Introduction

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Multiple forms of cytochrome P-450 (CYP)² exist in liver microsomes and these CYP forms play important roles in the oxidation of structurally diverse xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens as well as endobiotic chemicals, including steroids, fatty acids, fat-soluble vitamins, and prostaglandins (Gonzalez, 1990; Guengerich and Shimada, 1991). Major CYP enzymes in human livers identified to date include CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 (and 3A7 in fetal livers) (Shimada et al., 1994; Guengerich, 1995). CYP1A1 and 1B1 are the important enzymes that are expressed mainly in the extrahepatic tissues; these two forms have been shown to oxidize diverse procarcinogens and some of the endobiotic chemicals (Kawajiri and Fujii-Kuriyama, 1991; Hayes et al., 1996; Shimada et al., 1996a). Large interindividual variations exist in the levels of each of these CYP enzymes, and these variations are considered to be one of the major factors contributing to different susceptibilities of humans toward actions and toxicities of drugs, toxic chemicals, and carcinogens (Shimada et al., 1994; Guengerich, 1995).

Several studies have reported that two or more CYP enzymes are able to oxidize xenobiotic and endobiotic chemicals at the same position of the molecules with different affinities (i.e., V_max/K_m ratio) in human liver microsomes (Wrighton et al., 1995; Rendic and DiCarlo, 1997). For example, 5-hydroxylation of omeprazole has been shown to be catalyzed by CYP2C19 and 3A4 (Andersson et al., 1993; Chiba et al., 1993; Yamazaki et al., 1997a); metabolic activation of acetaminophen by CYP2E1 and 1A2 (Raucy et al., 1989; Patten et al., 1993; Snawger et al., 1994); O-deethylation of 7-ethoxycoumarin by CYP2E1, 1A1, and 1A2 (Yamazaki et al., 1996); 6-hydroxylation of chlorzoxazone by CYP2E1, 1A1, and 1A2 (Carriere et al., 1993; Ono et al., 1995); 5-hydroxylation of lansoprazole by CYP3A4 and 2C19 (Pichard et al., 1995; Pearce et al., 1996); oxidation of antipyrine by CYP1A2, 2C9, and 3A4 (Sharer and Wrighton, 1996; Engel et al., 1996); 1'-hydroxylation of bufuralol by CYP2D6, 1A1, and 1A2 (Yamazaki et al., 1994); 4-hydroxylation of tamoxifen by CYP2D6, 2C9, and 3A4 (Wiseman and Lewis, 1996; Crewe et al., 1997); and 3-hydroxylation and N-demethylation of diazepam by CYP2B6, 2C19, and 3A4 (Ono et al., 1996; Yang et al., 1998). The roles of these CYP enzymes in the xenobiotic oxidation reactions have been shown to be determined by the ratio of V_max to K_m of individual CYP forms and by the levels of expression of individual CYP forms in liver microsomes of human samples (Crespi and Penman, 1997; Iwatsubo et al., 1997a; Ito et al., 1998). We have recently shown that omeprazole 5-hydroxylation activities of different human samples could be predicted with kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and 3A4 enzymes (Yamazaki et al., 1997a).

In this study, we further examined the roles of individual forms of CYP enzymes in the O-deethylation of 7-ethoxycoumarin and 6-hydroxylation of chlorzoxazone, two model reactions suggested to be catalyzed by several CYP enzymes (Carriere et al., 1993; Ono et al., 1995; Yamazaki et al., 1996; Yang et al., 1999) in human liver microsomes. Kinetic parameters for these two reactions were determined in 14 forms of recombinant human CYP expressed in microsomes of Trichoplusia ni cells infected with a baculovirus containing human CYP and NADPH-CYP reductase cDNA inserts. The roles of several CYP enzymes in liver microsomes were predicted with these kinetic parameters of recombinant CYP enzymes and estimated contents of CYP proteins in liver microsomes of different human samples (Iwatsubo et al., 1997a,b; Yamazaki et al., 1997a).

	Materials and Methods

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Chemicals. 7-Ethoxycoumarin was obtained from Aldrich Chemical Co. (Milwaukee, WI) and 7-hydroxycoumarin from Katayama Chemical Co. (Osaka, Japan). Chlorzoxazone and its 6-hydroxylated metabolite were donated by Dr. F. P. Guengerich of Vanderbilt University. Other reagents and chemicals used in this study were obtained from sources as described previously or were of highest qualities commercially available (Shimada et al., 1994, 1998).

Enzyme Preparation. Human liver samples were obtained from organ donors or patients undergoing liver resection as described previously (Mimura et al., 1993; Shimada et al., 1994). Liver microsomes were prepared as described and suspended in 10 mM Tris-HCl buffer (pH 7.4) containing 1.0 mM EDTA and 20% glycerol (v/v) (Guengerich, 1994).

Enzyme Assays. 7-Ethoxycoumarin O-deethylation activities by CYP enzymes were determined by HPLC as described (Yamazaki et al., 1999). Briefly, incubation mixtures consisted of human liver microsomes (0.025 mg protein/ml) or recombinant CYP (5 pmol/ml) with several concentrations of 7-ethoxycoumarin in a final volume of 0.20 ml of 100 mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system (Shimada et al., 1996b). Incubations were carried out at 37°C for 10 min and terminated by adding 10 µl of 60% HClO₄ (w/v). Product formation was analyzed by HPLC with a C18 5-µm analytical column (Mightsil RP-18, 150 × 4.6 mm; Kanto Chemical Co., Tokyo, Japan) equipped with a C18 5-µm guard column (Mightsil RP-18, 5-4.6 mm; Kanto Chemical Co.). The eluent consisted of a mixture of 45% CH₃CN (v/v) containing 20 mM NaClO₄ (pH 2.5) and the fluorimetric detection was done at an excitation wavelength of 338 nm and an emission wavelength of 458 nm.

Statistical Analysis. Kinetic parameters for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation by human CYP enzymes were estimated with a computer program (KaleidaGraph; Synergy Software, Reading, PA) designed for nonlinear regression analysis.

Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes Based on Kinetic Parameters of Recombinant CYP Enzymes. To define the potential roles of individual forms of CYP enzymes in the oxidation of 7-ethoxycoumarin and chlorzoxazone by different human samples, we calculated the predicted activities of human liver microsomal 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation activities with kinetic parameters of recombinant human CYP enzymes and estimated contents of these CYP proteins by immunoblotting. The equation used for the prediction of expected activities was obtained from the previous method (Iwatsubo et al., 1997b):

(1)

where v = predicted activities of 7-ethoxycoumarin O-deethylation or chlorzoxazone 6-hydroxylation (nanomoles of product formed per minute per milligram protein); A, B, C, and D = contents of individual forms of CYP (nanomoles per milligram protein); S = substrate concentration; and K_{m1, 2, 3, and 4} and V_{max1, 2, 3, and 4} = kinetic parameters from individual forms (A, B, C, and D) of CYP, respectively.

	Results

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Kinetic Analysis of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by 14 Forms of Recombinant Human CYPs. Kinetic parameters for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation by 14 forms of recombinant human CYP enzymes (in microsomes of T. ni cells) were determined as described in Materials and Methods (Table 1).

Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes with Kinetic Parameters of Recombinant CYP Enzymes. We examined the 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation activities catalyzed by human liver microsomes with the kinetic parameters of recombinant human CYP enzymes described above and the estimated contents of immunochemically determined CYPs in human liver microsomes (Table 2). Three human samples, C-12, C-13, and C-16 were first selected for the analysis of prediction of these two activities, then correlations between them were measured and predicted rates of 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation in liver microsomes of 24 human samples were compared. Kinetic parameters of recombinant CYP1A2, 2A6, 2B6, and 2E1 were used for the prediction of 7-ethoxycoumarin O-deethylation activities and those of CYP1A2, 2D6, 2E1, and 3A4 were used for the prediction of chlorzoxazone 6-hydroxylation activities because these recombinant CYP enzymes had significant levels of these catalytic activities (Table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   A, prediction of 7-ethoxycoumarin O-deethylation by liver microsomes of human samples HL-12 (), HL-13 (), and HL-16 () with parameters shown in Table 1. Different (36 points of) concentrations of 7-ethoxycoumarin were used for determination of activities. Open symbols represent measured rates; lines with closed symbols represent predicated rates. B, relationship between predicted and measured rates of 7-ethoxycoumarin O-deethylation by liver microsomes of 24 human samples. The substrate concentration used was 100 µM.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   7-Ethoxycoumarin O-deethylation by liver microsomes of Hl-12 (A), Hl-13 (B), and HL-16 (C). Eadie-Hofstee plots are shown in (A) to (C). D, E, and F, predicted rates () of 7-ethoxycoumarin O-deethylation (with parameters shown in Table 1) in samples HL-12, HL-13, and HL-16, respectively. The effects of removal of components of CYP1A2 (), CYP2A6 (), CYP2B6 (), and CYP2E1 () from the equation also are shown in (D) to (F).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   A, prediction of chlorzoxazone 6-hydroxylation by liver microsomes of human samples HL-12 (), HL-13 (), and HL-16 () with parameters shown in Table 1. Different (10 points of) concentrations of chlorzoxazone were used for determination of activities. Open symbols represent measured rates; lines with closed symbols represent predicated rates. B, relationship between predicted and measured rates of chlorzoxazone 6-hydroxylation by liver microsomes of 24 human samples. The substrate concentration used was 500 µM.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of removal of components of CYP1A2 (), CYP2D6 (), CYP2E1 (), and CYP3A4 () from the equation of predicted rates () of chlorzoxazone 6-hydroxylation by liver microsomes in human samples HL-12 (A), HL-13 (B), and HL-16 (C). The parameters used for prediction are shown in Table 1.

	Discussion

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Present results showed that 7-ethoxycoumarin O-deethylation activities of human liver microsomes could be predicted with kinetic parameters of recombinant CYP1A2, 2E1, 2A6, and 2B6 and contents of these CYP forms in human liver microsomes. CYP1A2 and 2E1 were suggested to be the major enzymes involved in O-deethylation of 7-ethoxycoumarin in human livers. Contributions of these two CYP forms were, however, found to be dependent on the contents of CYP forms in the livers of human samples examined. When a human sample C-12 that contained very high levels of CYP2E1 in the liver was used, the activity of 7-ethoxycoumarin O-deethylation was largely dependent on CYP2E1 (Fig. 2D). The results are of interest because the ratio of V_max to K_m was ~2.5-fold higher in recombinant CYP1A2 than that of CYP2E1 (Table 1). It has widely been accepted that the intrinsic clearance may be determined by the ratio of V_max to K_m of CYP enzymes in xenobiotic oxidation reactions (Lin and Lu, 1997; Ito et al., 1998; Northrop, 1998). The present results support the view that the levels of expression of individual forms of CYP are also very important in understanding the basis of intrinsic clearance of xenobiotic oxidations by CYPs. However, when a human sample C-16 was used in which the levels of CYP1A2 and 2E1 expression were determined to be 16 and 5% of total CYP in liver microsomes, CYP1A2 contributed more evidently than CYP2E1 (Fig. 2C). In a preliminary account, we found that antihuman CYP1A2 IgG inhibited liver microsomal 7-ethoxycoumarin O-deethylation (at a substrate concentration of 100 µM) more strongly than anti-human CYP2E1 did in a human sample C-16, whereas the anti-CYP2E1 IgG inhibited the activities of a human sample C-12 by ~80%.

The minor roles of CYP2A6 and 2B6 in the 7-ethoxycoumarin O-deethylation were suggested in this study, probably due to the low-level expressions of these CYPs in human liver microsomes, except that CYP2B6 was suggested to be partly involved in the O-deethylation of 7-ethoxycoumarin in human sample C-13 in which the content of CYP2B6 was relatively high (~9% of total CYP) in liver microsomes (Table 1). Previously, we reported that the levels of CYP2B6 in human liver microsomes are <1% of total CYP examined in 30 Japanese and 30 Caucasian samples (Mimura et al., 1993; Shimada et al., 1994). In this study, we reexamined 16 Japanese and 8 Caucasian samples with anti-WB-2B6 and recombinant CYP2B6 (both from Gentest) for immunoblotting analysis and found that the average levels of CYP2B6 in liver microsomes were ~0.6% of total CYP for the Japanese and ~2% of total CYP for the Caucasians. Several recent studies have supported the view that CYP2B6 is expressed at significant levels in liver microsomes of humans (Code et al., 1997; Ekins et al., 1998; Stresser and Kupfer, 1999).

Chlorzoxazone was suggested to be oxidized mainly by CYP1A2, 2E1, and 3A4 in human liver microsomes. The V_max value of recombinant CYP2E1 for chlorzoxazone 6-hydroxylation was the highest among 14 forms of CYP; however, the K_m value of CYP2E1 was 38- and 7-fold higher that those of CYP1A2 and 3A4, respectively. As a result, the V_max/K_m ratio of CYP2E1 for the 6-hydroxylation of chlorzoxazone was ~3.6-fold lower than that of CYP1A2. However, the present results showed that CYP2E1 was the most active in catalyzing chlorzoxazone 6-hydroxylation activities in human sample C-12 in which the level of CYP2E1 expression was the highest in human samples examined (Fig. 4A). In samples C-13 and C-16, it was suggested that CYP1A2, 2E1, and 3A4 are all involved in the oxidation of chlorzoxazone, depending on the concentrations of these CYP levels in liver microsomes. It is also interesting to note that CYP1A2 was more active in catalyzing chlorzoxazone than CYP2E1 at low substrate concentrations in a sample C-16, probably because this enzyme has a low K_m component in human liver microsomes, as has been reported previously (Yamazaki et al., 1996, 1999).

It has previously been shown that CYP2E1 is a major enzyme in catalyzing chlorzoxazone 6-hydroxylation in human liver microsomes in vitro (Peter et al., 1990; Yamazaki et al., 1995; Kim et al., 1996), and in vivo when the drug is dosed to humans (O'Shea et al., 1994; Williams et al., 1994; Vesell et al., 1995; Chen and Yang, 1996; Kim et al., 1996). Roles of other CYP enzymes such as CYP1A2 (Berthou et al., 1995; Ono et al., 1995) and CYP3A4 (Gorski et al., 1997) in the reaction have recently been reported. Our present results supported the view that CYP1A2 and 3A4 as well as CYP2E1 actually involved in the oxidation of chlorzoxazone, largely depending on the contents of these CYP enzymes in liver microsomes of human samples.

CYP1A1 was found to be very active in catalyzing O-deethylation of 7-ethoxycoumarin and 6-hydroxylation of chlorzoxazone in recombinant (insect) enzymes. CYP1B1 also was shown to catalyze 7-ethoxycoumarin O-deethylation although was not as active in catalyzing chlorzoxazone. These two forms of CYP have been shown to be expressed in extrahepatic tissues in humans (Kawajiri and Fujii-Kuriyama, 1991; Shimada et al., 1992, 1996b), and thus may be important when in vivo clearance of several xenobiotic chemicals is considered.

CYP3A4 has been shown to be most abundantly present in human liver microsomes; ~30% of total CYP were suggested to be CYP3A4-related enzymes in liver microsomes of human adults (Shimada et al., 1994). Studies also have suggested that >50% of clinically used drugs are oxidized by CYP3A4 in humans (Guengerich, 1997; Rendic and DiCarlo, 1997). The importance of CYP3A4 in the chlorzoxazone 6-hydroxylation also is suggested in this study when human samples with high contents of CYP3A4 in the liver were used.

Several approaches have been applied to predict the human liver microsomal xenobiotic-oxidizing activities with kinetic parameters of recombinant CYP enzymes in vitro (Crespi, 1995; Crespi and Penman, 1997; Iwatsubo et al., 1997a; Rodrigues, 1999). Crespi and his associates have used the so-called relative activity factors (RAFs) for the prediction of human liver activities; the RAFs can be calculated from the ratio of activities between human liver microsomes and recombinant CYP enzymes toward specific (known) substrates for individual CYP enzymes (Crespi, 1995; Crespi and Penman, 1997). They showed that the relative contributions of CYP enzymes in the xenobiotic-oxidizing activities by human liver microsomes may be calculated by using these RAF values from individual forms of recombinant human CYP (Crespi, 1995; Crespi and Penman, 1997). Our present approach that was originally proposed by Iwatsubo et al. (1997a,b) is based on the prediction with kinetic parameters of recombinant CYP enzymes and contents of individual CYP forms in human liver microsomes. In both approaches it is very important to know whether recombinant CYP enzymes in several cDNA-based systems have similar catalytic activities and substrate specificities to the native CYP enzymes present in human liver microsomes. It also should be mentioned that the levels of expression of NADPH-CYP reductase in the recombinant CYP systems expressed in T. ni cells are always higher than those present in human liver microsomes; the molar ratio of the reductase to CYP1A2 and 2E1 was 1.2 and 0.6 (from the data sheet provided by the manufacturer), respectively, whereas in human liver microsomes the ratio of the reductase to total CYP was ~0.05 (Kim et al., 1996).

In conclusion, the present results support the view that the intrinsic clearance (V_max/K_m ratio) of individual CYP forms in the oxidation of xenobiotic chemicals is one of the important factors to predict the roles of individual forms of CYP in human liver microsomes. However, the levels of expression of CYP enzymes in liver microsomes of different human samples are also very important in understanding the basis for roles of individual forms of CYP in xenobiotic oxidation reactions.