The types of reactive intermediates generated upon reduction of chromium(VI) by glutathione or hydrogen peroxide and the resulting DNA damage have been determined. In vitro, reaction of chromium(VI) with glutathione led to formation of two chromium(V) complexes and the glutathione thiyl radical. When chromium(VI) was reacted with DNA in the presence of glutathione, chromium-DNA adducts were obtained, with no DNA strand breakage. The level of chromium-DNA adduct formation correlated with chromium(V) formation. Reaction of chromium(VI) with hydrogen peroxide led to formation of hydroxyl radical. No chromium(V) was detectable at 24°C (297 K); however, low levels of the tetraperoxochromium(V) complex were detected at 77 K. Reaction of chromium(VI) with DNA in the presence of hydrogen peroxide produced significant DNA strand breakage and the 8-hydroxydeoxyguanosine adduct, whose formation correlated with hydroxyl radical production. No significant chromium-DNA adduct formation was detected. Thus, the nature of chromium(VI)-induced DNA damage appears to be dependent on the reactive intermediates, i.e., chromium(V) or hydroxyl radical, produced during the reduction of chromium(VI).

Neural zinc finger factor 1 (NZF-1) is a member of a family of neural-specific transcription factors that contain multiple copies of a relatively uncharacterized zinc binding motif. We have studied the metal binding and DNA binding properties of a fragment of NZF-1 containing two adjacent zinc binding domains. Partial proteolysis with endoproteinase Lys-C identified metal-stabilized fragments containing either one or both of the zinc binding domains. Both domains were required for specific DNA binding to the beta-retinoic acid receptor element, producing a DNase I footprint covering predominantly one strand. The metal binding site was probed via cobalt(II) substitution. The visible absorption spectrum of the cobalt(II) complex is consistent with Cys-Cys-His-Cys coordination of the metal. The two domains appear to have similar affinities for metal and bind cobalt(II) and zinc(II) with dissociation constants of 4 (+/- 2) x 10(-)(7) M and 1.4 (+/- 0.8) x 10(-)(10) M, respectively. The domains fold upon the addition of zinc, as observed by (1)H NMR. However, an additional weak binding site causes line broadening in the presence of excess zinc, presumably due to aggregation.

The reaction of chromium(VI) with hydrogen peroxide was studied in the presence of glutathione. In vitro, reaction of chromium(VI) with hydrogen peroxide alone led to production of hydroxyl radical as the significant reactive intermediate, while reaction of chromium(VI) with glutathione led to formation of two chromium(V)-glutathione complexes and the glutathione thiyl radical. Incubation of chromium(VI) with glutathione prior to addition of hydrogen peroxide led to formation of peroxochromium(V) species and a dramatic increase in hydroxyl radical production over that detected in the reaction of chromium(VI) with hydrogen peroxide alone. In contrast, addition of chromium(VI) to a preincubated mixture of glutathione and hydrogen peroxide led to a decrease in hydroxyl radical production over that obtained in the reaction of chromium(VI) with hydrogen peroxide. When pBR322 DNA was added to the above reactions, the extent of chromium(VI)-induced DNA strand breakage correlated with the relative amount of hydroxyl radical formed. Reaction of chromium(VI) with calf thymus DNA in the presence of a preincubated mixture of glutathione and hydrogen peroxide led to detection of the 8-hydroxydeoxyguanosine adduct, whose formation correlated with that of hydroxyl radical production. No significant chromium-DNA adduct formation was detected. The results suggest that, in the cellular metabolism of chromium(VI), preformed chromium(V)-glutathione complexes may react with hydrogen peroxide in a Fenton-type manner to produce hydroxyl radical as the DNA-damaging agent. However, if glutathione reacts with hydrogen peroxide prior to exposure to chromium(VI), the amount of hydroxyl radical generated may not be sufficient to cause significant DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)

The types of reactive intermediates generated upon reduction of chromium(VI) by glutathione or hydrogen peroxide and the resulting DNA damage have been determined. In vitro, reaction of chromium(VI) with glutathione led to formation of two chromium(V) complexes and the glutathione thiyl radical. When chromium(VI) was reacted with DNA in the presence of glutathione, chromium-DNA adducts were obtained, with no DNA strand breakage. The level of chromium-DNA adduct formation correlated with chromium(V) formation. Reaction of chromium(VI) with hydrogen peroxide led to formation of hydroxyl radical. No chromium(V) was detectable at 24 degrees C (297 K); however, low levels of the tetraperoxochromium(V) complex were detected at 77 K. Reaction of chromium(VI) with DNA in the presence of hydrogen peroxide produced significant DNA strand breakage and the 8-hydroxydeoxyguanosine adduct, whose formation correlated with hydroxyl radical production. No significant chromium-DNA adduct formation was detected. Thus, the nature of chromium(VI)-induced DNA damage appears to be dependent on the reactive intermediates, i.e. chromium(V) or hydroxyl radical, produced during the reduction of chromium(VI).