Ascorbate is the primary reductant of the phenoxyl radical of etoposide in the presence of thiols both in cell homogenates and in model systems

Biochemistry. 1994 Aug 16;33(32):9651-60. doi: 10.1021/bi00198a034.


Phenoxyl radicals are intermediates in the oxidation of phenolic compounds to quinoid derivatives (quinones, quinone methides), which are known to act as ultimate mutagenic, carcinogenic, and cytotoxic agents by directly interacting with macromolecular targets or by generating toxic reactive oxygen species. One-electron reduction of phenoxyl radicals may reverse oxidative activation of phenolic compounds to quinoids, thus preventing their cytotoxic effects. In the present work, we studied interactions of ascorbate, thiols (glutathione, dihydrolipoic acid, and metallothioneins), and combinations thereof with the phenoxyl radical generated by tyrosinase-catalyzed oxidation of VP-16 [etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside)], a hindered phenol widely used as an antitumor drug. We found by liquid chromatography-ionspray mass spectrometry and electron spin resonance (ESR) that tyrosinase caused oxidation of VP-16 to its o-quinone and aromatized derivative via intermediate formation of the phenoxyl radical. Both ascorbate and thiols (GSH, dihydrolipoic acid, and metallothioneins) were able to directly reduce the VP-16 phenoxyl radical and prevent its oxidation. 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 signals in the ESR spectra. In combinations, ascorbate plus GSH and ascorbate plus metallothionein acted independently and additively in reducing the VP-16 phenoxyl radical. Ascorbate was more reactive: the VP-16-dependent oxidation of GSH or metallothionein commenced only after complete oxidation of ascorbate. The semidehydroascorbyl radical ESR signal preceded the quenching of the VP-16 phenoxyl radical by GSH and metallothionein. In the presence of ascorbate plus dihydrolipoic acid, ascorbate 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 phenoxyl radical signal. When VP-16 and tyrosinase were incubated in the presence of retina or hepatocyte homogenates, a two-phase lag period was observed by ESR for the appearance of the VP-16 radical signal: an ascorbate-dependent part (semidehydroascorbyl radical observable, sensitive to ascorbate oxidase) and thiol-dependent part (no radical signals in the spectra, sensitive to mersalyl acid). About 50% of the thiol-dependent part of the lag period could be accounted for by endogenous GSH (as revealed by treatment with GSH peroxidase+cumene hydroperoxide).(ABSTRACT TRUNCATED AT 400 WORDS)

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ascorbic Acid / metabolism*
  • Electron Spin Resonance Spectroscopy
  • Etoposide / analogs & derivatives
  • Etoposide / chemistry
  • Etoposide / metabolism*
  • Free Radicals
  • Liver / metabolism
  • Male
  • Monophenol Monooxygenase / metabolism
  • Oxidation-Reduction
  • Phenols / chemistry
  • Phenols / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Retina / metabolism
  • Subcellular Fractions / metabolism
  • Sulfhydryl Compounds / metabolism*
  • Thioctic Acid / analogs & derivatives
  • Thioctic Acid / metabolism


  • Free Radicals
  • Phenols
  • Sulfhydryl Compounds
  • phenoxy radical
  • Etoposide
  • Thioctic Acid
  • dihydrolipoic acid
  • Monophenol Monooxygenase
  • Ascorbic Acid