EFFECT OF PHENOBARBITAL ON 7-ETHOXYCOUMARIN-O-DEETHYLASE AND MICROSOMAL EPOXIDE HYDRASE ACTIVITIES IN COLLAGEN GEL CULTURES OF RAT HEPATOCYTES

Ethoxycoumarin-O-deethylase (ECOD) and microsomal epoxide hydrase (mEH ) activities have been studied in collagen gel cultures of rat hepatoc ytes under control conditions and after exposure to phenobarbital. The hepatocytes were either sandwiched between two layers of extracellula r matrix (type I collagen) or directly mixed with the same type of col lagen gel. In preliminary experiments it was first tested whether it w as possible to measure ECOD activity directly in intact hepatocytes th at were embedded in a collagen gel matrix. Probably by a delay of subs trate penetration through the collagen gel, the latter had to be remov ed by a collagenase digestion before ECOD activity measurements look p lace. When cultured in control medium, the activity of both phase I en zymes studied decreased as a function of culture time in sandwich as w ell as in immobilization cultures. After 4 days, however, a steady-sta te situation was reached and this level of enzymatic activity was obse rved for at least 2 wk. When the cells were exposed to 3.2 mM phenobar bital, an increased ECOD activity remained detectable for at least 14 days. In sandwich cultures a two-to threefold increase of ECOD activit y was observed, whereas in immobilization cultures, increases of only 30 to 46% of the values observed in the control medium were measured. After exposure of the collagen gel cultures to 3.2 mM phenobarbital, t he mEH activity was increased to the same extent (factor 2 to 2.5) in both culture models. (C) 1997 Published by Elsevier Science Ltd.