Kinetics of tienilic acid bioactivation and functional generation of drug-protein adducts in intact rat hepatocytes.

Drug-induced autoimmune hepatitis is among the most severe hepatic idiosyncratic adverse drug reactions. Considered multifactorial, the disease combines immunological and metabolic aspects, the latter being to date much better known. As for many other model drugs, studies on tienilic acid (TA)-induced hepatitis have evidenced the existence of bioactivation during the hepatic oxidation of the drug, allowing the identification of the neoantigen of anti-LKM2 autoantibodies and the pathway responsible for its formation. However, most of these results are based on the use of microsomal fractions whose relevance to the liver in vivo still needs to be established. In the more complex intact cell environment, several endogenous processes may play a significant role on triggering the reaction and should therefore be considered. In this work we have characterised the kinetics of TA biotransformation in metabolically competent hepatocytes, the influence of TA bioactivation on physiological GSH levels, and the qualitative and quantitative profile of drug-protein conjugates generated in situ, as a function of exposure time. Results confirm that intact hepatocytes reproduce in vitro the metabolic sequence that leads to the functional generation of drug-protein adducts, in conditions that simulate clinical human exposure to TA. Metabolically competent cultured hepatocytes appear as a very promising approach to investigate the early preimmunological events of drug-induced autoimmune hepatitis, adequate to identify the conditions that may modulate the formation and specificity of drug-protein adducts in vivo, to study the hepatic disposition of the TA-protein targets, and to define the specific role of the hepatocyte in the origin of this adverse reaction.