Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes

Cristina Trambitas, Alison Holloway, Denis Crankshaw
McMaster University, Hamilton, Ontario, Canada

Cytochromes P450 (CYP) are key enzymes involved in the oxidative biotransformation of xenobiotics and endogenous substances. Several methods are available to assess activity of these enzymes, but among them, the use of fluorogenic substrates has gained popularity because such assays are homogeneous, sensitive, easy to perform, and have a relatively high throughput. These properties are well-suited to our aim of studying developmental changes in the activity of CYPs in rats. Unfortunately, the selectivity of most fluorogenic substrates for rat CYPs is not known. We therefore set out to characterize the biotransformation of 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC), 7-benzyloxyquinoline (BQ), 7-methoxy-4-(trifluoromethyl)-coumarin (MFC), 7-ethoxyresorufin (ERES), and 7-methoxyresorufin (MRES) by liver microsomes from adult male Wistar rats (400 – 550 g) and to determine their sensitivities to a panel of putative inhibitors of rat or human CYP isoforms. Compounds with purported selective activity against rat CYPs were: 3-methylcholanthrene (CYP1A1), chlorzoxazone (CYP2E1), clotrimazole (CYP3A), diethyldithiocarbamate (CYP2E1 & 3A) fluoxetine (CYP2E11), furafylline (CYP1A2), isoniazid (CYP2E1), ketoconazole (non-selective), quinidine (CYP2C6), quinine (CYP2D1), and sulfaphenazole (CYP2C11 & 2C6). Compounds with purported selectivity against human CYPs, but unknown inhibitory activity in rats were: nicotine (CYP2A6 & 2A13), nifedipine (CYP3A4), propranolol (CYP2D6), ticlopidine (CYP2B6) and tranylcypromine (CYP2A6 & CYP2C19).

Incubations of substrates with rat liver microsomes were performed in black 96-well plates under linear conditions at 37°C in the presence of an NADPH regenerating system. The formation of product was monitored kinetically by measurement of fluorescence intensity at the appropriate wavelengths.

Biotransformation of ERES fit a two enzyme model with Km values of 0.05 and 3.5 μM respectively, but activity was only detectable in 2 of 6 animals. MRES biotransformation showed substrate inhibition, with a Km of 1.3 μM but was also not consistently expressed. Biotransformation of BFC was best fit by a cooperative model with a Km of 25 μM, Vmax of 57 pmol/min/mg protein and a Hill coefficient of 1.3. BFC hydrolase activity was partially inhibited (26 – 60% activity remaining) by clotrimazole, fluoxetine, isoniazid, ketoconazole, propranolol and quinine with pKi values greater than 6.5. Biotransformation of BQ followed Michaelis-Menten kinetics with a Km of 35 μM and a Vmax of 272 pmol/min/mg. BQase was inhibited to 40% of its control value by clotrimazole and ketoconazole with pKis of 8.9 and 7.9, respectively. MFC was biotransformed according to Michaelis-Menten kinetics with a Km of 69 μM and a Vmax of 190 pmol/min/mg. MFCase was inhibited to 20-30% of control by clotrimazole and ketoconazole with pKis of 6.9 and 6.4, respectively and to 70-80% of control by fluoxetine, tranylcypromine and quinine with pKis of 7.9, 6.9 and 7.3, respectively. Enzyme kinetics are means of triplicates in a single experiment. Competition data are means from 3 – 4 experiments in duplicate.

We conclude that ERES and MRES are unreliable fluorogenic substrates in adult male rats that have not been exposed to enzyme inducers. BFC appears to be biotransformed simultaneously by several CYP isoforms whereas BQ is a selective probe for CYP3A and another CYP that is not sensitive to any of the inhibitors we tested. MFC also appears to be biotransformed by CYP3A in addition to at least one other isoform that is sensitive to fluoxetine, tranylcypromine and quinine. Supported by CIHR.