An In Vitro Cytotoxicity Assay Using Rat Hepatocytes and MTT and Coomassie Blue Dye as Indicators

Abstract

Isolated hepatocytes from male Sprague-Dawley rats suspended in culture medium supplemented with either 0.2 or 2% bovine serum albumin (BSA) were allowed to attach to collagen coated 96-well dishes. Ten test chemicals from the MEIC list and salicylic acid were added individually to the dishes, and at the end of 24 and 48 hours, cytotoxicity was determined by measuring MTT (tetrazolium salt) reduction (mitochondrial integrity) and total cellular protein using Coomassie blue dye (reflecting cell number). Total cellular lactate dehydrogenase activity was also determined in some experiments, as an indicator of plasma membrane integrity. The relative toxicities of the test chemicals were quantified by the estimation of EC10, EC20 and EC50 values for each parameter. Except for one chemical, digoxin, in the MTT assay, cytotoxic potency increased with incubation time. The hepatocytes tended to be more sensitive to the chemicals in medium containing 0.2% BSA than in medium containing 2% BSA. Simple linear regression analyses of the log transformed data from the MTT assay versus log oral LD50 in rats for the test chemicals gave the best results using EC10 at 24 hours (r2 = 0.86). With protein as the cytotoxic indicator, the best results were obtained with EC values in the medium containing 2% BSA, again at 24 hours (r2 = 0.83). These results suggest that the MTT and Coomassie blue dye assays could be useful indicators for testing the cytotoxic potential of chemicals in rat hepatocyte cultures.