Inactivation of Cytochrome P450 2E1 by tert-Butylisothiocyanate

Anna L. Blobaum, Yipin Lu, Ute M. Kent, Shaomeng Wang, and Paul F. Hollenberg ... Ute M. Kent, Elizabeth S. Roberts-Kirchhoff, Namdoo Moon, William R...
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1154

Chem. Res. Toxicol. 1998, 11, 1154-1161

Inactivation of Cytochrome P450 2E1 by tert-Butylisothiocyanate Ute M. Kent, Elizabeth S. Roberts,† Jarin Chun,‡ Kimberly Hodge,§ Jenny Juncaj,| and Paul F. Hollenberg* Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109 Received June 5, 1998

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 °C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 µM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 µM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.

Introduction Cytochrome P450s (see ref 1 for nomenclature) play a major role in the detoxification of xenobiotics. Although, in some instances, P450s can activate certain environmental or dietary substances to carcinogens. Of particular interest in this respect is P450 2E1 since it metabolizes ethanol and many other low-molecular weight compounds that are potentially toxic or carcinogenic such as nitrosamines, halogenated alkanes, acetaminophen, and aromatic compounds such as benzene and styrene (2-4). P450 2E1 also reduces dioxygen to reactive oxygen radicals that may play a role in lipid peroxidation and cytokine production. P450 2E1 is a constitutively expressed and inducible P450 isoform in the endoplasmic reticulum of mammalian hepatocytes. Lower levels of this enzyme are also found in kidney, lung, intestine, nasal mucosa, lymphocytes, and brain. The sequences of rabbit, rat, mouse, and human P450 2E1 have been determined and are well-conserved among species (1, 5). Human and rat P450 2E1 show 78% homology at the amino acid level (6). P450 2E1 can be induced by ethanol and starvation (7, 8), and is believed to have an important * To whom correspondence should be addressed at Department of Pharmacology, Medical Science Research Building III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: [email protected]. † Present address: Department of Chemistry and Biochemistry, University of Detroit Mercy, Detroit, MI 48219-0900. ‡ J.C. was a student in the Undergraduate Research Opportunities Program at the University of Michigan. § K.H. was from Meharry Medical College and a recipient of a Charles Ross Fellowship Award. | J.J. was from the University of Detroit Mercy and was a recipient of a Summer ASPET Undergraduate Research Fellowship Award.

role in the pathogenic progression of alcoholic liver disease (9, 10). P450 2E1 has been implicated in the causation of alcohol- and tobacco-associated cancers (11). Villard et al. (12) have also shown that P450 2E1 was induced in mouse lung after exposure to cigarette smoke. An important role for P450 2E1 has been postulated in the metabolic activation of the tobacco-specific procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1 (5, 13) as well as in the metabolism of the tobacco smoke component 3-hydroxypyridine (10). NNK was found to induce lung tumors in rats, mice, and hamsters, as well as liver, nasal, and pancreatic tumors in rats (1316). The occurrence of NNK-derived DNA and hemoglobin adducts in smokers indicated that NNK is also associated with tobacco-related lung cancers in humans (17-20). Isothiocyanates are cancer chemopreventive agents found primarily in cruciferous vegetables such as broccoli, cabbage, and watercress (21, 22). When these vegetables are chewed, the glucosynolated precursors are enzymatically processed by a thioglucoside glucohydrolase. The resulting intermediates rearrange to form isothiocyanates, hydrogen sulfate, and glucose (21, 22). Wattenberg demonstrated that benzyl- (BITC) and phenethylisothiocyanate (PEITC) inhibited the carcinogenic effects of 1 Abbreviations: pNP, p-nitrophenol; reductase, NADPH-cytochrome P450 reductase; BSA, bovine serum albumin; DLPC, dilauroylL-R-phosphatidylcholine; PCN, pregneolone-16R-carbonitrile; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; tBITC, tert-butylisothiocyanate; BITC, benzylisothiocyanate; PEITC, phenethylisothiocyanate; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; βNF, β-naphthoflavone; PB, phenobarbital; AHH-1, AHH-1 human lymphoblastoid cell line.

10.1021/tx980130+ CCC: $15.00 © 1998 American Chemical Society Published on Web 08/29/1998

Inactivation of 2E1 by tert-Butylisothiocyanate

polycyclic aromatic hydrocarbons (23). PEITC was shown to inhibit N-nitrosobenzylmethylamine-induced esophageal cancers in rats (24). Extensive studies have been conducted with PEITC and its effect on NNK-induced carcinogenesis. When PEITC was added to the diet before and during administration of NNK, a 50% reduction in lung tumors in F344 rats was observed (25). Furthermore, a decrease in the level of methyl- and pyridyloxobutyl-DNA adducts was found in the lungs, suggesting that the effect of PEITC was due to an inhibition of the metabolic activation of NNK (25). In lung microsomes, PEITC was found to be a competitive inhibitor of NNK metabolic activation with an IC50 of 150-210 nM (26). The rat liver microsomal N-nitrosodimethylamine demethylase activity could be inhibited by both BITC and PEITC with an IC50 of 8-9 µM (27). In this report, the inactivation of P450 2E1 by tertbutylisothiocyanate (tBITC) both in microsomes and in a reconstituted system was characterized. The findings indicated that this novel isothiocyanate inactivated P450 2E1 in a mechanism-based manner. In addition, tBITC was found to be more specific for inactivating P450 2E1 compared to other isozymes than the previously studied isothiocyantes.

Experimental Procedures Materials. Dilauroyl-L-R-phosphatidylcholine (DLPC), NADPH, BSA, p-nitrophenol (pNP), 4-nitrocatechol, erythromycin, β-naphthoflavone (βNF), pregnenolone-16R-carbonitrile (PCN), and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC), resorufin, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin were obtained form Molecular Probes Inc. (Eugene, OR), and 7-hydroxy-4-(trifluoromethyl)coumarin was from Enzyme Systems Products (Livermore, CA). HPLC-grade acetonitrile was from Fisher (Pittsburgh, PA) and trifluoroacetic acid from Pierce (Rockford, IL). Isothiocyanates were purchased from Trans World Chemicals (Rockville, MD). P450 2B6 and 3A4 microsomes from AHH-1 human lymphoblastoid cells were obtained from Gentest (Woburn, MA). Purification of P450 and Reductase. Rat NADPHcytochrome P450 reductase (reductase) was purified from Escherichia coli as previously described (28). Shortened bacterially expressed rabbit P450 2E1 and rabbit P450 2E1 T303A were purified as described by Larson et al. (29) with minor modifications. Bacterial membranes were solubilized in 0.7% Tergitol NP10, 1 mM EDTA, 40 µM 4-methylpyrazole, and 20% glycerol in 10 mM potassium phosphate buffer (pH 6.4). After SSepharose chromatography, the P450-containing fractions were applied to a hydroxyapatite (Bio-Rad, Hercules, CA) column equilibrated with 10 mM potassium phosphate buffer (pH 7.7), 0.2% Tergitol NP10, 50 µM 4-methylpyrazole, and 20% glycerol (equilibration buffer). The column was washed with equilibration buffer containing 30 mM potassium phosphate. P450 2E1 was eluted with equilibration buffer containing 400 mM potassium phosphate. Samples were dialyzed overnight against 10 mM potassium phosphate (pH 7.7), 0.1 mM EDTA, 50 µM 4-methylpyrazole, 0.5% Tergitol NP10, and 20% glycerol. The dialyzed protein was applied to a DEAE-Sepharose column equilibrated with 3 mM potassium phosphate (pH 7.7), 0.1 mM EDTA, 50 µM 4-methylpyrazole, 0.5% Tergitol NP10, and 20% glycerol. P450 2E1 was eluted with a gradient of 10 to 100 mM potassium phosphate buffer (pH 7.7) containing 0.1 mM EDTA, 50 µM 4-methylpyrazole, 0.5% Tergitol NP10, and 20% glycerol. The detergent was removed from the P450 samples by chromatography over a hydroxyapatite column equilibrated with 10 mM potassium phosphate buffer (pH 7.4), containing 50 µM 4-methylpyrazole and 20% glycerol. P450 2E1 was eluted with 500

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1155 mM potassium phosphate (pH 7.4), containing 50 µM 4-methylpyrazole and 20% glycerol, and dialyzed against 100 mM potassium phosphate (pH 7.4), 1 mM EDTA, and 20% glycerol. Microsome Preparation. Microsomal membranes were prepared from the livers of fasted male Fischer rats (175-190 g, Harlan Sprague-Dawley, Indianapolis, IN) according to Saito and Strobel (30). P450 2E1 was induced by ip injection of 100 mg/kg pyridine in water for 3 days. P450 2B1 was induced by ip injection of 100 mg/kg phenobarbital in water for 3 days. P450 1A1 and 1A2 were induced by ip injection of 80 mg/kg β-naphthoflavone in corn oil for 3 days. P450 3A2 was induced by ip injection of 50 mg/kg PCN in corn oil for 3 days. Enzyme Activity Assays. Microsomal P450 2E1 activity was determined using pNP as a substrate (31). Each primary reaction mixture contained 7 µM P450 from microsomes of pyridine-induced rats in 100 mM potassium phosphate (pH 6.8), 4, 8, 16, or 32 µM isothiocyanate (in 1 µL of CH3OH/100 µL), and 1.2 mM NADPH. To control samples was added 1 µL of CH3OH. After incubation for 0 or 5 min with NADPH, the P450 2E1 activity was measured by transferring 50 µL of the primary reaction mixture into 450 µL of a secondary reaction mixture containing 1 mM pNP, 10 mM ascorbate, 100 mM potassium phosphate buffer (pH 6.8), and 1 mM NADPH. The samples were incubated for 10 min at 30 °C, and the reactions were stopped with 100 µL of 1.5 M perchloric acid. The catechol product was quantitated spectrophotometrically at 490 nm. Microsomal P450 1A1 activity was measured with 7-ethoxyresorufin as the substrate, and P450 1A2 activity was measured with 7-methoxyresorufin as the substrate (32, 33). Each primary reaction mixture contained 10 µM P450 from microsomes of βNF-treated rats in 50 mM Tris-HCl (pH 7.5), 50 mM MgCl2, 0.1 or 1 mM tBITC (in 1 µL of CH3OH/100 µL), and 1.2 mM NADPH. To control samples was added 1 µL of CH3OH/100 µL. After incubation for 0 or 5 min at 30 °C with NADPH, the P450 1A1 or P450 1A2 activity was measured by transferring 20 µL of the primary reaction mixture into 980 µL of a secondary reaction mixture containing either 5 µM 7-ethoxyresorufin or 7-methoxyresorufin, 50 mM Tris-HCl (pH 7.5), and 0.2 mM NADPH. The increase in fluorescence was monitored at room temperature on a SLM-Aminco model SPF-500 C spectrofluorometer with excitation at 522 nm and emission at 586 nm. Microsomal P450 3A2 activity was determined using erythromycin as a substrate. N-Demethylation of erythromycin was quantified by measuring the formation of formaldehyde (34). Each primary reaction mixture contained 4.1 µM P450 from microsomes of PCN-induced rats in 50 mM potassium phosphate (pH 7.4), 0.1 or 1 mM tBITC (in 1 µL of CH3OH/100 µL), and 1.2 mM NADPH. To control samples was added 1 µL of CH3OH/100 µL. After incubation with NADPH for 0 or 5 min, the P450 3A2 activity was measured by transferring 20 µL of the primary reaction mixture into 480 µL of a secondary reaction mixture containing 1 mM erythromycin, 50 mM potassium phosphate buffer (pH 7.4), and 1 mM NADPH. After incubation for 10 min at 30 °C, the reactions were stopped by adding 250 µL of 60% trichloroacetic acid, and the amount of formaldehyde that was generated was measured spectrophotometrically according to Nash at 412 nm (34). The amount of formaldehyde produced was quantitated from a standard curve. Microsomal P450 2B1 activity was assayed with 7-(benzyloxy)resorufin as the substrate (35). Each primary reaction mixture contained 1.5 µM P450 from microsomes of phenobarbital-treated rats in 50 mM potassium phosphate buffer (pH 7.4), 0, 0.004, 0.008, 0.016, 0.032, 0.1, or 1 mM tBITC, and 1.2 mM NADPH. After incubation with NADPH for 0 or 5 min at 30 °C, 25 µL aliquots were transferred to 975 µL of a secondary reaction mixture containing 5 µM (benzyloxy)resorufin and 0.2 mM NADPH in 50 mM Tris-HCl buffer (pH 7.4). The reaction was quenched after 3 min with 750 µL of CH3OH. The resorufin product was measured spectrofluorometrically with excitation at 522 nm and emission at 586 nm.

1156 Chem. Res. Toxicol., Vol. 11, No. 10, 1998 P450 2B6 activity was determined using 7-EFC as a substrate (36). Each primary reaction mixture contained 160 nM P450 2B6 in 50 mM potassium phosphate buffer (pH 7.4), 0, 0.1, or 1 mM tBITC, and 1.2 mM NADPH. After incubation for 0 or 5 min at 30 °C, 25 µL (4 pmol) was transferred into 975 µL of a secondary assay mixture, and the 7-EFC activity was measured as previously described (37). P450 3A4 activity was measured with erythromycin as a substrate (34). Each primary reaction mixture contained 250 nM P450 3A4, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.5), 20% glycerol, 0.5 mM EDTA, 30 mM MgCl2 (microsomal suspension buffer), 0, 0.1, or 1 mM tBITC, and 1.2 mM NADPH. After incubation with NADPH for 0 or 5 min at 37 °C, the P450 3A4 activity was measured by transferring 40 µL (10 pmol) into 460 µL of a secondary reaction mixture containing 1 mM erythromycin and 1 mM NADPH in the microsomal suspension buffer. After incubation for 30 min at 37 °C, 250 µL of the NASH reagent (34) was added, and the reaction mixture was heated at 60 °C for 10 min. The amount of formaldehyde that was generated was measured by fluorescence with excitation at 410 nm and emission at 510 nm (38). For the determination of the kinetic parameters, the P450 2E1 activity was measured with pNP as a substrate using HPLC and electrochemical detection of the catechol product (39). For microsomal P450 2E1, each primary reaction mixture contained 0.15 mg of the microsomal protein (0.8 nmol of P450/mL), 100 mM potassium phosphate buffer (pH 6.8), 1 mM NADPH, and 1 µL of tBITC in CH3OH in a total volume of 250 µL. At the indicated times, 25 µL (20 pmol) of the primary reaction mixture was transferred into 475 µL of a secondary reaction mixture containing 0.2 mM pNP, 100 mM potassium phosphate buffer (pH 6.8), 5 mM MgCl2, 0.5 mM NADPH, and 40 µg of BSA/mL. The samples were then incubated for 15 min at 30 °C, and the reactions were quenched with 25 µL of trifluoroacetic acid. For purified reconstituted P450 2E1, each primary reaction mixture contained either 60 pmol of 2E1, 60 pmol of 2E1 T303A, or 60 pmol each of 2E1 and b5, 120 pmol of reductase, 20 µg of lipid, 50 units of catalase, tBITC in 1 µL of CH3OH, and 1.5 mM NADPH in a total volume of 150 µL. To control samples was added 5 µM tBITC in 1 µL of solvent and an equal volume of H2O instead of NADPH. At 0, 1, 2, 3, 4, 6, and 8 min, 25 µL (6 pmol of P450 2E1) of the primary reaction mixture was transferred into a secondary reaction mixture and the mixture incubated as described above for microsomes. Between 10 and 20 µL of each sample was injected onto a C18 reversed-phase column equilibrated with 25% CH3CN containing 0.1% trifluoroacetic acid. The flow rate was 1.2 mL/min, and the catechol product was detected electrochemically. The 7-EFC activity of purified reconstituted P450 2E1 was measured essentially as previously described for 2B1 (37). P450 2E1, reductase, and in some assays cytochrome b5 were reconstituted with lipid for 1 h at 4 °C. Primary incubation mixtures contained 1 nmol of P450 2E1, 1 nmol of reductase, 200 µg of DLPC, 250 units of catalase, tBITC (in 1 µL of CH3OH/mL) or 1 µL/mL CH3OH, and 1.2 mM NADPH in a total reaction volume of 1 mL of 150 mM potassium phosphate buffer (pH 7.4) containing 40 µg of BSA/mL. The 7-EFC activity was measured in a secondary reaction mixture as previously described, except that the samples were incubated for 15 min at 30 °C (37). Partition Coefficient. For the estimation of the partition coefficient, each primary reaction mixture contained P450 2E1 (1 µM), reductase (1 µM), 200 µg of DLPC/mL, 250 units of catalase/mL, 50 mM potassium phosphate buffer (pH 7.4), 1.2 mM NADPH, 40 µg of BSA/mL, and 2-46 µM tBITC (in 1 µL of CH3OH/150 µL). The samples were incubated for 10 min at 30 °C, and the residual 7-EFC activity was determined by transferring 20 µL aliquots into a secondary reaction mixture as described above. Substrate Protection. Substrate protection from tBITCdependent inactivation of P450 2E1 was tested by including 5 µM tBITC together with 7-ethoxycoumarin at molar ratios of 1:0, 1:2, and 1:20 tBITC:7-ethoxycoumarin in the primary

Kent et al. Table 1. Effect of Isothiocyanates on the pNP Activity of P450 2E1 Microsomesa % activity remaining isothiocyanate tert-butyl

isobutyl

benzyl

benzhydryl

concentration (µM)

0 min

5 min

0 4 8 16 32 0 4 8 16 32 0 4 8 16 32 0 4 8 16 32

100 97 77 58 21 100 90 73 58 41 100 77 56 39 29 100 106 92 90 68

89 74 53 29 18 94 74 74 52 43 87 57 44 32 20 92 85 81 58 44

a Assay conditions were as described in Experimental Procedures. The values shown represent the means from three or four experiments.

reaction mixture. At the indicated times, duplicate 25 µL aliquots were removed and assayed for pNP activity as described above for the purified enzyme. Effect of Exogenous Nucleophiles. The 7-EFC activity of reconstituted P450 2E1 was measured as described above except that some of the primary reaction mixtures also contained 1 mM DTT, 1 mM glutathione, or 1 mM methylamine together with 20 µM tBITC. Spectrophotometric Quantitation of 2E1. P450 2E1 and reductase were reconstituted with lipid for 1 h at 4 °C. The primary reaction mixture contained 1 µM 2E1, 1 µM reductase, 20 µg of DLPC, 125 units of catalase, 1 mM tBITC (in 5 µL of CH3OH/500 µL), and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 500 µL. Reactions were initiated with 1.2 mM NADPH. To control samples was added an equal volume of H2O instead of NADPH. After 10 min at 30 °C, 10 µL aliquots of the primary reaction mixtures were assayed for 7-EFC activity, and 100 µL of the primary reaction mixture was diluted with 900 µL of ice cold 50 mM potassium phosphate (pH 7.4), containing 40% glycerol and 0.6% Tergitol NP-40 (quench buffer). Dithionite and CO were added, and the reduced carbonyl spectra were recorded from 400 to 500 nm on a DW2 UV/VIS spectrophotometer (SLM Aminco, Urbana, IL) equipped with an OLIS spectroscopy operating system (On-Line Instrument Systems, Inc., Bogart, GA) (40). HPLC Analysis of P450 2E1. HPLC analysis of the native and modified proteins was performed as previously described (41).

Results Effect of Isothiocyanates on P450 2E1 Activity in Microsomes. Several isothiocyanates were tested for their ability to inactivate the pNP activity of cytochrome P450 2E1 in microsomes from pyridine-induced rats. Of the compounds tested, propyl-, isopropyl-, isobutyl-, and benzylisothiocyanate have been identified in cruciferous vegetables such as cabbage, broccoli, and horseradish (42). As shown in Table 1, tBITC was the most effective compound tested that inactivated P450 2E1 in a timedependent manner. A concentration as low as 4 µM resulted in 26% inactivation within 5 min. More com-

Inactivation of 2E1 by tert-Butylisothiocyanate

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1157

Table 2. Effect of tBITC on Microsomal P450sa microsomes major P450

substrate

PCN PB

3A2 2B1

erythromycin 7-(benzyloxy)resorufin

AHH-1

2B6

7-EFC

[tBITC] (mM) % inactivation 1.0 0.1

10 34

1.0 0.1 1.0

83 65 77

a Assay conditions were as described in Experimentl Procedures. The values shown represent the means from two or three different experiments.

plete inactivation was seen at higher concentrations of tBITC. However, at a concentration of 8 µM, a significant decrease in the pNP activity at 0 min was also observed. This decreased activity was probably due to a competitive inhibition of pNP metabolism by the tBITC carried over into the secondary reaction mixture. The final concentrations of tBITC in the secondary reaction mixtures were 0.4, 0.8, 1.6, and 3.2 µM, respectively. Two other naturally occurring isothiocyanates, isobutylisothiocyanate and benzylisothiocyanate, were also tested. Timedependent inactivation was observed with both compounds at 4 µM. However, at higher concentrations of both isothiocyantes, the competitive inhibition of the pNP activity by the isothiocyanate carried over into the secondary reaction prevented observation of any inactivation. Benzhydrylisothiocyanate was also found to inactivate the P450 2E1 activity in microsomes at concentrations above 16 µM. Incubations with isopropylor propylisothiocyanate (32 µM) resulted in losses of activity of 28 and 26%, respectively (data not shown). No inactivation of P450 2E1 activity was seen in experiments with 4-32 µM crotonyl-, naphthyl-, 2-biphenylyl-, or 1,2diphenylethylisothiocyanate (data not shown). Effect of tBITC on the Activities of Other P450s. The effect of tBITC on the activities of P450 1A1, 1A2, 3A2, 2B1, 2B6, and 3A4 was examined (Table 2). No inactivation of P450 1A1, 1A2, or 3A4 was observed at concentrations of 0.1 or 1 mM tBITC. The P450 3A2 activity in microsomes was not affected by 0.1 mM tBITC, but a 10% reduction in the erythromycin demethylation activity occurred with 1 mM tBITC. P450 2B1 was not inactivated at the low micromolar tBITC concentrations (0-32 µM) that inactivated P450 2E1. However, at 0.1 mM tBITC a 34% decrease and at 1 mM a 83% decrease in the benzyloxyresorufin O-deethylation activity were observed. Similarly, the 7-EFC O-deethylation activity of human P450 2B6 was inactivated with 0.1 and 1 mM tBITC by 65 and 77%, respectively. These results indicate that tBITC was a more specific inactivator for P450 2E1 than the other P450s that were tested. Inactivation of P450 2E1 by tBITC in Microsomes. The inactivation of rat liver P450 2E1 in microsomes with tBITC was time- and concentration-dependent (Figure 1). Pseudo-first-order kinetics were observed between 2 and 20 µM tBITC. The kinetic constants shown in Table 3 for the inactivation of rat microsomal P450 2E1 were determined by plotting the reciprocals of the initial rates of inactivation as a function of the reciprocals of the tBITC concentrations as shown in the inset of Figure 1. The maximal rate of inactivation at saturation (kinactivation) was 0.72 min-1. The concentration required for the halfmaximal rate of inactivation (KI) was 11 µM, and the time required for one-half of the enzyme molecules to become inactivated (t1/2) was 1.0 min at 30 °C.

Figure 1. Time- and concentration-dependent inactivation of P450 2E1 pNP activity in pyridine-induced microsomes by tBITC. Duplicate samples were removed from the primary reaction mixtures at the indicated time points and assayed for pNP activity as described in Experimental Procedures. The concentrations of tBITC were (0) 0, ([) 2, (O) 4, (2) 8, (4) 12, (b) 16, and (]) 20 µM. The inset shows the double-reciprocal plot of the rates of inactivation of the pNP hydroxylation activity as a function of the tBITC concentration. Data points represent averages from two separate experiments. Table 3. Kinetic Constants for the Inactivation of P450 2E1 with tBITC in Microsomes and in the Reconstituted Systema system rat microsomes rabbit 2E1 rabbit 2E1 T303A rabbit 2E1 and b5

kinetic constant

pNP assay

7-EFC assay

KI kinactivation t1/2 KI kinactivation t1/2 KI kinactivation t1/2 KI kinactivation t1/2

11 µM 0.72 min-1 1.0 min 7.6 µM 0.27 min-1 2.6 min 6.6 µM 0.28 min-1 2.5 min 14 µM 0.38 min-1 1.9 min

ndb nd nd 9.5 µM 0.25 min-1 2.8 min nd nd nd 9.1 µM 0.18 min-1 3.9 min

a Assay conditions and the kinetic analyses were as described in Experimental Procedures. The kinetic constants were determined from the rates of inactivation from duplicate samples of two or three separate experiments. b nd, not determined.

Inactivation of Purified Reconstituted P450 2E1 by tBITC. Although P450 2E1 activity is the major activity observed with pNP in microsomes from pyridineinduced rats, the possibility that other microsomal proteins might contribute to the metabolism of tBITC could not be ruled out. Therefore, the effect of tBITC on purified rabbit P450 2E1 in a reconstituted system was examined. tBITC inactivated purified P450 2E1 in a time- and concentration-dependent manner (data not shown). The kinetic constants were determined from the rates of inactivation with 1, 2, 4, 6, 8, 10, and 12 µM tBITC as described above for microsomes. The KI was 7.6 µM, the kinactivation 0.27 min-1, and the t1/2 2.6 min (Table 3). Inactivation of Purified Reconstituted P450 2E1 T303A by tBITC. Previous observations suggested that T303 plays a crucial role in the proton donation step (43). This residue was also implicated in the inactivation of P450 2B4 with 2-ethynylnaphthalene (44). Therefore, the effect of this amino acid substitution on the ability of the mutant enzyme to be inactivated by tBITC was tested. The rates of inactivation of the P450 2E1 T303A mutant were determined with 1, 2, 4, 6, 8, 10, and 12

1158 Chem. Res. Toxicol., Vol. 11, No. 10, 1998

µM tBITC. The kinetic constants for the inactivation of 2E1 T303A with tBITC were similar to those seen with the wild type enzyme, suggesting that T303 in P450 2E1 does not play a role in the mechanism of tBITC inactivation. The KI was 6.6 µM, the kinactivation 0.28 min-1, and the t1/2 2.5 min (Table 3). Effect of b5 on the Kinetics of Inactivation of P450 2E1 with tBITC. Cytochrome b5 has been shown to enhance the activity of P450 2E1 with certain substrates (45). Furthermore, anti-b5 immunoglobulin inhibited P450 2E1 activity in human microsomes (46). Therefore, purified P450 2E1 was reconstituted together with b5 and reductase, and the kinetics of inactivation by tBITC were analyzed. The inactivation of the P450 2E1 pNP activity with tBITC was again time- and concentration-dependent (data not shown). Pseudo-first-order kinetics were observed between 10 and 40 µM tBITC. The rates of inactivation did not increase above 50 µM tBITC (data not shown). The kinetic constants for P450 2E1 inactivation in the presence of b5 were determined as described previously. The KI was 14 µM, the kinactivation 0.38 min-1, and the t1/2 1.9 min (Table 3). With b5, the KI was more similar to that observed for tBITC inactivation of P450 2E1 in microsomes. Effect of tBITC on the 7-EFC O-Deethylation Activity of P450 2E1 and P450 Heme Content. pNP has been used as the substrate of choice to measure P450 2E1 activity in microsomes (31), although recently it was shown that P450 3A enzymes also O-hydroxylate pNP (47). The conventional spectrophotometric pNP assay suffers from a lack of sensitivity. Therefore, a HPLC assay that detects the catechol product electrochemically has been described (39). However, this HPLC assay is rather time-consuming. For the subsequent studies of the reduced CO complex, it was desirable to determine the degree of inactivation immediately prior to these spectrophotometric analyses. Therefore, the P450 2E1 activity was evaluated with 7-EFC as a substrate. Kinetic analyses were carried out to confirm that 7-EFC was a suitable substrate for the purified reconstituted system, that it was metabolized, and that 7-EFC metabolism was inactivated by tBITC with KI and kinactivation values similar to those seen with pNP. With purified P450 2E1, the KI for tBITC was 9.5 µM, the kinactivation 0.25 min-1, and the t1/2 2.8 min when the 7-EFC assay was used to assess inactivation (Table 3). When P450 2E1was reconstituted together with b5, the 7-EFC assay gave a KI for tBITC of 9.1 µM, a kinactivation of 0.18 min-1, and a t1/2 of 3.9 min (Table 3). These values were reasonably similar to those seen with pNP and indicated that 7-EFC could be used as a substrate with the purifed enzyme system to monitor the loss of P450 2E1 enzymatic activity. When P450 2E1 was inactivated by incubation with 1 mM tBITC for 10 min, a 75% decrease in the 7-EFC O-deethylation activity was observed (Table 4). No significant loss of activity was observed in samples incubated only with tBITC but without NADPH. This demonstrated the absolute requirement for a catalytic step, since inactivation was only seen in the presence of both NADPH and tBITC. Concurrent with the 75% loss in activity, there was a 63% loss of heme as measured by the reduced CO difference spectrum. Control samples incubated with tBITC only, as well as samples inactivated with tBITC in the presence of NADPH, were analyzed by reversed-phase chromatography. The P450

Kent et al. Table 4. Effect of tBITC on P450 2E1 Activity, P450 Concentration, and the Amount of Heme Remaininga P450 concentration heme recovered 0 min 10 min (µM) (area units × 106) % activity

without NADPH with NADPH

100 88

94 25

0.83 0.31

10.4 9.4

a Assay conditions were as described in Experimental Procedures. The P450 concentration was measured by reduced CO difference spectroscopy. Heme recovery was determined by integrating the area under the heme peak of the HPLC elution profile at 405 nm.

Figure 2. Loss of P450 2E1 7-EFC O-deethylation activity as a function of the ratio of tBITC:2E1. Duplicate samples were removed and assayed as described in Experimental Procedures. Each data point represents the average from two separate experiments. The extrapolated partition ratio was determined from the intercept of the linear regression line derived from the lower tBITC concentrations and the straight line obtained from the higher ratios.

heme in both samples eluted at the same time (data not shown), and the recovery of the intact heme from the inactivated sample was similar to that of the control (90%). These observations suggested that the tBITCinactivated P450 2E1 was unable to form a reduced CO complex. However, the heme moiety of the inactive sample was not modified or the heme adduct was not stable to the acidic HPLC conditions. Partition Ratio. The number of molecules of inactivator metabolized per molecule of enzyme inactivated was estimated as previously described (37). Purified reconstituted P450 2E1 was incubated with different concentrations of tBITC, and the reactions were allowed to reach completion. The turnover number was determined by plotting the percent activity remaining as a function of the molar ratio of tBITC:P450 2E1 (Figure 2). The extrapolated turnover number was determined from the intercept of the linear regression line derived from the lower tBITC concetrations with the straight line obtained from the higher ratios of tBITC:P450 2E1. The partition ratio was 7. Substrate Protection. Simultaneous incubations of 5 µM tBITC with increasing concentrations of 7-ethoxycoumarin in the primary reaction mixture decreased the rate of P450 2E1 inactivation by tBITC (Figure 3). These observations suggest that tBITC inactivated P450 2E1 rather than reductase and that the inactivation was most likely due to the binding of a tBITC metabolite to the P450 2E1 active site.

Inactivation of 2E1 by tert-Butylisothiocyanate

Figure 3. Substrate protection against P450 2E1 inactivation by 5 µM tBITC. Duplicate samples were removed from the reaction mixtures at the indicated times and assayed as described in Experimental Procedures. Each point represents the average from two separate experiments. The primary reaction mixtures contained molar ratios of tBITC:7-ethoxycoumarin of (0) 0:0, (b) 1:0, (4) 1:2, and ([) 1:20.

Figure 4. Effect of exogenous nucleophiles on the rate of inactivation of P450 2E1 by tBITC. P450 2E1 was incubated with (b) 20 µM tBITC, (1) 20 µM tBITC and 1 mM DTT, (]) 20 µM tBITC and 1 mM glutathione, or (0) without any additions and assayed for residual 7-EFC activity as described in Experimental Procedures.

Effect of Exogenous Nucleophiles. The effect of exogenous nucleophiles on the rate of inactivation of P450 2E1 by tBITC was investigated by simultaneously incubating P450 2E1 with 20 µM tBITC and 1 mM DTT or 1 mM glutathione. Figure 4 shows that DTT or glutathione slowed the rate of inactivation of P450 2E1 by tBITC, but did not prevent inactivation by tBITC as had been observed with other isothiocyanates where a glutathioneisothiocyanate complex was formed.2

Discussion P450 2E1 activates a number of xenobiotics to toxins and carcinogens. This detrimental activity can be further enhanced by induction of P450 2E1 with cigarette smoke or chronic ethanol ingestion. Therefore, specific P450 2E1 inhibitors or inactivators could be beneficial agents for preventing the metabolic activation of these xenobiotics. 2

T. Goosen, manuscript in preparation.

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Several inhibitors of P450 2E1 such as the drug disulfiram, the sedative and anticonvulsant chlormethiazole, and dietary compounds such as PEITC from watercress, BITC from broccoli, and diallylsulfide from garlic have been identified (48-50). Chlormethiazole was found to be a noncompetitive inhibitor of chlozoxazone hydroxylation in humans with a KI of 12 µM (49). Diallylsulfide was shown to inhibit the pNP hydroxylase activity in microsomes with a KI of 188 µM and a maximal rate of inactivation of 0.31 min-1. The inactivation was both competitive and time-dependent. Isothiocyanates have been shown to inhibit P450 2B1, 2A1, 1A1, 1A2, and 2E1 activities (51). Conaway et al. (52) reported an IC50 of 1.8 µM for the PEITC inhibition of the pentoxyresorufin activity of P450 in liver microsomes from phenobarbital-induced rats. PEITC was also shown to inhibit P450 1A2 primarily in a competitive manner (53). P450 1A2 appears to be the major human liver enzyme that is responsible for the bioactivation of cigarette smoke carcinogens (54) In this report, we describe the mechanism-based inactivation of P450 2E1 by tBITC. Initially, several isothiocyanates were tested in an attempt to find a P450 2E1-specific mechanism-based inactivator. Except for tBITC and BITC, most of the isothiocyanates either showed little inactivation or exhibited significant inhibition without a further time-dependent loss in activity. tBITC was more selective in inactivating the enzymatic activity of P450 2E1 compared to other P450s that were studied. Little or no inactivation was seen for P450 1A1/ 2, 3A2, and 3A4 at 0.1 and 1 mM tBITC. Inactivation of P450 2B isoforms was observed at concentrations of g0.1 mM. In the reconstituted system, BITC and PEITC inactivated P450 2E1 with kinetics similar to those of tBITC.3 The KI for PEITC inactivation of reconstituted P450 2E1 was 3 µM, and the kinactivation was 0.14 min-1 (data not shown). Furthermore, the KI for inactivating P450 2B1 in the reconstituted system with BITC or PEITC was similar to that observed for 2E1 inactivation.2 These observations indicate that although BITC and PEITC are as effective as tBITC in inactivating P450 2E1, tBITC is more selective for P450 2E1 since higher concentrations were required to inhibit the P450 2B1 activity. The inactivation of P450 2E1 was time- and concentration-dependent and required both NADPH and tBITC. Linear pseudo-first-order kinetics were observed for P450 2E1 activity in microsomes or the reconstituted system with either pNP or 7-EFC as a substrate. The observed KI values were all in the low micromolar range and similar to the IC50 values reported by Jiao et al. (27) for PEITC (8.3 µM) and BITC (9 µM) inhibition in rat and human liver microsomes. Reduced rates of inactivation of P450 2E1 were observed when 7-ethoxycoumarin was incubated together with tBITC. The ability of 7-ethoxycoumarin to protect P450 2E1 suggested that tBITC inactivated P450 2E1 by binding to the active site of the P450 and not to the reductase. Adding fresh reductase to dialyzed inactivated P450 2E1 samples did not restore the P450 activity, further indicating that the effect of tBITC was on P450 2E1 (data not shown). A decreased rate of inactivation in the presence of exogenous nucleophiles has been interpreted as being due 3

R. Garcia, manuscript in preparation.

1160 Chem. Res. Toxicol., Vol. 11, No. 10, 1998

to the ability of the nucleophile to trap reactive intermediates that escape from the active site. However, in the case of isothiocyanates, the formation of a complex between the sulfur moiety of the isothiocyanate and sulfhydryl-containing reagents has been demonstrated.2 Therefore, the decrease in the rate of inactivation by tBITC in the presence of sulfur-containing nucleophiles may be indicative of a requirement for the sulfur in the inactivation process by tBITC. Additional support that the reactive intermediates formed from tBITC bind primarily to the active site of P450 2E1 came from observations of a low partition ratio and the ability of alternate substrates to protect P450 2E1 from inactivation. Conclusive evidence for a 1:1 stoichiometry of reactive tBITC intermediate:P450 will require synthesis of radiolabeled tBITC. These studies are in progress. Including 1 mM methylamine together with tBITC had no effect on the rate of inactivation, suggesting that at the pH and with the tBITC concentrations used in these studies a modification of amine side chains was unlikely. Initial spectral studies indicated that the inactivation of P450 2E1 by tBITC in the presence of NADPH resulted in a modified 2E1 protein that was unable to form a reduced CO complex. Several studies with mechanismbased inactivators of P450s have resulted in a loss of the P450 reduced CO spectrum. This has generally been caused by heme alkylation (41, 55, and references therein). Alkylated hemes would be expected to elute with retention times different from those of the unmodifed heme under the reversed-phase HPLC conditions used in this study; however, no significant difference in the elution time or the recovery of the heme from tBITCinactivated samples compared to control was observed. This suggested either that the heme was not modified or that the modification was not stable to the acidic HPLC conditions. The further characterization of the role of tBITC in the loss of the P450 2E1 activity and reduced CO spectrum is under investigation.

Acknowledgment. This work was supported in part by NIH Grant CA 16954 (P.F.H.).

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