Bulky DNA-adduct formation induced by Ni(II) in vitro and in vivo as assayed by 32P-postlabeling.
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 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 microM). 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 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 mumols/kg NiAc2. The effects of NiAc2 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.
Chang
J
J
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Watson
W P
WP
Randerath
E
E
Randerath
K
K
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D015197:Carcinogenicity Tests / Q000379:methods; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry / Q000187:drug effects*; D003903:Deuterium / Q000378:metabolism; D017666:Deuterium Oxide; D005290:Ferric Compounds / Q000633:toxicity; D016166:Free Radical Scavengers; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000166:cytology; D008168:Lung / Q000166:cytology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009153:Mutagens / Q000633:toxicity*; D009532:Nickel / Q000633:toxicity*; D010759:Phosphorus Isotopes; D017382:Reactive Oxygen Species / Q000378:metabolism; D014867:Water / Q000378:metabolism
D005290:Ferric Compounds; D016166:Free Radical Scavengers; D009153:Mutagens; D010759:Phosphorus Isotopes; D017382:Reactive Oxygen Species; D014867:Water; C024823:ferric sulfate; C022838:nickel chloride; D009532:Nickel; D004247:DNA; D003903:Deuterium; D017666:Deuterium Oxide; D001205:Ascorbic Acid