α-Asarone toxicity in long-term cultures of adult rat hepatocytes

In this work we studied the toxic effects of α-asarone, a hypolipidemic active principle of Guatteria gaumeri Greenman, on long-term cultures of adult rat hepatocytes cultivated on a feeder layer of 3T3 cells. The exposure for one and two weeks to α-asarone (1-50 μg/ml) produced intracytoplasmic lipid droplets and at higher concentrations (25-50 μg/ml) retraction of the hepatocyte cords and cell detachment. Ultrastructurally, the treated cultures (10 μg/ml) showed enlargement and vacuolization of the mitochondria in addition to lipid droplets. The triacylglycerol content increased up to 2.3-fold in the cultures treated for one week with 50 μg/ml, whereas the protein content per culture, a rough estimate of cell number and viability, decreased by up to 53% in the cultures treated for two weeks with 50 μg/ml. The synthesis and secretion of proteins, measured by the incorporation of [³H]-leucine into cellular and secreted macromolecules, decreased also in the cultures exposed. After one and two week exposure to 50 μg/ml of α-asarone, the secretion of labeled proteins decreased by 53 and 67%, respectively, whereas the synthesis of cellular and total proteins decreased by 48-67%, respectively. The secretion of proteins was the most sensitive parameter of α-asarone toxicity. The mean inhibitory dose (ID₅₀), i.e., that producing 50% inhibition in the incorporation of the labeled precursor, was 22.12 and 5.04 μg/ml after one and two weeks exposure, respectively. Our results show that long-term exposure to micromolar concentrations of α-asarone produces morphologic and ultrastructural alterations, triacylglycerol accumulation (fatty liver), and inhibition of protein synthesis and secretion. Our results also suggest that this culture system could be a suitable in vitro model for studying long-term effects of hepatotoxic chemicals.