Derivatives of dehydrocrotonin (DHC; Compound 1) with different anti-ulcerogenic properties but less toxicity were produced by reducing the cyclohexenone moiety of DHC with NaBH,, (Compound 11), reducing the cyclohexenone and lactone moieties with LiAlH4 (Compound 111) and transforming the lactone moiety into an amide (Compound IV) using dimethylamine. Derivatives of DHC were assayed in cultured hepatocytes and V79 fibroblasts. Three independent endpoints assays for cytotoxicity were used, namely, the DNA content, tetrazolium reduction (MTT) and neutral red uptake (NRU). Compound III was less toxic than the other DHC derivatives in both cell cultures. IC50 values ranging from 250 to 600 MM were obtained for Compounds 11 and IV in the NRU and DNA content tests evaluated in 4-hour hepatocyte cultures. Although Compound 11 showed relatively low cytotoxicity in rat hepatocytes based on the NRU and DNA content assays, a very high toxicity (IC50=10 muM) was observed in the MTT test. Metabolites of Compound 11 in conditioned medium from 4-hour old hepatocyte cultures enhanced the MTT-reducing ability of V79 fibroblasts. The cytotoxicity of the derivatives was greater in recently isolated hepatocytes (only a 4-hour incubation for cell attachment prior to treating with the derivatives) than in hepatocytes previously cultured (24-hour incubation) before the treatment. Thus, aging reduced the cytotoxic effects of DHC derivatives in isolated hepatocytes, suggesting that P450-mediated biotransformation of such derivatives may lead to the formation of more toxic metabolites.215281288