Chem. Res. Toxicol. 2004, 17, 1151-1156
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Identification of the Main Human Cytochrome P450 Enzymes Involved in Safrole 1′-Hydroxylation Yune-Fang Ueng,*,†,‡ Chih-Hang Hsieh,§ Ming-Jaw Don,† Chin-Wen Chi,§,| and Li-Kang Ho§ National Research Institute of Chinese Medicine, 155-1, and Department of Pharmacology, National Yang-Ming University, 155, Li-Nong Street, Section 2, Taipei 112, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu Hsing Street, Taipei 110, and Department of Medical Research and Education, Taipei Veterans General Hospital, Shih Pai, Taipei 112, Taiwan, Republic of China Received November 19, 2003
Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1′-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1′-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n ) 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 ( 0.7 nmol/min/mg protein. In human liver (n ) 3) microsomes, the mean Km and Vmax values of SOH were 5.7 ( 1.2 mM and 0.14 ( 0.03 µmol/min/nmol P450, respectively. The mean intrinsic clearance (Vmax/Km) was 25.3 ( 2.3 µL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r ) 0.569) and chlorzoxazone hydroxylation (r ) 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH.
Introduction Safrole (4-allyl-1,2-methylenedioxybenzene) is a natural plant constituent, mainly present in sassafras oil and some other essential oils. A major source of human exposure to safrole is through the consumption of spices, such as nutmeg, cinnamon, and black pepper. It is also found in high amounts (15 mg safrole/g) in Piper betel inflorescence, which has been commonly chewed together with areca quid in Taiwan (1). Areca quid chewers had greater chronic liver disease risks than the group without areca quid chewing in Taiwanese aborigines (2). Safrole is classified as a weak hepatocarcinogen in rodents and possibly in humans (3). In oral cancer patients with the areca quid chewing history, safrole-like DNA adducts were found in the oral tissue (4). The concentration of safrole in saliva could be as high as 420 µM during the chewing of areca quid (5). Safrole is oxidized by P4501 to form 1′-hydroxysafrole (Scheme 1), which forms DNA adducts after O-sulfonation (6). The urinary glucuronide conjugate of 1′-hydroxysafrole ac* To whom correspondence should be addressed. Tel: 886-228201999 Ext. 6351. Fax: 886-2-28264266. E-mail: ueng@ cma23.nricm.edu.tw. † National Research Institute of Chinese Medicine. ‡ Taipei Medical University. § National Yang-Ming University. | Taipei Veterans General Hospital. 1 Abbreviations: P450, cytochrome P450; SOH, safrole 1′-hydroxylation.
Scheme 1
counted for 1-3% of an intraperitoneal dose of safrole administered to rats, hamsters, and guinea pigs (7). In male mice, 30% or more of a dose of safrole is excreted as the glucuronide conjugate of 1′-hydroxysafrole. The excretion of 1′-hydroxysafrole conjugates was stimulated in rats treated with P450 inducers, phenobarbital and 3-methylcholanthrene (7). However, human hepatic SOH was not reported. To identify the main P450 involved in the human metabolic activation of safrole, the present study determined the SOH activity of human liver microsomes and Escherichia coli-expressing bicistronic human P450s.
Materials and Methods Chemicals. Chlorzoxazone, coumarin, dextromethorphan, dextrorphan, diethyldithiocarbamate, 7-ethoxyresorufin, 7-pentoxyresorufin, ketoconazole, 4-methylpyrazole, β-NADP, R-naphthoflavone, orphenadrine, quinidine, safrole, sulfaphenazole, and tolbutamide were purchased from Sigma-Chemico Inc. (St.
10.1021/tx030055p CCC: $27.50 © 2004 American Chemical Society Published on Web 07/29/2004
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Louis, MO). 6-Hydroxychlorzoxazone and 4-hydroxytolbutamide were purchased from Sigma/RBI (Natick, MA). 1′-Hydroxysafrole was synthesized using piperonal following the method of Borchert et al. (7). The structure of 1′-hydroxysafrole was identified by 1H and 13C NMR analyses. The purity of 1′hydroxysafrole was g99% as analyzed by HPLC. Liver Microsomal Preparations. The nontumor Chinese liver (ChL) samples were obtained from patients who underwent liver resection in Taipei Veteran General Hospital (Taipei). All liver samples were kept at -70 °C immediately after surgery according to a protocol approved by the medical committee for conducting human research at the hospital. The experiments using human liver samples were under the agreement of the Department of Surgery, Taipei Veteran General Hospital (Taipei). Human liver microsomes were prepared from the frozen liver samples following the method of Guengerich (8). Male C57BL/ 6J mice (5 weeks, 13-15 g) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan, Republic of China). The mice were allowed a 1 week acclimation in the animal center with air conditioning and an automatically controlled photoperiod of 12 h light daily. Mouse liver microsomes were prepared from nonfrozen mouse livers following the method of Alvares and Mannering (9). The microsomal pellets and suspension were stored at -80 °C in a deep freezer before use. Enzyme Preparations. Rabbit cytochrome b5 and the constructed plasmids of bicistronic human P450s were kindly provided by Dr. F. Peter Guengerich (Nashville, TN). Bicistronic human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 constructs consisting of the coding sequence of a P450 followed by that of NADPH-P450 reductase were transformed to E. coli DH5R by electroporation (Gene pulser II, Bio-Rad, Hercules, CA). Bacterial membrane fractions were prepared by differential centrifugation following the method of Parikh et al. (10). Bacterial membrane fractions were stored at -80 °C until use. SOH Assay. The stock solution of safrole was prepared in methanol for assays with various concentrations. The final concentrations of methanol in assays were less than 2%. The incubation mixture of human liver microsomes consisted of 0.2 mg/mL microsomal protein, 10 mM Tris-HCl buffer, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 200 µM to 100 mM safrole, and a NADPH-generating system in the final volume of 0.5 mL. The NADPH-generating system consisted of 5 mM glucose-6phosphate, 0.5 mM β-NADP, and 0.25 unit/mL glucose-6phosphate dehydrogenase. For recombinant P450 enzymes, 100 pmol P450/mL was added in the assays. The reaction was carried out at 37 °C for 20 min with horizontal shaking and then stopped by the addition of 1.2 mL of methanol. After centrifugation at 2500 rpm for 15 min, the supernatant was injected into HPLC. A Microsorb-MV C18 column (100 Å, 4.6 mm × 250 mm, Varian, Inc., Palo Alto, CA) was eluted with 55% (v/v) methanol (in water) at 1 mL/min. The absorbance at 280 nm was monitored using a HP1100 UV detector (Agilent/ Hewlett-Packard GmbH, Bo¨blingen, Germany). The formation of 1′-hydroxysafrole was determined using synthetic standard. Enzyme Contents and P450 Model Reaction Assays. P450 and cytochrome b5 contents were determined by the spectrophotometric method of Omura and Sato (11). Coumarin hydroxylation and dextromethorphan O-demethylation activities were determined by HPLC with a fluorescence detector (12, 13). The O-dealkylations of 7-ethoxyresorufin and pentoxyresorufin were determined by measuring the fluorescence of resorufin (14, 15). The hydroxylations of chlorzoxazone and tolbutamide and the oxidation of nifedipine were determined by HPLC with an UV detector (16-18). The substrate concentrations were 0.5 mM chlorzoxazone, 20 µM coumarin, 200 µM dextromethorphan, 2 µM 7-ethoxyresorufin, 0.2 mM nifedipine, 10 µM pentoxyresorufin, and 2.5 mM tolbutamide. The microsomal protein concentration was determined by the method of Lowry et al. (19).
Figure 1. HPLC chromatogram of the safrole oxidation assay of human liver microsomes. Human liver microsomes were incubated with safrole in the presence (+NADPH) or absence (-NADPH) of a NADPH-generating system. 1′-Hydroxysafrole and safrole appeared at 6.2 and 18.0 min, respectively.
Figure 2. Relationship of SOH activity and microsomal protein concentration in the assay. The SOH activity was determined using 2 mM safrole in the reaction mixture. A solid line between 0 and 0.3 mg protein/mL represents the line of best fit as analyzed by linear regression using SigmaPlot (Jandel Scientific, San Rafael, CA). Data Analysis. The kinetic parameters of SOH were analyzed using the software Sigma Plot (Jandel Scientific, San Rafael, CA). The values of hydroxylation velocity (v) at various safrole concentrations (S) were fitted by nonlinear least-squares regression without weight due to the equation according to the equation v ) (VmaxSn)/(Kmn + Sn), where Vmax is the maximal velocity and n value is the measure of cooperativity. The n value was initially calculated from the plots of log[v/(Vmax - v)] vs log S (Hill plot) by linear regression. The estimates of variance (denoted by () are presented from the analysis of an individual set of data. The pairs of variables show that a significant relationship was analyzed by Spearman rank correlation test using the software Sigma Plot. A p value smaller than 0.05 was considered statistically significant.
Results SOH in Human Liver Microsomes. HPLC analysis of oxidation products of safrole showed that the incubation of microsomes with safrole and the NADPH-generating system generated a peak of 1′-hydroxysafrole, which did not appear in the absence of the NADPH-generating system (Figure 1). The amount of 1′-hydroxysafrole less than 1 nmol was considered not detectable by HPLC. In microsomes of human liver sample ChL7, the SOH activity was in the linear range of 0.1-0.3 mg microsomal protein/mL in assay (r2 ) 0.999, Figure 2). The reaction rate became a plateau at the protein concentration between 0.6 and 1.4 mg/mL. Thus, 0.2 mg microsomal
Safrole Hydroxylation by Human P450
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Table 1. Enzyme Kinetic Analyses of the Formation of 1′-Hydroxysafrole by Human Liver Microsomesa sample
Km (mM)
Vmax (µmol/min/ nmol P450)
Clint (Vmax/Km) (µL/min/nmol P450)
ChL1 ChL2 ChL3
3.8 ( 0.6 5.7 ( 2.3 7.7 ( 1.7
0.11 ( 0.01 0.12 ( 0.02 0.20 ( 0.02
28.9 21.1 26.0
a The activities were determined using 0.2 mg microsomal protein/mL and 2 mM safrole in the incubation mixture. The reaction was performed at 37 °C for 20 min in a shaking water bath. The results represent means ( SE of the parameter estimates determined using the nonlinear regression without weight as described in the Materials and Methods.
Table 2. Effects of P450 Selective Inhibitors on SOH Activity in Human Liver Microsomesa inhibitors (P450 form)
% of control
SKF525A R-naphthoflavone (CYP1A2) orphenadrine (CYP2B6) sulfaphenazole (CYP2C9) quinidine (CYP2D6) 4-methylpyrazole (CYP2E1) diehtyldithiocarbamate (CYP2E1) ketoconazole (CYP3A4)
15 ( 6 101 ( 7 88 ( 4 42 ( 6 96 ( 7 55 ( 6 26 ( 5 91 ( 2
a The ChL microsomal SOH activity was determined as described in Figure 1. The SOH activities were in the range of 0.83 to 2.38 nmol/min/mg protein in the presence of 200 µM safrole. The data represent means ( SE of six human liver samples. The concentrations of R-naphthoflavone, orphenadrine, sulfaphenazole, quinidine, 4-methylpyrazole, and ketoconazole added in the assay were 10, 300, 1, 1, 20, and 1 µM, respectively. For SKF525A (500 µM) and diethyldithiocarbamate (100 µM), the microsomes were preincubated with inhibitors in the presence of a NADPHgenerating system at 37 °C for 10 min.
protein/mL was used in the following SOH activity determination. The SOH activities of 18 human liver samples were in the range of 3.5-16.9 nmol/min/mg protein, and the mean (( SE) value was 8.7 (( 0.7) µmol/ min/mg protein. The kinetic parameters of microsomal SOH were analyzed using three samples (Table 1). The apparent Km values of SOH activity in ChL1, ChL2, and ChL3 were 3.8, 5.7, and 7.7 mM, respectively. The Vmax values of SOH activity in ChL1, ChL2, and ChL3 were 0.11, 0.12, and 0.20 µmol/min/nmol P450, respectively. The mean Km and Vmax values (( SE, n ) 3) were 5.7 (( 1.2) mM and 0.14 (( 0.03) µmol/min/nmol P450 (28.7 ( 3.5 nmol/min/mg protein), respectively. Hill and EadieHofstee polts showed a monophasic nature (result not shown). The Hill coefficients of SOH activity in ChL1, ChL2, and ChL3 determined by nonlinear regression were 1.13 ( 0.12, 0.98 ( 0.31, and 1.18 ( 0.24. The intrinsic clearances (Vmax/Km) of safrole in three human samples were in the range of 21.1-28.9 µL/min/nmol P450. Inhibition by Selective P450 Inhibitors. The concentration used for each inhibitor is supposed to selectively inhibit about 60-80% of the P450 form specific reaction with a model substrate (20). The inhibitor concentration used in the inhibition study was in the range of 1-500 µM. Thus, 200 µM safrole was added in the assay. The SOH activities of six human liver samples were in the range of 0.83-2.38 nmol/min/mg protein at 200 µM safrole. The preincubation of microsomes with SKF525A caused a 85% inhibition of SOH activity (Table 2). To identify the major P450 form(s) responsible for SOH, R-naphthoflavone, orphenadrine, sulfaphenazole, quinidine, and ketoconazole were used as selective in-
Table 3. Correlation of SOH Activity with P450 Isoform Specific Activities in 18 Human Liver Microsomesa assay (P450 form)
correlation coefficient (r)
7-ethoxyresorufin O-deethylation (CYP1A2) coumarin hydroxylation (CYP2A6) 7-pentoxyresorufin O-dealkylation (CYP2B6) tolbutamide hydroxylation (CYP2C9) dextromethophan O-demethylation (CYP2D6) chlorzoxazone hydroxylation (CYP2E1) nifedipine oxidation (CYP3A4)
0.417 -0.096 0.255 0.569* 0.299 0.770* 0.305
a The ranges (mean ( SE) of activities were 7-ethoxyresorufin O-deethylation, 8.5-33.2 (23.5 ( 2.4) pmol/min/mg protein; coumarin hydroxylation, 0.01-0.53 (0.17 ( 0.03) nmol/min/mg protein; pentoxyresorufin O-dealkylation, 0.03-17.8 (5.7 ( 1.1) pmol/ min/mg protein; tolbutamide hydroxylation, 0.06-0.50 (0.22 ( 0.04) nmol/min/mg protein; dextromethophan O-demethylation, 0.003-0.15 (0.07 ( 0.01) nmol/min/mg protein; chlorzoxazone hydroxylation, 0.12-0.62 (0.39 ( 0.04) nmol/min/mg protein; and nifedipine oxidation, 0.05-0.66 (0.22 ( 0.04) nmol/min/mg protein. An asterisk represents that pairs of variables show a significant relationship as analyzed by the Spearman rank correlation test; p < 0.05.
hibitors for hepatic CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4, respectively (Table 2). The CYP2E1 substrate 4-methylpyrazole was used as a competitive inhibitor, and diethyldithiocarbamate was used as a mechanism-based inhibitor of CYP2E1 (21, 22). The addition of 10 µM R-naphthoflavone, 300 µM orphenadrine, 1 µM quinidine, and 1 µM ketoconazole had no effects on SOH activities of human liver microsomes. The addition of 1 µM sulfaphenazole and 20 µM 4-methylpyrazole caused 58 and 45% decreases of microsomal SOH activity, respectively. Preincubation of microsomes with 100 µM diethyldithiocarbamate caused a 74% inhibition of SOH activity. Correlation Analyses. The human liver preparations used in this study showed variability in the activities of various P450 forms toward model reactions. The ranges (mean ( SE) of activities are listed in the legend of Table 3. Microsomal SOH activities of some liver samples were not detectable at safrole concentrations less than 2 mM. Thus, liver microsomal activities of 18 samples were determined using 2 mM safrole. This concentration was higher than the saliva concentration in humans chewing areca quid but lower than the Km values determined in three samples. The SOH activity had a significant correlation with tolbutamide hydroxylation (r ) 0.569) and chlorzoxazone hydroxylation activities (r ) 0.770), which were marker reactions catalyzed by CYP2C9 and CYP2E1, respectively (Table 3). However, the SOH activity showed no significant correlation with 7-ethoxyresorufin O-deethylation (r ) 0.417), coumarin hydroxylation (r ) -0.096), pentoxyresorufin O-dealkylation (r ) 0.255), dextromethorphan O-demethylation (r ) 0.299), and nifedipine oxidation (r ) 0.305) activities, which were marker reactions catalyzed by CYP1A2, CYP2A6, CYP2B6, CYP2D6, and CYP3A4, respectively. SOH by Bicistronic Human P450s. The SOH activities of E. coli membranes expressing bicistronic human P450s were determined using 2 mM safrole. The CYP2E1catalyzed SOH activity showed a good linear relationship with a P450 concentration in the range of 10-200 pmol/ mL (r2 ) 0.990, Figure 3). Our results showed that CYP2C9 and CYP2E1 had the highest SOH activities (Figure 4). CYP2C9 had a SOH activity 2-, 35-, 4-, 3-, 2-, and 10-fold greater than CYP1A1, CYP1A2, CYP1B1,
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Ueng et al. Table 5. SOH Activities of Human, Mouse, and Rat Liver Microsomesa species humanb C57BL/6J mouse CD-1 mousec Fischer rat
Figure 3. Relationship between SOH and CYP2E1 concentration in assay. The membrane fraction of E. coli expressing P450 2E1 was prepared as described in the Materials and Methods. The SOH activity was determined using various P450 concentrations in the presence of 2 mM safrole and a NADPHgenerating system as described in the Materials and Methods.
Figure 4. SOH activities of bacterial membranes expressing bicistronic human P450 enzymes. The 1′-hydroxylation activity was determined using 100 pmol/mL P450 and 2 mM safrole in the assay. The results represent means ( SE of three separate determinations with duplicates. Table 4. Enzyme Kinetic Analyses of the Formation of 1′-Hydroxysafrole by Bicistronic Human CYP2C9 and CYP2E1 Expressed in E. colia CYP form
Km (mM)
Vmax (µmol/min/ nmol P450)
Clint (Vmax/Km) (mL/min/nmol P450)
CYP2C9 CYP2E1
2.5 ( 0.6 1.2 ( 0.3
0.93 ( 0.07 1.15 ( 0.08
0.37 0.96
a The activities were determined using 10 pmol P450/mL in the assay. The results represent means ( SE of the parameter estimates calculated as described in Table 1.
CYP2A6, CYP2D6, and CYP3A4, respectively. CYP2E1 had a SOH activity 3-, 41-, 5-, 3-, 3-, and 11-fold greater than CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2D6, and CYP3A4, respectively. The addition of 200 pmol rabbit cytochrome b5/mL had no stimulatory effect on the SOH activities of CYP2E1 and CYP3A4 (results not shown). For the kinetic analyses, the concentrations of safrole in the assays were in the range of 25 µM to 10 mM. CYP2C9 and CYP2E1 had Km values of 2.5 ( 0.6 and 1.2 ( 0.3 mM and the Vmax values of 0.93 ( 0.07 and 1.15 ( 0.08 µmol/min/nmol P450, respectively (Table 4). The measure of cooperativity showed that the n values of CYP2C9- and CYP2E1-catalyzed SOH were 0.88 ( 0.13 and 0.94 ( 0.16, respectively.
gender
number
SOH (nmol/min/ mg protein)
ref
male male female male female
18 6 3 5 4 4
8.7 ( 0.7 2.33 ( 0.17 1.42 ( 0.20 1.37 ( 0.30 0.38 ( 0.03 0.28 ( 0.02
25 25 25 25
a Human and C57BL/6J mouse hepatic SOH activities were determined using 0.2 mg microsomal protein/mL and 2 mM safrole in the reaction mixture. The reaction was performed at 37 °C for 20 min in a shaking water bath. The formation rate of 1′hydroxysafrole was measured as described in the Materials and Methods. The data represent the means ( SEM of liver microsomal activity. The SOH activities reported by Swanson et al. (25) were determined using 2 mM safrole. b The human samples include 17 males and one female. c The livers of 5-18 mice were pooled and used as one determination.
Discussion Safrole is capable of inducing chromosome aberrations and is a documented rodent hepatocarcinogen (23, 24). The treatment of rats with safrole caused the formation of stable safrole-DNA adducts in the liver (23). Swanson et al. (25) reported that hepatic microsomes from CD-1 mice had SOH activities 4-5-fold higher than those of rats. Male mice or rats had SOH activities similar to or slightly higher than the respective female animals. Our determination of the SOH activity showed that male C57BL/6J mice had SOH activities 60-70% greater than CD-1 mice (Table 5). Human liver microsomes had SOH activities 23-, 31-, 6-, 6-, and 4-fold greater than the male Fischer rat, female Fischer rat, male CD-1 mice, female CD-1 mice, and C57BL/6J mice, respectively (Table 5). In human, the mean SOH activity of 17 males was 8.7 ( 0.8 nmol/min/mg protein and the only female sample was 8.1 nmol/min/mg protein. These results revealed the variations of SOH activities in different species. Our SOH assay method is different from the method of Swanson et al. (25), and this difference may contribute to part of the variations in activity determination. Reports showed that the presence of methanol at a concentration g1% decreased CYP2C and CYP2E1 catalytic activities in human liver microsomes (26, 27). Thus, our results may be underestimated. The high SOH activities in human liver preparations suggested that there might be a higher toxicity risk for human exposed to safrole. Our results showed that human liver microsomal SOH was sensitive to the inhibition by CYP2C9 and CYP2E1 inhibitors (Table 2) and showed a significant correlation with tolbutamide hydroxylation and chlorzoxazone hydroxylation activities (Table 3). Bicistronic CYP2C9 and CYP2E1 had higher SOH activities than the other P450s determined in this study (Figure 4). These results indicated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH. Other methods including the use of recombinant P450 expressed in other systems and the immunoinhibition procedures could also provide additional evidence for the roles of P450 forms in safrole oxidations. The addition of cytochrome b5 had no stimulatory effect on CYP2E1-catalyzed SOH. This result agrees with that of a previous report of bicistronic CYP2E1-catalyzed chlorzoxazone hydroxylation (10). Surprisingly, the addition of cytochrome b5 had no stimulatory effect on CYP3A4-catalyzed SOH. This result was
Safrole Hydroxylation by Human P450
different from the report of Yamazaki et al. (28) who showed that bicistronic CYP3A4-catalyzed testosterone 6β-hydroxylation was enhanced by adding cytochrome b5. The reason for this difference needs further studies. The v vs substrate concentration plot of CYP2C9- and CYP2E1-catalyzed SOH showed a hyperbolic characteristic (results not shown). Kinetic analysis showed a Hill coefficient close to 1, suggesting that there was no cooperativity. Our results showed that both CYP2C9 and CYP2E1 had Km values higher than 1 mM. These Km values were lower than the values of human liver microsomes, and they were high as compared to other P450 substrates reported (29). The high Km value suggested the low affinity for safrole binding to the active site for 1′-hydroxylation (Table 4). Inadequate substrate concentration or product inhibition might cause the plateau of activity using a protein concentration higher than 0.3 mg/mL in the presence of 2 mM safrole, which was lower than the mean Km value of human liver microsomes. CYP2E1 had a Km value 52% lower than CYP2C9 and a Vmax value 24% higher than CYP2C9. The intrinsic clearance of safrole by CYP2E1 was 3-fold greater than CYP2C9. These results suggested that CYP2E1 had a higher safrole binding affinity and catalytic capacity of SOH than CYP2C9. The SOH activity of CYP2E1 was also higher than CYP2C9 at 0.1 and 0.3 mM safrole, which were close to the concentrations that humans might be exposed to (results not shown). To calculate the contribution of P450 forms in microsomal SOH activity, the specific activities of CYP2C9 and CYP2E1 were multiplied by their microsomal contents. In human liver microsomes, CYP2C9 was considered to constitute about half to 75% of the total CYP2C content (30). Thus, the average contents of microsomal CYP2C9 and CYP2E1 were about 48 and 22 pmol/mg protein, respectively (30, 31). Thus, the contributions of CYP2C9 and CYP2E1 toward SOH are nearly equal. A previous rat study showed that phenobarbital and 3-methylcholanthrene treatment stimulated the urinary excretion of the glucuronide conjugate of 1′-hydroxysafrole (7). However, the effects of other P450 inducers on safrole oxidation were not reported. Rat CYP2B1 and CYP1A1 are the P450 forms highly induced by phenobarbital and 3-methylcholanthrene, respectively (32, 33). Our human study indicated that CYP2B6 and CYP1A1 did not play major roles in the SOH (Tables 2 and 3 and Figure 4). This may be due to the relatively small content of CYP2B6 in human liver, and CYP1A1 is essentially an extrahepatic P450 (31). The stimulation of rat excretion of glucuronide conjugate of 1′-hydroxysafrole might be due to the species difference or elevation of other phenobarbital and 3-methylcholanthrene inducible P450s and cnonjugation enzymes. The human and rat CYP2C subfamily is responsive to the induction by phenobarbital (34, 35). UDP-glucuronosyl transferase can be induced by both phenobarbital and 3-methylcholanthrene (36). Induction of these drug-metabolizing enzymes might also be able to elevate the excretion of safrole metabolites. The induction of CYP2E1 and CYP2C9 might increase the toxicity of safrole through the stimulation of SOH. CYP2E1 was inducible by ethanol and cigarette smoke in rodents (37). In humans, the clearance of CYP2E1 substrate chlorzoxazone and the lymphocyte CYP2E1 protein level are higher in alcoholics than in the control group (38). However, the acute exposure of ethanol decreased human CYP2E1 activity (39). Thus, the effect
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of ethanol on safrole toxicity in occasional drinkers may be different from alcoholics. Besides the liver, human buccal tissue is possibly exposed to a high concentration of safrole. In human buccal tissue and the immortalized SVpgC2a buccal cell line, CYP2C and CYP2E1 mRNA were detectable as analyzed by the reverse transcriptase polymerase chain reaction (40). In the SVpgC2a cell line, the CYP2E1 activity was detectable but the CYP2C9 activity was under the detection limit (40). However, the effects of ethanol and other P450 inducers on buccal P450 were not reported. In summary, our results showed that human liver microsomes had high SOH activities. CYP2E1 and CYP2C9 were the main hepatic P450 forms involved in human SOH. It will be of interest to study the effects of ethanol consumption and cigarette smoking on safroleassociated oral and hepatic toxicities in the future.
Acknowledgment. We appreciate Dr. F. Peter Guengerich (TN) for kindly providing rabbit cytochrome b5 and the plasmids of recombinant human P450s. This work was supported by NSC91-3112-B077-001. The tissue samples used in this study were supported by a grant from the National Science and Technology Program in Pharmaceuticals and Biotechnology (NSC91-2323-B073001).
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