Characterization of biotransformation enzyme activities in primary rat proximal tubular cells.
The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed 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 toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.
Schaaf
G J
GJ
Department of Veterinary Pharmacology, Pharmacy and Toxicology (VFFT), Faculty of Veterinary Medicine, Utrecht University, PO Box 80152 NL 3508 TD, Utrecht, The Netherlands. g.schaaf@vfft.vet.uu.nl
de Groene
E M
EM
Maas
R F
RF
Commandeur
J N
JN
Fink-Gremmels
J
J
D000818:Animals; D001711:Biotransformation; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity; D015262:Xenobiotics / Q000493:pharmacokinetics / Q000633:toxicity; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; D005723:gamma-Glutamyltransferase; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase