ESR Evidence for the Glutathionyl Radical in the Reduction of Chromium(VI) By Glutathione

"SUMMARYElectron spin resonance (ESR) measurements provide direct evidence for the involvement of the glutathionyl radical, as well as an isolable Cr(V)-glutathione intermediate in the Cr(VI) glutathione redox reactions. These results indicate a new mechanism for the reduction of Cr(VI) by glutathione in relationship to cellular environment and suggest that synergistic reactions of Cr(V) and GS· might be responsible for the (unexpected) increase in the Cr(VI)-induced DNA-strand breaks at high glutathione levels.INTRODUCTIONWhile the reduction of Cr(VI) by glutathione (GSH) is thought to be a critical' step in the pathogenesis of the Cr(VI)-exposure related carcinogenesis1- 4, the exact reduction mechanism is not yet understood. The following reaction steps have been suggested :(A) Initial step:Cr04 2- + GSH - GSCrOa - (1)(B) At high GSH levels:GSCroa- + GSH - Cr( IV) + GSSG (2)(C) At low GSH levels: GSC rO a - - Cr ( V ) + GS ·(3)Thus far, however, in these reactions, the GS· radical has eluded spectroscopic detection, while the evidence for the Cr(V) formation has been reported positives as well as negative2·. Her~ we present spin-trap ESR evidence for the formation of the GS· radical, and a definitive ESR detection of an isolable cr(V)-GSH intermediate in Cr(VI)-GSH reactions.MATERIALS. AND METHODSESR spectra were obtained at X-band (-9.7 GHz) using a Bruker ER200D ESR spectrometer. The magnetic field was calibrated with a self-tracking NMR Gaussmeter (Bruker, Model ER035M) and the microwave frequency was measured with a Hewlett-Packard (Model 5340A) frequency counter. The spin probes, a-(4-pyridyl-1-oxide) -N -tert-butylnitrone (4-POBN) and 5,5-dimethyl~1-pyrroline-1-ox1de (DMPO) were purchased from Aldrich, K2Cr201 from Fisher and used without further purification. All experiments were done at room temperature."