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 g_∥ = 1.9831 and g_⊥ = 1.9772, indicating that the unpaired electron occupies the d_z² orbital of the Cr(V) ion, with square-pyramidal geometry. Addition of hydrogen peroxide (H₂O₂) to the NAD(P)H-Cr(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 α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin traps. The dependence of Cr(V) and .OH radical formation on the H₂O₂ and Cr(VI) concentrations indicates that the Cr(V) species react with H₂O₂ to generate the .OH radicals. Similar results were obtained by using various diols (arabinose, cellobiose, FAD, fructose, glyceraldehyde, ribose, and tartaric acid), α-hydroxycarboxylic acids, and glutathione. Investigations with superoxide dismutase showed no significant participation of O₂^- 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 H₂O₂ to generate .OH radicals, which might be the initiators of the primary events in the Cr(VI) cytotoxicity.