title: Quantitative classification of DNA damages induced by submicromolar cadmium using oligonucleotide chip coupled with lesion-specific endonuclease digestion.
abstract: Implementation of proper analytical tool for systematic investigation and quantitative determination of different classes of cadmium ion-induced DNA damages, especially at low metal ion concentrations, is still lacking. Using lesion-specific enzymes that cleave DNA at specific classes of damage and a fluorometric approach developed for quantifying fluorophore-labeled oligonucleotides bound to chip surfaces, we determined the frequencies of different lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites) induced by submicromolar Cd(2+). Cd(2+)-treated oligonucleotide chips were digested with various endonucleases (Fpg protein, endonuclease III, endonuclease IV), producing a de novo single strand break (SSB) at their substrate modifications. The frequency of SSB and double strand break (DSB) was computed from the difference of pre- and post-Cd(2+)-treatment oligonucleotide coverage on the chip. While the frequency of SSBs and oxidized bases were successfully quantified even at 0.5 μM of Cd(2+), DSB frequency could be easily quantitated at 8.7 μM [Cd(2+)]. The numbers of abasic sites were below the oligonucleotide detection limit (2.4 amole; equivalent to 0.24 fM for a reaction volume of 100 μL). SSBs were found to constitute about 85-90% of single strand damages, while oxidized bases comprise only 4-7% of the total at 0.9 to 8.7 μM [Cd(2+)].
authors.affiliation: Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea.
authors.forename: Sukdeb
authors.initials: S
authors.lastname: Pal
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Kim
authors.forename: Sun Hee
authors.initials: SH
authors.lastname: Park
authors.forename: Heung Bin
authors.initials: HB
authors.lastname: Lim
authors.forename: Kyeong-Hee
authors.initials: KH
authors.lastname: Lee
authors.forename: Joon Myong
authors.initials: JM
authors.lastname: Song
mesh_terms: D002104:Cadmium / Q000633:toxicity*; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects; D004249:DNA Damage; D004720:Endonucleases / Q000378:metabolism*; D004785:Environmental Pollutants / Q000633:toxicity*; D029968:Escherichia coli Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D004785:Environmental Pollutants; D029968:Escherichia coli Proteins; D002104:Cadmium; D013482:Superoxide Dismutase; D004720:Endonucleases