E.P.R. Spectroscopic Studies of the Reactions of Chromium(VI) with L-Ascorbic Acid in Buffer Solutions: Implications for Understanding the Roles of Chromium(V) and Chromium(IV) Species and the Buffer Dependences of In Vitro DNA Damage

Abstract

The reaction of chromium(VI) with L-ascorbic acid (AsA) in buffer solutions was investigated by e.p.r. spectroscopy. Chromium(V)/ascorbate complexes with signals at giso = 1.9791 and chromium(VI)/ascorbate/peroxo complexes with signals at g_iso = 1.9819 and g_iso = 1.9824 were observed in all buffers. New signals at g_iso values of 1.9765 and 1.9781 were observed in Tris–HCl [tris(hydroxymethyl)aminomethane hydrochloride] buffer and were assigned to a mixed-ligand ascorbate/Tris complex of chromium(V), [CrO(ascorbate)(Tris)]^2– , and a Tris/peroxo species, [CrO(O2)(Tris)]^2– , respectively. The speciation of the e.p.r.-active chromium(V) complexes detected from solutions with other buffers, such as HEPES, cacodylate and phosphate, was not influenced by the buffer type. Preincubation of catalase with ascorbate solutions inhibited the formation of the chromium(V) peroxo species in all buffers. Manganese(II) reduces the chromium(V) species produced in the reaction, which has shown that it is inappropriate to use manganeses(II) as a selective reagent for monitoring the concentrations of chromium(IV) in such reactions. In particular, manganeses(II) reacts more efficiently with the chromium(V) species that are most damaging to DNA in vitro, viz., the mixed-ligand chromium(V)/ascorbate/peroxo complexes. The correlation of the present results with those of in vitro DNA damage experiments reported in the literature has revealed that the chromium(V)/ascorbate/peroxo species are the major species responsible for the in vitro DNA strand breaks in all of the buffer systems. These species are not expected to be as important in vivo.