6J mice

Life Sciences 67 (2000) 2189Ð2200

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice Yune-Fang Uenga,*, Chi-Chuo Shyub, Yun-Lian Lina, Sang Shin Parkc, Jyh-Fei Liaoc, Chieh-Fu Chena a

National Research Institute of Chinese Medicine, 155-1, Li-Nong Street, Sec. 2, Taipei 11221, Taiwan, R. O. C. b Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, R. O. C. c Ilchun Molecular Medicine Institute, Medical Research Center, Seoul National University, Seoul, Korea Received 30 November 1999; accepted 15 March 2000

Abstract Effects of baicalein and wogonin, the major ßavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresoruÞn O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresoruÞn O-deethylation (EROD), and MROD activities were increased by both ßavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonintreatments. However, cytosolic GST activity was not affected by ßavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue speciÞcity. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Baicalein; Wogonin; Cytochrome P450; UGT; GST

* Corresponding author. Tel.: 886-2-28201999, ext. 6351; fax: 886-2-2826-4266. E-mail address: [email protected] (Y.-F. Ueng) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 0 9 -2

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Introduction Phase I and phase II drug-metabolizing enzymes play pivotal roles in the determination of biological fates of xenobiotics [1]. Cytochrome P450 (CYP)-dependent monooxygenase is one of the major phase I enzymes catalyzing various oxidative and reductive metabolism. Microsomal monooxygenase system consists of a family of CYP enzymes, NADPH-CYP reductase (CYP reductase), and phospholipids. Due to the broad substrate speciÞcity of CYP enzymes, a series of evidence showed that alteration of the CYP pool could inßuence the biological effects of xenobiotics [2,3]. UDP-glucuronosyl transferase (UGT) and glutathione S-transferase (GST) are the major phase II enzymes involved in the conjugation metabolism of xenobiotics. UGT and GST catalyze the transfer of glucuronic acid and glutathione to a variety of endogenous and exogenous substrates, respectively. Glucuronides of drugs can accumulate during long term therapy and may cause toxicity [4]. CYP, UGT, and GST are responsive to the inductive and inhibitory effects of many endogenous and exogenous factors, such as hormone, growth factor, and nutrition [5]. Modulation of drug-metabolizing enzymes may change the pharmacological and toxicological effects of xenobiotics in humans and result in serious drug-drug interactions. Scutellariae radix has been commonly used in traditional Chinese medicine for inßammation, suppurative dermatitis, and allergic disease [6]. It is also a main component in Shosaiko-to, a Japanese Kampo medicine which showed protective effects against hepatocellular carcinoma and CCl4-, b-galactosamine-, and alcohol-induced liver damage [7,8]. Koizumi et al. [9] have reported that oral administration of hot water extract of Scutellariae radix resulted in changes of CYP reductase, aniline hydroxylase, and aminopyrine N-demethylase activities in rat. This change varied by varying dosages and time periods of treatment. Kang et al. [10] reported that microsomal CYP1A and benzo(a)pyrene hydroxylation (AHH) activity were slightly increased but CYP2B1 and pentoxyresoruÞn O-dealkylation activities were decreased by extract treatment. These reports suggested that constituents of Scutellariae radix might inßuence CYP-dependent monooxygenase. Baicalein and wogonin (Scheme 1) are the major ßavonoids of Scutellariae radix and mainly present as their glucuronide forms. Baicalein and wogonin glucuronides can constitute up to 20% and 3% of the dry weight of Scutellariae radix, respectively [11,12]. After digestion, the glucuronides are readily hydrolyzed by intestinal bacteria [13]. Several evidence suggested that baicalein and related ßavonoids are the major components responsible for the pharmacological effects of Scutellariae radix [6,14]. However, little information is available on the in vivo effects of these

Scheme 1. Structures of baicalein and wogonin.

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ßavonoids on drug-metabolizing enzymes. Therefore, the present study was carried out to delineate the dietary effects of baicalein and wogonin on CYPs, UGT, and GST in mouse liver, kidney, and lung. Materials and methods Materials Baicalein and wogonin were isolated from the root of Scutellariae baicalensis following the method of Kimura et al. [15]. The purity of ßavones was $97% as determined by HPLC and NMR analyses. NADH, NADPH, glutathione, benzo(a)pyrene, and nifedipine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pyridine metabolite of nifedipine was a gift from Dr. F. Peter Guengerich, Vanderbilt University, Nashville, TN, USA. Acrylamide was purchased from Bio-Rad PaciÞc Ltd. (Kowloon, Hong Kong). Horseradish peroxidase conjugated rabbit anti-mouse IgG was purchased from Pierce Chemical Co. (Rockford, IL, USA). Animal treatment and microsomal preparation Male C57BL/6J mice (5 weeks old, weighing 12 z 15 g) were purchased from the National Animal Center in Taiwan. Before experimentation, mice were allowed a one-week acclimation period at the animal quarters with air conditioning and an automatically controlled photoperiod of 12 hr light daily. A liquid diet was prepared according to the diet formulations of Lieber and DeCarli [16]. Baicalein and wogonin were dissolved in the oil mixture as previously described [17]. Control group was fed with a control diet containing same constituents without ßavonoids. Mice (n56 per group) were fed ad libitum and the daily dietary intake was monitored. Tissues were removed and washed microsomes and cytosols were prepared by differential centrifugation 16 hr after the last feeding [17]. Contents of monooxygenase components and activities of CYP, UGT, and GST were determined within two weeks. For immunoblot analysis, mice were treated with a single injection of 3-methylcholanthrene (3-MC) at 80 mg/kg intraperitoneally and liver microsomes were prepared after 48 hr. Enzyme assays Microsomal contents of CYP and cytochrome b5 (b5) were determined by the method of Omura and Sato [18]. CYP reductase activity was determined following the method of Phillips and Langdon [19] using cytochrome c as a substrate. AHH activity was assayed by ßuorometric determination of the formation of 3-hydroxybenzo(a)pyrene [20]. The O-dealkylations of 7-ethoxyresoruÞn (EROD) and 7-methoxyresoruÞn (MROD) were determined by measuring ßuorescence of resoruÞn [21]. Caffeine 3-demethylation (CDM) activity was assayed following the method of Lee et al. [22]. N-demethylation of benzphetamine (BDM), N-nitrosodimethylamine (NDM), and erythromycin (EMDM) were determined by measuring the formation of formaldehyde using NashÕs reagent [23]. Nifedipine oxidation (NFO) was determined following the method of Guengerich et al. [24]. Microsomal UGT activity was assayed following the method of Bock et al. [25] using p-nitrophenol as a substrate. Cytosolic GST activity was determined using 1-chloro-2,4-dinitrobenzene as a substrate in the

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presence of GSH by monitoring the increase of absorbance at 340 nm [26]. Microsomal and cytosolic protein concentrations were determined by the method of Lowry et al. [27]. Immunoblot analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out using the discontinuous system of Laemmli [28]. In general, microsomal proteins were electrophoresed on 7.5% (w/v) polyacrylamide gels. For distinction between CYP1A1 and CYP1A2, 10% polyacrylamide gel was used. Electrophoresis was carried out at 4oC and at 20 mamp/gel during stacking and 40 mamp/gel during separation. Following electrophoresis, microsomal proteins were transferred from the slab gel to a nitrocellulose membrane using the method of Towbin et al. [29]. Immunodetection of P450s was performed using monoclonal antibodies against rat CYP1A1 (MAb 1-7-1), CYP2E1 (MAb 1-98-1) and CYP3A (MAb 2-13-1) [30]. Polyclonal rabbit anti-rat CYP 2B1 was purchased from Daiichi Pure Chemical Co., Ltd. (Tokyo, Japan). Immunorelated microsomal protein was detected by rabbit anti-mouse IgG conjugated with horseradish peroxidase and then stained using a chemiluminescence detection kit from Amersham (Buckinghamshire, UK). The protein band density was analyzed by densitometry using ImageMaster (Pharmacia Biotech Ltd., Uppsala, Sweden). Statistical analysis The statistical signiÞcance of differences between control and treated animals was evaluated by the StudentÕs t-test. A p value , 0.05 was considered as statistically signiÞcant. Results Mice were fed with liquid diets with or without ßavonoids for one week. The effects of diets containing increasing concentrations of ßavonoids on mouse hepatic AHH activities are presented in Fig. 1. Feeding mice with a diet containing 5 mM or 7.5 mM baicalein or wogonin

Fig. 1. Dose-response of effects of baicalein and wogonin on AHH activities in mouse liver. AHH activities were determined after one-week treatment of liquid diets containing increasing concentrations of ßavonoids as indicated. * Asterisks represent value signiÞcantly different from the control value, p , 0.05.

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Table 1 Dietary intake and body and tissue weights of control, baicalein-, and wogonin-treated mice Dietary intake (ml/mouse/day) Body weight (g) Liver weight (g) Kidney weight (g) Lung weight (g)

Control

Baicalein

Wogonin

14.0 6 0.6 19.7 6 0.4 1.05 6 0.05 0.30 6 0.02 0.17 6 0.01

14.3 6 0.5 20.3 6 0.4 0.98 6 0.02 0.29 6 0.02 0.17 6 0.01

14.5 6 0.5 19.2 6 0.2 1.05 6 0.01 0.30 6 0.02 0.15 6 0.01

Mice were administered liquid diets containing 5 mM ßavonoids for one week. Control mice received a control diet. Data represent mean 6 SEM of six mice at least.

resulted in signiÞcant decreases of AHH activities. Thus, mice were fed with a liquid diet containing 5 mM baicalein or wogonin in the following studies. Control, baicalein-, and wogonin-treated groups ingested similar amounts of liquid diet (Table 1). There were no signiÞcant differences in body and tissue weights between control and ßavonoid-treated groups (Table 1). Both ßavonoid treatments had no signiÞcant effects on hepatic CYP and b5 contents or CYP reductase activity (Table 2). Table 3 shows the results of monooxygenase activity determinations. Treatment of mice with baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, baicalein and wogonin showed different modulation of CYP1A2-catalyzed activities of MROD and CDM. Baicalein-treatment caused a 31% increase of MROD activity in mouse liver. In contrast, wogonin-treatment caused a 55% decrease of MROD activity. Consensus with the MROD determination, baicalein caused a slight increase of CDM activity whereas wogonin-treatment caused a 55% decrease of CDM activity. There were no signiÞcant changes in the hepatic EROD activity by baicalein- and wogonin-treatments. Effects of ßavonoids on renal and pulmonary monooxygenases were studied (Table 4). Extrahepatic CYP contents and CYP reductase activities were not affected by the ßavonoid treatments. Wogonin-treatment resulted in a 58% decrease of AHH activity in kidney microsomes. Baicalein- and wogonin-treatments caused 2- and 4-fold increases of pulmonary AHH activity, respectively. Baicalein- and wogonin-treatments increased pulmonary MROD activity. Increase of pulmonary EROD activity was also observed in baicalein- and wogonintreated groups.

Table 2 Effects of baicalein and wogonin on components of monooxygenase system in mouse liver Assay CYP (nmol/mg protein) b5 (nmol/mg protein) CYP reductase (nmol/min/mg protein)

Control

Baicalein

Wogonin

0.44 6 0.05 0.25 6 0.03 81.7 6 10.5

0.42 6 0.04 0.30 6 0.03 100.2 6 13.1

0.35 6 0.05 0.34 6 0.04 103.6 6 21.2

Mice were administered liquid diets containing 5 mM ßavonoids for one week. Control mice received a control diet. Data represent mean 6 SEM of six mice.

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Table 3 Effects of baicalein and wogonin on monooxygenase activities in mouse liver Assay Benzo(a)pyrene hydroxylation (pmol/min/mg protein) 7-Ethoxyresorufin O-deethylation (pmol/min/mg protein) 7-Methoxyresorufin O-demethylation (pmol/min/mg protein) Caffeine 3-demethylation (pmol/min/mg protein) Benzphetamine N-demethylation (nmol/min/mg protein) N-Nitrosodimethylamine N-demethylation (nmol/min/mg protein) Nifedipine oxidation (nmol/min/mg protein) Erythromycin N-demethylation (nmol/min/mg protein)

Control

Baicalein

Wogonin

301.7 6 20.6

213.6 6 13.7*

171.3 6 19.1*

21.5 6 5.3

20.0 6 3.5

22.6 6 2.9

365.3 6 39.1

479.3 6 28.2*

163.2 6 22.2*

43.0 6 3.8

56.0 6 3.6

27.6 6 4.3*

3.12 6 0.11

2.69 6 0.13*

2.45 6 0.22*

2.85 6 0.17

1.82 6 0.27*

2.21 6 0.17*

0.53 6 0.04

0.38 6 0.03*

0.40 6 0.04*

0.85 6 0.06

0.46 6 0.06*

0.55 6 0.05*

Mice were administered liquid diets containing 5 mM ßavonoids for one week. Control mice received a control diet. Data represent mean 6 SEM of six mice. * Asterisks represent values signiÞcantly different from the respective control values, p , 0.05.

Table 4 Effects of baicalein and wogonin on monooxygenases in mouse kidney and lung Kidney Assay CYP (nmol/mg protein) CYP reductase (nmol/min/mg protein) Benzo(a)pyrene hydroxylation (pmol/min/mg protein)

Lung

Control

Baicalein

Wogonin

Control

Baicalein

Wogonin

0.063

0.074

0.073

0.054

0.048

0.055

63.3 6 8.8 83.6 6 4.6 56.3 6 11.0 110.9 6 6.1 101.2 6 6.8

109.8 6 7.7

5.2 6 1.1

4.2 6 1.1

2.2 6 0.3*

4.3 6 0.4

8.8 6 0.8*

16.5 6 2.2*

7-Ethoxyresorufin O-deethylation (pmol/min/mg protein)

n.d.**

n.d.

n.d.

2.7 6 0.6

5.8 6 0.8*

28.0 6 3.0*

7-Methoxyresorufin O-demethylation (pmol/min/mg protein)

n.d.

n.d.

n.d.

3.1 6 0.6

5.3 6 1.5

n.d.

Mice were administered liquid diets containing 5 mM ßavonoids for one week. Control mice received a control diet. Extrahepatic tissues from two mice within the same group were pooled, microsomes were prepared, and enzyme activities were determined. Two or three microsomes were combined for CYP content determination. Data represent mean 6 SEM for Þve determinations, except CYP content determinations (n52). * Asterisks represent values signiÞcantly different from the control values, p , 0.05. ** n.d.: not detectable.

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Table 5 Effects of baicalein and wogonin on UGT and GST activities in mouse liver, kidney, and lung Assay

Tissue

Control

Baicalein

Wogonin

UDP-glucuronosyl transferase (nmol/min/mg protein)

Liver Kidney Lung

53.9 6 3.8 5.07 6 0.47 6.59 6 3.38

32.7 6 2.2* 2.00 6 0.45* 5.49 6 0.33

29.7 6 5.3* 3.00 6 0.61* 5.54 6 3.07

Glutathione S-transferase (mmol/min/mg protein)

Liver Kidney Lung

7.20 6 0.23 3.48 6 0.46 3.41 6 0.20

8.42 6 0.42 3.61 6 0.3 2.82 6 0.26

7.65 6 0.49 3.38 6 0.33 3.33 6 0.48

Microsomes and cytosols of individual mouse liver and pooled kidneys and lungs from two mice were prepared and conjugative activities were determined as described in Materials and methods. Data represent mean 6 SEM of six and three determinations for hepatic and extrahepatic tissues, respectively. * Asterisks represent values signiÞcantly different from the control values, p , 0.05.

Table 5 shows the results of UGT and GST conjugation activity determinations. Baicaleintreatment resulted in 39% and 61% decreases of UGT activities in liver and kidney, respectively. Wogonin-treatment resulted in 45% and 41% decreases of UGT activities in liver and kidney, respectively. However, pulmonary UGT activity was not changed by baicalein- and wogonin-treatments. Cytosolic GST activity was not affected by both ßavonoids in liver, kidney, and lung. Immunoblot analysis of hepatic CYP hemoproteins was carried out to determine the effects of baicalein and wogonin on CYP proteins, and the results are presented in Fig. 2. Im-

Fig. 2. Immunoblot analysis of microsomal CYP enzymes in mouse liver. Lane 1 contained 0.5 mg of microsomal protein from 3-MC-treated mice; lanes 2, 3, and 4 contained hepatic microsomal protein from control (C), baicalein (B)-, and wogonin (W)-treated mice. Twenty Þve mg microsomal protein was loaded for immunoreaction with mouse anti-rat CYP1A1 (MAb 1-7-1) and anti-rat CYP3A (MAb 2-13-1). Fifty mg microsomal protein was loaded for immunoreaction with mouse anti-rat CYP2E1 (MAb 1-98-1). Electrophoresis and immunodetection were carried out as described in the Materials and methods section.

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munoblot analysis of microsomal proteins from control and ßavonoid-treated mice using MAb 1-7-1 against rat CYP1A1 showed that a CYP1A protein was increased and decreased by baicalein and wogonin, respectively (Fig. 2, top, lanes 2 z 4). The mobility of the CYP1A protein was similar to the mobility of CYP1A2 inducible by 3-MC (top, lanes 1 and 2). Quantitative analysis of protein band intensity was performed using densitometry as described in the section of Materials and methods. The results showed that baicalein- and wogonin-treatments caused a 37% increase and a 44% decrease of CYP1A2 protein level in mouse liver, respectively. Immunoblots probed using mouse MAb 1-98-1 showed that baicalein- and wogonin-treatments caused 50% and 54% decreases of the levels of CYP2E1immunorelated protein, respectively (middle, lanes 2 z 4). Protein blots analyzed using mouse MAb 2-13-1 showed that the intensities of a CYP3A-immunorelated protein in baicalein- and wogonin-treated mice were 60% and 48% of the intensity in control mice, respectively (bottom, lanes 2 z 4). However, CYP2B-immunoreactive protein level was not affected by both ßavonoids as analyzed by immunoblot using rabbit anti-rat CYP2B1 antibody (data not shown). To reveal the modulatory effects of ßavonoids on extrahepatic CYP1A, microsomal proteins of kidney and lung were subjected to the immunoblot analysis probed using MAb 1-7-1. There was an immunorelated protein detected in kidneys of control mice. Treatments with baicalein and wogonin had no effect on the protein (data not shown). In control mice, there was an immunorelated protein present in lung (Fig. 3, lane 1). After baicalein- and wogonin-treatments, two CYP1A-immunorelated proteins were strongly induced in lung (lanes 2 and 3). Discussion Modulation of drug-metabolizing enzymes plays a crucial role in the determination of the pharmacological effects of drugs. Alteration of drug-metabolizing activities may cause serious drug-drug interactions in a patient under herbal medicine and other drug treatments. Kang et al. [10] reported that rat CYP2B1 was decreased by the crude extract of Scutellariae radix. Our results also showed that BDM activity, mainly catalyzed by CYP2B, was decreased by both baicalein- and wogonin-treatments. However, immunoblot analysis showed that there were no changes in the protein intensity of CYP2B-immunoreactive protein by the ßavonoid treatments (data not shown). Besides CYP2B, CYP2C and CYP3A can also cata-

Fig. 3. Immunoblot analysis of microsomal CYP1A in mouse lung. Fifty mg microsomal protein from control (C), baicalein (B)-, and wogonin (W)-treated mice were loaded. CYP1A-immunorelated proteins were detected by using mouse anti-rat CYP1A1 (MAb 1-7-1).

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lyze BDM reaction [31,32]. CYP3A is the most abundant hepatic CYP enzyme in human and experimental animals [33]. Both baicalein- and wogonin-treatments decreased CYP3A protein level and activities. Thus, the decrease of CYP3A might be at least one of the causes of BDM inhibition by ßavonoids. In contrast to the AHH activity induction and CYP reductase changes in previous crude extract reports [9,10], our results showed that AHH activity was signiÞcantly reduced and CYP reductase activity was not affected by both ßavonoids in mouse liver (Tables 1 and 2). These discrepancies between our ßavonoid study and previous crude extract reports can be attributed to several possibilities including the effects of other CYP modulators present in the crude extract and differences in species, antibody, and treatment regimen used. Several human and rodent CYP enzymes including CYP1A1, CYP1A2 and CYP3A are responsible for AHH activity [34]. CYP1A1, essentially an extrahepatic CYP, has the highest AHH activity. CYP1A2 is the most abundant CYP1A member in the liver [33]. Therefore, changes of CYP1A2 and CYP3A are important factors in the modulation of AHH activity in liver. Wogonin decreased both CYP1A2 and CYP3A whereas baicalein increased CYP1A2 but decreased CYP3A. The sum of CYP1A2 and CYP3A inhibition might result in the greater AHH inhibition by wogonin than by baicalein. Aromatic hydrocarbon receptor (AhR) is an important factor involved in CYP1A induction [35]. Both baicalein and wogonin have the basic planar polycyclic aromatic hydrocarbon-like structure of AhR ligands. However, our results demonstrated that baicalein and wogonin show inductive and inhibitory effects on CYP1A2 in mouse liver, respectively (Table 2 and Fig. 2). Strong CYP1A induction by baicalein and wogonin was observed in mouse lung (Table 3 and Fig. 3). This is the Þrst report of tissue-speciÞc regulation of CYP1A by natural ßavonoids. MROD and CDM activities were mainly catalyzed by CYP1A2 in liver. Baicalein-treatment caused increases of MROD activities and CYP1A2 protein level. In contrast, wogonin reduced MROD and CDM activities. This decrease was consensus with the suppressed CYP1A2 protein level. The actual mechanism for this dramatic difference of these two ßavonoids on the modulation of hepatic CYP1A2 is unknown. However, the substitutional differences of ßavonoids may affect the formation of optimal AhR-ligand complex to trigger the induction effect. Reports on the modulation of constitutive CYP1A2 by ßavonoids are rare. Recently, Ciolino et al. [36] have suggested that different CYP1A1 modulation by ßavonoids might be attributed to the substitutional differences of ßavonoids. Both quercetin and kaempferol are ligands of AhR by gel shifting assay. Quercetin- but not kaempferoltreatment strongly induced CYP1A1 in human breast cancer MCF-7 cells. The absence of 59-hydroxyl substitution of kaempferol might have resulted in the disability of CYP1A1 induction. However, galangin which has no hydroxy groups on the B-ring induced EROD activity and CYP1A1 mRNA level [37]. The number of hydroxyl substitutions of ßavones does not to be related to the inductive or inhibitory action. The substitution position and the interaction between substitutions may be more important. Similar variations have been reported for the relation between substitutions and the activities of Ah receptor-binding and differentiationinduction of ßavonoids [38,39]. Both baicalein and wogonin have no hydroxy groups in the B-ring but only baicalein shows inductive effect on CYP1A2 and related activity in mouse liver. Thus, the additional 8-methoxy and the absence of 6-hydroxy substitutions in the benzo-g-pyrone moiety of wogonin may be important in its inhibitory action. Further studies of the structure and activity relationship of ßavonoids will be required to address this question.

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We previously reported that naringin suppressed CYP1A2 protein level and MROD activity without affecting CYP3A and 2E1 [17]. Our present results showed that hepatic CYP 1A2 was induced and inhibited by baicalein and wogonin, respectively. However, catalytic activities of CYP2B (BDM), CYP2E1 (NDM), and CYP3A (NFO and EMDM) were generally decreased by both ßavonoids (Table 2 and Fig. 2). Since the modulation was not restricted to one CYP form, a general mechanism but not a gene speciÞc modulation mechanism was likely contributing to this inhibition. Thus, regulation of CYP enzymes by ßavonoids may involve a speciÞc receptor-mediated pathway as described above for CYP1A and a general mechanism for other CYPs. The ßavonoid antioxidant has been reported to affect cytokines and protein kinase [30]. The inßuence of cellular redox potential, cytokines, and protein phosphorylation can be responsible for the modulation of ßavonoids on monooxygenase. In addition to these pathways, direct interactions of CYP-catalyzed oxidations and a cross talk between regulatory factors may also be involved in the multiple effects of ßavonoids. In our study, the inhibition of hepatic CYP2E1 and CYP3A catalytic activities was in company with the reduction of CYP protein level as analyzed by immunoblot. Possible mechanisms for the decreased protein level include protein stability reduction and protein synthesis inhibition. To assess a possible CYP destruction mechanism, we have determined heme oxygenase activity in mouse liver. There were no signiÞcant changes after ßavonoid-treatments (data not shown). Further investigations are required to elucidate the modulatory mechanism of ßavonoids. In vitro, the presence of baicalein resulted in a decrease of UGT conjugation activity toward SN-38, the active metabolite of an anticancer drug CPT-11 [40]. Baicalein decreased b-glucuronidase activity toward SN-38-glucuronide and may prevent the enterohepatic circulation of SN-38 and protect against intestinal toxicity of CPT-11 [41]. Our in vivo results showed that both baicalein and wogonin suppressed UGT conjugation activity (Table 4). Therefore, administration of baicalein and wogonin may prevent enterohepatic circulation of drugs mainly metabolized by glucuronide conjugation. These results indicated that attention should be paid to possible drug-drug interactions of patients concomitantly treated with natural products mainly containing baicalein or wogonin and other medicines metabolized by CYP and UGT enzymes. The estimated human daily intake of baicalein from Kampo medicine is relative low and is roughly about 0.5% of the dosage used in the present mouse studies [42]. However, the usual regimen for Kampo medicine treatment in humans takes a long period of time. It will be interesting to further examine the effects of long term treatment with lower dosage of baicalein or wogonin. Acknowledgments This work was supported by National Research Institute of Chinese Medicine and a grant NSC 88-2314-B077-010 from the National Science Council, Executive Yuan, R. O. C. References 1. Guengerich FP. Catalytic selectivity of human cytochrome P450 enzymes: relevance to drug metabolism and toxicity. Toxicology Letters 1994;70(2):133Ð8. 2. Guengerich FP. Roles of cytochrome P-450 enzymes in chemical carcinogenesis and cancer chemotherapy. Cancer Research 1988;48(11):2946Ð54.

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