COMPARISON OF DNA-DAMAGE FROM GENOTOXICANTS USING THE MICROGEL ELECTROPHORESIS ASSAY WITH PRIMARY CULTURES OF RAT AND HUMAN HEPATOCYTES

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