Measurement of DNA oxidation in human cells by chromatographic and enzymic methods

Andrew Collins, Catherine Gedik, Nicholas Vaughan, Sharon Wood, Ann White, Jacques Dubois, Jean François Rees, Steffen Loft, Peter Møller, Henrik Poulsen, Jean Cadet, Thierry Douki, Jean Luc Ravanat, Sylvie Sauvaigo, Henry Faure, Isabelle Morel, Bénédicte Morin, Bernd Epe, Nicole Phoa, Andrea HartwigTanja Schwerdtle, Piero Dolara, Lisa Giovannelli, Maura Lodovici, Ryszard Olinski, Karol Bialkowski, Marek Foksinski, Daniel Gackowski, Zdena Duračková, Lucia Hlinčiková, Peter Korytar, Monika Sivonová, Mária Dušinská, Csilla Mislanová, José Viña, Lennart Möller, Tim Hofer, Jonas Nygren, Eric Gremaud, Karl Herbert, Joseph Lunec, Chris Wild, Laura Hardie, Jo Olliver, Elizabeth Smith

Research output: Contribution to journalArticlepeer-review

258 Scopus citations

Abstract

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 106 guanines for chromatographic methods, and 0.53 per 106 guanines for techniques based on FPG.

Original languageEnglish
Pages (from-to)1089-1099
Number of pages11
JournalFree Radical Biology and Medicine
Volume34
Issue number8
DOIs
StatePublished - 15 Apr 2003
Externally publishedYes

Keywords

  • 8-Oxo-7,8-dihydroguanine
  • DNA oxidation
  • Free radicals
  • Method validation

Fingerprint

Dive into the research topics of 'Measurement of DNA oxidation in human cells by chromatographic and enzymic methods'. Together they form a unique fingerprint.

Cite this