ASCORBATE IS THE PRIMARY REDUCTANT OF THE PHENOXYL RADICAL OF ETOPOSIDE IN THE PRESENCE OF THIOLS BOTH IN CELL HOMOGENATES AND IN MODEL SYSTEMS

Phenoxyl radicals are intermediates in the oxidation of phenolic compo unds to quinoid derivatives (quinones, quinone methides), which are kn own to act as ultimate mutagenic, carcinogenic, and cytotoxic agents b y directly interacting with macromolecular targets or by generating to xic reactive oxygen species. One-electron reduction of phenoxyl radica ls may reverse oxidative activation of phenolic compounds to quinoids, thus preventing their cytotoxic effects. In the present work, we stud ied interactions of ascorbate, thiols (glutathione, dihydrolipoic acid , and metallothioneins), and combinations thereof with the phenoxyl ra dical generated by tyrosinase-catalyzed oxidation of VP-16 [etoposide, otoxin-9-(4,6-O-ethylidene-beta-D-glucopyranoside) ], a hindered phen ol widely used as an antitumor drug. We found by liquid chromatography -ionspray mass spectrometry and electron spin resonance (ESR) that tyr osinase caused oxidation of VP-16 to its o-quinone and aromatized deri vative via intermediate formation of the phenoxyl radical. Both ascorb ate and thiols (GSH, dihydrolipoic acid, and metallothioneins) were ab le to directly reduce the VP-16 phenoxyl radical and prevent its oxida tion. The characteristic ESR signal of the VP-16 phenoxyl radical was quenched by the reductants. The semidehydroascorbyl radical ESR signal was detected in the presence of ascorbate; thiols did not produce sig nals in the ESR spectra. In combinations, ascorbate plus GSH and ascor bate plus metallothionein acted independently and additively in reduci ng the VP-16 phenoxyl radical. Ascorbate was more reactive: the VP-16- dependent oxidation of GSH or metallothionein commenced only after com plete oxidation of ascorbate. The semidehydroascorbyl radical ESR sign al preceded the quenching of the VP-16 phenoxyl radical by GSH and met allothionein. In the presence of ascorbate plus dihydrolipoic acid, as corbate was also more reactive toward the VP-16 phenoxyl radical than dihydrolipoic acid, but the ascorbate concentration was maintained at the expense of its regeneration from dehydroascorbate by dihydrolipoic acid. In ESR spectra, the semidehydroascorbyl radical ESR signal was continuously detected and then was abruptly substituted by the VP-16 p henoxyl radical signal. When VP-16 and tyrosinase were incubated in th e presence of retina or hepatocyte homogenates, a two-phase lag period was observed by ESR for the appearance of the VP-16 radical signal: a n ascorbate-dependent part (semidehydroascorbyl radical observable, se nsitive to ascorbate oxidase) and thiol-dependent part (no radical sig nals in the spectra, sensitive to mersalyl acid). About 50% of the thi ol-dependent part of the lag period could be accounted for by endogeno us GSH (as revealed by treatment with GSH peroxidase + cumene hydroper oxide). Homogenates prevented VP-16 oxidation by tyrosinase. The abili ty of ascorbate and thiols, the two major water-soluble intracellular antioxidants, to directly reduce phenoxyl radicals may be an important mechanism of their protective function against cytotoxicity of phenol ic/quinoid redox couples.