Inactivation of Cytochrome P450 2B1 by Benzyl ... - ACS Publications

Chem. Res. Toxicol. 2000, 13, 1349-1359

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Inactivation of Cytochrome P450 2B1 by Benzyl Isothiocyanate, a Chemopreventative Agent from Cruciferous Vegetables Theunis C. Goosen,†,‡ Ute M. Kent,‡ Linda Brand,† and Paul F. Hollenberg*,‡ Department of Pharmacology, Potchefstroom University for Christian Higher Education, Potchefstroom 2520, South Africa, and Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109 Received June 28, 2000

A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities. The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a timeand concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl and phenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitors of P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivated in a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for halfmaximal inactivation (KI) was 5.8 µM, and the maximal rate constant for inactivation was 0.66 min-1 at 23 °C. BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost within 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption spectrum of the inactivated protein did not decrease significantly, and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC and protein precipitation analyses indicated that the P450 apoprotein was covalently modified by a metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 ( 0.16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1 and that inactivation occurs primarily through protein modification.

Introduction 1

Mammalian cytochrome P450 (P450) monooxygenases comprise a superfamily of membrane-bound hemeproteins which catalyze the metabolism of a wide variety of endogenous and exogenous compounds, including steroids, therapeutic drugs, and carcinogens (1, 2). The resultant increases in polarity usually facilitate excretion and are considered to be a detoxification process, but in some instances, foreign compounds are converted to * To whom correspondence should be addressed: Department of Pharmacology, Medical Science Research Bldg. III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: [email protected]. Phone: (734) 764-8166. Fax: (734) 763-5387. † Potchefstroom University for Christian Higher Education. ‡ The University of Michigan. 1 Abbreviations: BaP, benzo[a]pyrene; BCA, bicinchonic acid; BITC, benzyl isothiocyanate; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; DLPC, l-R-phosphatidylcholine dilauroyl; DTNB, 5,5′dithiobis(2-nitrobenzoate); DTT, dithiothreitol; 7-EC, 7-ethoxycoumarin; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; 7-HFC, 7-hydroxy-4(trifluoromethyl)coumarin; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone; P450, cytochrome P450; P450 2B1, major P450 from liver microsomes of phenobarbital-treated rats; PB, phenobarbital; PEITC, 2-phenethyl isothiocyanate; PHITC, 6-phenylhexyl isothiocyanate; PPITC, 3-phenylpropyl isothiocyanate; reductase, NADPH-cytochrome P450 reductase; TFA, trifluoroacetic acid.

products with much greater cytotoxicity, mutagenicity, or carcinogenicity (3). In some cases, the formation of a reactive intermediate by P450 may also lead to the inactivation of the enzyme. P450 substrates, which are metabolized to reactive intermediates that inactivate the enzyme, are classified as mechanism-based inactivators (4). Several methods have been used to elucidate regions of the protein critical for substrate binding and catalysis (5-8). Since some mechanism-based inactivators are able to covalently modify the P450 apoprotein, these inactivators may be used as probes for identifying important active-site amino acid residues (9, 10). Several sulfurcontaining compounds have been shown to function as mechanism-based inactivators by binding to the P450 apoprotein, including tienilic acid, spironolactone, diallyl sulfide, and parathion (11-14). Isothiocyanates (Figure 1) are potent and selective inhibitors of carcinogenesis induced by a variety of chemical carcinogens (15-18). The inhibition of P450 enzymes, in addition to induction of certain phase II metabolic enzymes, has been implicated in the chemopreventative action of isothiocyanates (16, 21). In most studies, the chemopreventative activity of isothiocyanates

10.1021/tx000133y CCC: $19.00 © 2000 American Chemical Society Published on Web 11/18/2000

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Figure 1. Structures of the isothiocyanates tested as inactivators of purified P450 2B1. (f) Location of the 14C label on BITC.

required administration either before or during exposure to the carcinogen. However, Wattenberg (19) reported that benzyl isothiocyanate (BITC) was involved in tumor suppression when administered 1 week after carcinogen exposure by an undefined mechanism. The combination of BITC and phenethyl isothiocyanate (PEITC) has recently also been proposed for the chemoprevention of lung cancer in humans (20, 22). Isothiocyanates are released upon chewing or maceration of cruciferous vegetables, such as cabbage, cauliflower, broccoli, and watercress where they occur as thioglucoside conjugates called glucosinolates (23, 24). The enzyme myrosinase is released at the same time and hydrolyzes the glucosinolate with rearrangement of intermediates producing the isothiocyanates, hydrogen sulfate, and glucose as the major products. The administration of isothiocyanates to rodents produced either increases or decreases in P450 content and activity (2527), depending on the experimental conditions, the isothiocyanate being studied, the target tissue being examined, and the specific P450 being assessed. The inhibition of P450s by isothiocyanates could result from three distinct mechanisms. The first would be a purely competitive inhibition of substrate metabolism at the active site of P450 (25, 27). Second, depending on the reactivity of the isothiocyanate involved, it could react directly with nucleophilic residues that are important in substrate metabolism with a resultant loss in enzyme activity (28, 29). A third mechanism would involve the metabolic activation of isothiocyanates (27, 30), presumably to a reactive isocyanate intermediate (31), which could ultimately bind to the heme moiety or apoprotein, thereby inactivating the enzyme. Oxidative desulfuration of the isothiocyanate could also release elemental sulfur, which could further contribute to enzymatic modification and possibly inactivation, as in the case of parathion (14). In this study, the inhibition of P450 2B1 by naturally occurring and synthetic isothiocyanates is reported. This is the first report where the inactivation of P450 2B1, the major phenobarbital-inducible isoform from rat liver, by BITC was elucidated using the reconstituted system. The findings indicate that BITC inactivates P450 2B1 in a mechanism-based manner. This study also demonstrates that the P450 2B1 apoprotein is specifically and covalently modified in a stoichiometric manner by a reactive intermediate formed during BITC metabolism.

Experimental Procedures Materials. Phenobarbital (PB), l-R-phosphatidylcholine dilauroyl (DLPC), NADPH, GSH, Tergitol NP-10, bovine serum albumin (BSA), and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). Benzyl isothiocyanate (BITC), 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 6-phenylhexyl isothiocyanate (PHITC), 7-ethoxycou-

Goosen et al. marin (7-EC), DMSO, sodium dithionite, and potassium cyanide were purchased from Aldrich Chemical Co. (Milwaukee, WI). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was purchased from Molecular Probes Inc. (Eugene, OR), and 7-hydroxy-4(trifluoromethyl)coumarin (7-HFC) was from Enzyme Systems Products (Livermore, CA). Bicinchonic acid (BCA) reagent, trifluoroacetic acid (TFA), and Slide-A-Lyzer cassettes were from Pierce (Rockford, IL). Ultima Gold liquid scintillation cocktail was obtained from Packard (Meridien, CT), and dithiothreitol (DTT) was from Bio-Rad (Hercules, CA). Escherichia coli Topp3 cells were obtained from Stratagene (La Jolla, CA). [14C]BITC labeled at the R-carbon with a specific activity of 56 mCi/mmol and a chemical purity of >97% by HPLC was kindly provided by F.-L. Chung (American Health Foundation, Valhalla, NY). All other materials were reagent grade and obtained from commercial sources. Purification of P450 and Reductase. P450 2B1 was prepared from liver microsomes of fasted male Long Evans rats (175-190 g; Harlan Sprague-Dawley, Indianapolis, IN) according to the method of Saito and Strobel (32). These rats were treated with 0.1% PB in the drinking water for 12 days. The cDNA for rat NADPH-cytochrome P450 reductase within the expression plasmid pOR263 (33) was expressed in E. coli Topp3 cells. The rat liver P450 reductase was expressed and purified as described previously (34). Inactivation of P450 2B1 by Isothiocyanates. The deethylation of 7-EFC to 7-HFC (35) was used to assess the inactivation of purified P450 2B1 by selected isothiocyanates in a two-stage incubation protocol. Purified P450 2B1 and reductase were reconstituted with lipid for 1 h at 4 °C. The primary reaction mixture contained varying concentrations of isothiocyanate in DMSO, 0.5 µM P450 2B1, 0.5 µM reductase, 200 µg/mL DLPC, 210 units of catalase, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 0.1 mL. Solvent (2 µL of DMSO) was added to incubations with no isothiocyanate present. The primary mixture was incubated at 30 °C for 3 min before initiation of the reaction with 1.2 mM NADPH or water in reactions without NADPH. Zero and 5 min after addition of NADPH, 10 µL aliquots (5 pmol of P450 2B1) were taken from the primary reaction mixture and added to a secondary reaction mixture containing 100 µM 7-EFC, 40 µg of BSA, 0.2 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 1 mL. The secondary reaction was allowed to proceed for 5 min at 30 °C and quenched by the addition of 334 µL of ice-cold acetonitrile. The formation of the deethylation product was assessed spectrofluorometrically at room temperature on an SLM-AMINCO model SPF-500C spectrofluorometer with excitation at 410 nm and emission at 510 nm. For the determination of the kinetic parameters with BITC, the primary mixture was preincubated for 3 min at 23 °C before initiation of the reaction with 1.2 mM NADPH or water in reactions without NADPH. Twenty, 40, 60, 80, and 100 s after addition of NADPH, 10 µL aliquots (5 pmol of P450 2B1) were removed and added to a secondary reaction mixture for the determination of residual 7-EFC O-deethylation activity as described above. Substrate Protection. In studies involving protection from BITC-dependent inactivation of P450 2B1 by the competitive substrate 7-EC, the primary incubation mixtures were reconstituted as described above with 10 µM BITC and increasing concentrations of 7-EC (50-500 µM). Control reaction mixtures contained either solvent or 7-EC to study the effect of 7-EC alone on 7-EFC O-deethylation activity. At the indicated time points, 10 µL aliquots were removed for determination of 7-EFC O-deethylation activity as described above. This activity was compared to the activity remaining when no 7-EC was present in the primary reaction mixture. Partition Ratio. The partition ratio for the inactivation of P450 2B1 by BITC was determined by incubating the enzyme with increasing concentrations of BITC and allowing the inactivation to go to completion. The reconstitution was carried out as described above. Primary reaction mixtures were incu-

Inactivation of P450 2B1 by Benzyl Isothiocyanate bated for 12 min at 30 °C, and the activity remaining was determined by measuring the 7-EFC O-deethylation activity as previously described. The percent activity remaining was plotted as a function of the molar ratio of BITC to P450 2B1 to determine the turnover number (4). Spectrophotometric Quantitation of P450 2B1. The ability of CO to bind to reduced P450 and exhibit a characteristic absorption maximum at 450 nm (36) was used to study the loss of spectrally detectable P450 after inactivation of P450 2B1 by BITC. P450 2B1 and reductase were reconstituted with lipid for 1 h at 4 °C. The primary reaction mixture contained 1.0 µM P450 2B1, 1.0 µM reductase, 200 µg/mL DLPC, 110 units of catalase, 20 µM BITC (in DMSO) or solvent control, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 0.5 mL. Reactions were initiated with 1.2 mM NADPH or water to control samples. After 12 min at 30 °C, 5 µL aliquots (5 pmol of P450 2B1) of the primary reaction mixtures were assayed for 7-EFC activity as described above, and 200 µL of the primary reaction mixture was diluted with 800 µL of ice-cold quench buffer that contained 40% glycerol, 0.6% Tergitol NP-10, and 50 mM potassium phosphate (pH 7.4). The samples were reduced with dithionite and split into two cuvettes. The sample in the sample buffer cuvette was gently bubbled with CO for 60 s, and the reduced carbonyl difference spectrum was 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). The absolute spectrum of P450 2B1 was studied to determine the fate of the heme after inactivation. For the absolute spectral studies, reactions were initiated by addition of 1.2 mM NADPH to the reconstituted mixture in a final volume of 1.0 mL. Spectra were recorded between 350 and 600 nm using diluting buffer in the reference cuvette on a DW2-OLIS spectrophotometer in the split beam mode. Effect of External Nucleophiles. The 7-EFC O-deethylation assay described previously was used with minor modifications to follow the inactivation in the presence of external nucleophiles. The primary reaction mixtures contained 10 µM BITC and either 10 mM GSH or 50, 100, or 500 µM KCN (titrated to neutral pH with HCl). Control incubations, which contained the nucleophile but no BITC, were used to study the effect of the nucleophile itself on the 7-EFC O-deethylation reaction. The chemical reactivity between BITC and GSH was studied by incubating 10 µM BITC with 100 µM GSH in 1.0 mL of water. Difference spectra were recorded from 200 to 340 nm on a DW2 UV/vis spectrophotometer (SLM Aminco) after addition of BITC. Alternatively, the amount of free GSH thiols remaining after incubation with BITC was determined by the reaction of GSH with DTNB. For the determination of the total sulfhydryl group content, 50 µM GSH was dissolved in 50 mM Tris-HCl buffer (pH 7.4) and incubated with 0 or 50 µM BITC at 30 °C for 20 min in a final volume of 1.0 mL. DTNB dissolved in 50 mM sodium phosphate buffer (pH 7.4) was then added to a final concentration of 1.0 mM. The number of free sulfhydryl groups was calculated using the extinction coefficient of reduced DTNB at 412 nm (E412 ) 1.36 × 104 M-1 cm-1) (37). Irreversibility of the Inactivation of P450 2B1 by BITC. P450 2B1 (1.0 nmol) was reconstituted and inactivated with 20 µM BITC in the primary reaction mixture as described above. Control samples were incubated with BITC but without NADPH. After incubation for 12 min at 37 °C, the samples (1.0 mL) were dialyzed overnight at 4 °C in Slide-A-Lyzer cassettes against 3 × 500 mL of 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 0.1 mM EDTA. Some samples were dialyzed against the same buffer containing 1.0 mM DTT. An aliquot equivalent to 150 pmol of P450 2B1, as determined by absorbance at 418 nm, was removed after dialysis and reconstituted with 20 µg of lipid for 30 min at 4 °C. Some of the dialyzed samples also received fresh reductase (150 pmol) during the reconstitution after dialysis. Enzymatic activity was assayed with 7-EFC as described above.

Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1351 Stoichiometry of Inactivation. The stoichiometry of binding was determined using a reconstituted reaction mixture containing 2 µM P450 2B1, 2 µM reductase, 240 µg/mL DLPC, 40 µM [14C]BITC, 150 units of catalase, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 375 µL. The mixture was incubated at 30 °C for 3 min before initiation by the addition of 1.2 mM NADPH or water to the incubation without NADPH. The NADPH- and time-dependent loss of activity was determined at 0 and 20 min as described above. Samples were dialyzed in Slide-A-Lyzer cassettes against 3 × 500 mL of dialysis buffer as described above. Aliquots were removed, and the amount of radioactivity remaining after dialysis was determined by liquid scintillation counting. Protein recovery was determined using the BCA reagent (38). The stoichiometry of binding was calculated after subtracting the background counts from dialyzed samples incubated with [14C]BITC in the absence of NADPH. For protein precipitation studies, BSA was added as a carrier to give a final total protein concentration of 20 mg/mL and the reaction mixtures were precipitated using 5% H2SO4 in methanol on ice for 1 h followed by centrifugation at 16000g for 15 min and repetitive H2SO4/methanol washes to remove unbound radioactive label (39). The stoichiometry of binding was determined after subtracting background counts from the [14C]BITCincubated sample in the absence of NADPH. HPLC Analysis for Specificity of Binding. A reaction mixture containing 1.0 µM P450 2B1, 1.0 µM reductase, 200 µg/mL DLPC, 20 µM [14C]BITC, 90 units of catalase, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 0.5 mL was incubated at 30 °C for 3 min before initiation by the addition of 1.2 mM NADPH or water to the incubation without NADPH. The NADPH- and time-dependent loss of activity was determined at 0 and 12 min by adding 5 µL aliquots (5 pmol of P450 2B1) to a secondary reaction mixture for the determination of 7-EFC O-deethylation activity as described above. The samples were dialyzed overnight at 4 °C against 3 × 500 mL of dialysis buffer as described above. The dialyzed samples were then analyzed by HPLC to determine binding specificity. The sample (0.5 nmol of P450 2B1) was injected onto a 4.6 mm × 100 mm POROS R1/H perfusive-particle column from Perseptive Biosystems (Framingham, MA). The solvent system consisted of solvent A (0.1% TFA) and solvent B (95% acetonitrile/5% water/0.1% TFA). Initial conditions were 20% B with a linear gradient to 75% B over the course of 18 min at a flow rate of 3.0 mL/min and then to 100% B over the course of 2.0 min. Fractions were collected every 0.4 min and monitored by liquid scintillation counting on a Beckman model LS-5801 liquid scintillation counter. The samples were monitored at 220, 280, and 405 nm with a Waters 490E detector. Retention times for heme, catalase, short reductase (proteolytically cleaved), reductase, and P450 2B1 were 6.2, 8.5, 9.8, 10.5, and 12.8 min, respectively, and were determined by injecting each of the purified proteins separately. Protein recovery was determined from standard curves generated with purified protein reconstitution mixtures.

Results Effect of Isothiocyanates on P450 2B1 Activity. As shown in Table 1, both of the naturally occurring isothiocyanates, benzyl (BITC) and phenethyl isothiocyanate (PEITC), inactivated P450 2B1 in a time- and concentration-dependent manner. The inactivation of P450 2B1 by BITC and PEITC was also NADPHdependent (5 min data). A BITC concentration as low as 1 µM resulted in 45% inactivation within 5 min, with more complete inactivation at higher concentrations. PEITC also exhibited effective inactivation of P450 2B1; however, at higher PEITC concentrations (5-10 µM), a significant decrease in the 7-EFC activity was also observed at 0 min. This decrease in activity was probably

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Table 1. Effect of Isothiocyanates on the 7-EFC O-Deethylation Activity of Purified P450 2B1a % activity remaining isothiocyanate benzyl

2-phenethyl

3-phenylpropyl

6-phenylhexyl

concentration (µM)

0 min

5 min

0 0.1 1 5 10 100 0 0.1 1 5 10 0 0.1 1 5 10 0 0.1 1 5 10

100 97 99 101 94 64 100 99 102 75 61 100 99 46 12 9 100 99 85 11 5

96 87 55 35 28 9 94 89 78 24 13 101 90 40 7 3 97 98 77 7 3

Assay conditions were as described in Experimental Procedures. Primary incubation mixtures contained BITC in the reconstituted system. The activity remaining after 0 min represents the activity measured in a secondary reaction prior to addition of NADPH, while 5 min represents the activity measured after a 5 min incubation of the primary reaction mixture with NADPH. The values shown represent the means from two or three experiments that did not differ by more than 6%.

Figure 2. Time- and concentration-dependent inactivation of reconstituted P450 2B1 by BITC following incubation with NADPH at 23 °C. Incubation conditions were as described in Experimental Procedures. The concentrations of BITC that were used were ([) 0, (O) 2, (2) 2.5, (0) 3.5, (b) 5, (4) 10, and (9) 20 µM. The data shown represent the average of six experiments which did not differ by more than 5%. The inset shows the double-reciprocal plot of the rates of inactivation as a function of the BITC concentration.

due to a competitive inhibition of 7-EFC metabolism by the PEITC carried over to the secondary reaction mixture. The final concentrations of the isothiocyanates in the secondary reaction mixtures were 1, 10, 50, and 100 nM, respectively. A similar observation was made with 100 µM BITC (1 µM in secondary) which also resulted in a >90% NADPH-dependent inactivation in 5 min. The synthetic isothiocyanates, PPITC and PHITC, were shown to be very potent inhibitors (NADPHindependent) of P450 2B1 activity. As shown in Table 1, PPITC was the most effective inhibitor of P450 2B1 activity, with a concentration of 1 µM resulting in an approximately 60% decrease in activity at 0 and 5 min compared to a 20% inhibition of activity by PHITC at the same concentration. At a concentration of 10 µM in the primary reaction, PPITC and PHITC both inhibited >95% of the 7-EFC activity in the secondary reaction mixture. However, both PPITC and PHITC exhibited little time- or NADPH-dependent inactivation, when comparing the activity remaining at 0 and 5 min, and the loss of activity appeared to be mostly due to competitive inhibition. We therefore decided to further characterize the inactivation of P450 2B1 with BITC, the most potent naturally occurring isothiocyanate. Inactivation of Purified P450 2B1 by BITC. Purified rat liver P450 2B1 in the reconstituted system was inactivated by BITC in a time- and concentrationdependent manner (Figure 2). The inactivation followed pseudo-first-order kinetics with respect to time between 2 and 20 µM BITC. The requirement for NADPH indicated that BITC had to be metabolized to a reactive species which was responsible for the inactivation. There was no lag time associated with the inactivation as can be seen in the time-dependent loss of EFC O-deethylation activity with increasing BITC concentrations (Figure 2). The rate of P450 2B1 inactivation by BITC was very

rapid, and kinetic determinations were performed at 23 °C rather than at 30 or 37 °C to decrease the inactivation rates. Linear regression analysis of the data in Figure 2 was used to determine the rate constants (kobserved) of inactivation. The plot of the reciprocals of the initial rate constants as a function of the reciprocals of the inactivator concentrations (Figure 2, inset) was used to determine the kinetic constants. The concentration of BITC required for the half-maximal rate of inactivation (KI) was 5.8 µM, and the time required for one-half of the enzyme molecules to become inactivated (t1/2) was 1.05 min at 23 °C. The maximal rate of inactivation at saturation (kinact) was 0.66 min-1. Substrate Protection. Simultaneous incubations of 10 µM BITC with increasing concentrations of 7-EC in the primary reaction mixture decreased the rate of P450 2B1 inactivation by BITC (Figure 3). The incubation of P450 2B1 for 1.67 min with BITC and 7-EC at a molar ratio of 1:50 resulted in a 12% loss in 7-EFC activity, while 58% of the activity was lost in the absence of 7-EC. The incubation of P450 2B1 with 500 µM 7-EC alone had no effect on 7-EFC deethylation activity. These observations suggest that inactivation was most likely due to the binding of a BITC metabolite to the P450 2B1 active site. Partition Ratio. The partition ratio (r), a measure of the efficiency of an inactivator, is defined as the number of product molecules produced per inactivation event (4). As shown in Figure 4, the turnover number (r + 1) was derived from the intercept between the line obtained by linear regression (lower BITC concentrations) and the average straight line at higher BITC concentrations. The turnover number is approximately 10, and because this number includes the one molecule of inactivator which inactivates the enzyme, assuming a 1:1 stoichiometry for the inactivation, the value for the partition ratio is 9.

a

Inactivation of P450 2B1 by Benzyl Isothiocyanate

Figure 3. Substrate protection against P450 2B1 inactivation by BITC. Samples were removed from the primary reaction at the indicated time points and assayed as described in Experimental Procedures. The primary reaction mixtures contained BITC:7-EC molar ratios of ([) 0:0, (O) 0:50, (2) 1:0, (0) 1:5, (b) 1:10, and (4) 1:50. The data shown are representative of three separate experiments.

Figure 4. Loss of P450 2B1 7-EFC O-deethylation activity as a function of the BITC:P450 2B1 concentration ratio. Assay conditions were as described in Experimental Procedures. The percent of P450 2B1 7-EFC O-deethylation activity remaining after complete inactivation was plotted against the molar ratio of BITC to P450 2B1. The data shown represent the mean of three separate experiments.

Spectrophotometric Quantitation of P450 2B1. The loss in catalytic activity of the protein could result either from modification of the apoprotein or from modification of the prosthetic heme group. The amounts of spectrally detectable P450 remaining after inactivation were determined from the reduced carbon monoxide difference spectra (36). As shown in Table 2, approximately 45% of the spectrally detectable P450 was lost in 12 min, taking into account the loss in control incubations

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over time. By comparison, >80% of the catalytic activity was lost after inactivation for 12 min compared to control. The maximum absorption of the absolute spectrum of the BITC-inactivated P450 2B1 was shifted by about 2 nm to a longer wavelength (420 nm) when compared to P450 2B1 in the presence of BITC but without NADPH added (Figure 5). There was a small decrease in the absorbance of the modified P450 which corresponded with the HPLC analysis of modified P450 2B1 (Figure 6), indicating that there was no significant heme destruction. Effect of External Nucleophiles. According to Silverman (4), a mechanism-based inactivator is chemically transformed by the target enzyme into the actual inactivating species and inactivation must occur by the reactive intermediate reacting at the active site prior to its release. Therefore, the presence of an excess of an external nucleophile, which can act as a trapping agent of reactive electrophilic intermediates, should not affect the rate of inactivation. The presence of 10 mM GSH totally inhibited the inactivation of P450 2B1 by BITC. The reaction between BITC and GSH gave rise to a characteristic thiocarbamate with an increased UV absorption maximum at 270 nm and a broad shoulder near 250 nm. At a BITC:GSH molar ratio of 1:1, only 10.6% of the sulfhydryl groups from GSH could be titrated with DTNB. This finding implies that almost 90% of the BITC reacted with GSH. The inclusion of KCN in the reaction mixture decreased the rate of P450 2B1 inactivation by BITC (data not shown). A KCN concentration of 50 or 500 µM decreased the rate of BITC inactivation by 18 or 42%, respectively. One possibility for a slower rate of inactivation in the presence of cyanide could be competition of the cyanide anion at the active site of P450 (40, 41). This was confirmed when separate experiments were conducted, using a different substrate (7-EFC), to study the effect of KCN on substrate metabolism. The inclusion of 500 µM, 1 mM, or 10 mM KCN decreased the rate of 7-EFC metabolism (studied as the rate of 7-HFC formation) by 15, 22, or 54%, respectively (data not shown). However, as shown in Table 3, KCN did not affect the extent of P450 2B1 inactivation. In the absence of KCN, approximately 75% of the enzyme activity was lost within 4 min, while in the presence of a 10-fold excess KCN, the corresponding loss in enzyme activity was observed after 12 min. Irreversibility of Inactivation of P450 2B1 by BITC. Inactivation of P450 2B1 by BITC was found to be irreversible by dialysis (Table 2). BITC-inactivated samples or control samples containing BITC, but no NADPH, were dialyzed extensively either with or without 1 mM DTT in the analysis buffer as described in Experimental Procedures. The samples were tested for 7-EFC O-deethylation activity, protein concentration by absorbance at 418 nm to correct for volume changes during dialysis, and the amount of P450 that could be detected by reduced CO difference spectroscopy. Table 2 shows the percent activity in samples prior to and after dialysis. Little activity was lost in control samples containing BITC but no NADPH during the 12 min incubation period as compared to time zero (93 vs 94% of the activity remaining). After removal of free BITC by dialysis, 87% of the activity remained compared to control. Reconstitution with fresh reductase did not increase the activity significantly (89% compared to 87%). BITC-inactivated samples lost almost 80% of their activity during the 12 min incubation period compared

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Table 2. Effect of BITC on 7-EFC O-Deethylation Activity of Purified P450 2B1 and Cytochrome P450 Contenta % activity remaining primary reaction

0 min

12 min

after dialysis

without BITC and without NADPH with BITC and without NADPH with BITC and with NADPH

100 97 ( 3 95 ( 2

94 ( 3 93 ( 1 18 ( 2

100 87 ( 4 23 ( 4b

% P450 remaining fresh

reductasec

100 89 ( 3 29 ( 2

0 min

12 min

after dialysis

100 99 ( 2 93 ( 3

95 ( 3 87 ( 4 54 ( 4

100 ndd 56 ( 3

a Assay conditions were as described in Experimental Procedures. b Inclusion of 1 mM DTT in the dialysis buffer did not significantly recover activity compared to values reported in the absence of DTT. c Samples were first dialyzed before reconstitution with fresh reductase. d Not determined. The values shown represent the means ( SD from four experiments.

Figure 5. UV/visible spectra of reconstituted P450 2B1 incubated with BITC in the presence of NADPH (s), with BITC in the absence of NADPH (- - -), and without BITC in the presence of NADPH (- - -). P450 2B1 was incubated with 20 µM BITC in the reconstituted system at 30 °C for 12 min as described in Experimental Procedures.

to the activity at time zero. After dialysis, 23% of the P450 2B1 activity could be recovered compared to 18% of the activity before dialysis. Inclusion of 1 mM DTT in the dialysis buffer gave similar results, 29% of the activity after dialysis compared to 22% before dialysis. Reconstitution of inactivated samples with fresh reductase did not bring about a significant increase in catalytic activity. The percentage of P450 that could be detected by spectral assessment of the reduced CO complex did not increase after removal of excess BITC by dialysis. After dialysis, 56% of the reduced CO spectrum remained, compared to 54% after a 12 min incubation (Table 2). These results indicate that inactivation of P450 2B1 by BITC is not reversible under these conditions and that reductase activity is not affected by inactivation. Stoichiometry of Inactivation. The stoichiometry of binding of the radiolabeled metabolite to P450 2B1 was determined by incubating the P450 in the reconstituted system with [14C]BITC with or without NADPH as described in Experimental Procedures. In three separate experiments, after subtraction of the background counts from samples incubated with [14C]BITC without NADPH, 0.88 nmol of labeled metabolite was incorporated into 1.0 nmol of inactivated P450 2B1. Protein precipitation studies were performed to confirm covalent binding of a radioactive BITC metabolite to P450 2B1 as described in Experimental Procedures. The binding of a radioactive BITC metabolite to P450 2B1 was found to be irreversible under these conditions. In three separate experiments,

Figure 6. HPLC analysis of the reconstituted system after incubation with [14C]BITC in the absence (A) or presence (B) of NADPH. The eluate was monitored at 220 (s) and 405 nm (- - -) and by liquid scintillation counting of fractions (b). Incubation conditions, sample preparation for HPLC, and chromatographic conditions were as described in Experimental Procedures. Peaks 1-5 represent heme, catalase, short reductase, reductase, and P450 2B1, respectively. Table 3. Effect of KCN on the Extent of P450 2B1 Inactivation by BITCa % activity remaining 4 min

12 min

with BITC and without KCN 85 ( 4 37 ( 4 25 ( 2 with BITC and with KCN 91 ( 3 61 ( 4 37 ( 3

primary reaction

0 min

1 min

13 ( 3 28 ( 3

a Assay conditions were as described in Experimental Procedures. P450 2B1 in the primary reaction was incubated with 10 µM BITC with or without 100 µM KCN at 30 °C. The values shown represent the mean ( SD of two experiments.

after subtraction of the background counts from samples incubated with [14C]BITC without NADPH, 0.92 nmol of labeled metabolite was incorporated into 1.0 nmol of P450 2B1. The stoichiometry results from the protein precipitation study and dialysis did not differ significantly, as determined by a Student’s t test. The data from these two experimental approaches were pooled, and the stoichiometry of binding was found to be 0.90 ( 0.16 mol (mean ( SD) of radiolabeled metabolite bound per mole of P450 2B1 inactivated. Specificity of Labeling. The specificity of the radioactive labeling of the proteins in the incubation mixture was investigated by HPLC analysis. As shown in Figure 6, the radioactivity was primarily associated with the P450 (eluting at 12.8 min) in the samples with NADPH.


In the samples without NADPH, there was also some radioactivity associated with the P450 as would be expected from the reactivity of isothiocyanates toward nucleophilic groups (22). Stoichiometric calculations revealed that 0.77 ( 0.11 mol (mean ( SD) of radiolabel was associated with each mole of P450 in samples without NADPH. However, these samples retained catalytic activity, and one could therefore assume binding to surface amino acid residues of P450 2B1. The increases in the amount of radiolabel associated with P450 in samples with NADPH were stoichiometric. The values that were obtained were slightly lower than but not statistically different from those reported above. Small amounts of radioactivity are associated with reductase and catalase, but these did not increase significantly in samples incubated with NADPH. No radioactivity was associated with the heme peak that eluted at 6.2 min, detected by measuring the absorbance at 405 nm. The 10-15% loss in the HPLC heme peak in samples with NADPH corresponded to the loss in heme absorbance of the absolute spectrum (Figure 5), and no other heme peaks were detected at 405 nm. Similar results were obtained by injecting samples onto a Vydac C4 protein and peptide column (data not shown). The recoveries of inactivated P450 were 20% lower than those of samples with no NADPH. When the C4 HPLC column was employed, the recovery of P450 was approximately 30% lower compared to that with control samples.

Discussion The results reported here demonstrate that BITC is a mechanism-based inactivator of P450 2B1. One of the main objectives of this study was to identify a suitable mechanism-based inactivator of P450 2B1 that would react specifically with the P450 apoprotein and prove useful in the elucidation of critical active site amino acid residues. Both of the naturally occurring isothiocyanates that have been studied, BITC and PEITC, inactivated purified P450 2B1 in a time- and NADPH-dependent manner. The potency of the isothiocyanates as inhibitors of 7-EFC O-deethylation activity increased with an increase in the alkyl chain length. PPITC and PHITC significantly inhibited P450 2B1 7-EFC O-dealkylation activity at lower concentrations. However, they did not inactivate in a mechanism-based manner, and inhibition appeared to be mostly competitive. The increased lipophilicity of PPITC and PHITC, when compared to that of BITC and PEITC, could favor interaction with the lipophilic regions of the protein or altered substrate orientation, precluding metabolism to a reactive intermediate (Figure 7) that would inactivate the enzyme as in the case of BITC and PEITC. A significant degree of structural specificity has been noted in the effects of isothiocyanates on enzymes that are involved in carcinogen activation and detoxification. Our observations are consistent with the increased chemopreventative potency of arylalkyl isothiocyanates in rodents treated with the tobacco-specific nitrosamine, NNK (15-17). PEITC and longer-chain isothiocyanates strongly inhibited NNK metabolic activation and tumorigenicity in mouse lung, while BITC had no effect. In turn, BITC strongly inhibits BaP-mediated carcinogenesis, while PEITC is ineffective. These effects might be related to the inhibitory mechanisms and potencies for P450 2B1 and P450 1A, respectively. However, in vivo


Figure 7. Proposed reaction scheme for the metabolism of BITC by P450 2B1.

effects of isothiocyanates are also largely dependent on the treatment protocol, the carcinogen that was examined, and the target tissue that was involved. Irreversible inhibitors, such as PEITC, are able to induce P450 2B1 and P450 2E1 after chronic administration with related increases in toxicity (25), and the conjugation with GSH has been shown to decrease potency, with shorter-chain isothiocyanates being more reactive toward GSH. Although the metabolism-dependent inactivation and competitive inhibition of P450 2E1 by PEITC and BITC have been previously reported (27), this is the first report of metabolism-dependent inactivation of the P450 2B isozyme by BITC in a reconstituted system. The BITC-dependent inactivation of P450 2B1 exhibited a number of characteristics consistent with mechanism-based inactivation. The inactivation of 7-EFC Odeethylation activity was time- and concentrationdependent, followed pseudo-first-order kinetics, was saturable, and required NADPH. BITC was an effective mechanism-based inactivator, with KI and kinact values of 5.8 µM and 0.66 min-1, respectively. The KI in the low micromolar range was similar to the IC50 values (5.0 µM) reported for inhibition of pentoxyresorufin (P450 2B1) activity in microsomes from PB-induced rats (42). When higher concentrations of BITC were used, there was a concomitant NADPH-independent decrease in the P450 2B1 activity assayed at 0 min with a >90% loss of activity assayed at 5 min. BITC therefore also appears to be a competitive inhibitor of P450 2B1. It is therefore important to realize that multiple processes are involved in the inhibition of substrate metabolism when IC50 values from the literature are reported and interpreted for BITC. The low partition ratio of BITC indicates that it is an efficient inactivator of P450 2B1. The partition ratio was estimated by titration of the enzyme with increasing concentrations of BITC so the number of inactivator molecules required to inactivate the enzyme completely could be measured (turnover number). At the highest concentrations that were used, the maximal level of inactivation that was obtained was approximately 80%. It is unlikely that the lack of complete inactivation was due to product inhibition since the samples were diluted

1356


100-fold in the secondary reaction and dialysis of the inactivated sample did not restore catalytic activity. One explanation could be that the modification of a critical amino acid residue(s) could only partially compromise either substrate binding or catalysis and subsequently would not result in complete inactivation of the 7-EFC O-deethylation activity. This is substantiated by the fact that the modification of critical amino acid residues can influence the rate of catalysis or metabolites formed by P450s (9, 34, 43). The mechanism of BITC-mediated inactivation of P450 2B1 was also explored in this study. The inactivation of hepatic microsomal P450s is thought to proceed through one of three characterized pathways (44). The first pathway involves covalent modification of the heme to products which can dissociate from the enzyme. This mechanism is involved in the inactivation of P450 by terminal olefins and acetylenes (45), as characterized by visible spectroscopy, HPLC, and mass spectrometry. A second pathway for inactivation involves destruction of the heme, in some cases to products which modify the protein. This pathway has been described for inactivators such as CCl4 (44), spironolactone (46), and cumene hydroperoxide (47). The third pathway involves the covalent modification of the protein moiety by a reactive metabolite, as in the case of the inactivation of P450 2B1 by chloramphenicol (10), 2-ethynylnaphthalene (48), and 9-ethynylphenantherene (49). The magnitude of the reduced-CO difference spectrum of P450 2B1 decreased by approximately 45% following the inactivation by BITC in a reconstituted system. The 45% loss in the ability of the BITC-modified P450 2B1 to bind CO did not correlate with the 82% loss in 7-EFC O-deethylation activity. In addition, the magnitude of the UV/visible absorption spectrum of the BITC-inactivated P450 2B1 did not decrease significantly even though the sample lost 82% of the 7-EFC O-deethylation activity. Therefore, although there is some loss in spectrally detectable P450, there are definite indications that the heme itself was not covalently altered or destroyed. Evidence to substantiate this includes the fact that the retention time of the heme peak of the modified protein on HPLC was not altered, the absence of radioactivity associated with heme when incubated with [14C]BITC and analyzed by HPLC, and the lack of significant change in the absorbance of the heme at 405 nm. Because there was no evidence for heme destruction or heme adduct formation, it seems likely that the BITC-derived metabolite may be covalently attached to a critical amino acid residue in proximity to the heme in such a way that it interferes with the interaction of CO with ferrous heme. The inactivation resulting in covalent modification of the apoprotein in the active site with concomitant loss in the reduced-CO spectrum of P450 has also been described for tert-butyl isothiocyanate (50), bergamottin (51), and methoxalen (52), but in most cases, there is little loss in the level of CO binding with protein modification. It has also been shown that the metabolism of 1-naphthyl isothiocyanate causes a 35% decrease in the amount of spectrally detectable P450 in microsomes, while incubation with its oxygen analogue, 1-naphthyl isocyanate, causes a similar metabolism-independent loss in P450 (53). The oxidative desulfuration of isothiocyanates to isocyanates has recently been described (54), and it is therefore possible that the loss in the level of spectrally detectable P450 could be due to the covalent interaction

Goosen et al.

of the isocyanate with P450 apoprotein which interferes with CO binding (Figure 7). The loss in the level of spectrally detectable P450 2B1 due to inactivation by BITC was also shown to be irreversible by dialysis. The inactivation of P450 2B1 was investigated using [14C]BITC, and the inactivated enzyme was analyzed by HPLC and liquid scintillation counting. There was a marked increase in the amount of radioactivity associated with the P450 apoprotein and a concomitant decrease in the amount of P450 apoprotein eluting when incubations were carried out in the presence of NADPH (Figure 6). The observation that inactivation results in the inability of P450 apoprotein to elute from the reverse-phase HPLC column seems to be a common feature of P450 enzymes inactivated by modification of the apoprotein (55, 56). It might be expected that conformational changes induced by covalent modification of the apoprotein could expose hydrophobic amino acid residues which could result in aggregation of the P450 and decreased recovery from the HPLC column. The results described in this study indicate that the P450 apoprotein is modified by a metabolite of BITC, which is specifically associated with the apoprotein. The modification by a radiolabeled reactive BITC intermediate appeared to be covalent since extensive dialysis did not lead to recovery of enzyme activity. Furthermore, the precipitation of protein followed by extensive organic solvent washes could not dissociate the radioactivity from the protein. Measurements of stoichiometry revealed that 0.90 ( 0.16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated. These data suggest that a metabolite of BITC is bound in the active site of the enzyme as would be expected with a 1:1 stoichiometry of inactivation. The absolute requirement for NADPH for the inactivation and the protection against inactivation offered by 7-EC confirmed the need for BITC to be metabolically activated. Several other inactivators have been shown to react with P450 in a 1:1 stoichiometry, and in some cases, the involvement of one critical amino acid has been demonstrated (9, 10). It would therefore appear from these results that the inactivation occurred at the active site. It was further shown by HPLC analysis that BITC could also react with P450, reductase, and catalase in the absence of NADPH. This is not surprising because of the reactivity of BITC toward nucleophilic protein groups, in some cases leading to the inactivation of enzymes which require sulfhydryl groups for their catalytic activity (29, 57). However, the metabolism-independent reaction of BITC with P450 2B1 did not affect the 7-EFC O-deethylation activity. One could therefore assume that the reaction of BITC with exposed surface amino acid residues was not involved in substrate catalysis. Although it has been shown that the modification of rabbit P450 2B4 by fluorescein isothiocyanate caused a 25% loss in benzphetamine N-demethylation activity due to modification of the -amino group of lysine382 (28), the result described in this study is similar to other reports where enzyme activity was not affected even though enzyme modification clearly occurred (29, 58). The possibility that BITC affected the enzymatic activity of reductase was examined. As shown in Figure 6, even though BITC reacted with reductase and catalase in the absence of NADPH, there was no significant increase in the amount of radioactive metabolite associ-


ated with reductase after addition of NADPH. Furthermore, reconstitution with fresh reductase after extensive dialysis did not restore catalytic activity (Table 2). These data clearly indicate that reductase activity was not affected by a reactive metabolite escaping the active site and that the loss in 7-EFC activity was entirely due to inactivation of P450 2B1. Nucleophilic scavengers such as GSH and DTT are used to trap electrophilic species that diffuse from the active site and may then possibly react with amino acid residues outside of the active site, leading to inactivation. The reactivity of isothiocynates toward nucleophiles is known (22), and it was therefore expected that GSH might react with BITC to form the corresponding thiocarbamate. The nonenzymatic reaction between BITC and GSH has been described previously (59) and was confirmed in this study. The protective effect of GSH against inactivation by BITC is thus due to formation of a chemical complex even before metabolism and not due to trapping of an electrophilic intermediate escaping the active site. The inactivation of P450 2E1 by tert-butyl isothiocyanate was not prevented by inclusion of GSH, but resulted in a decreased rate of inactivation (50). This reaction would therefore not appear to be universal as some isothiocyanates have a much lower reaction rate toward nucleophiles, depending on the electrophilicity of the alkyl substituent (29). The inclusion of potassium cyanide in the inactivation reaction mixture reduced the rate of BITC-mediated inactivation of P450 2B1, but did not affect the extent of inactivation. The mechanism of enzyme inactivation probably involves the oxidative desulfuration of BITC to the reactive benzyl isocyanate intermediate, which would react with nucleophilic amino acid residues in proximity to the active site (Figure 7). This reaction is thought to proceed in a manner analogous to that of the oxidative desulfuration of parathion, R-naphthylthiourea, and diallyl sulfide (60), releasing elemental sulfur which could contribute to enzyme inactivation through formation of hydrodisulfides. One possible explanation for the decreased inactivation rate in the presence of KCN would be the trapping of sulfur by the cyanide anion as thiocyanates. However, cyanide can also bind to the ferric form of P450 (40, 41), resulting in a decreased rate of reduction of P450 (40). In this study, cyanide decreased the rate of 7-EFC deethylation to HFC, and cyanide could therefore also decrease the rate of BITC metabolism that would ultimately be seen as a reduced rate of inactivation. Although a 1:1 stoichiometric binding of radiolabeled BITC metabolite per enzyme molecule inactivated was observed, conclusive evidence for the lack of sulfur binding would require the use of [35S]BITC. However, dialysis of the inactivated enzyme under reductive conditions in the presence of DTT did not restore catalytic activity. It was initially thought that the oxidative desulfuration of parathion resulted in the inactivation of P450 by sulfur since heme destruction was necessary in addition to sulfur modification (14), suggesting that a second species might be involved. The BITC-mediated inactivation of P450 2B1 may therefore proceed primarily through formation of the reactive benzyl isocyanate intermediate as indicated by the stoichiometric protein modification by the radiolabeled BITC metabolite. The results of this study demonstrated that the naturally occurring isothiocyanate, BITC, functions as a mechanism-based inactivator of P450 2B1 and that the


inactivation proceeds through covalent modification of the P450 apoprotein. These observations suggest that BITC would be a useful tool in the labeling and exploration of the P450 2B1 active site. Current studies are directed at identifying the labeled peptide and the critical amino acid(s) involved in inactivation. Studies aimed at identifying the reactive intermediate formed during the metabolism of BITC, which is responsible for the inactivation of P450 2B1, are described in another paper (61).

Acknowledgment. We are grateful to Hsia-lien Lin for purification of P450 2B1 and to Dr. Fung-Lung Chung who provided us with the [14C]BITC. This publication was supported in part by grants from the Foundation for Pharmaceutical Education (SA Druggist), the Potchefstroom University for CHE (T.C.G.), and NIH Grants CA 16954 (P.F.H.) and CA 46535 (Fung-Lung Chung) from the National Cancer Institute.

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(37)

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(47)

(48)

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(54)

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