Stearns D M, Courtney K D, Giangrande P H, Phieffer L S, Wetterhahn K E
Department of Chemistry, Dartmouth College, Hanover, New Hampshire.
Environ Health Perspect. 1994 Sep;102 Suppl 3(Suppl 3):21-5. doi: 10.1289/ehp.94102s321.
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.
在室温下，于0.10 M（N-[2-羟乙基]哌嗪-N'-[2-乙磺酸]）（HEPES）和0.05 M三（羟甲基）氨基甲烷盐酸盐（Tris-HCl）缓冲液（pH 7.0）中，在0.10 M 5,5-二甲基-1-吡咯啉-1-氧化物（DMPO）存在的情况下，通过电子顺磁共振光谱研究了铬（VI）与一当量抗坏血酸盐的反应。观察到了铬（V）、抗坏血酸自由基和碳基DMPO-自由基加合物。在HEPES缓冲液中观察到较高水平的铬（V），在Tris-HCl缓冲液中观察到较高水平的DMPO-自由基加合物。在37℃下，在两种缓冲液中对与铬（VI）和抗坏血酸盐一起孵育的小牛胸腺DNA进行了体外铬-脱氧核糖核酸结合研究。在HEPES缓冲液中观察到较高的铬-脱氧核糖核酸结合。在37℃下，在两种缓冲液中对与铬（VI）和抗坏血酸盐一起孵育的pBR322 DNA进行了体外DNA链断裂研究。在Tris-HCl缓冲液中观察到较高的切口百分比。添加DMPO降低了Tris-HCl缓冲液中的切口水平。这些结果表明，在产生DNA链断裂方面，自由基比铬（V）更具反应性，并且铬（V）将与DNA反应生成铬-脱氧核糖核酸加合物。缓冲液影响铬（VI）还原时产生的反应中间体的性质这一事实，可能与铬（VI）在细胞内代谢的差异以及在各种细胞培养系统和动物组织体内观察到的DNA损伤有关。
