Bulky DNA-adduct formation induced by Ni(II) in vitro and in vivo as assayed by 32P-postlabeling

Abstract

Various small oxidation products (e.g. 8-hydroxydeoxyguanosine) can be induced in DNA by nickel compounds. In this study, the 32P-postlabeling assay was applied to determine whether Ni(II) compounds are able to induce bulky DNA-adduct formation in vitro and in vivo. In vitro studies detected two major and several minor adducts in DNA incubated with NiCl 2 and H 2O 2 at 37°C for 1 h. Formation of the two major adducts increased with incubation time (0–24 h) and NiCl 2 concentration (0–800 μM). Adduct levels were greatly reduced by hydroxyl free-radical scavengers, i.e. 0.4 M sodium formate or 0.05 M p-nitrosodimethylaniline, and by a singlet oxygen scavenger, 0.05 M sodium azide. The in vitro effects of NiCl 2 on DNA were significantly enhanced by (1) addition of 3 mM ascorbic acid, (2) replacement of H 2O with D 2O in the reaction, and (3) prior denaturation of DNA. Adduct formation presumably involved a Fenton-type reaction, in which DNA crosslinks may arise by reaction with hydroxyl free radicals and singlet oxygen. For in vivo studies, male 6–8 wk old B6C3F1 mice were used. In untreated mice, several I-compounds (putative indigenous DNA modifications that increase with age) were detected in liver, kidney, and lung. Two of these spots (1 and 2) were chromatographically identical to the two major spots induced by Ni(II) in vitro. The intensities of spots 1 and 2 in kidney and of some other spots in liver and lung were increased 1 and 2 h after i.p. injection with a single dose of 170 μmoles/kg NiAc 2. The effects of NiAc 2 were reduced or undetectable in the three tissues 24 h after treatment. These observations indicate the capacity of Ni(II) to induce and modulate bulky DNA modifications both in vitro and in vivo.