In addition the kidney is

In addition, the kidney is also capable of actively metabolizing many drugs, hormones and xenobiotics [4], [5], [6], [7]. Several compounds are known to be activated locally by renal biotransformation processes. For example, in male mice, chloroform, acetaminophen and 1,1-dichloroethene are bioactivated locally by renal cytochrome P450s (CYP450s) [3]. In addition, the kidney is susceptible to bioactivation by phase II enzymes, in particular to the toxicity caused by glutathione (GSH) derived S-conjugates. GSH-conjugation has been considered to be one of the most important detoxicification pathways for electrophilic and lipophilic compounds. The GSH-conjugates or their successive products (cysteine-conjugates, mercapturates) are actively transported into the kidney and further metabolized by several enzymes including N-acetyltransferase (NAT) and beta-lyase to mercapturic Iberiotoxin and other thiol compounds, respectively [8]. The mercapturates can be readily excreted, and the route catalysed by NAT is thus considered to be a detoxifying pathway. However, N-deacetylases, particularly in the kidney, may antagonize the action of N-acetyltransferases by catalyzing the deacetylation of mercapturic acids. The resulting cysteine-S-conjugates may subsequently be activated by cysteine-S-conjugate β-lyase into reactive thiols. Thus, the balance between detoxification and activation defines predominantly the site-specific nephrotoxicity of a particular compound. The PT expresses the complete set of these enzymes important in the biotransformation pathways of GSH-conjugates at relatively high activities, as compared to other tissue sites [8]. The aim of this study was to characterize the expression and activity of biotransformation enzymes in primary cultures of rat PT cells, in order to assess their applicability in nephrotoxicity studies. PT cells in primary culture have been used in many studies, but most of them have focused on transport-related mechanisms [9]. The rat was chosen as an experimental model, as the large database available on biotransformation enzymes, together with the knowledge of rat renal biotransformation processes, is advantageous [10], [11]. The selected enzymes represent typical phase I or phase II biotransformation enzymes. The CYP450 isoenzymes addressed here are often referred to as drug metabolizing enzymes (DMEs), whilst GGT, β-lyase and UGT play an important role in the PT specific toxicity of xenobiotics. The substrates that were used for assessment of enzyme activities include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CYP2B/C/3A), dextromethorphan (CYP2D1) and 1-chloro-2,4-dinitrobenzene (GST), l-glutamic acid gamma-(7-amido-4-methyl-coumarin) (GGT), S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (beta-lyase) and 1-naphthol (UGT). Western blot analysis and microsomal incubations were performed to support the CYP450 isoenzyme expression pattern in freshly isolated and primary cultured PT cells. Time dependency was assessed by determining the biotransformation activity directly after plating and at three following time points of culture, at day 1, day 4 and day 7 after isolation.