P-450 Mediated Dehydrocrotonin Toxicity On Rat Hepatocyte Cultures

Dehydrocrotonin (DHC) is a diterpene with antiulcerogenic properties isolated from Croton cajucara Benth barks. Since subchronic (35 days) DHC treatment induced hepatotc0dcity in rats, we studied the DHC cytotoxicity on r&t hepatocytes. Hepatocytes were isolated from adult male Wistar rats by the two step collagenase liver perfusion. Cells were seeded at 3 × 10 5. cells/well in 24 well tissue culture plates in Ham F-12 supplemented medium, incubated at 37° C in a humidified atmosphere of 5% CO2 in air. Four hours after seeding, the unattached cells Were discarded and 1 ml/well of fresh medium (without serum) containing various dilutions of DHC was added and incubated for 20 h. Three independent endpoints, MTT (tetrazolium salt) reduction, neutral red uptake (NRU) and nucleic acid content (NAC) were measured for relative toxicity assessment. We determine the metabolic capacity of primary cultured r&t hepatocytes with 4, 24 and 48 h old. A very high toxicity (IC50 6 uM) was a/forded by MTT test, nevertheless for NAC and NRU was only slight (IC50 290 - 390 uM). A marked age culture dependent cytotoxicity reduction of DHC was observed suggesting a P-450 mediated DHC biotransformation leading to more toxic metabolites. Supported by CAPES, CNPq, FAPESP and FINEP.11