ATLA::Alternatives to Laboratory Animals

Volume 31, Number 3

Use of the PFGE. assay for studies of DNA breakage induced by toxic chemicals.

Eva Markova1, Cecilia Clededson2 and Ada Kolman3

1Department of Molecular Genetics, Cancer Research Institute, 833 91 Bratislava 37, Slovak Republic; 2Expertrådet AB, Högklintavägen 7, 172 64 Sundbyberg, Sweden; 3Department of Molecular Biology and Functional Genomics, Stockholm University, 106 91 Stockholm, Sweden

SUMMARY

The relevance of the pulsed field gel electrophoresis (PFGE) assay for the estimation of the DNA damaging effects of chemicals was studied. Four chemicals were randomly chosen from the list of 50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals with known human acute systemic toxicity: acetylsalicylic acid, paracetamol, ethylene glycol and sodium chloride. Human fibroblasts (VH-10) were used as a model system. For the estimation of cytotoxic effect, cell monolayers were treated with chemicals for 24 hours. Cloning efficiency (colony-forming ability) at different concentrations of the test chemicals was estimated, and the 50% inhibitory concentration (IC50) was determined. The IC50 values obtained demonstrated a correlation with human lethal blood concentrations. The induction of DNA double-strand breaks, measured by PFGE as the fraction of activity released, was detected after treatment with paracetamol. However, the other three chemicals tested mainly induced DNA degradation.

Keywords: cytotoxicity, DNA degradation, double-strand breaks, fraction of activity released, human diploid fibroblasts, MEIC chemicals, pulsed field gel electrophoresis