Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to i t's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation e nzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proxima l tubular cells (PT cells). Specific marker substrates for determining cyto chrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxy resorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbuta mide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenz ymes decreased considerably during culture with the greatest loss in activi ty within 24 h of culture. In addition, expression of CYP450 apoprotein, in cluding CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes fr om freshly isolated PT cells by immunoblotting using specific antibodies. C YP2B and CYP3A apoprotein could not be detected. Activity of the phase II b iotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1 -chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumari n). S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and l-naphthol, respectively, a s marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still express ed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated to xicity, the strong time dependency of especially phase I and. to a lesser e xtent, phase II biotransformation activities confers limitations to their a pplication. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.