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

Free Radic Biol Med. 2003 Apr 15;34(8):1089-99. doi: 10.1016/s0891-5849(03)00041-8.

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 10(6) guanines for chromatographic methods, and 0.53 per 10(6) guanines for techniques based on FPG.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Chromatography / methods*
  • Chromatography, High Pressure Liquid / methods
  • DNA / drug effects
  • DNA / metabolism*
  • DNA Damage
  • DNA Repair
  • DNA-Formamidopyrimidine Glycosylase / pharmacology
  • Dose-Response Relationship, Drug
  • Free Radicals
  • Gas Chromatography-Mass Spectrometry / methods
  • Guanine / analogs & derivatives*
  • Guanine / pharmacology
  • HeLa Cells
  • Humans
  • Hydrolysis
  • Light
  • Oxygen / metabolism*
  • Photosensitizing Agents / pharmacology
  • Pyrrolidines / pharmacology
  • Quinolizines / pharmacology

Substances

  • Free Radicals
  • Photosensitizing Agents
  • Pyrrolidines
  • Quinolizines
  • Ro 19-8022
  • 8-hydroxyguanine
  • Guanine
  • DNA
  • DNA-Formamidopyrimidine Glycosylase
  • Oxygen