Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus.
The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10(6) guanines, with a median of 10.47 per 10(6) guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10(6) guanines with a median of 5.23 per 10(6) guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.988-oxoGua per 10(6) guanines, with a median of 0.79 per 10(6) guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.
D000818:Animals; D002624:Chemistry, Clinical; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D013058:Mass Spectrometry; D012016:Reference Values; D015203:Reproducibility of Results; D013552:Swine
C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA