BULKY DNA-ADDUCT FORMATION INDUCED BY NI(II) INVITRO AND INVIVO AS ASSAYED BY P-32 POSTLABELING

Various small oxidation products (e.g. 8-hydroxydeoxyguanosine) can be induced in DNA by nickel compounds. In this study, the P-32-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 NiCl2 and H2O2 at 37-degrees-C for 1 h. Formation of the two major adducts increased with incubation time (0-24 h) and NiCl2 concentration (0-800 muM). 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 NiCl2 on DNA were significantly enhanced by (1) addition of 3 mM ascorbic acid, (2) replacement of H2O with D2O 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 1-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 mumoles/kg NiAc2. The effects of NiAc, 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.