0022-3565/01/2961-198 –206$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 296:198–206, 2001
Vol. 296, No. 1
0/872060
Printed in U.S.A.
Effects of Benzyl Isothiocyanate on Rat and Human
Cytochromes P450: Identification of Metabolites Formed by
P450 2B1
THEUNIS C. GOOSEN, DANIELLE E. MILLS, and PAUL F. HOLLENBERG
Department of Pharmacology, Potchefstroom University for CHE, Potchefstroom, South Africa (T.C.G.); and Department of Pharmacology, The
University of Michigan, Ann Arbor, Michigan (T.C.G., D.E.M., P.F.H.)
Received June 26, 2000; accepted September 25, 2000
This paper is available online at http://jpet.aspetjournals.org
The cytochromes P450 (P450s) play an important role in
the oxidative metabolism and detoxification of various drugs
and carcinogens (Guengerich, 1991). P450 enzymes are able
to incorporate one of the two atoms of an O2 molecule into a
broad variety of substrates with concomitant reduction of the
other oxygen atom by two electrons to produce H2O (Groves
and Han, 1995). The resultant increases in polarity of the
metabolites usually facilitate excretion or further detoxification of the products formed. Since P450s have also been
shown to play key roles in the activation of a variety of
carcinogens (Guengerich, 1991), the inhibition of P450-dependent carcinogen activation, especially by dietary substances, has been extensively studied. Naturally occurring
isothiocyanates are released from their glucosinolate precurThis publication was supported in part by grants from the Foundation for
Pharmaceutical Education (SA Druggist) (to T.C.G.), the Potchefstroom University for Christian Higher Education (to T.C.G.), and National Institutes of
Health Grants CA 16954 (to P.F.H.) and CA 46535 (to F.-L.C.) from the
National Cancer Institute.
respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of P450 2B1 inactivated by [14C]BITC indicated
specific and covalent modification of the P450 apoprotein by a
metabolite of BITC. High-performance liquid chromatography
analysis of the BITC metabolites revealed that benzylamine was
the major metabolite and there were lesser amounts of benzoic
acid, benzaldehyde, N,N⬘-di-benzylurea, and N,N⬘-di-benzylthiourea. Presumably, BITC was metabolized to the reactive
benzyl isocyanate intermediate that covalently modified the
P450 apoprotein or hydrolyzed to form benzylamine. BITC was
an efficient inactivator of P450 2B1 with a partition ratio of
approximately 11:1. This irreversible inactivation of P450s by
BITC could contribute significantly to its chemopreventative
action.
sors through the activity of the enzyme myrosinase after
chewing or maceration of cruciferous vegetables such as cabbage, cauliflower, and broccoli (Fenwick et al., 1983). Benzyl
isothiocyanate (BITC) is released in significant concentrations from cabbage, radishes, Indian cress, garden cress, and
mustard spinach (Fenwick et al., 1983).
Isothiocyanates have been shown to be potent and selective
inhibitors of carcinogenesis induced by a variety of chemical
carcinogens such as tobacco-derived nitrosamines and polycyclic aromatic hydrocarbons (Chung, 1992). These effects
are partly due to the direct inhibition and/or down-regulation
of the P450 responsible for carcinogen activation, resulting in
decreased amounts of ultimate carcinogens formed (Zhang
and Talalay, 1994). In addition, isothiocyanates have been
shown to induce certain phase II enzymes responsible for the
detoxification of electrophilic intermediates formed during
phase I metabolism (Zhang and Talalay, 1994). The relative
importance of these two mechanisms might differ among
ABBREVIATIONS: P450s, cytochromes P450; BaP, benzo(a)pyrene; BITC, benzyl isothiocyanate; DLPC, L-␣-phosphatidylcholine dilauroyl;
7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; GC-MS, gas chromatography-mass spectrometry; GSH, glutathione; 7-HFC, 7-hydroxy-4-(trifluoromethyl)- coumarin; reductase, NADPH-cytochrome P450 reductase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PEITC, 2-phenethyl isothiocyanate; PAGE, polyacrylamide gel electrophoresis.
198
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ABSTRACT
Naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are potent and selective inhibitors of carcinogenesis induced by a variety of chemical carcinogens. These effects appear to be mediated through favorable modification of
both phase I and II enzymes involved in carcinogen metabolism. The inactivation of rat and human cytochromes P450
(P450s) in microsomes and the reconstituted system by BITC
was investigated. BITC is a mechanism-based inactivator of rat
P450s 1A1, 1A2, 2B1, and 2E1, as well as human P450s 2B6
and 2D6. BITC was most effective in inactivating P450s 2B1,
2B6, 1A1, and 2E1, whereas the activities of human P450 2C9
and rat P450 3A2 were not altered. The concentrations required
for half-maximal inactivation (KI) of P450s 1A1, 1A2, 2B1, and
2E1 were 35, 28, 16, and 18 ␮M, respectively. The corresponding values for kinact were 0.26, 0.09, 0.18, and 0.05 min⫺1,
Inactivation of Cytochromes P450 by Benzyl Isothiocyanate
Materials and Methods
Chemicals. Phenobarbital, pyridine, ␤-napthoflavone, pregnenolone-16␣-carbonitrile, p-nitrophenol, 4-nitrocatechol, erythromycin, dilauroyl L-␣-phosphatidylcholine (DLPC), NADPH, bovine
serum albumin (BSA), and catalase were purchased from Sigma
Chemical Co. (St. Louis, MO). Benzyl isothiocyanate (BITC), benzylamine, benzoic acid, 7-ethoxycoumarin, dimethyl sulfoxide , and
sodium dithionite were purchased from Aldrich Chemical Co. (Milwaukee, WI). Resorufin, 7-ethoxyresorufin, 7-methoxyresorufin,
7-benzyloxyresorufin, and 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) were purchased from Molecular Probes Inc. (Eugene, OR).
7-Hydroxy-4-(trifluoromethyl)coumarin (7-HFC) was from Enzyme
Systems Products (Livermore, CA). N,N⬘-Di-benzylurea and N,N⬘-dibenzylthiourea were provided by Dr. M.-S. Lee (Wayne State University, Detroit, MI). HPLC-grade acetonitrile, ethyl acetate, and
methanol were purchased from Fisher (Pittsburgh, PA). Hyperfilm-MP was obtained from Amersham Pharmacia Biotech (Cleveland, OH). Topp3 Escherichia coli cells were obtained from Stratagene (La Jolla, CA). [14C]BITC labeled at the ␣-carbon with a
specific activity of 56 mCi/mmol and chemical purity ⬎97% by HPLC
was kindly provided by Dr. F.-L. Chung (American Health Foundation, Valhalla, NY). All other materials were of reagent grade and
obtained from commercial sources.
Purification of P450 2B1 and P450 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 (1981). These rats were treated with
0.1% phenobarbital in their drinking water for 12 days. The cDNA
for rat NADPH-cytochrome P450 reductase (hereafter referred to as
“reductase”) within the expression plasmid pOR263 was expressed in
E. coli Topp3 cells. The rat liver reductase was expressed and purified as described (Hanna et al., 1998).
Preparation of Microsomes. Microsomes were prepared from
liver homogenates of male Fisher 344 rats (175–190 g; Harlan
Sprague-Dawley) as described previously (Coon et al., 1978). P450
2B1 was induced by i.p. injection of 100 mg/kg phenobarbital in
water for 3 days. P450 2E1 was induced by i.p. injection of 100 mg/kg
pyridine in water for 3 days. P450s 1A1 and 1A2 were induced by i.p.
injections of 80 mg/kg ␤-napthoflavone in corn oil for 3 days. P450
3A2 was induced by i.p. injection of 50 mg/kg pregnenolone-16␣carbonitrile in corn oil for 3 days. Animals were fasted for 18 h after
the last dose and sacrificed.
Microsomes from human lymphoblastic cells expressing P450 2B6
were from Gentest Corp. (Woburn, MA). Human liver P450s 2C9 and
2D6 were coexpressed with reductase in E. coli (He et al., 1999). The
pCW vectors containing P450s 2C9 and 2D6 and the pACYC vectorcontaining reductase provided by Dr. Thomas Friedberg (University
of Dundee, UK) were transformed into JM109 cells provided by Dr.
Kan He (Parke-Davis Pharmaceutical Research, Ann Arbor, MI).
The P450 concentrations of all preparations were determined from
the reduced carbon monoxide difference spectra (Omura and Sato,
1964), recorded on a DW2 UV-visible spectrophotometer (SLM
Aminco, Urbana, IL) equipped with an OLIS spectroscopy operating
system (On-Line Instrument Systems, Inc., Bogart, GA).
Microsomal Enzyme Activity Assays. Microsomal P450 1A1
activity was measured with 7-ethoxyresorufin as the substrate, and
P450 1A2 activity was measured with 7-methoxyresorufin as the
substrate (Burke et al., 1994). The primary reaction mixtures contained 10 ␮M P450 from microsomes of ␤-napthoflavone-treated rats
in 50 mM Tris-HCl (pH 7.5); 50 mM MgCl2; 1, 10, or 100 ␮M BITC (in
1 ␮l of CH3OH/100 ␮l) or solvent in control samples. The primary
microsomal incubation mixtures were all preincubated for 3 min at
30°C before initiation of the reactions with NADPH or water in
reactions without NADPH. At 0 and 10 min after addition of NADPH
(0.8 mM), the P450 1A1 or 1A2 activity was measured by transferring 20 ␮l (P450 1A1) or 40 ␮l (P450 1A2) of the primary reaction
mixture into 980 ␮l or 960 ␮l, respectively, of a secondary reaction
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isothiocyanates, depending on their specificity to influence
the specific enzymes involved, and must be determined individually. The important role of P450 enzymes in the metabolism of endogenous compounds, drug metabolism, and the
detoxification of numerous xenobiotics (Guengerich, 1991)
also has practical implications for this approach to chemoprevention. A third mechanism involving suppression of tumor promotion by an undefined mechanism has also been
reported for BITC (Wattenberg, 1981).
Yang and coworkers (1994) have described the inhibition of
several P450s, including P450s 1A1, 1A2, 2A1, 2B1, and 2E1,
involved in carcinogen activation by isothiocyanates. BITC
and phenethyl isothiocyanate (PEITC) have been shown to
inhibit N-nitrosodimethylamine demethylation activity with
IC50 values of 8 to 9 ␮M. The mechanism of inhibition by
PEITC involves both competitive and metabolism-dependent
inhibition of P450 2E1 (Ishizaki et al., 1990). BITC and
PEITC have also been studied extensively for their role in the
prevention of lung cancer. PEITC inhibits lung carcinogenesis induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), whereas BITC is
ineffective in this regard (Chung, 1992). In turn, BITC effectively inhibits benzo(a)pyrene (BaP)-induced lung tumors in
A/J mice, whereas PEITC is ineffective (Lin et al., 1993). The
synergistic combination of isothiocyanates in the prevention
of human cancer such as lung cancer in individuals resistant
to smoking cessation is also under investigation (Hecht,
1997).
The administration of isothiocyanates to rodents may produce either increases or decreases in microsomal P450 content and activity. The effects appear to be dependent on the
experimental conditions, the isothiocyanate used, the treatment regimen, the target tissue examined, and the specific
monooxygenase measured (Zhang and Talalay, 1994). Acute
administration of PEITC to rats decreased liver P450 2E1
activity, whereas P450 2B1 activity and content increased
approximately 10-fold, without any significant effect on lung
P450 2B1 and P450 1A2 activities (Guo et al., 1992). However, chronic administration of PEITC increased levels of
both P450 2B1 and P450 2E1 with related increases in carcinogen toxicity (Smith et al., 1993). Epidemiological data
suggest that the consumption of fruit and vegetables decreases the risk for cancer development (Block et al., 1992),
and isothiocyanates might therefore contribute significantly
to these effects through modulation of P450 activities.
The mechanism of P450 inhibition by isothiocyanates is
thought to involve reversible competitive inhibition as well
as metabolism-dependent inhibition of P450 2E1 (Ishizaki et
al., 1990) and P450 1A2 (Smith et al., 1996) by PEITC. We
have recently also reported the mechanism-based inactivation of P450 2B1 (Goosen et al., 2000) and P450 2E1 (Moreno
et al., 1999) by BITC. The inactivation of P450s 2B1 and 2E1
involves the formation of a reactive intermediate that covalently modifies the P450 apoprotein.
In the current report, the mechanism-based inactivation of
several P450 isozymes by BITC, both in microsomes and in
the reconstituted system, is described. In addition, BITC was
found to be metabolized to the reactive benzyl isocyanate
intermediate by P450 2B1. This intermediate then covalently
modifies the P450 apoprotein.
199
200
Goosen et al.
30°C. After 10 min the reactions were quenched with 100 ␮l of 1.5 M
perchloric acid and left on ice for 20 min. The samples were centrifuged at 16000g at 4°C. The supernatant was carefully removed
before addition of NaOH (1 M final) and spectrophotometric quantitation of the product at 490 nm. For the determination of the kinetic
parameters with BITC, the primary mixtures were incubated with
increasing concentrations of BITC, and aliquots were removed at the
indicated times for the determination of residual p-nitrophenol activity remaining as described above.
Microsomal P450 3A2 activity was determined using erythromycin as a substrate. N-Demethylation of erythromycin was quantified
by measuring the formation of formaldehyde (Nash, 1953). The primary reaction mixtures contained 4.1 ␮M P450 from microsomes of
pregnenolone-16␣-carbonitrile-treated rats in 50 mM potassium
phosphate buffer (pH 7.4); 1, 10, or 100 ␮M BITC (in 1 ␮l of CH3OH/
100 ␮l); or solvent in control samples. At 0 and 10 min after addition
of NADPH (1.2 mM), the P450 3A2 activity was assayed by transferring 20 ␮l of the primary reaction mixture into 480 ␮l of a secondary reaction mixture containing 1 mM erythromycin and 1 mM
NADPH in 50 mM potassium phosphate buffer (pH 7.4). After incubation for 10 min at 30°C, the reactions were stopped by adding 250
␮l of 60% trifluoroacetic acid and the amount of formaldehyde
formed was measured spectrophotometrically according to Nash
(1953).
SDS-PAGE Analysis for Specificity and Irreversibility of
Binding. Inactivation of purified P450 2B1 by BITC was investigated using a reconstituted mixture containing 4 ␮M P450 2B1, 4
␮M reductase, 200 ␮g/ml DLPC, 55 ␮M [14C]BITC, 90 U of catalase,
and 50 mM Tris-HCl (pH 7.4) in a total volume of 85 ␮l. The primary
incubation 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. At 0 and 12 min after addition of NADPH, aliquots of 5 ␮l
(20 pmol of P450 2B1) were taken from the primary reaction and
added to a secondary reaction similar to that used for the determination of residual P450 2B6 activity. The formation of 7-HFC was
measured as described for P450 2B6.
Aliquots containing 0.1 nmol of P450 2B1 were removed at 0 and
12 min and diluted with sample loading buffer, boiled for 3 min, and
loaded on a 10% polyacrylamide gel and electrophoresed with the
buffer system described by Laemmli (1970). After electrophoretic
separation of the proteins, the gels were either stained with 0.25%
Coomassie Blue R-250 or transferred to a Immobilon-PSQ polyvinylidene difluoride microporous membrane with 25 mM Tris base containing 192 mM glycine, and 30% methanol at 100 mV for 1 h.
Autoradiography was performed by exposing the membrane to Biomax MR film supplemented with a Biomax intensifying screen
(Eastman Kodak Co., Rochester, NY) for 1 week at ⫺80°C.
Metabolism of BITC by P450 2B1. Metabolites formed during
incubation with P450 2B1 were analyzed by HPLC and gas chromatography-mass spectrometry (GC-MS). Purified P450 2B1 and reductase were reconstituted with DLPC for 1 h at 4°C. The reaction
mixture contained 95.8 ␮M [14C]BITC, 2 ␮M P450 2B1, 2 ␮M reductase, 200 ␮g/ml DLPC, 400 U of catalase, and 50 mM potassium
phosphate buffer (pH 7.4) in a total volume of 0.5 ml. The reactions
were initiated with 1.2 mM NADPH or by adding water to the
reactions without NADPH. After 30 min at 30°C the reactions were
stopped by addition of 1 ml of ice-cold ethyl acetate. The samples
were extracted two more times with ethyl acetate, and the organic
phases were pooled. The remaining reaction mixture was adjusted to
pH 11.0 with NaOH and extracted twice with 1-ml portions of ethyl
acetate. The reaction mixture was re-adjusted to pH 3.0 with HCl
and extracted twice with 1 ml of ethyl acetate. Approximately 85% of
the 14C label was recovered. The extracts were combined and dried
using a Speed-Vac (Savant, Farmingdale, NY). To prevent loss of the
volatile metabolites, 50 ␮l of dimethyl sulfoxide was added and the
residual ethyl acetate was evaporated completely. Metabolites were
resolved by HPLC on a 5-␮m reversed-phase C18 column (4.6 ⫻ 250
mm, Rainin, Ultrasphere-ODS) (Varian, Walnut Creek, CA). The
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mixture containing either 5 ␮M 7-ethoxyresorufin (for 1A1) or 7-methoxyresorufin (for 1A2), 50 mM Tris-HCl (pH 7.5), 50 mM MgCl2,
and 0.2 mM NADPH. Secondary reaction mixtures were quenched
after 5 min (1A1) or 15 min (1A2) with 334 ␮l of ice-cold CH3CN
before determining the fluorescence at room temperature on a SLMAminco model SPF-500 C spectrofluorometer with excitation at 522
nm and emission at 586 nm. For the determination of the kinetic
parameters with BITC, the primary mixtures were incubated with
increasing concentrations of BITC, and aliquots were removed at the
indicated times. The residual 7-ethoxyresorufin or 7-methoxyresorufin activity remaining was measured as described above.
Microsomal P450 2B1 activity was assayed with 7-benzyloxyresorufin as the substrate (Nerarkur et al., 1993). The primary
reaction mixtures contained 1.5 ␮M P450 2B1 from microsomes of
phenobarbital-treated rats in 50 mM Tris-HCl buffer (pH 7.4); 1, 10,
or 100 ␮M BITC (in 1.0 ␮l of CH3OH/100 ␮l); or solvent in control
samples. At 0 and 10 min after addition of NADPH (1.2 mM), the
P450 2B1 activity was assayed by transferring 25-␮l aliquots to 975
␮l of a secondary reaction mixture containing 5 ␮M benzyloxyresorufin and 0.2 mM NADPH in 50 mM Tris-HCl buffer (pH 7.4). The
reaction was quenched after 3 min with 750 ␮l of ice-cold CH3OH.
The resorufin product was measured spectrofluorometrically with
excitation at 522 nm and emission at 586 nm. For the determination
of the kinetic parameters with BITC, the primary mixtures were
incubated with increasing concentrations of BITC. Aliquots were
removed at the indicated times, and residual 7-benzyloxyresorufin
activity was measured as described above.
The activities of P450s 2B6, 2C9, and 2D6 were determined using
7-EFC as a substrate (Buters et al., 1993; He et al., 1999). The
primary reaction mixtures contained 160 nM P450 2B6 or 2.2 ␮M
P450 2C9 in 50 mM potassium phosphate buffer (pH 7.4); 1, 10, or
100 ␮M BITC (in 1 ␮l of CH3OH/100 ␮l); or solvent in control
samples. Primary reactions for P450 2D6 contained 1.1 ␮M P450
2D6 in 50 mM HEPES buffer (pH 7.5) containing 20% glycerol, 0.5
mM EDTA, and BITC as described before. Microsomal activity was
assayed at 0 and 10 min after addition of 1.2 mM NADPH. The P450
2B6 activity was assayed by transferring 40 ␮l of the primary reaction mixture into 960 ␮l of a secondary reaction mixture containing
100 ␮M 7-EFC, 40 ␮g of BSA, 0.2 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4). The secondary reaction was allowed
to proceed for 10 min at 30°C and was quenched by the addition of
334 ␮l of ice-cold CH3CN. The P450 2C9 activity was assayed by
transferring 50 ␮l of the primary reaction to 250 ␮l of a secondary
reaction mixture containing 100 ␮M 7-EFC, 120 ␮g/ml BSA, 10 mM
MgCl2, and 0.2 mM NADPH in 50 mM potassium phosphate buffer
(pH 7.4). After incubation for 10 min at 30°C, the reactions were
stopped with 100 ␮l of ice-cold 30% CH3CN in 0.1 M Tris-HCl (pH
9.0) and centrifuged at 16,000g for 10 min before removal of the
supernatant. The P450 2D6 activity was assayed by transferring 52
␮l of the primary reaction to 948 ␮l of a secondary reaction mixture
containing 200 ␮M 7-EFC, 40 ␮g/ml BSA, 0.2 mM NADPH in 50 mM
HEPES buffer (pH 7.5) as above. After incubation for 15 min at 30°C,
the reactions were stopped with 334 ␮l of ice-cold CH3CN. The
formation of the deethylation product (7-HFC) was measured spectrofluorometrically at room temperature with the excitation at 410
nm and emission at 510 nm. The product formed was quantified
based on a standard curve constructed using known amounts of
7-HFC.
Microsomal P450 2E1 activity was determined using p-nitrophenol as the substrate. The primary reaction mixtures contained 5.5
␮M P450 from microsomes of pyridine-treated rats in 100 mM potassium phosphate buffer (pH 6.8); 1, 10, or 100 ␮M BITC (in 1 ␮l of
CH3OH/100 ␮l); or solvent in control samples. At 0 and 10 min after
addition of NADPH (1 mM), the P450 2E1 activity was assayed by
transferring 50-␮l aliquots of the primary reaction mixture into 450
␮l of a secondary reaction mixture containing 0.1 mM p-nitrophenol,
10 mM ascorbate (fresh), and 1 mM NADPH in 100 mM potassium
phosphate buffer (pH 6.8). The reaction mixtures were incubated at
Inactivation of Cytochromes P450 by Benzyl Isothiocyanate
concentrations of BITC (Fig. 1). Both P450 1A1 and P450 1A2
were inactivated in a time- and concentration-dependent
manner. A concentration of 100 ␮M BITC resulted in 62%
inactivation of the 7-ethoxyresorufin (P450 1A1) or 52% inactivation of the 7-methoxyresorufin (P450 1A2) activity, following 10-min incubations (Fig. 2). Kinetic studies revealed
that, although the concentrations of BITC required for halfmaximal inactivation (KI) of P450 1A1 and P450 1A2 were
similar, the rate of inactivation of P450 1A1 was much faster
than that of P450 1A2 (Table 1). This difference was also seen
in Fig. 2 where 10 ␮M BITC resulted in a statistically significant loss of P450 1A1 activity with no significant loss in
P450 1A2 activity. With P450 1A1 and especially P450 1A2 a
loss in activity at time 0 was observed suggesting inhibition
due to carryover into the secondary reaction with higher
concentrations of BITC (Fig. 1). These data indicate that
BITC is more effective as an inactivator of P450 1A1 activity
when compared with inactivation of P450 1A2.
P450s 2B1 and 2B6 were both inactivated by BITC to
similar extents in time- and concentration-dependent manners. A concentration of 100 ␮M BITC resulted in an 88% and
70% inactivation of the 7-benzyloxyresorufin (P450 2B1) and
the 7-EFC (P450 2B6) activity, respectively (Fig. 2). There
was no statistically significant difference in the amount of
P450 2B1 or 2B6 activity remaining following incubation
with 1 or 10 ␮M BITC. A concentration as low as 10 ␮M
already resulted in approximately 50% loss in activity of both
these enzymes following a 10-min incubation. P450 2E1 activity was inactivated by 71% when incubated with 100 ␮M
BITC and by 27% when incubated with 10 ␮M BITC (Fig. 2).
Results
Effect of BITC on the Activities of P450s. The effects of
BITC on the activities of P450s 1A1, 1A2, 2B1, 2B6, 2C9,
2D6, 2E1, and 3A2 were examined. Microsomes were incubated with different concentrations of BITC or solvent added
to control samples as described under Materials and Methods. Kinetic parameters (Table 1) were calculated from the
rates of inactivation of the P450s incubated with increasing
TABLE 1
Kinetic constants for the inactivation of P450s 1A1, 1A2, 2B1, and 2E1
by BITC in microsomes
Assay conditions and the kinetic analyses were as described under Materials and
Methods. The kinetic constants were determined from double-reciprocal plots of the
initial rates of inactivation (Fig. 1) against BITC concentrations.
P450
KI
␮M
1A1
1A2
2B1
2E1
35
28
16
18
kinact
min
⫺1
0.26
0.09
0.18
0.05
t1/2
min
2.7
7.7
3.8
14
Fig. 1. Time- and concentration-dependent inactivation of microsomal
P450s 1A1, 1A2, 2B1, and 2E1 by different concentrations of BITC following incubation with NADPH at 30°C. Incubation conditions were as
described under Materials and Methods. Reaction mixtures contained
varying concentrations of BITC as follows. P450 1A1: 0 ␮M (f), 25 ␮M
(E), 33 ␮M (Œ), 50 ␮M (䡺), 75 ␮M (F), and 100 ␮M (⌬); P450 1A2: 0 ␮M
(f), 25 ␮M (E), 33 ␮M (Œ), 50 ␮M (䡺), and 75 ␮M (F); P450 2B1: 0 ␮M (f),
7.5 ␮M (E), 10 ␮M (Œ), 15 ␮M (䡺), 25 ␮M (F), and 35 ␮M (⌬); P450 2E1:
0 ␮M (f), 15 ␮M (E), 20 ␮M (Œ), 40 ␮M (䡺), and 50 ␮M (F). The data
shown represent the average of duplicates from two or three separate
experiments that did not differ by more than 6%.
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HPLC system consisted of a Waters (Milford, MA) 490E programmable variable wavelength detector, Waters 501 HPLC pumps, a
Waters system interface module, and a fraction collector (model 201,
Gilson, Middleton, WI). The system was operated through the Maxima 820 chromatography workstation from Waters. The solvent system consisted of solvent A (5% CH3CN/1% acetic acid/H2O) and
solvent B (80% CH3CN/1% acetic acid/H2O) adjusted to pH 4.2 with
5 M potassium hydroxide. Initial conditions were 5% B at a flow rate
of 1 ml/min, increasing to 35% B in 5 min, then to 65% B in 30 min,
and finally 90% B in 5 min. The solvent was maintained at 90% B for
5 min before returning to initial conditions. Fractions were collected
every 0.6 min and monitored by liquid scintillation counting on a
liquid scintillation counter (model LS-5801, Beckman, Berkeley,
CA).
For GC-MS analysis of metabolites, purified P450 2B1 and reductase were reconstituted as before. The reaction mixtures contained
50 ␮M BITC, 3 ␮M P450 2B1, 3 ␮M reductase, 200 ␮g/ml DLPC, 400
U of catalase, and 50 mM potassium phosphate buffer (pH 7.4) in a
total volume of 1.0 ml. The reactions were initiated by addition of 1.4
mM NADPH and incubated for 30 min at 30°C. The reactions were
stopped by the addition of 1.5 ml of methylene chloride on ice. This
was followed by two extractions with 1.5 ml of methylene chloride,
the pH of the mixtures was adjusted to 3.0 and then 11.0, and the
mixtures were re-extracted twice with 1.5 ml of methylene chloride
at each pH. The extracts were combined and dried using a Speed-Vac
(Savant) to approximately 500 ␮l. The extracts from five separate
reactions were combined and dried over anhydrous sodium sulfate
before final evaporation in the Speed-Vac to approximately 5 ␮l.
Analysis by GC-MS was carried out on a Finnigan MAT 4500 mass
spectrometer (ThermoQuest, San Jose, CA) coupled to a HP 5890 gas
chromatograph via a heated interface. This GC-MS system employed
a Galaxy data system, manufactured by LGC Inc. (Los Gatos, CA).
Gas chromatographic separation employed a DB-5 capillary column
(30 m ⫻ 0.32 mm i.d. ⫻ 1.0-␮m film thickness) purchased from
Altech Associates, Inc. (Deerfield, IL). Helium gas flow was maintained at approximately 10-psi head pressure, and the column was
installed in a splitless configuration. The gas chromatograph temperature program was initiated at 50°C and raised at 10°C/min to
275°C. Mass spectrophotometric conditions were as follows: electron
impact ionization, with 70-eV electron energy. The ion source temperature was maintained at 150°C.
Statistics. Data were analyzed where appropriate using the Student’s t test. A p value of ⬍0.05 was considered to be a statistically
significant difference.
201
202
Goosen et al.
As shown in Table 1, the KI value for the inactivation of P450
2E1 was similar to that required for the inactivation of P450
2B1. However, the rate of inactivation (kinact) was much
slower for P450 2E1 than the rate of inactivation of P450
2B1.
Human P450 2D6 was also inactivated by BITC in a mechanism-based manner, but the rate of inactivation appeared to
be very slow since approximately 75% of the activity remained following a 10-min incubation with 100 ␮M BITC. As
shown in Fig. 2, BITC did not cause a statistically significant
metabolism-dependent inhibition of human P450 2C9 or rat
P450 3A2 activities when incubated with up to 100 ␮M BITC.
Specificity of BITC Binding. The specificity of the radioactive labeling of the proteins in the reconstituted incubation mixture by BITC was investigated by separation of
the proteins using SDS-PAGE followed by autoradiography
as described under Materials and Methods. Radiolabeled
BITC was bound to all proteins in the reconstituted system
(Fig. 3A) in the absence of NADPH. This nonspecific labeling
of proteins did not result in any loss of catalytic activity.
Samples incubated with BITC and NADPH under conditions
where more than 80% of the 7-EFC O-deethylation activity
was lost showed a marked increase in radiolabel only on the
P450 band (Fig. 3A, lane 4). That this increase was NADPHand BITC-specific and not due to uneven loading of samples
can be seen from the Coomassie Blue stain shown in Fig. 3B.
These data indicate that the labeling of P450 2B1 by a metabolite of BITC was covalent and specific for P450 2B1.
Analysis of Metabolites of BITC. The metabolites
formed by incubating radiolabeled BITC with P450 2B1 in
the reconstituted system were analyzed by reversed-phase
HPLC with UV detection at 254 and 286 nm or by liquid
scintillation counting as described under Materials and
Methods. Seven metabolites were separated from the ethyl
acetate extracts of samples incubated with NADPH as detected by liquid scintillation counting of fractions (Fig. 4).
The peak retention time of the major metabolite, which accounted for almost 50% of the total product formed, corre-
Fig. 4. HPLC elution profile monitored by liquid scintillation counting of
a reaction mixture containing [14C]BITC and reconstituted P450 2B1,
incubated with (f) or without (E) NADPH. The chromatographic conditions were as described under Materials and Methods. Peaks 2, 4, 5, 6,
and 7 were identified by coelution with authentic standards and GC-MS
analyses and represent benzylamine (6.7 min), benzoic acid (14.5 min),
benzaldehyde (19.8 min), N,N⬘-di-benzylurea (29.1 min), and N,N⬘-dibenzylthiourea (35.6 min), respectively. BITC (peak 8) eluted at 39.9 min.
Peaks 1 and 2 represent unidentified metabolites. aImpurities from
[14C]BITC present in all samples; the purity of [14C]BITC was ⬎97% by
HPLC.
sponded to benzylamine (Table 2). The other metabolites
identified by coelution with authentic standards were identified as benzoic acid, benzaldehyde, N,N⬘-di-benzylurea, and
N,N⬘-di-benzylthiourea. In control samples without NADPH,
two products corresponding to benzylamine and N,N⬘-di-benzylthiourea were also detected (Fig. 4). However, the amount
of benzylamine formed in the control reaction was less than
30% of the amount formed in the experimental reactions
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Fig. 2. Inactivation of microsomal P450s 1A1, 1A2, 2B1, 2B6, 2C9, 2D6,
2E1, and 3A2 activity by BITC. Experimental details are described under
Materials and Methods. The liver microsomes were incubated in the
presence of 1, 10, or 100 ␮M BITC with NADPH. Residual activities of the
P450s were determined after 10 min. The results are reported as the
mean ⫾ S.D. of three experiments. The Student’s t test was used to
indicate significant differences from control values at the corresponding
times, *p ⬍ 0.05, **p ⬍ 0.001.
Fig. 3. SDS-PAGE analysis of the P450 2B1 in the reconstituted system
after incubation with [14C]BITC. A, autoradiography of the gel; B, Coomassie Blue-stained gel. The experimental details were as described
under Materials and Methods. Samples in lanes 1 and 2 were incubated
without NADPH for 0 or 12 min, respectively. Samples in lanes 3 and 4
were incubated with NADPH for 0 or 12 min, respectively.
Inactivation of Cytochromes P450 by Benzyl Isothiocyanate
203
TABLE 2
Product formation by P450 2B1 incubated with [14C]benzyl isothiocyanate
Assay conditions were as described under Materials and Methods. The values shown represent the mean ⫾ S.D. from four separate experiments.
Product
Complete System
⫺NADPH Control
NADPH-Dependent Metabolite Formation
nmol product/nmol P450 2B1/30 min
Unknown 1a
Benzylamine
Unknown 3
Benzoic acid
Benzaldehyde
N,N⬘-Di-benzylurea
N,N⬘-Di-benzylthiourea
Benzyl isothiocyanate
Total extracted
0.84 ⫾ 0.03
6.98 ⫾ 0.08
1.32 ⫾ 0.01
0.28 ⫾ 0.03
1.62 ⫾ 0.26
1.29 ⫾ 0.13
1.83 ⫾ 0.23
25.5 ⫾ 0.58
39.7
N.D.b
1.94 ⫾ 0.23
N.D.
N.D.
N.D.
N.D.
1.65 ⫾ 0.26c
36.8 ⫾ 0.55
40.4
0.84
5.04
1.32
0.28
1.62
1.29
0.18
10.6
a
See Fig. 4.
b
S.D., not detected (⬍0.1 nmol).
c
Not statistically different from the complete system.
Discussion
The results reported here demonstrate that BITC is a
mechanism-based inactivator of rat P450s 1A1, 1A2, 2B1,
and 2E1, and human P450s 2B6 and 2D6. Human P450 2C9
and rat P450 3A2 were not inactivated by BITC. P450s 1A1
and 1A2 were inactivated in a time- and NADPH-dependent
manner. BITC was shown to be an efficient inactivator of
P450 1A1 with KI and kinact values of 35 ␮M and 0.26 min⫺1,
respectively. The KI for P450 1A2 (28 ␮M) was similar to the
KI value obtained for P450 1A1, but the rate of inactivation
was much slower than that of P450 1A1. These results are in
good agreement with the IC50 values reported for the inhibition of ethoxyresorufin activity (54 ␮M) by BITC in microsomes from 3-methylcholanthrene-induced rats (Conaway et
al., 1996). It is possible that the reactive intermediate responsible for the inactivation of P450 1A2 is released more
readily from the active site of the enzyme, thereby increasing
the partition ratio for inactivation and effectively decreasing
the rate of inactivation (Silverman, 1996). It is also possible
that BITC is preferentially oxidized to a different product,
possibly benzaldehyde, instead of being desulfurated to benzyl isocyanate as shown in Fig. 5. One component of the
inhibition of P450 1A1 and P450 1A2 by BITC was also seen
to be metabolism-independent as evidenced by the inhibition
of catalytic activity when assayed at time 0 (Fig. 1). Both of
these enzymes have been investigated extensively for their
roles in carcinogen activation and metabolism. Many reports
implicate a role for increased P450 1A1 levels in lung cancer
(McLemore et al., 1990), and the inhibition of P450 1A1 may
be involved in the inhibition of BaP-induced lung carcinogenesis by BITC (Lin et al., 1993). P450 1A2 is involved in the
metabolic activation of tobacco-derived NNK (Smith et al.,
1996), aflatoxin B1, and other carcinogenic aryl amines and
heterocyclic amines (Guengerich, 1995).
The inactivation of P450s 2B1 and 2B6 displayed characteristics of mechanism-based inactivation, including a timeand concentration-dependent loss in catalytic activity. The
dependence on NADPH for inactivation indicated that BITC
had to be metabolized to a reactive intermediate responsible
for the inactivation process. BITC was shown to be the most
selective in inactivating P450 2B1 and 2B6 compared with
the other P450s examined in this study. The KI for the
inactivation of P450 2B1 in microsomal preparations was 16
␮M, and kinact was 0.18 min⫺1. These results were comparable to the KI and kinact of 5.8 ␮M and 0.66 min⫺1 determined
using purified P450 2B1 (Goosen et al., 2000). The inactivation of human P450 2B6 by BITC has biological importance,
because this enzyme has been shown to activate several
carcinogens, including BaP, NNK, and aflatoxin B1 (Code et
al., 1997). Although P450 2B6 is expressed in low levels in
human liver (Guengerich, 1995), it has been found to be
expressed in lung and uterine endometrium and is induced in
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
(Table 2). There was no significant difference between the
amount of N,N⬘-di-benzylthiourea formed in reactions with
or without NADPH. Two additional products eluting at 4.8
and 12.7 min were detected by liquid scintillation counting
and accounted for less than 16% of the total products formed
in the samples incubated with NADPH.
The amounts of product formed, as shown in Table 2, were
quantified by integration of the liquid scintillation data since
only benzaldehyde and N,N⬘-di-benzylthiourea were formed
in sufficient amounts to be quantified by UV detection. The
average yield of product specifically formed in the NADPHdependent reaction was 10.6 nmol of product/nmol of P450
2B1. The enzyme lost more than 90% of its original activity
under the conditions used to determine product formation.
Therefore, the partition ratio, defined as the number of metabolite molecules produced per enzyme molecule inactivated
(Silverman, 1996), is approximately 11:1.
The identities of benzylamine, benzoic acid, benzaldehyde,
and N,N⬘-di-benzylurea were confirmed by GC-MS analysis
as described under Materials and Methods. The product ion
spectra and GC retention times were compared with the
authentic standards. Benzylamine eluted at 7.6 min and
gave characteristic ion fragments at m/z (relative %) 107
(M⫹, 61.1%), 106 (100%), 91 (6.2%), and 77 (41.3%) by loss of
H, NH, and CH2, respectively. Benzoic acid eluted at 9.4 min
and gave characteristic ion fragments at m/z (relative %) 122
(M⫹, 45.5%), 105 (54.6%), and 77 (47.9%) by loss of OH and
CO, respectively. Benzaldehyde eluted at 7.1 min and gave
characteristic ion fragments at m/z (relative %) 106 (M⫹,
69.2%), 105 (54.6%), and 77 (100%) by loss of H and CO,
respectively. N,N⬘-Di-benzylurea eluted at 8.3 min and gave
no parent ion fragment with characteristic ion fragments at
m/z (relative %, fragment lost) 163 (4.5%, M⫹-C6H5), 134
(100%, M⫹-PhCH2NH), 106 (50.5%, M⫹-PhCH2NHCO), 91
(35.6%, M⫹-PhCH2(NH)2CO), and 77 (13.4%, M⫹Ph(CH2)2(NH)2CO). BITC eluted at 11.2 min under these
conditions.
204
Goosen et al.
patients with breast cancer (Hellmold et al., 1998). One
would therefore expect tissue-specific activation of carcinogens.
The KI and kinact for P450 2E1 inactivation in microsomal
preparations were 18 ␮M and 0.05 min⫺1, respectively, and
this is in accordance with values obtained using purified
P450 2E1 (Moreno et al., 1999). It appears that BITC is a
more efficient inactivator of P450 2B1 than P450 2E1 as
reflected in the slower inactivation rate and larger partition
ratio for inactivation of P450 2E1. The partition ratio for
inactivation of P450 2B1 determined from the amount of
product released per mole of enzyme inactivated is approximately 11 compared with 27 for P450 2E1. This could explain
the in vivo effects on these isozymes described earlier. P450
2D6 is involved in the metabolism of a significant number of
drugs, and different phenotypes may be associated with diseases such as Parkinsonism and various cancers
(Guengerich, 1995). BITC also inactivated P450 2D6 in a
mechanism-based manner. However, the rate of inactivation
was slow since 100 ␮M BITC resulted in only approximately
20% loss of activity in 10 min. Human P450 2C9 and rat P450
3A2 were not inactivated by BITC.
The mechanism of inactivation of hepatic microsomal
P450s is thought to proceed through one of three characterized pathways (Osawa and Pohl, 1989): formation of a reactive intermediate that covalently modifies the heme moiety,
destruction of the heme with cross-linking to the apoprotein,
or covalent modification of the P450 apoprotein. The separation of P450 2B1 by SDS-PAGE followed by autoradiography,
indicated a specific increase in radiolabeled metabolite associated with the P450 apoprotein. Modification of the heme is
not implicated, because the heme would be dissociated from
the protein under the conditions of SDS-PAGE and previous
studies indicated no apparent heme modification (Goosen et
al., 2000). The radiolabel remained bound to the protein
14
C label on BITC.
under denaturing conditions, and this indicates a covalent
modification of the apoprotein by a metabolite of BITC. Stoichiometric calculations revealed that approximately 1 mol of
radiolabeled metabolite was associated per mole of enzyme
inactivated (Goosen et al., 2000). Taken together, these data
suggest that a critical amino acid in the active site of the
enzyme is modified during metabolism, which then prevents
further binding or catalysis of substrate.
In an attempt to identify the reactive intermediate involved in enzyme inactivation, the metabolites formed by this
reaction were analyzed by HPLC and GC-MS. As shown in
Table 2, the major metabolite identified was benzylamine.
The formation of isocyanates from isothiocyanates was first
reported by Lee (1992), who described the conversion of
2-naphthyl isothiocyanate to 2-naphthyl isocyanate, and subsequently the formation of benzyl isocyanate from BITC (Lee,
1996). Presumably, BITC is oxidatively desulfurated to the
putative product benzyl isocyanate (Fig. 5). The greater electronegativity of oxygen compared with sulfur implies increased reactivity of the central carbon atom on the isocyanate functionality. Accordingly, hydrolysis of benzyl
isocyanate to benzylamine is rapid and complete. Alternatively, benzyl isocyanate could also react with benzylamine to
form the corresponding carbamate, N,N⬘-di-benzylurea.
BITC in turn can also react with benzylamine to yield the
thiocarbamate, N,N⬘-di-benzylthiourea. The formation of
N,N⬘-di-benzylurea is dependent on the formation of the benzyl isocyanate intermediate since it has been shown that
diarylthioureas do not desulfurate readily in vivo, whereas
monoarylthioureas desulfurate considerably (Lee, 1996). It is
further believed that benzyl isocyanate partitions between
hydrolysis or alternatively reacts with nucleophilic amino
acid residues in the active site of the enzyme. This is evident
from the covalent attachment of a 14C-labeled metabolite of
BITC to the apoprotein of P450 2B1. These observations are
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Fig. 5. Proposed reaction scheme for the metabolism of BITC by purified P450 2B1. 夝, site of the
Inactivation of Cytochromes P450 by Benzyl Isothiocyanate
Acknowledgments
We thank Dr. Ute M. Kent and Hsia-Lien Lin for preparation of
rat liver microsomes and purification of P450 2B1 and reductase. We
are also grateful to Dr. Fung-Lung Chung, who provided us with the
[14C]BITC, and Dr. Mei-Sie Lee, who provided some of the authentic
standards. We thank Dr. Alfin D. N. Vaz for help with the GC-MS
analyses and for helpful discussions and suggestions.
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similar to those made by El-Hawari and Plaa (1977), who
showed that protein binding of 1-[3H]naphthyl isothiocyanate (labeled at the 4-position of the ring) or 1-[14C]naphthyl
isothiocyanate (labeled in the isothiocyanate moiety) to rat
liver microsomes was NADPH-dependent. However, the reactive binding species was not identified, presumably
1-naphthyl isocyanate by analogy to the present findings. In
control reactions without NADPH, the formation of benzylamine from spontaneous hydrolysis of BITC and the formation of N,N⬘-di-benzylthiourea was also observed. The
amount of N,N⬘-di-benzylthiourea was similar to that formed
in experimental reactions, whereas there was an almost
4-fold increase in the formation of benzylamine in these
reactions, as would be expected by the formation of the benzyl isocyanate intermediate.
A second pathway for the metabolism of BITC by cytochrome P450 2B1 was also identified in this study. BITC
could be oxidized at the ␣-carbon to yield benzaldehyde and
the thiocyanate anion. Alternatively, benzylamine could be
deaminated to give benzaldehyde and ammonia. The mechanism for the formation of benzaldehyde was not investigated here, but the possible role for the formation of the
thiocyanate anion in the tumor suppression by BITC
(Wattenberg, 1981) favors this pathway. The subsequent oxidation of benzaldehyde would yield benzoic acid. The formation of benzoic acid was also observed in dogs, where administration of BITC resulted in the excretion of hippuric acid,
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(Brüsewitz et al., 1977). This second pathway might also be
important in the metabolism of BITC by P450s which are not
inactivated by BITC.
The extensive conjugation of BITC with GSH (Brüsewitz et
al., 1977) might also be important for the in vivo effects of
BITC. Conjugation is usually considered to be a detoxification process but might also act as a transport mechanism for
BITC with subsequent cleavage at peripheral organs (Meyer
et al., 1995). The half-life of the isocyanate is extremely short
and would therefore reduce any local tissue reactions. However, conjugation of the isocyanate product with GSH could
contribute to mutagenicity, because some isocyanates are
known to be mutagenic and toxic (Raulf-Heimsoth and Baur,
1998). Therefore, local release of the conjugated product
might also contribute to the toxicity of BITC (Hirose et al.,
1998) and should be evaluated when these compounds are
considered for dietary supplementation to prevent cancer.
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human P450s 2B6 and 2D6. The inactivation of purified P450
2B1 probably proceeded through metabolism of BITC to the
reactive benzyl isocyanate intermediate, which covalently
modified the P450 apoprotein. It is believed that this inactivation of several P450s involved in carcinogen activation
might contribute significantly to its chemopreventative effect.
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Send reprint requests to: Dr. Paul F. Hollenberg, Dept. of Pharmacology,
Medical Science Research Bldg. III, 1150 West Medical Center Dr., Ann Arbor,
MI 48109-0632. E-mail: [email protected]
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