In vitro toxicology in hepatocyte bioreactors-extracellular acidification rate (EAR) in a target cell line indicates hepato-activated transformation of substrates

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)I 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 mi recirculating perfusion system including 30 mug/ml CYCL. Tn 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 mul/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 <mu>g/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. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.