Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells.
7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [(14)C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 10(6) nucleotides, 5-6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.
Mundt
Janna M
JM
Chemistry, Materials, Earth and Life Sciences Directorate, Lawrence Livermore National Laboratory, 7000 East Avenue, L-452, Livermore, CA 94551, USA.
Hah
Sang Soo
SS
Sumbad
Rhoda A
RA
Schramm
Vern
V
Henderson
Paul T
PT
D000080242:8-Hydroxy-2'-Deoxyguanosine; D002250:Carbon Radioisotopes; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000378:metabolism
D002250:Carbon Radioisotopes; D012313:RNA; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D012264:Ribonucleotide Reductases; D011683:Purine-Nucleoside Phosphorylase; D003842:Deoxycytidine Kinase; D003849:Deoxyguanosine