Potential artifacts in the measurement of DNA deamination.
Attack on DNA by some reactive nitrogen species results in deamination of adenine and guanine, leading to the formation of hypoxanthine and xanthine, respectively. Published levels of these products in cellular DNA have varied widely. Although these two deamination products are often measured by GC-MS analysis, the procedure of acid hydrolysis to release DNA bases for derivatization poses a risk of artifactual deamination of the DNA. In this study, we demonstrated the artifactual formation of these two deamination products during acid hydrolysis and hence developed a method for detecting and measuring 2'-deoxyinosine, the nucleoside of hypoxanthine. Our assay for 2'-deoxyinosine employs nuclease P1 and alkaline phosphatase to achieve release of the nucleosides from DNA, followed by HPLC prepurification with subsequent GC-MS analysis of the nucleosides. This assay detected an increase in the levels of 2'-deoxyinosine in DNA when commercial salmon testis DNA was treated with nitrous acid. We also used it to measure levels in various rat tissues of both normal and endotoxin-treated rats, but could not find increased 2'-deoxyinosine formation in tissues even though *NO production was substantially increased.
Lim
Kok Seong
KS
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597.
Huang
Shan Hong
SH
Jenner
Andrew
A
Wang
Huansong
H
Tang
Soon Yew
SY
Halliwell
Barry
B
D000586:Amination; D000818:Animals; D016477:Artifacts; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D004195:Disease Models, Animal; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006868:Hydrolysis; D019271:Hypoxanthine / Q000378:metabolism; D019169:Jurkat Cells; D008070:Lipopolysaccharides / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D018384:Oxidative Stress; D051381:Rats; D018805:Sepsis / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism
D008070:Lipopolysaccharides; D019820:Xanthine; D019271:Hypoxanthine; D009569:Nitric Oxide; D004247:DNA