REDUCTION OF PHENOXYL RADICALS OF THE ANTITUMOR AGENT ETOPOSIDE (VP-16) BY GLUTATHIONE AND PROTEIN SULFHYDRYLS IN HUMAN LEUKEMIA-CELLS - IMPLICATIONS FOR CYTOTOXICITY

Phenoxyl radicals are inevitable intermediates in the oxidative enzyma tic metabolism of a phenolic antitumour drug, etoposide (VP-16), by pe roxidases, cytochrome P-450, prostaglandin synthetase and tyrosinase, as well as in its interactions with oxygen and peroxyl radicals. It ha s been shown that one-electron reduction of the VP-16 phenoxyl radical by ascorbate and thiols prevents/delays its oxidative metabolism by t yrosinase both in model systems and in cell homogenates. To elucidate the role of endogenous thiols in the reduction of VP-16 phenoxyl radic als, K562 human leukaemia cells grown in Dulbecco's modified Eagle's m edium which does not contain vitamin C (ascorbate) were used, thus exc luding the ascorbate-dependent reduction of VP-16 phenoxyl radicals. V P-16 phenoxyl radicals were reduced by endogenous reductants in K562 c ell homogenates, intracellular thiols mainly being responsible. Deplet ion 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 thi ol-dependent reduction of VP-16 phenoxyl radicals: (1) K562 cell homog enates depleted or supplemented with glutathione (GSH) in vitro; (2) h omogenates derived from K562 cells with a decreased level of endogenou s thiols and GSH (using a specific inhibitor of gamma-glutamyl cystein e synthetase, buthionine-S,R-sulfoximine; BSO) and (3) homogenates der ived 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 hom ogenates to reduce VP-16 phenoxyl radicals. Similarly, depletion or su pplementation of K562 cells or cell homogenates with GSH proportionall y decreased or increased the ability to reduce VP-16 phenoxyl radicals . Reduction of VP-16 phenoxyl radicals by K562 cell homogenates was si milar to that obtained from cell homogenates isolated from K/VP.5 cell s, a VP-16 resistant cell line derived from K562 cells. Elevation of e ndogenous thiols by cadmium chloride increased the ability of homogena tes to reduce VP-16 phenoxyl radicals but did not reveal any significa nt difference in the ability of the two types of cells to interact wit h VP-16 radicals. Finally, BSO treatment of K562 cells led to potentia tion of VP-16-induced DNA damage and to an increase in VP-16-induced g rowth inhibition, suggesting that, in the absence of ascorbate, modula tion of endogenous thiols may be an important factor determining the o xidative metabolism and cytotoxic activity of VP-16 in tumour cells.