Studies of cytotoxicity of T-2 and drug metabolizing enzymes using primary cultures of adult rat hepatocytes

Abstract

Primary culture of adult rat hepatocytes has been considered as a ideal model for toxicological studies because cultures hepatocytes maintained many liver-specific functions. The most well characterized one is hormonal response. In this study this system was applied to toxicological study of T-2 toxin which is mycotoxin produced mainly by Fusaria. T-2 toxin caused severe inhibition effect on hormonal induction of $\alpha$-aminoisobutyric acid (AIB) transport and tyrosine aminotransferase (TAT) activity in culture hepatocytes. This effect was observed at low concentration of toxin (up to 100 ng/ml) and dose-response relationship was shown. Pretreatment also elicit the inhibition but the extent was decreased. Along with these inducible parameters, as noninducible parameter, ouabain uptake which is active membrane transport was also tested. But at 100ng/ml, this was not effected by T-2 toxin even if treatment duration was increased. To elucidate the mechanism by which T-2 toxin cause damage in inducible functions, cellular macromolecular synthesis was measured. Protein synthesis was decreased to about half at concentration of 25 ng/ml but RNA synthesis was not significantly inhibited. Because hormonal induction of AIB uptake and TAT activity require de novo synthesis of protein, protein synthesis inhibition was proposed as the mechanism of hepatotoxicity. When the treatment time was elongated, culture hepatocytes overcame protein synthesis inhibition and its rate was accelerated by removing toxin from culture medium. This phenomenon was reappeared by preincubation of T-2 toxin with S-9 fractions. In this repair process, carboxyesterase seemed to function mainly because incubation without NADPH exerted major portion of activity. But the inclusion of conjugation substrates, UDP-glucuronic acid and glutathione did not show any effect. In standpoint of repair, liver seems to be active because liver microsome has 10 times stronger carboxyesterase activity than that of ...