Chromium(VI) reduction by ascorbate: role of reactive intermediates in DNA damage in vitro

Reaction of chromium(VI) with one equivalent of ascorbate was studied by electron paramagnetic resonance spectroscopy in the presence of 0.10 M 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) at room temperature in 0.10 M (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES) and 0.05 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffers (pH 7.0 room temperature). Chromium(V), ascorbyl radical, and carbon-based DMPO-radical adducts were observed. A higher level of Cr(V) was observed in HEPES buffer and a higher level of the DMPO-radical adducts was observed in Tris-HCl buffer. Chromium-DNA binding studies were carried out in vitro for calf thymus DNA incubated with Cr(VI) and ascorbate in both buffers at 37 degrees C. Higher Cr-DNA binding was observed in HEPES buffer. DNA strand-break studies were carried out in vitro on pBR322 DNA incubated with Cr(VI) and ascorbate in both buffers at 37 degrees C. Higher percent nicking was observed in Tris-HCl buffer. Addition of DMPO decreased nicking levels in Tris-HCl buffer. These results suggest that free radicals are more reactive than Cr(V) in producing DNA strand breaks and that Cr(V) will react with DNA to produce Cr-DNA adducts. The fact that buffer affects the nature of the reactive intermediates produced upon reduction of Cr(VI) may be related to differences in intracellular metabolism of Cr(VI) and resulting DNA damage observed in various cell culture systems and animal tissues in vivo.