of K562 cells led to potentiation of VP-16-induced DNA damage and to an increase in VP-16-induced growth inhibition, suggesting that, in the absence of ascorbate, modulation of endogenous thiols may be an important factor determining the oxidative metabolism and cytotoxic activity of VP-16 in tumour cells.