Evidence of oligonucleotides containing 8-hydroxy-2'-deoxyguanosine in human urine.
The product of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG), when detected in urine, is considered to be a global, noninvasive biomarker of in vivo oxidative DNA damage. In this paper we describe a novel approach to confirm the presence of oligonucleotides containing 8-OHdG in human urine. Fractions of urine were prepared by gel-filtration chromatography, and the presence of oligonucleotides was confirmed by ELISA using a monoclonal anti-(single-stranded DNA) antibody. Pools of urine fractions were subsequently prepared according to ELISA reactivity, each containing oligonucleotides with a known range of base numbers. The level of 8-OHdG in each pool was subsequently determined using a commercial ELISA kit. Results confirmed that oligonucleotides containing 8-OHdG are present in urine and, most significantly, oligomers of <30-55 bases were found to be associated with 8-OHdG. This finding strongly supports the involvement of nucleotide excision repair (NER) in the removal of 8-OHdG from the cell. The novel approach adopted in this study was validated using cell culture supernatant obtained from an in vitro model comprising CCRF cells exposed to vitamin C; this model has previously been shown to stimulate removal of 8-OHdG from the cell by an NER-dependent process.
Patel
Parul R
PR
Radiation and Oxidative Stress Group, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester Royal Infirmary, University Hospitals of Leicester NHS Trust, Leicester LE2 7LX, UK.
Bevan
Ruth J
RJ
Mistry
Nalini
N
Lunec
Joseph
J
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005612:Freeze Drying; D006801:Humans; D008297:Male; D009841:Oligonucleotides / Q000737:chemistry / Q000652:urine*
D009841:Oligonucleotides; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine