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ToxSci Advance Access published online on October 25, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp255
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© The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes and cDNA-expressed rat cytochrome P450 isoforms

Emilija Makaji*, Cristina S. Trambitas{dagger}, Pamela Shen{dagger}, Alison C. Holloway* and Denis J. Crankshaw*,{dagger},{ddagger}

* Department of Obstetrics & Gynecology {dagger} Honours Biology & Pharmacology Program McMaster University, Hamilton, Ontario, Canada

{ddagger} Corresponding author: E-mail address: cranksha{at}mcmaster.ca, Telephone: +1 905 525 9140 ext 22759, Fax: +1 905 524 2911, Mailing address: Department of Obstetrics & Gynecology, McMaster University, HSC 3N52F, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5

Cristina S. Trambitas E-mail address: trambics{at}mcmaster.ca Emilija Makaji E-mail address: makajie{at}mcmaster.ca, Pamela Shen E-mail address: shenp{at}mcmaster.ca, Alison C. Holloway E-mail address: hollow{at}mcmaster.ca

Received September 2, 2009; revision received October 13, 2009; accepted October 13, 2009


  Abstract

We have evaluated the use of a panel of six fluorogenic cytochrome P450 substrates as a potential tool for rapid screening for global changes in CYP activity in rats under different physiological conditions. The biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin, 7-benzyloxy-4-(trifluoromethyl)-coumarin ,7-benzyloxyquinoline, 3-cyano-7-ethoxycoumarin, 7-methoxy-4-(trifuoromethyl)-coumarin and 7-ethoxy-4-trifluoromethyl-coumarin by microsomes from adult male rat liver were characterized, their sensitivities to fifteen putative inhibitors were determined and compared to similar experiments using nine different cDNA-expressed rat CYPs. Inhibitory profiles of the substrates in microsomes were different from each other, with some overlap, suggesting that each substrate is to some extent biotransformed by a different CYP isoform. Ketoconazole and clotrimazole were non-selective inhibitors while ticlopidine selectively inhibited biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin. CYP 2A1 did not biotransform any of the substrates and CYP 2E1 was insensitive to all the inhibitors tested. Some inhibitors did not affect the biotransformation of the fluorogenic substrates by cDNA-expressed isoforms as predicted by their effects on conventional substrates, for example chlorzoxazone and diethyldithiocarbamate were inactive against CYP2E1, and CYP2C6 was not inhibited by sulfaphenazole. When results in microsomes and cDNA-expressed CYPs were compared, only the majority of the biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin by microsomes could be assigned with full confidence to a specific CYP isoform, namely CYP2D2. Nevertheless different inhibitory profiles of the substrates indicate that the panel will be useful for rapid functional quantification of global CYP activity in rats under different experimental conditions. Our results also demonstrate the inappropriateness of extrapolating inhibitory data between conventional and fluorogenic CYP substrates.

Key Words: cytochrome P450; rat; liver; cDNA-expressed, fluorogenic substrate; inhibitor.


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