On the Hydroxyl Radical Formation in the Reaction between Hydrogen Peroxide and Biologically Generated Chromium(V) Species

"Electron spin resonance (ESR) measurements on solutions and isolated powders provide direct evidence for the involvement of Cr(V) species in the reduction of Cr(VI) by NAD(P)H. ESR analysis of an isolated Cr(V)NAD( P)H solid yields 6 11 = 1.9831 and 6.i. = 1.9772, indicating that the unpaired electron occupies the d22 orbital of the Cr(V) ion, with square-pyramidal geometry. Addition of hydrogen peroxide (H2 0 2 ) to the N AD(P)HCr( VI) reaction mixtures suppresses the Cr(V) species and generates hydroxyl (""OH) radicals. The ""OH radicals were detected via ESR spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide and a-(4-pyridyl-1- oxide)-N-tert-butylnitrone as spin traps. The dependence of Cr(V) and ""OH radical formation on the H20 2 and Cr(VI) concentrations indicates that the Cr(V) species react with H20 2 to generate the ""OH radicals. Similar results were obtained by using various diols (arabinose, cellobiose, FAD, fructose, glyceraldehyde, ribose, and tartaric acid), a-hydroxycarboxylic acids, and glutathione. Investigations with superoxide dismutase showed no significant participation of 02 in the generation of OH radicals. These results thus indicate that the Cr(V) complexes, produced in the reduction of Cr(VI) by cellular reductants, react with H20 2 to generate ""OH radicals, which might be the initiators of the primary events in the Cr(VI) cytotoxicity.In this paper we report an electron spin resonance (ESR)3 spectroscopic detection of Cr(V) species in the reduction of potassium dichromate, henceforth called Cr(VI), by NAD(P)H and other diols. Also reported here is the detection of the hydroxyl (""OH) radical in the reaction between hydrogen peroxide (H2O 2) and a variety of Cr(V) complexes. This work was undertaken because while the reduction of Cr(VI) to lower oxidation states has been suggested as a key step m the mechanism of Cr(VI) toxicity, mutagenicity, and DNA alteration, the details are not yet clear (1-5).Earlier studies on the mechanism of Cr(VI) reduction in cellular systems include those on the reduction of chromate by microsomes (6-8), rat liver DT-diaphorase (9), and aldehyde oxidase (10). All these studies imply that Cr(VI) can be reduced to Cr(Ill) by cellular constituents. Regarding the mechanism of Cr(Vl) carcinogenicity, Jennette postulated that Cr(V) is the ultimate carcinogenic form of chromium compounds (8). This conclusion has led to many further investigations of the formation of Cr(V) in the reduction of Cr(VI) under biologically relevant conditions (11-33). More recently, however, Kawanishi and co-workers (34) have investigated the mechanism of DNA cleavage induced by Cr(VI) in the presence of H20 2 and have argued that it is not Cr(V) itself that is carcinogenic, but it is the reactive oxygenated species (02, 10 2 , ""OH) (produced by the decomposition of the Crv(02) ¾ - ion) that cause the DNA alteration."