DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation.
Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells. By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10(6) bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10(6) bp in mitochondrial DNA from rat and porcine liver. Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitive modifications. Here, we summarize our indications that all Fpg-sensitive modifications are recognized under the assay conditions and that on the other hand there is no artifactual generation of oxidative damage during the analysis. In addition, we show that the steady-state levels of Fpg-sensitive modifications in human lymphocytes and in two mammalian cell lines were higher in proliferating than in resting (confluent) cells. Only some of the Fpg-sensitive base modifications induced by various oxidants are 8-oxoG residues, as demonstrated for the damage under cell-free conditions. The percentage was dependent on the species ultimately responsible for the DNA damage and was approx. 40% in the case of hydroxyl radicals and peroxynitrite, 75% for type II photosensitizers (reacting via singlet oxygen) and only 20-30% in the case of type I photosensitizers such as riboflavin and acridine orange, which are assumed to react directly with the DNA.
Pflaum
M
M
Institute of Pharmacy, University of Mainz, Germany.
Will
O
O
Mahler
H C
HC
Epe
B
B
D000818:Animals; D020029:Base Pairing / Q000187:drug effects; D001671:Biochemistry / Q000379:methods*; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism; D002455:Cell Division; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043382:DNA-Formamidopyrimidine Glycosylase; D003676:Deferoxamine / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D006151:Guanosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008322:Mammals; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D017319:Photosensitizing Agents / Q000494:pharmacology
D029968:Escherichia coli Proteins; D016877:Oxidants; D017319:Photosensitizing Agents; D006151:Guanosine; C046215:8-hydroxyguanosine; D004247:DNA; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003676:Deferoxamine; D004121:Dimethyl Sulfoxide