A biohybrid system constituted of primary pig hepatocytes and microporous synthetic membranes was used to investigate the diclofenac biotransformation and its long-term effect on hepatocyte specific functions. The maintenance of drug biotransformation functions and the effect of diclofenac continuous administration on the viability and metabolic functions of hepatocytes have been explored. Pig hepatocytes were isolated from slaughterhouse organs and were cultured on polyethersulfone membranes and on collagen. The ability of cells to eliminate diclofenac and to synthesise metabolites was evaluated by an HPLC analysis. Urea and albumin productions were established as indicators of differentiated hepatocyte functions. Pig hepatocytes eliminated diclofenac and produced two main metabolites: 4'-hydroxydiclofenac (4'-OHdic) and N,5-dihydroxydiclofenac (N,5-(OH)(2)dic). Cells continuously exposed to subtoxic drug concentrations maintained their biotransformation functions until 10 days of culture. The rate of diclofenac elimination increased according to a saturation kinetics. Diclofenac showed a cytotoxic effect at a concentration higher than 100 mu M as confirmed by analysis of DNA fragmentation. The urea and albumin synthesis as well as the total protein production were also affected by diclofenac concentrations. An important concentration-dependent effect of diclofenac on protein synthesis was observed. The results give information concerning the effect of diclofenac on specific functions of pig hepatocytes continuously exposed to the drug and on the long-term ability of cells to metabolise diclofenac in a membrane biohybrid system. (c) 2005 Elsevier B.V. All rights reserved.