Detection of DNA-crosslinking agents with the alkaline comet assay

Environ Mol Mutagen. 1996;27(3):196-201. doi: 10.1002/(SICI)1098-2280(1996)27:3<196::AID-EM4>3.0.CO;2-D.

Abstract

The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37 degrees C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Cell Survival
  • Cisplatin / pharmacology
  • Cross-Linking Reagents / analysis*
  • Cross-Linking Reagents / pharmacology
  • DNA / analysis
  • DNA / drug effects*
  • DNA Damage*
  • Drug Synergism
  • Electrophoresis, Agar Gel / methods*
  • Evaluation Studies as Topic
  • Formaldehyde / pharmacology
  • Humans
  • Hydrogen-Ion Concentration
  • Lymphocytes / chemistry
  • Lymphocytes / drug effects*
  • Male
  • Methyl Methanesulfonate / pharmacology
  • Methylurea Compounds / pharmacology
  • Mitomycin / pharmacology
  • Urea / analogs & derivatives
  • Urea / pharmacology

Substances

  • Cross-Linking Reagents
  • Methylurea Compounds
  • Formaldehyde
  • Mitomycin
  • Urea
  • DNA
  • Methyl Methanesulfonate
  • diazolidinylurea
  • carbamol
  • Cisplatin