Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein.
This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.
Potts
R J
RJ
Department of Biochemistry, C-440 Given Medical Building, University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.
Bespalov
I A
IA
Wallace
S S
SS
Melamede
R J
RJ
Hart
B A
BA
D000818:Animals; D002104:Cadmium / Q000633:toxicity*; D002478:Cells, Cultured; D020552:Comet Assay; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004347:Drug Interactions; D004720:Endonucleases / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D008668:Metallothionein / Q000302:isolation & purification / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051381:Rats
D002104:Cadmium; D008668:Metallothionein; D006861:Hydrogen Peroxide; D004720:Endonucleases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase