Detection of DNA-crosslinking agents with the alkaline comet assay.
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.
Pfuhler
S
S
Department of Pharmacology and Toxicology, University of Ulm, Germany.
Wolf
H U
HU
D000328:Adult; D002470:Cell Survival; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000032:analysis* / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000187:drug effects*; D004249:DNA Damage; D004357:Drug Synergism; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005069:Evaluation Studies as Topic; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000737:chemistry / Q000187:drug effects*; D008297:Male; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008782:Methylurea Compounds / Q000494:pharmacology; D016685:Mitomycin / Q000494:pharmacology; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology
D003432:Cross-Linking Reagents; D008782:Methylurea Compounds; D005557:Formaldehyde; D016685:Mitomycin; D014508:Urea; D004247:DNA; D008741:Methyl Methanesulfonate; C044982:diazolidinylurea; C006892:carbamol; D002945:Cisplatin