Abstract
Genotoxic agents can be detected by measuring DNA single-strand breaks which result in DNA migration from single cells in agarose using an electrophoretic field, further migration indicates an increase in DNA strand breaks. This alkaline microgel electrophoresis technique (comet assay) was applied to primary cultures of rat and human hepatocytes that had been exposed to direct-acting (ethylmethane sulfonate, EMS) and secondary (benzo[ a]pyrene, BP; cyclophospamide, CP) genotoxicants, as well as a liver tumor promoter (phenobarbital, PB). Cell viabilities in all studies were greater than 85% by a fluorochrome-mediated viability assay. BP and CP exhibited a moderate effect on DNA migration which did not vary greatly between rat and human hepatocytes, while treatment of hepatocytes with EMS resulted in the greatest degree of DNA damage and neither rat or human hepatocytes appeared more sensitive. PB did not increase DNA migration. The microgel electrophoresis assay demonstrated the ability to detect DNA damage which correlated with the induction of DNA repair in hepatocytes with these test compounds. This study indicates the potential utilitarian use of hepatocytes, which are a metabolically competent cell, for detection of DNA damage by both direct and secondary (those requiring bioactivation) genotoxicants.
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