Introduction

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Phenethyl isothiocyanate (PEITC¹) (Fig. 1) is a constituent of cruciferous vegetables, including horseradish, cabbage, cauliflower, brussels sprouts, radishes, and watercress (Fenwick et al., 1983). PEITC occurs as its thioglucoside conjugate, called glucosinolate. When the vegetable is chewed, myrosinase is released from a separate cellular compartment and hydrolyzes the glucosinolate to produce isothiocyanate as well as other products (Hecht, 2000). Various epidemiological studies have demonstrated that the consumption of vegetables is associated with lower risk for cancers of various types, such as lung, esophagus, stomach, etc. (Steinmetz and Potter, 1991). The remarkable ability of some isothiocyanates to prevent cancer in laboratory animals treated with carcinogens was reported (Hecht, 2000). Among a wide variety of isothiocyanates that are naturally occurring, PEITC was the most extensively studied. PEITC was reported to inhibit lung carcinogenesis by the tobacco-specific nitrosamines, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in mice (Morse et al., 1992) and rats (Hecht et al., 1996), liver tumor by N-nitrosodiethylamine in mice (Pereira, 1995), and esophageal tumor by N-nitrosobenzylmethylamine in rats (Stoner et al., 1991). The consumption of average portions of the appropriate vegetables can result in the intake of tens of milligrams of isothiocyanates. For example, when 57 g of watercress is consumed, ~12 mg of PEITC is released (Chung et al., 1992). Thus, consumption of even moderate amounts of cruciferous vegetables entails the uptake of chemopreventive PEITC in great excess to carcinogens.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Structure of PEITC.

Most carcinogens require enzymatic transformation by cytochrome P450 (CYP) to exert their carcinogenic effects. Some of the intermediates formed in this process may be electrophiles, which can react with nucleophilic sites in critical macromolecules such as DNA, RNA, and protein. NNK requires metabolic activation (-hydroxylation) by CYP to express its carcinogenic activity. The mechanism of chemoprevention by PEITC is considered to be the inhibition of CYPs involved in the activation of carcinogens. In humans, it has been reported that NNK is metabolized by CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 (Crespi et al., 1991; Smith et al., 1992; Patten et al., 1996). Although the inhibition of those CYP isoforms by PEITC was suggested by in vivo studies, there is no in vitro study that directly reports the inhibitory effects of PEITC on human CYP isoforms except CYP1A2 (Smith et al., 1996). Recently, it has been reported that benzyl isothiocyanate (BITC), another naturally occurring isothiocyanate, is a mechanism-based inactivator of rabbit CYP2E1 (Moreno et al., 1999), rat CYP2B1 (Goosen et al., 2000), and human CYP2B6 and CYP2D6 (Goosen et al., 2001). Because of the structural similarity, the possibility of inactivation of human CYP isoforms by PEITC is surmised. In the present study, we investigated the inhibition and mechanism-based inactivation potencies of PEITC for human CYP isoforms using microsomes from baculovirus-infected insect cells expressing human CYP.

	Experimental Procedures

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Materials. PEITC was purchased from Aldrich (Milwaukee, WI). Phenacetin, acetaminophen, and caffeine were purchased from Wako Pure Chemical Industries (Osaka, Japan). 7-Benzyloxyresorufin, resorufin, coumarin, and chlorzoxazone were obtained from Sigma (St. Louis, MO). 7-Hydroxycoumarin sulfate sodium salt, 6-hydroxypaclitaxel, S-()-warfarin, 7-hydroxywarfarin, S-(+)-mephenytoin, (±)-4'-hydroxymephenytoin, (±)-bufuralol hydrochloride, (±)-1'-hydroxybufuralol maleate, and 6-hydroxychlorzoxazone were purchased from Ultrafine Chemicals (Manchester, UK). Paclitaxel was kindly provided by Bristol-Myers Squibb Pharmaceutical (Tokyo, Japan). Testosterone, 6-hydroxytestosterone, and 11-hydroxytestosterone were from Steraloids, Inc. (Wilton, NH). NADP⁺, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were from Oriental Yeast (Tokyo, Japan). Microsomes from baculovirus-infected insect cells expressing human CYP1A2, CYP2A6 + cytochrome b₅ (b₅), CYP2B6 + b₅, CYP2C8 + b₅, CYP2C9(Arg) + b₅, CYP2C19 + b₅, CYP2D6(Val), CYP2E1 + b₅, and CYP3A4 + b₅ were obtained from Gentest (Woburn, MA). All enzymes were coexpressed with NADPH-cytochrome P450 oxidoreductase. Other chemicals were of the highest grade commercially available.

Enzyme Assays. Phenacetin O-deethylase (POD) activity in CYP1A2-expressed microsomes, coumarin 7-hydroxylase (COH) activity in CYP2A6-expressed microsomes, benzyloxyresorufin O-dealkylase (BROD) activity in CYP2B6-expressed microsomes, paclitaxel 6-hydroxylase (PTXOH) activity in CYP2C8-expressed microsomes, S-warfarin 7-hydroxylase (WFOH) activity in CYP2C9-expressed microsomes, S-mephenytoin hydroxylase (MPOH) activity in CYP2C19-expressed microsomes, bufuralol 1'-hydroxylase (BFOH) activity in CYP2D6-expressed microsomes, chlorzoxazone 6-hydroxylase (CZXOH) activity in CYP2E1-expressed microsomes, and testosterone 6-hydroxylase (TESOH) activity in CYP3A4-expressed microsomes were determined as described previously (Nakajima et al., 1999a; Ohyama et al., 2000). For inhibition study, these activities were determined without or with various concentrations of PEITC. PEITC and the substrates were dissolved in methanol so that the final concentration of solvent in the incubation mixture was <1%.

Kinetic Analysis. The concentrations of the substrates ranged as follows: phenacetin (5-100 µM), coumarin (0.2-5 µM), 7-benzyloxyresorufin (0.1-2 µM), paclitaxel (10 µM), S-warfarin (0.25-10 µM), S-mephenytoin (10-100 µM), bufuralol (0.2-5 µM), chlorzoxazone (20-250 µM), and testosterone (10-250 µM), respectively. Kinetic parameters were estimated from the fitted curves using a computer program K·cat (BioMetallics, Princeton, NJ) designed for nonlinear regression analysis. All data were analyzed using the average of duplicate determinations. The variances between the duplicate determinations were <10%.

Mechanism-Based Inactivation of Human CYP Activities. Microsomes from baculovirus-infected insect cells expressing human CYPs were preincubated at 37°C for 5, 10, 15, and 20 min for the inactivation of CYP activities with various concentrations of PEITC in the presence of an NADPH-generating system (NADPH for CYP2B6). After the preincubation, the corresponding marker activity was measured according to the method described in the previous section. Kinetic parameters of the inactivation process, k_inact and K_i, were calculated as described previously (Nakajima et al., 1999b).

	Results

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Inhibition of Human CYP Activities by PEITC. The inhibitory effects of PEITC on the activity of each CYP isoform were investigated (Table 1). The inhibition type and K_i value were determined by Lineweaver-Burk plots (Fig. 2). PEITC competitively inhibited POD activity catalyzed by CYP1A2 (K_i = 4.5 ± 1.0 µM) and COH activity catalyzed by CYP2A6 (K_i = 18.2 ± 2.5 µM). BROD activity catalyzed by CYP2B6 was the most strongly inhibited by PEITC. The inhibition type was noncompetitive and the K_i was 1.5 ± 0.0 µM. PTXOH activity catalyzed by CYP2C8 was not affected by PEITC up to 100 µM (data not shown). PEITC noncompetitively inhibited WFOH activity catalyzed by CYP2C9 (K_i = 6.5 ± 0.9 µM), MPOH activity catalyzed by CYP2C19 (K_i = 12.0 ± 3.2 µM), BFOH activity catalyzed by CYP2D6 (K_i = 28.4 ± 7.9 µM), and CZXOH activity catalyzed by CYP2E1 (K_i = 21.5 ± 3.4 µM). The inhibition for TESOH activity catalyzed by CYP3A4 was a mixed-type of competitive (K_i = 34.0 ± 6.5 µM) and noncompetitive (K_i = 63.8 ± 12.5 µM) inhibition.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Lineweaver-Burk plots for inhibition of human CYP activities by PEITC. POD activity catalyzed by recombinant CYP1A2 (A), COH activity catalyzed by recombinant CYP2A6 (B), BROD activity catalyzed by recombinant CYP2B6 (C), WFOH activity catalyzed by recombinant CYP2C9 (D), MPOH activity catalyzed by recombinant CYP2C19 (E), BFOH activity catalyzed by recombinant CYP2D6 (F), CZXOH activity catalyzed by recombinant CYP2E1 (G), and TESOH activity catalyzed by recombinant CYP3A4 were determined without or with various concentrations of PEITC as described under Experimental Procedures. Data represent the average of duplicate determinations.

Mechanism-Based Inactivation of Human CYP Activities by PEITC. The possibility that PEITC is a mechanism-based inactivator of human CYP1A2 (POD), CYP2A6 (COH), CYP2B6 (BROD), CYP2D6 (BFOH), CYP2E1 (CZXOH), and CYP3A4 (TESOH) was investigated. As shown in Fig. 3, the NADPH-, time- and concentration-dependent inactivation of CZXOH activity catalyzed by CYP2E1 by PEITC was observed. The K_i was 9.98 µM and the k_inact value was 0.339 min¹. The other activities were not inactivated by PEITC (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Time- and concentration-dependent inactivation of CZXOH activity catalyzed by recombinant CYP2E1 by PEITC. The recombinant CYP2E1 was preincubated with various concentrations of PEITC at 37°C for 5, 10, 15, and 20 min in the presence of an NADPH-generating system. The preincubation mixtures were mixed with 50 µM chlorzoxazone and incubated at 37°C for 5 min. The 6-hydroxychlorzoxazone was determined by high-performance liquid chromatography. Data represent the average of duplicate determinations.

	Discussion

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Among the many CYP families, the metabolism of xenobiotics in humans is mainly catalyzed by isoforms belonging to the CYP1, CYP2, and CYP3 families (Spatzenegger and Jaeger, 1995). In the present study, the selectivity of inhibition or mechanism-based inactivation of these human CYP isoforms by PEITC was investigated. POD activity catalyzed by CYP1A2 was competitively inhibited by PEITC. The result is consistent with a previous report by Smith et al. (1996) that keto alcohol formation from NNK catalyzed by a CYP1A2 reconstituted system was competitively inhibited (K_i = 0.18 µM). COH activity catalyzed by CYP2A6 and BFOH catalyzed by CYP2D6 were inhibited by PEITC competitively and noncompetitively, respectively. CYP2A6 is a principal enzyme that catalyzes nicotine metabolism to cotinine (Nakajima et al., 1996a) and cotinine to trans-3'-hydroxycotinine (Nakajima et al., 1996b). However, it has been reported that the consumption of 2 ounces (56.8 g) of watercress at each meal for 3 days did not alter the urine levels of nicotine, cotinine, and trans-3'-hydroxycotinine (Hecht et al., 1999). It has also been reported that the consumption of 50 g of watercress did not affect the metabolism of debrisoquine (a probe drug of CYP2D6) in humans (Caporaso et al., 1994). Therefore, the inhibition potency of PEITC for CYP2A6 and CYP2D6 observed in this in vitro study might not be clinically significant in vivo. On the other hand, it has been reported that 50 g of watercress consumption caused a decrease in the levels of oxidative metabolites of acetaminophen, which is catalyzed by CYP2E1 (Chen et al., 1996). In another study, the area under the curve of chlorzoxazone (an in vivo probe of CYP2E1 in humans) was significantly increased after 50 g of watercress ingestion (Leclercq et al., 1998). Those in vivo inhibitions agreed with our in vitro inhibition study. CYP3A4 was inhibited by PEITC, although the inhibition potency was not strong. The observed inhibition of human CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 by PEITC is consistent with reports that those enzymes participate in the activation of nitrosamines (Crespi et al., 1991; Smith et al., 1992; Patten et al., 1996).

CYP2B6 was most effectively inhibited by PEITC. CYP2B6 has been reported to catalyze the metabolism of cyclofosfamide and ifosfamide (Rendic and Di Carlo, 1997). In addition, PEITC exhibited inhibitory effects on CYP2C9 and CYP2C19, which catalyze many drugs clinically used, such as warfarin, phenytoin, and omeprazole, etc. (Rendic and Di Carlo, 1997). An investigation of whether the consumption of moderate amounts of cruciferous vegetables affects the pharmacokinetics of drugs metabolized by these CYP isoforms is needed. Furthermore, when the phenotyping is performed using probe drugs for CYP isoforms to determine the ability of metabolism of individuals, the possibility should be kept in mind that the consumption of cruciferous vegetables might influence the phenotype determination.

It was clearly shown that human CYP2E1 was inactivated by PEITC in a time-, concentration-, and NADPH-dependent manner. The other isoforms investigated were not inactivated. Recently, it has been reported that BITC, another isothiocyanate, could inactivate human CYP2B6 and CYP2D6, although the kinetic parameters were not determined (Goosen et al., 2001). Rat CYP1A1 (K_i = 35 µM and k_inact = 0.26 min¹), CYP1A2 (K_i = 28 µM and k_inact = 0.09 min¹), CYP2B1 (K_i = 16 µM and k_inact = 0.18 min¹), and CYP2E1 (K_i = 18 µM and k_inact = 0.05 min¹) were also reported to be inactivated by BITC (Goosen et al., 2001). The kinetics of inactivation for rabbit CYP2E1 and rat CYP2B1 by BITC were K_i = 13 µM and k_inact = 0.09 min¹ (Moreno et al., 1999), and K_i = 5.8 µM and k_inact = 0.66 min¹(Goosen et al., 2000), respectively. Accordingly, it is suggested that the inactivation of human CYP2E1 by PEITC (K_i = 9.98 µM and = 0.339 min¹) observed in the present study would be comparable with the inactivation by BITC reported previously. However, the inactivation selectivity of PEITC would be different from BITC.

It has been reported that BITC is metabolized to the reactive benzyl isocyanate or benzylamine, which covalently modify the CYP apoprotein (Goosen et al., 2000, 2001). In humans and rats, BITC is also metabolized through conjugation with glutathione and finally excreted as mercapturic acid (Brüsewitz et al., 1977). On the other hand, when PEITC was administered to mice, more than 80% of the urinary metabolites were PEITC conjugates derived from glutathione conjugation. The two major urinary metabolites were N-acetylcysteine conjugate of PEITC and a cyclic mercaptopyruvate conjugate, which accounted for approximately 25% and 10%, respectively, of the PEITC administered (National Cancer Institute, Chemoprevention Branch and Agent Development Committee, 1996). In human urine, N-acetylcysteine conjugate was completely excreted within 24 h of ingestion (Chung et al., 1992). When rats were administered PEITC orally, it was rapidly converted to phenethylamine (National Cancer Institute, Chemoprevention Branch and Agent Development Committee, 1996). Although the reactive metabolites that modify CYP enzymes have not been identified yet, similar metabolic pathways with BITC might possibly exist in PEITC.

In conclusion, it was clearly demonstrated that the naturally occurring isothiocyanate, PEITC, acts as an inhibitor of human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 and is a mechanism-based inactivator of human CYP2E1. Since NNK is believed to play a significant role as a cause of lung cancer in smokers and it has been confirmed that NNK metabolism is modified by the consumption of PEITC in humans (Hecht et al., 1995), PEITC is being developed as a chemopreventive agent (National Cancer Institute, Chemoprevention Branch and Agent Development Committee, 1996). The inhibition and inactivation of human CYP isoforms by PEITC might contribute significantly to drug-drug interactions.