Longterm stability of phase I and phase II enzymes of porcine liver cells in flat membrane bioreactors.
Recently, researchers have focused on the use of bioartificial liver devices to support patients with fulminant hepatic failure. Our team developed a cell-based flat membrane bioreactor (FMB). In this, porcine liver cells were maintained in 3D-coculture between two gel layers in a sandwich configuration for 3 weeks to study the influence of this bioreactor technique on the preservation of basic, not induced activities of phase I and phase II enzymes. First, the time and substrate dependencies of the following enzymes were measured: ethoxyresorufin-O-deethylase (EROD, CYP 1A1/1A2) and ethoxycoumarin-O-deethylase (ECOD, CYP 2B6) as phase I enzymes, and glutathione-S-transferase (GST), UDP-glucuronosyltransferase (UGT) and sulfotransferase (ST) as phase II enzymes. To find optimal test conditions Michaelis-Menten kinetics were calculated. Next, different potential inducers were tested to find out the most effective compounds. Based on these results, the basic, not induced levels of the different enzymes were determined in the flat membrane bioreactor. Furthermore, the response of these enzyme activities to the chosen inducers was investigated to examine whether the cells keep their ability for drug-drug interactions. Basic, not induced activities of both phase I enzymes and the phase II enzymes GST and UGT were maintained at nearly the initial levels during the complete period of study. In addition, it was possible to induce these enzymes twice or three times in a weekly interval. In contrast, the basic, not induced activity of ST increased during the first 10 days of culture. It stabilized then and was maintained steady. As in short-term investigations, no reaction of the ST-activity towards any inducer could be obtained. These results prove that porcine liver cells preserve their phase I and phase II activities and respond to inducing drugs over 3 weeks in culture. Therefore, the flat membrane bioreactor is not only suitable for investigating drug metabolism, drug-drug interactions, and enzyme induction but also for supporting liver functions.
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Leibniz Research Laboratories for Biotechnology and Artificial Organs, Medical School, Forschungszentrum der MHH, Podbielskistrasse 380, D-30659 Hannover, Germany.
Bader
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D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism*; D000818:Animals; D019149:Bioreactors; D001711:Biotransformation / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D004795:Enzyme Stability; D005260:Female; D014453:Glucuronosyltransferase / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D015262:Xenobiotics / Q000494:pharmacology
D015262:Xenobiotics; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase; D015238:Sulfotransferases