title: In vitro toxicology in hepatocyte bioreactors-extracellular acidification rate (EAR) in a target cell line indicates hepato-activated transformation of substrates.
abstract: In this article we introduce an in vitro model for hepato-mediated toxicity testing consisting of a Hepatocyte-Bioreactor connected to a microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells. The EAR in this system represented the metabolic activity of a tested cell line under the influence of bioreactor supernatant. Cyclophosphamide (CYCL), a well-known hepato-activated cytostatic drug was used as a model substrate because of its widespread clinical use. The predrug CYCL needed CYP 450 dependent activation to its active cytotoxic metabolite 4-OH cyclophosphamide. Primary pig hepatocytes from slaughterhouse organs were cultured in a collagen sandwich configuration in specially designed flasks and after 3 days introduced into a 50 ml recirculating perfusion system including 30 microg/ml CYCL. In a parallel open circuit, this bioreactor was connected to three perfusion chambers of a microphysiometer system housing 1.5 x 10(5) ZR 751 cells (breast tumor cell line). Bioreactor supernatant including CYCL was pumped at 150 microl/min into the microphysiometer. The recorded EARs under CYCL influence were correlated to controls, which were set to be 100%. After 1 and 7 h of bioreactor supernatant perfusion, including activated CYCL, the ZR 751 cell line showed an EAR of 98.99%+/-3.15 (mean+/-SD) and 81. 32%+/-10.18 (P<0.05), respectively, as compared to controls (bioreactor supernatant from the identical set-up without CYCL). The inactivated predrug CYCL showed no effect on the EAR: Perfusion of medium with 30 microg/ml CYCL alone, excluding the bioreactor activation, resulted in an EAR of 100. 11%+/-4.74 (mean+/-SD) after 7 h. Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of the predrug by the bioreactor.
authors: Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University, D-81366 Munich, Germany. koebe@gch.med.uni-muenchen.de
authors: H G
authors: HG
authors: Koebe
authors: C J
authors: CJ
authors: Deglmann
authors: R
authors: R
authors: Metzger
authors: S
authors: S
authors: Hoerrlein
authors: F W
authors: FW
authors: Schildberg
mesh_terms: D000418:Albumins / Q000032:analysis / Q000378:metabolism; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D019149:Bioreactors; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000633:toxicity*; D004247:DNA / Q000032:analysis; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D005470:Fluorometry; D022781:Hepatocytes / Q000737:chemistry* / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D008297:Male; D009153:Mutagens / Q000633:toxicity; D009994:Osmolar Concentration; D010477:Perfusion / Q000379:methods; D013552:Swine; D018675:Toxicity Tests / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014468:Umbelliferones / Q000032:analysis
chemical_list: D000418:Albumins; D018906:Antineoplastic Agents, Alkylating; D009153:Mutagens; D014468:Umbelliferones; C087936:maphosphamide; C031477:7-hydroxycoumarin; D003520:Cyclophosphamide; D004247:DNA; D007770:L-Lactate Dehydrogenase