Determination of the CYP2B6 Component of 7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation Activity in Human Liver Microsomes

Abstract

CYP2B6 protein has been isolated and purified from human liver (1, 2). In a panel of 60 individual human liver microsome samples, this P450 accounts for <l% of total P450 content in human liver (2). However, a large mtermdlvldual variability has been reported for the hepatic levels of CYP2B6 mRNA (3) and protein (1, 2, 4). Experiments with primary cultures of human hepatocytes have shown that CYP2B6 is inducible by treatment of the cells with phenobarbital (5), dexamethasone (5, 6), or rifampm (rifampicm) ($6). By contrast, the level of this P450 in hepatocyte cultures does not appear to be altered by other P450 mducers such as /3-naphthoflavone (5), 2,3,7,8-tetrachlorodibenzo-p-dloxm (5) or pregnenolone 16a-carbonitnle (6). Several substrates have been ldentlfied for cDNA-expressed CYP2B6, including I-l-ethoxycoumarin (3, 7, 8), benzo[a]pyrene (9), phenanthrene (10), and methoxychlor (11). However, little is known regardmg the role of this P450 in drug metabolism, although cDNA-expressed CYP2B6 is an active catalyst of lidocaine N-deethylatlon (12) and human hver microsomal CYP2B6 appears to be a high-Km catalyst of cyclophosphamlde 4-hydroxylation (8). Detailed mvestlgations of hepatic mlcrosomal CYP2B6 have been limited because: 1 Many of the heterologous anti-CYP2B antibody preparations are not useful owing to their slgmficant cross-reactivity with other human P450 enzymes; 2 A CYP2B6-specific chemical inhibitor has yet to be found; and 3 A dlagnostlc catalytic activity for human hepatlc mlcrosomal CYP2B6 has not been identified.

1 Introduction

CYP2B6 protein has been isolated and purified from human liver (1, 2). In a panel of 60 individual human liver microsome samples, this P450 accounts for <l% of total P450 content in human liver (2). However, a large interindividual variability has been reported for the hepatic levels of CYP2B6 mRNA (3) and protein (1, 2, 4). Experiments with primary cultures of human hepatocytes have shown that CYP2B6 is inducible by treatment of the cells with phenobarbital (5), dexamethasone (5, 6), or rifampm (rifampicin) (5, 6). By contrast, the level of this P450 in hepatocyte cultures does not appear to be altered by other P450 inducers such as β-naphthoflavone (5), 2,3,7,8-tetrachlorodibenzo-p-dioxin (5) or pregnenolone 16a-carbonitnle (6). Several substrates have been identified for cDNA-expressed CYP2B6, including 7-ethoxycoumarin (3, 7, 8), benzo[a]pyrene (9), phenanthrene (10), and methoxychlor (11). However, little is known regarding the role of this P450 in drug metabolism, although cDNA-expressed CYP2B6 is an active catalyst of lidocaine N-deethylation (12) and human liver microsomal CYP2B6 appears to be a high-Km catalyst of cyclophosphamide 4-hydroxylation (8). Detailed investigations of hepatic mlcrosomal CYP2B6 have been limited because:

• 1. Many of the heterologous anti-CYP2B antibody preparations are not useful owing to their significant cross-reactivity with other human P450 enzymes;

• 2. A CYP2B6-specific chemical inhibitor has yet to be found; and

• 3. A diagnostic catalytic activity for human hepatic microsomal CYP2B6 has not been identified.

Recent studies have indicated the involvement of CYPlA, CYP2B6, CYP2C, and CYP2El in 7-ethoxy-4-trifluoromethylcoumarin O-deethylation in human liver microsomes (4, 13). However, selectivity of this reaction for CYP2B6 can be achieved by use of a low substrate concentration (5μ mM) and by preincubation of human liver microsomes with a mixture of inhibitory antibodies to block the activities of the CYPlA, CYP2C, and CYP2El enzymes (4). This chapter describes a spectrofluorometric method for the determination of the CYP2B6 component of human liver microsomal 7-ethoxy-4-trifluoro-methylcoumarin O-deethylation activity. Simple modifications of this assay method can be introduced to measure cDNA-expressed CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (4).

2 Materials

• 1. Assay buffer: 100 mM potassium phosphate, pH 7.4.

• 2. Substrate 7-ethoxy-4-trifluoromethylcoumarin (MW = 2582) (Enzyme Systems Products, Livermore, CA) (see Note 1 ) Prepare a 25 mM (6.4 mg/mL) stock solution dissolved in methanol Dilute to 50 μ mM with assay buffer (see Notes 2 and 3 ).

• 3. Metabohte standard 7-hydroxy-4-trifluoromethylcoumarin (MW = 2302) (Enzyme Systems Products) (see Note 1 ).

• 4. Cofactor generating system: 26 mM (20 mg/mL) NADP⁺ (see Note 4 ), 66 mM (20 mg/mL) D-glucose-6-phosphate, 66 mM(13.3 mg/mL) magnesium chloride (MgC1₂. 6H₂0). Glucose-6-phosphate dehydrogenase (40 U/mL) in 5 mM (1.5 mg/mL) sodium citrate (C₆H₅0₇Na_s. 2H₂O)

• 5. Enzymes: Dilute human liver microsomes in assay buffer to a working concentration of 1 mg microsomal protein/ml and keep on ice (see Note 5 ) cDNA-expressed BY2B6 (GENTEST, Woburn, MA).

• 6. Antibodies:

  • a. Anti-CYPl A2 (polyclonal antibody developed in rabbit, Danchi Pure Chemicals, Tokyo, Japan, and is available from GENTEST)

  • b Anti-CYP2C (polyclonal antibody developed in goat, Danchi Pure Chemicals, and is available from GENTEST)

  • c Anti-CYP2El (monoclonal antibody developed in mouse, GENTEST, Woburn, MA ) Premix the mhrbitory antibodies in the followmg proportions: 10 μL of monoclonal anti-CYP2E1, 20 μL of polyclonal anti-CYP1 A2 and 90 μL of polyclonal anti-CYP2C

• 7 Deproteinizing agent: 20% (w/v) trichloroacetic acid.

• 8 100 mM Tris-HCl, pH 9.0.

• 9 Equipment includes a spectrofluorometer

3 Methods

• 1. Mix 10 μL of the diluted human liver microsomes with 4 μL of the antibody mixture (see Subheading 2 , item 6). Incubate for 30 min on ice

• 2. Add the followmg to each incubation tube (total mcubation volume of 200 μL)

  • a. 154 μL of assay buffer

  • b 20 μL of 50 μ mM 7-ethoxy-4-trifluoromethylcoumarin (5 μ mM final concentration, see Note 6 )

  • c. 10 μL of a solution contaming 26 mM NADP⁺, 66 mM D-glucosed-phosphate, and 66 mM magnesium chloride

  • d 2 μL of glucose-6-phosphate dehydrogenase (40 U/mL) in 5 mM sodium citrate

• 3. Prewarm incubation tubes to 37°C and add 14 μL of the mixture containing the human liver microsomes and the inhibitory antibodies to initiate the enzymatic reaction (stagger each incubation with 15-s delay intervals) (see Note 7 )

• 4. Incubate samples at 37°C for 20–30 min in a water bath (see Note 8 ).

• 5. Add 40 μL of 20% (w/v) titchloroacetic acid to stop enzymatic reaction and place the incubation tube on ice (see Note 9 ).

• 6. Centrifuge reaction mixture at 10,000g for 3 min.

• 7. Transfer 100 μL of the supernatant to a clean test tube containing 1.9 mL of 100 mMTris-HCI, pH 9.0, and vortex.

• 8. Measure the fluorescence at an excitation wavelength of 410 nm and an emission wavelength of 510 nm (see Note 10 )

• 9. Prepare blank incubation tubes by adding the complete incubation mixture but with heat-mactivated microsomes Process the blank incubation tubes as per steps 4–8

• 10. Prepare standards by adding a known amount (e.g., 0.01,0.02,0.05,0.1, 0.2,0.5, and 1 nmol) of authentic 7-ethoxy-4-trifluoromethylcoumarin metabolite to tubes containing the complete incubation mixture but with heat-mactivated microsomes. Process the incubation tubes containing the standards as per steps 4–8

• 11. Calculate net fluorescence of each unknown sample and standard by subtracting the fluorescence reading of the blank from that of the unknown or standard.

• 12. Plot a standard curve of net fluorescence against amount of authentic 7-hydroxy-4-trifluoromethylcoumarin and determine the amount of product formation in each unknown sample by linear regression analysis.

• 13. Calculate 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity and express it as nmol product formed/min/mg microsomal protein or as nmol product formed/min/nmol total P450 (see Note 11 )

4 Notes

• 1. 7-Ethoxy-4-trifluoromethylcoumarin and 7-hydroxy-4-trifluoromethylcoumarin are light sensitive

• 2. The limit of solubility of 7-ethoxy-4-trifluoromethylcoumarin in potassium phosphate buffer is ~50 μ mM, but this can be increased to ~100 μ mM by using dimethylsulfoxide (DMSO) (0.2%, v/v final concentration) as a vehicle. This concentration of DMSO results in an ~5% decrease in enzyme activity (14) Ethanol is highly inhibitory to CYP2B6.

• 3. Stock solutions of 7-ethoxyd-trifluoromethylcoumarin are stable for at least 1 yr when stored at−20°C (14).

• 4. 7-Ethoxy-4-trifluoromethylcoumarin 0-deethylase assays can also be carried out with nicotinamide adenine dinucleotide phosphate (NADPH) (e.g., 1 mM final concentration) instead of an NADPH-generating system (e.g., NADP⁺, D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase).

• 5. Dilute only sufficient amounts of microsomes for each experiment The remamder of the undiluted microsomes can be stored at −80°C for future use (15).

• 6. The apparent Km value for cDNA-expressed CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation is ~3 μ mM (4). The assay is conducted at 5 μ mM substrate concentration in order to faclhtate the efficacy of the antibodies against the higher Km forms such as CYPZC19 (apparent Km = 17 μ mM), CYP2C9 (apparent Km = 35 μ mM), CYP2El (apparent Km = 46 μ mM) and CYP2A6 (apparent Km >50 μ mM).

• 7. Conduct preliminary experiments to ensure that the assay is linear with respect to microsomal protein concentration.

• 8. Conduct preliminary experiments to ensure that the assay is linear with respect to incubation time.

• 9. The 7-ethoxy-4-trifluoromethylcoumarin O-deethylation assay can also be performed using a direct continuous spectrofluorometric method (13, 14)

• 10. Determine the optimal excitation wavelength and emission wavelength to be used with each particular spectrofluorometer by exammlng the excitation and emission spectra (14) of 7-ethoxy-4-trifluoromethylcoumarin and 7-hydroxy-4-trifluoromethylcoumarin generated by that instrument.

• 11. The CYP2B6 component of human liver microsomal 7-ethoxy-4-tnfluoro-methylcoumarin 0-deethylation activity at 5 μ mM substrate concentration in a panel of 17 individual human liver microsome samples ranged from 0.02–0.5 nmol/min/mg microsomal protein (4).