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.

Hormonal and embryonic regulation of chemokines IL-8, MCP-1 and RANTES in the human endometrium during the window of implantation.

Chemokines are a family of small polypeptides which specialize in the attraction of leukocytes. The presence of specific leukocyte subsets at the implantation site is an important element of the complex, and not completely understood, process of embryonic implantation. This report includes the investigation of the in-vivo immunolocalization and hormonal regulation of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and RANTES (regulated upon activation normal T-cell expressed and secreted) in the human endometrium during hormone replacement therapy cycles for oocyte recipients in an IVF programme. In addition, we have analysed the embryonic regulation of these endometrial epithelial chemokines (IL-8 and MCP-1) using an in-vitro model for the apposition phase of human implantation by co-culturing single human embryos until the blastocyst stage with human endometrial epithelial cells (EEC). IL-8 and MCP-1 were immunolocalized in the human endometrium to the glandular and lumenal epithelium as well as to the endothelial cells. RANTES was mainly localized to the stromal compartment and endothelial cells. The immunoreactive levels of endometrial IL-8 and MCP-1 were up-regulated by the administration of progesterone during the receptive phase of the cycle. Furthermore, it was demonstrated that, in vitro, the human blastocyst does not produce measurable amounts of IL-8, MCP-1 or RANTES; however, it does up-regulate EEC IL-8 mRNA expression (P < 0.05) and protein production (P < 0.05), but not IL-8 secretion. The human embryo did not regulate EEC MCP-1 expression. These results provide evidence of hormonal and embryonic regulation of specific endometrial chemokines, suggesting two different but related mechanisms to induce the production of chemokines by the EEC, thus contributing to the attraction of specific leukocyte populations during the peri-implantation phase.