Detection of DNA effects in human cells with the comet assay and their relevance for mutagenesis.
The single cell gel test (SCG-test or comet assay) is a rapid and sensitive method for measuring DNA damage and repair in individual cells. A wide variety of mutagens have been shown to cause DNA alterations detectable with the comet assay, but it is not yet clear whether a relationship exists between the DNA effects and the induction of mutations. We are therefore investigating in a cell culture system with human cells (MRC5CV1) the induction of DNA damage by environmental mutagens and the formation of mutations at the HPRT gene. In the present study we investigated benzo[a]pyrene (BP), an environmental mutagenic and carcinogenic polycyclic aromatic hydrocarbon, and its reactive metabolite (+)-anti-benzo[a]pyrene-7,8-diol 9, 10-oxide ((+)-anti-BPDE). S9 mix activated BP and the direct acting mutagen (+)-anti-BPDE caused a concentration-related increase in DNA migration in the comet assay. Postincubation experiments indicated that induced DNA effects are eliminated by DNA repair within 24 h. BP-treatment caused a strong genotoxic effect in the comet assay but had only a marginal effect on the frequency of gene mutations. When cells were treated with BP in the presence of cadmium sulphate, a clear increase in genotoxicity was observed while the effect on mutations was unchanged. Our results indicate that DNA alterations detected with the comet assay do not necessarily relate to mutagenesis. The absence of a close relationship between DNA migration in the comet assay and mutagenesis may be explained by the fact that some effects seen in the comet assay occur as a consequence of an error free DNA repair process.
Speit
G
G
Universität Ulm, Abteilung Medizinische Genetik, Germany. p.borm/grat/rulimburg.nl
Hanelt
S
S
Helbig
R
R
Seidel
A
A
Hartmann
A
A
D001564:Benzo(a)pyrene / Q000378:metabolism / Q000633:toxicity; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D003584:Cytological Techniques; D004249:DNA Damage / Q000187:drug effects*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004785:Environmental Pollutants / Q000633:toxicity; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D016296:Mutagenesis / Q000187:drug effects*
D004785:Environmental Pollutants; D001564:Benzo(a)pyrene; D007041:Hypoxanthine Phosphoribosyltransferase