MECHANISMS OF DRUG-INDUCED TOXICITIES

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This year we have continued our studies on the mechanism of inhalation-induced hepatitis. Our goal has been to determine the molecular basis of the hepatotoxicity produced by halothane in the guinea pig model and to define the route by which the immune system comes into contact with the trifluoroacetylated (TFA)-antigens associated with this toxicity. Male outbred Hartley guinea pigs were administered halothane, and livers and blood were collected after 8 and 48 hours. Based on serum ALT levels and livers histology, treated animals could be designated as being either susceptible or nonsusceptible to halothane-induced liver injury. Immunoblot and immunohistochemistry studies indicated that the metabolism of halothane in livers of susceptible guinea pigs resulted in the formation of much higher levels of TFA-protein adducts than those of resistant animals. The level of TFA adducts in rat liver was approximately an order of magnitude lower than those of the susceptible guinea pigs which may explain why rats are resistant to the hepatotoxic effects of halothane. When TFA adducts were immunopurified from the sera, it was found that a susceptible guinea pig had much higher serum levels of TFA-protein adducts than those of a resistant animal. These adducts were likely derived from damaged hepatocytes because a correlation existed between ALT and serum TFA-adducts levels. Surprisingly, P4502A levels, but not those of other P450s, correlated with the high amounts of TFA-protein adducts detected in the livers of two guinea pigs. Moreover, when P4502A6, the human orthologue of guinea pig P4502A, was over-expressed in HepG2 cells, it was able to metabolize halothane to form TFA-antigens as were cells containing over-expressed P4502E1. The results of these studies strongly indicate that the level of TFA-adducts is an important factor in determining whether halothane causes liver damage in guinea pigs and possibly in humans, and that both P4502E1 and P4502A6 likely have an important role in TFA-protein adduct formation in humans. The finding of relatively high levels of intact TFA-protein adducts in the serum of susceptible guinea pigs, indicates that these adducts have been released from damaged hepatocytes and suggests that they may be able to induce immune reactions in tissues far removed from the liver.