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Carcinogenesis Advance Access originally published online on December 19, 2005
Carcinogenesis 2006 27(4):782-790; doi:10.1093/carcin/bgi301
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Effects of benzyl and phenethyl isothiocyanate on P450s 2A6 and 2A13: potential for chemoprevention in smokers

Linda B. von Weymarn 1, {ddagger}, Jamie A. Chun 1 and Paul F. Hollenberg 1, *

1 Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA

* To whom correspondence should be addressed. Tel: +1 734 764 8166; Fax: +1 734 763 5387; Email: phollen{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isothiocyanates have been shown to be potent inhibitors of carcinogenesis in animals exposed to a number of chemical carcinogens including the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study the effects of benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), two naturally occuring isothiocyanates, on P450 2A6 and 2A13 were investigated. P450s 2A6 and 2A13 are thought to be the primary human P450 enzymes responsible for the in vivo metabolism of nicotine and NNK, respectively. In vitro, BITC and PEITC efficiently inhibited P450 2A6- and 2A13-mediated coumarin 7-hydroxylation. The inhibition of P450 2A6 and 2A13 by BITC was non-competitive with KI's of 4.1 and 1.3 µM, respectively. PEITC was a more potent inhibitor of both enzymes than BITC, with a KI of 0.37 µM for P450 2A6 and 0.03 µM for P450 2A13. P450 2A6-mediated metabolism of nicotine and P450 2A13-mediated {alpha}-hydroxylation of NNK were also inhibited significantly by these two isothiocyanates. Both BITC and PEITC were able to inactivate P450 2A6 and 2A13 in an NADPH-dependent manner potentially through the formation of adducts to the apoprotein. The potent inhibition of P450 2A6- and 2A13-mediated metabolisms together with the ability of BITC and PEITC to inactivate the enzymes suggests the possibility that these isothiocyanates could be developed as chemopreventive agents to protect smokers who are unwilling or unable to quit smoking against lung cancer.

Abbreviations: BITC, benzylisothiocyanate; BSA, bovine serum albumin; DLPC, dilauroyl-L-{alpha}-phosphatidylcholine; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; 7-OHC, 7-hydroxycoumarin; OPBA, 4-oxo-4-(3-pyridyl)butyric acid; P450, cytochrome P450; PEITC, phenethyl isothiocyanate; reductase, NADPH-P450 oxidoreductase; TCA, trichloroacetic acid; TFA, trifluoroacetic acid


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
It is well established that the leading cause of lung cancer in humans is smoking. Tobacco-derived nitrosamines are present in significant quantities in both unburned tobacco and in tobacco smoke and they have been shown to be potent carcinogens in animal models (1). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most abundant and potent pulmonary carcinogens present in tobacco and, like many chemical carcinogens, requires metabolic activation by enzymes from the cytochrome P450 (P450) superfamily of enzymes to exert its tumorigenic potential. Enzymes from the P450 2A subfamily efficiently catalyze the metabolic activation of a number of nitrosamines, including NNK, in vitro (2–6). However, the ability of individual P450 2A enzymes to metabolize nitrosamines to their ultimate carcinogenic forms differs significantly among the P450 2Aenzymes.

There are three members in the human P450 2A gene family, two of which are proven to be functional enzymes (5,7,8). P450 2A6 is a hepatic enzyme that constitutes ~1–10% of the total P450 content in human livers. P450 2A6, like many other P450 2A enzymes, is a specific and efficient coumarin 7-hydroxylase (9–11). In addition, P450 2A6 is the primary enzyme responsible for the conversion of nicotine to cotinine, an inactive metabolite of nicotine, in vivo (12–14). P450 2A13 is an extra-hepatic P450 enzyme that is expressed in significant levels in human lung and trachea. It is 95% identical in its sequence to P450 2A6, differing by only 32 amino acids. There are distinct overlaps in the substrate specificities of P450s 2A6 and 2A13. However, the rates of substrate metabolism and the stereo-selectivities differ significantly between the two enzymes (5,11,15). Of potential importance for human lung cancer, NNK is bioactivated much more efficiently by P450 2A13 than by 2A6. The Vmax/Km for methylene hydroxylation of NNK, which is believed to be an obligatory step in its activation to the ultimate carcinogen form, by 2A6 is 0.008 compared with 0.36 for 2A13 (5). The high rate of NNK bioactivation by P450 2A13 together with the higher level of expression of P450 2A13 in the human lung suggests that P450 2A13 could be the primary enzyme in human lung responsible for the local metabolic activation of NNK. The metabolism of nicotine by P450 2A13 in vitro is also more efficient than by P450 2A6 (16).

Isothiocyanates are potent and selective inhibitors of carcinogenesis in rodents induced by a number of chemical carcinogens including NNK (17). Isothiocyanates such benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) are found in high levels in cruciferous vegetables, such as watercress, gardencress, cabbage, cauliflower and broccoli, where they occur as thioglucoside conjugates called glucosinolates (18,19). The glucosinolates are hydrolyzed upon chewing or maceration by the enzyme myrosinase to release the isothiocyanate as well as other products. Inhibition of P450 enzymes and induction of phase II enzymes, among other things, have been implicated in the chemopreventive action of the isothiocyanates. There are three possible mechanisms that can lead to the inhibition of the P450 enzymes by isothiocyanates: (i) the isothiocyanates may inhibit carcinogen activation by competitive inhibition of the P450 enzymes involved in the bioactivation; (ii) depending on the reactivity of the particular isothiocyanate, the isothiocyanate may react directly with one or more nucleophilic amino acids in the P450 active site that are crucial for P450 catalysis; and (iii) metabolic activation of the isothiocyanate to a reactive intermediate may result in covalent binding of the isothiocynate to the heme moiety or to the apoprotein resulting in inactivation of the enzyme.

The chemopreventive properties of BITC and PEITC in animal models have been extensively studied [reviewed in (17)]. BITC is an inhibitor of lung tumor formation in mice treated with polyaromatic hydrocarbons such as benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene (20–22), but it is not a very potent inhibitor of tumor formation attributed to exposure of the mice to nitrosamines such as NNK (23,24). In contrast, PEITC has been reported to be a potent inhibitor of lung tumor formation by NNK in mice and rats, esophageal tumor formation in rats treated with N-nitrosobenzylmethylamine and liver tumors in rats treated with N-nitrosodiethylamine (24–27). The difference in specificity between the two isothiocyanates is most likely due to their ability to inhibit/inactivate different P450 enzymes. BITC and PEITC are structurally very similar, differing by only one carbon in the chain that connects the isothiocyanate functional group to the phenyl ring (Figure 1). PEITC is reported to be a competitive inhibitor of P450s 1A2 and 2A6, a non-competitive inhibitor of P450s 2B6, 2C9, 2C19, 2E1 and 2D6, but it did not inhibit P450 2C8 (28). BITC is a mechanism-based inactivator of P450s 1A1, 1A2, 2B1, 2E1, 2B6 and 2D6, but not of P450s 3A2 and 2C9 (29). Both BITC and PEITC have been reported to be mechanism-based inactivators of P450s 2B1 and 2E1 (29–32). Interestingly, only PEITC was able to inactivate a mutant form of P450 2E1, P450 2E1 T303A (32). Currently, there are very little data available on the effects of BITC and PEITC on the two human P450 2A enzymes 2A6 and 2A13.


Figure 1
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  		Fig. 1. Structures of the two isothiocyanates (BITC and PEITC) used in the studies.

 
In this report we have evaluated the ability of BITC and PEITC to inhibit P450 2A6- and 2A13-mediated coumarin 7-hydroxylation as well as P450 2A6-mediated nicotine oxidation and P450 2A13-mediated NNK metabolism. The ability of BITC and PEITC to inactivate P450s 2A6 and 2A13 in a time-, concentration- and NADPH-dependent manner was also investigated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Dilauroyl-L-{alpha}-phosphatidylcholine (DLPC), NADPH, bovine serum albumin (BSA), coumarin, 7-hydroxycoumarin (7-OHC) and catalase were purchased from Sigma Chemical Co. (St Louis, MO). BITC and PEITC were purchased from Trans World Chemicals (Rockville, MD). [5-3H]-(S)-Nicotine and 3H-NNK were gifts from Sharon Murphy at the University of Minnesota Cancer Center (Minneapolis, MN). Trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL). All other reagents were purchased from Sigma Chemical Co. and were of analytical grade.

Enzyme expression and purification
The cDNA for P450 2A13 was a gift from Xinxin Ding (Wadsworth Center, New York State Department of Health, Albany, NY) and the expression vector containing His-tagged P450 2A6 was a gift from Fred Guengerich (Vanderbilt University, Nashville, TN). Expression of P450 2A6 in DH5{alpha} cells was accomplished using essentially the same protocol that has been used previously for the expression of P450 2A6 and P450 3A4 (33,34). Transformed cells were grown in 10 ml Luria–Bertani media containing 100 µg/ml ampicillin at 37°C overnight prior to expansion into 1 liter of TB peptone containing ampicillin (100 µg/ml), 1 mM thiamine and trace elements. The cells were grown at 32°C until the OD at 600 nm reached 0.6, and at that time {delta}-aminolevulinic acid (0.5 mM) was added. The cells were induced with 1 mM isopropyl-ß-D-thiogalactoside (IPTG) when the OD at 600 nm was ~1.2 AU and then allowed to grow for 20 h while shaking at 200 r.p.m. at 32°C. The cells were harvested and purified according to the published protocols (35). The purity of the enzyme was determined by LC/MS. The HPLC and MS conditions used were the same as described in the Whole protein LC/MS analysis section.

The conditions for the expression of P450 2A13 were modified from a published protocol previously (36). The use of C41(DE3) cells significantly increased the expression of P450 2A13 compared with DH5{alpha} cells. In addition, inducing the cells with IPTG for 72 h while shaking at 120 r.p.m. at 24°C increased the yield significantly. The NADPH-P450 oxidoreductase (reductase) used was expressed in Escherichia coli Topp 3 cells and purified as described previously (37). All enzymes were purified according to the protocols published previously (34,35,37).

Effects of BITC and PEITC on P450s 2A6 and 2A13
P450 2A6 and P450 2A13 were reconstituted with reductase and lipid (DLPC) for 45 min at 4°C (36). After the reconstitution, catalase and Tris buffer were added to the reconstituted enzymes to give a reconstituted enzyme solution containing 1 pmol/µl P450 2A13, 2 pmol/µl reductase, 0.1 µg/µl lipid and 26 U/µl catalase in 50 mM Tris buffer, pH 7.4. The molar ratio of P450 to reductase was 1:2 unless otherwise noted.

Inhibition of P450 2A6- and 2A13-mediated coumarin 7-hydroxylation
The formation of 7-OHC was measured as described previously (6). Aliquots of the reconstituted enzyme solution containing 5 pmol P450 were added to reaction mixtures containing coumarin (0.4–20 µM), BITC or PEITC and NADPH (0.2 mM) in 50 mM Tris buffer, pH 7.4. The concentrations of BITC used were 3, 5 and 25 µM for P450 2A6 and 0.6, 1 and 3 µM for P450 2A13. The PEITC concentrations used were 3, 6 and 9 µM for P450 2A6 and 0.03, 0.06 and 0.1 µM for P450 2A13. The final reaction volume was 300 µl. After a 10 min incubation at 30°C the reaction was terminated by the addition of 20 µl of 15% trichloroacetic acid (TCA). The 7-OHC formed was analyzed by HPLC using HPLC System I (described below) with fluorescence detection (excitation wavelength, 350 nm, and emission wavelength, 453 nm) as described previously (38). Kinetic parameters were determined using the Ez-Fit 7 kinetics program from Perrella Scientific (Amherst, NH) (39). This program uses non-linear regression to calculate kinetic constants.

Inhibition of P450 2A6-mediated nicotine metabolism
Aliquots of the reconstituted enzyme solution described above containing 15 pmol P450 2A6 were added to reaction mixtures containing [5-3H]-(S)-nicotine (200 µM), BITC (25 µM) or PEITC (3 µM) and NADPH (0.2 mM) in 100 mM Tris buffer, pH 7.4. The final reaction volume was 200 µl. After a 20 min incubation at 30°C, the reaction was terminated by the addition of 20 µl of 10% trifluoroacetic acid (TFA). The samples were centrifuged for 15 min at 1500 g and the supernatant was analyzed by HPLC using HPLC System II.

Inhibition of P450 2A13-mediated NNK metabolism
Aliquots of the reconstituted enzyme solution described above containing 15 pmol P450 2A13 were added to reaction mixtures containing [3H]NNK (10 µM), BITC (1 µM) or PEITC (0.3 µM) and NADPH (0.2 mM) in 100 mM Tris buffer, pH 7.4. The final reaction volume was 200 µl. After a 20 min incubation at 30°C, the reactions were terminated by the addition of 20 µl each of 0.3 M Ba(OH)2 and ZnSO4. The samples were centrifuged at 1500 g for 15 min and the supernatant was analyzed by HPLC using HPLC System III. The standards, 4-oxo-4-(3-pyridyl)butyric acid (OPBA) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), were added to the samples before HPLC analysis.

Inactivation of P450s 2A6 and 2A13 by BITC and PEITC
A primary reaction mixture containing 1 pmol/µl P450, 2 pmol/µl reductase, 0.1 µg/µl lipid, 26 U/µl catalase, BITC or PEITC (0, 10 or 100 µM), and 1 mM NADPH in 50 mM Tris buffer (pH 7.4) was incubated for 5 min at 30°C before the addition of NADPH. Aliquots (4 µl) were removed before the addition of NADPH and at 10 min after the addition of NADPH and added to a secondary reaction mixture containing coumarin (20 µM) and NADPH (0.2 mM) in 50 mM Tris buffer, pH 7.4, in a final volume of 300 µl. The amount of catalytic remaining activity was determined using the coumarin 7-hydroxylation assay described above. The kinetics for the inactivation of P450 2A6 and 2A13 by BITC were determined in a similar manner except that the volume of the secondary reaction (the coumarin 7-hydroxylation assay) was increased from 300 to 800 µl and the concentration of coumarin was increased from 20 to 40 µM. For PEITC, an aliquot of each sample was run through a G50 Sephadex spin-column to remove PEITC. This was performed to eliminate any competitive inhibition of the P450s by PEITC in the secondary reaction mixture. The remaining activity before and after the spin-column runs was determined for 0 and 100 µM PEITC, at 0, 2 and 10 min time-points.

The amount of catalytic remaining activity, spectrally detectable P450 remaining, and native heme remaining were determined to aid in the determination of the extent of inactivation as well as the mechanism of inactivation. Aliquots containing 5 pmol P450 (5 µl) were removed from the primary reactions before the addition of NADPH and then at 10 min after the addition of NADPH and assayed for coumarin 7-hydroxylation remaining activity as described above. The flow-through from the spin-column was also assayed for coumarin 7-hydroxylation remaining activity.

At the 0 and 10 min time-points, aliquots of the primary reaction mixture (100 pmol P450) were added to 900 µl of an ice-cold quench buffer containing 40% glycerol and 0.6% NP-40 in 50 mM potassium phosphate, pH 7.4. The reduced CO spectra were measured on a DW2 UV/VIS spectrophotometer (SLM Aminco, Urbana, IL) with an OLIS spectroscopy operating system (On-Line Instrument Systems, Bogart, GA) using the method of Omura and Sato (40).

At the 10 min time-point, 100 µl aliquots of both the inactivated and control samples (100 pmol P450) were analyzed by reversed-phase HPLC using HPLC System IV and the elution of heme and protein were monitored using a diode array detector. Heme was monitored at 405 nm and protein was monitored at 260 nm.

The flow through for each sample from the spin-columns was also assayed for remaining activity, amount of heme remaining as measured by the reduced CO spectrum and the amount of native heme remaining as measured by HPLC.

Whole protein LC/MS analysis
ESI-LC/MS of the proteins was carried out on a ThermoQuest (Thermoquest, Schaumburg, IL) ion trap mass spectrometer interfaced with a Hewlett Packard 1100 series HPLC system (Hewlett Packard, Palo Alto, CA). P450 2A6 or 2A13 (0.4 nmol) was reconstituted with reductase (0.4 nmol) and lipid (30 µg) for 45 min at 4°C. The primary reaction mixture contained 1 pmol/µl P450, 1 pmol/µl reductase, 75 ng/µl lipid, 26 U/µl catalase, 100 µM BITC or PEITC, and 1 mM NADPH (water in control samples) in 50 mM Tris buffer, pH 7.4, in a total volume of 200 µl. The P450/reductase ratio was 1:1. The samples were incubated for 5 min at 30°C before the addition of NADPH. At 10 min after the addition of NADPH the exposed control (–NADPH, +BITC or PEITC) and the inactivated (+NADPH, +BITC or PEITC) samples (50 pmol P450) were separated by reversed-phase HPLC using HPLC System V and analyzed on the ion trap mass spectrometer. MS scans were acquired with the sheath gas set to 90 (arbitrary units) and the auxillary gas set to 30 (arbitrary units). The spray voltage was 3.5 kV, the capillary voltage 45 V and the capillary temperature was set at 200°C. The protein spectra were deconvoluted using the Bioworks Browser 3.1 (ThermoFinnigan Corp., Woburn, MA).

HPLC analysis
HPLC System I for the separation and detection of 7-OHC
The HPLC system used consisted of a Waters 600 system controller, Waters 501 series pumps, a Waters 474 fluorescence detector and a Waters 717 autosampler (Waters Corp., Milford, MA). 7-OHC was eluted isocratically with 65% H2O/34% methanol/1% acetic acid on a Varian Microsorb-MV C18, 5 µm, 100 Å column (250 x 4.6 mm). The flow rate was 0.8 ml/min.

HPLC System II for the separation and quantification of nicotine metabolites
The HPLC system consisted of a Waters 600 gradient controller, two Waters 510 pumps, a Waters 441 absorbance detector and a ß-ram radioflow detector (IN/US Systems, Tampa, FL). The nicotine and nicotine metabolites were separated on a Luna C18 reversed-phase HPLC column (0.46 x 25 cm, 5 µm; Phenomenex, Torrance, CA) and were eluted isocratically (0.2% TFA in water) over 30 min at a flow rate of 0.7 ml/min (16). The scintillant (Monoflow X; National Diagnostics, Atlanta, GA) flow rate was 2.1 ml/min.

HPLC System III for the separation and quantification of NNK metabolites
The HPLC system used was the same as described for the nicotine metabolites. NNK and its metabolites were separated on a Gemini C18 reversed-phase HPLC column (0.46 x 25 cm, 5 µm, 110 Å; Phenomenex). The metabolites were eluted using a gradient from 100% A (20 mM sodium phosphate and 1 mM sodium bisulfate, pH 7) to 30% B (5% water and 95% methanol) in 60 min, and then to 50% B in 10 min. The flow rate was 0.7 ml/min. The scintillant (Pico-Fluor 40; PerkinElmer, Boston, MA) flow rate was 3.0 ml/min.

HPLC System IV for heme analysis
The samples were separated on a reversed-phase Phenomenex Jupiter C4, 5 µm, 300 Å column (150 x 2.0 mm; Phenomenex). The mobile phase consisted of 0.1% TFA in water (A) and 0.05% TFA in acetonitrile (B). The separation was accomplished by holding the mobile phase at 70% A/30% B for 5 min followed by a linear gradient to 80% B in 25 min and then to 95% B in 5 min. The elution of heme containing peaks was monitored at 405 nm using a Waters diode array detector. The flow rate was 1 ml/min.

HPLC System V for LC/MS analysis of adducts to the apoprotein
The samples were injected onto an Agilent Zorbax 300SB-C3 reversed-phase HPLC column that was equilibrated with 60% water containing 0.1% TFA (A) and 40% acetonitrile containing 0.1% TFA (B). The protein components were eluted by maintaining the initial concentrations of the mobile phase at 60% A/40% B for 5 min followed by a linear gradient to 80% B in 25 min and then to 90% B in 5 min, followed by holding at 90% B for 15 min. The flow rate was 0.3 ml/min.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
P450 2A6 and P450 2A13 expressed in E. coli and partially purified were used to study the effects of two naturally occurring isothiocyanates, BITC and PEITC, on P450 2A6- and 2A13-mediated coumarin 7-hydroxylation activity. A new protocol for the expression of P450 2A13 in E.coli was developed that significantly increased the level of expression (~50–70 nmol purified P450/liter culture) compared with the protocol published previously (~10–30 nmol purified P450/liter culture) (36). Overall, the expression of P450 2A13 was less efficient than the expression of P450 2A6 (~500 nmol purified P450/liter culture) and the conditions for maximal expression differ significantly between the two highly homologous enzymes.

Both BITC and PEITC were able to inhibit P450 2A6 and 2A13-mediated coumarin 7-hydroxylation (Figure 2 and Table I). The inhibition of P450 2A6-mediated coumarin 7-hydroxylation by BITC had a KI of 4.1 µM and was non-competitive, whereas the non-competitive inhibition by PEITC had a KI of 0.37 µM. The inhibition of P450 2A13-mediated coumarin 7-hydroxylation by PEITC was extremely potent, with an apparent KI for the uncompetitive inhibition of 30 nM. BITC was also a potent non-competitive inhibitor of P450 2A13-mediated coumarin 7-hydroxylation exhibiting a KI of ~1.3 µM.


Figure 2
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  		Fig. 2. Inhibition of P450 2A6 and 2A13-mediated coumarin 7-hydroxylation by BITC and PEITC. The inhibition of P450 2A6 and 2A13-mediated coumarin 7-hydroxylation was determined as described in the Materials and methods section. (A) Inhibition of P450 2A6 by 0 µM (closed squares), 3 µM (closed triangles), 5 µM (closed inverted triangles), and 10 µM (closed diamonds) BITC. (B) Inhibition of P450 2A6 by 0 µM (closed squares), 3 µM (closed triangles), 6 µM (closed inverted triangles) and 9 µM (closed diamonds) PEITC. (C) Inhibition of P405 2A13 by 0 µM (closed squares), 0.6 µM (closed triangles), 1.0 µM (closed inverted triangles) and 3.0 µM (closed diamonds) BITC. (D) Inhibition of P450 2A13 by 0 µM (closed squares), 0.03 µM (closed triangles), 0.06 µM (closed inverted triangles) and 0.1 µM (closed diamonds) PHITC. Curves were generated using non-linear regression analysis. The data shown represent the mean and standard deviations from three experiments performed in duplicate.

 

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  		Table I. Inhibition of P450 2A6- and 2A13-mediated coumarin 7-hydroxylationa

 
P450 2A6 is thought to be one of the key enzymes involved in the metabolism of nicotine in smokers. Therefore, the ability of BITC and PEITC to inhibit P450 2A6-mediated nicotine metabolism was determined. BITC (25 µM) and PEITC (3 µM) significantly inhibited P450 2A6-mediated nicotine metabolism, and the inhibition was 84 and 75%, respectively (Table II). The concentration of nicotine used in this assay, 200 µM, is approximately the Km for nicotine 5'-oxidation by P450 2A6 in the absence of cytosol (16).


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  		Table II. Inhibition of P450 2A6-mediated nicotine metabolism and P450 2A13-mediated NNK metabolism by BITC and PEITCa

 
BITC and PEITC both inhibited the bioactivation of the tobacco-specific carcinogen NNK by P450 2A13. The P450 2A13-mediated metabolism of NNK was inhibited ~40% by 1 µM BITC and 32% by 0.3 µM PEITC (Figure 3 and Table II). The two major pathways for the bioactivation of NNK, resulting in {alpha}-methylene hydroxylation (measured as OPBA) and {alpha}-methyl hydroxylation (measured as HPB), were inhibited to similar extents (Figure 3).


Figure 3
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  		Fig. 3. HPLC chromatograms for the inhibition of P450 2A13-mediated {alpha}-hydroxylation of NNK by BITC and PEITC. The inhibition of NNK metabolism by BITC and PEITC was determined as described in the Materials and methods section. P450 2A13 was incubated with NNK (11 µM) in the absence of isothiocyanate (A) or in the presence of 1 µM BITC (B) or 0.3 µM PEITC (C) for 20 min at 30°C. The metabolites were identified based on co-elution with authentic standards. The chromatograms are representative of two independent experiments run in duplicate.

 
PEITC and BITC have previously been shown to be mechanism-based inactivators of several different P450 enzymes (28,30,31). The ability of PEITC and BITC to inactivate P450s 2A6 and 2A13 in a time-, concentration- and NADPH-dependent manner was investigated. The normal protocol for studying mechanism-based inactivation includes a pre-incubation step in the presence of the inactivator and NADPH followed by an incubation step that measures the remaining activity. However, this protocol could not be used to study the mechanism-based inactivation of P450s 2A6 and 2A13 by PEITC since significant losses (25%) in activity were observed in the control samples containing 10 µM isothiocyanate (Table III). These losses in the exposed control samples are due to inhibition of the coumarin 7-hydroxylation activity by the isothiocyanate transferred from the primary to the secondary reaction along with the P450 to be analyzed for the remaining activity. However, the losses in activity in the inactive samples (+BITC/PEITC, +NADPH) were greater than the losses in the exposed controls (+BITC/PEITC, –NADPH), suggesting that there is an NADPH-dependent mechanism-based inactivation (Table III). For PEITC, with both P450 2A6 and 2A13, there was almost a complete loss in activity in the exposed controls (100 µM), hence any mechanism-based inactivation may have been completely masked by inhibition.


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  		Table III. Effects of BITC and PEITC on P450 2A6- and 2A13-mediated coumarin 7-hydroxylationa

 
We were able to determine the kinetics for the NADPH-dependent inactivation of P450 2A6 by BITC by increasing the volume of the secondary reaction in order to eliminate inhibition in the secondary reaction. The loss in activity was log-linear with time and concentration dependent (Figure 4A). The concentration of BITC required to obtain the half maximal rate of inactivation (KI) was 28 µM, the kinact. was 0.055 min–1 and the t1/2 was 12.6 min. The inactivation of P450 2A13 by BITC was very rapid (Figure 4B). The loss in activity was log-linear for the first minute at the 100 µM concentration. At lower concentrations the inactivation appeared to be virtually complete by the first time-point (0.5 min). At concentrations above 100 µM there was a significant inhibition in the secondary reaction even at a 200-fold dilution of BITC. There was a clear concentration-dependent inactivation of P450 2A13 by BITC and there is a time-dependent loss in activity at the high-BITC concentrations (Figure 4B).


Figure 4
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  		Fig. 4. Time- and concentration-dependent inactivation of P450 2A6 and P450 2A13 by BITC. Experimental conditions are described in the Materials and methods section. (A) Inactivation of P450 2A6 by BITC. (B) Inactivation of P450 2A13 by BITC. The BITC concentrations used were as follows: 0 µM (closed circles), 20 µM (open circles), 40 µM (closed inverted triangles), 60 µM (open inverted triangles) and 100 µM (closed squares) for P450 2A6 and 0 µM (closed circles), 10 µM (open circles), 20 µM (closed inverted triangles), 60 µM (open inverted triangles) and 100 µM (closed squares) for P450 2A13. % Act. Remaining refers to the amount of coumarin 7-hydroxylation activity remaining compared with control samples. The data shown represent the mean and standard deviation of three independent experiments. The inset represents the double-reciprocal plot generated from the slopes of the lines at the various concentrations.

 
In studies to investigate the mechanism-based inactivation by PEITC, inhibition of the secondary reaction was observed even at a 1:1000 dilution (data not shown). Higher dilutions were not possible due to the limits of detection of the assay. Therefore, in order to investigate whether or not PEITC is an inactivator of P450 2A6 and/or P450 2A13, we used spin-columns to remove PEITC prior to assaying for the remaining activity. Aliquots of the control samples incubated in the absence of PEITC and samples incubated in the presence of 100 µM PEITC and NADPH were removed at different time-points and the remaining activity was measured following elution from the spin-columns. As shown in Table IV, there was a time-dependent loss in activity for both enzymes when incubated with PEITC and NADPH. To determine whether there was NADPH-dependent loss in activity we compared losses in activity in samples incubated with PEITC and NADPH with the control samples incubated with PEITC and without NADPH. As a comparison, the same experiments were performed with BITC. Before applying the samples to spin-columns there was an apparent NADPH-dependent loss in activity for BITC (71%) and PEITC (43%) with P450 2A13 and for BITC (45%) with P450 2A6 (Table V). No loss in activity was observed following the incubation of PEITC with P450 2A6. Following elution of the samples from the spin-columns, no loss in activity was observed in the exposed control samples for either isothiocyanate with P450s 2A6 or 2A13 (data not shown). However, eluting the samples from the spin-columns did not restore any of the activity in the inactive samples, indicating that the enzymes were inactivated in an NADPH-dependent manner (Table V). A loss in the catalytic activity of P450 2A6 was observed following incubation with PEITC and NADPH after the spin-column elution but not before (Table IV).


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  		Table IV. PEITC- and time-dependent loss in P450 2A6 and 2A13 activitya

 

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  		Table V. Effect of BITC and PEITC on P450 2A6- and 2A13-mediated coumarin activity, reduced CO spectrum and HPLC-detected hemea

 
In order to get an indication of the mechanism of inactivation (formation of adducts with protein, heme or both), the amount of native unmodified heme and the amount of the P450 that gave a reduced CO spectra was determined after inactivation. As shown in Table V, there were no significant losses in the amount of native heme present after inactivation of P450 2A6 or 2A13 by either isothiocyanate. Neither BITC nor PEITC gave rise to heme adducts when the samples were analyzed by HPLC with monitoring at 405 nm (data not shown). Very different results were obtained when comparing the losses in the reduced CO spectrum observed with P450s 2A6 and 2A13. No significant loss in the reduced CO spectrum was observed when P450 2A6 was inactivated with BITC. For P450 2A13, inactivation by BITC resulted in a loss in the reduced CO spectrum; however, the loss in the reduced CO spectrum (57%) was less than the loss in activity (71%) (Table V). There was no observable loss in the reduced CO spectrum for P450 2A6 following inactivation by PEITC before running the samples through the spin-column. Upon running the samples through the spin-column, a loss in the reduced CO spectrum (37%) was observed that corresponded to the loss in activity (35%). The losses in the reduced CO spectra and in the activity corresponded well for P450 2A13 following inactivation by PEITC both before and after elution from the spin-column.

The lack of heme adducts and the fact that no losses in the amount of native heme were observed (Table V) suggested that the mode of inactivation was through the formation of protein adducts. ESI-LC/MS analysis was used to investigate the formation of BITC- and PEITC-derived adducts to the P450 2A6 and 2A13 apoproteins. The inactivation of P450 2A13 by both BITC and PEITC resulted in what appears to be adducts on the P450 apoprotein (Figure 5); however, the presence of non-specific binding of the isothiocyanates to the apoprotein in the absence of NADPH makes the data somewhat hard to interpret. The mass of unmodified P450 2A13 as determined by ESI-LC/MS was 56652 ± 5 mass units. (The theoretical mass based on the amino acid sequence is 56645.) In the BITC-inactivated samples, three peaks were observed in addition to that for the unmodified protein (Figure 5A). The mass differences between the unmodified protein and the additional peaks were 159 ± 6 mass units, which could correspond to the addition of one molecule of BITC with an attached oxygen atom (MW = 165); 293 ± 3, which could correspond to the non-specific binding of two BITC molecules (MW = 298); and 447, which potentially corresponds to the binding of three BITC molecules. However, this peak was not always observed. Some non-specific binding of BITC was also seen in the exposed control sample (inset of Figure 4A). A peak corresponding to binding of one BITC molecule was generally not observed in the control samples, while a peak corresponding to the binding of two BITC molecules was usually prominent in the control samples. In the inactive sample there was a prominent peak at 56 808, which was not seen in the exposed control sample, suggesting that this adduct is the product of the metabolism of BITC to a reactive intermediate that has bound to the enzyme. The amount of adducts relative to native protein was greater in the inactivated sample than in the exposed control, again suggesting that at least some of the adducts observed are due to NADPH-dependent binding.


Figure 5
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  		Fig. 5. ESI-LC/MS analysis of P450 2A13 apoprotein adducts following inactivation by BITC and PEITC. P450 2A13 was incubated in the reconstituted system with BITC or PEITC in the presence (inactive sample) or absence (exposed control) of NADPH (1 mM) and the proteins were then analyzed by ESI-LC/MS as described in the Materials and methods section. (A) Deconvoluted spectrum of the P450 2A13 peak from a sample inactivated by BITC, the exposed control sample is shown in the inset. (B) Deconvoluted spectrum of the P450 2A13 peak from a PEITC-inactivated sample, the exposed control sample is shown in the inset.

 
In the PEITC-inactivated P450 2A13 samples the presence of a peak with a mass difference of 187 ± 8 mass units compared with the un-adducted protein potentially suggested the presence of an adducted protein that has one molecule of PEITC (163 mass units) and one or two oxygen atoms attached (Figure 4B). The additional mass difference of 329 mass units (56 981) corresponds to the non-specific binding of two molecules of PEITC. The non-specific binding of one and two molecules of PEITC was also seen in the control sample, but no peak corresponding to PEITC plus one or more oxygen was observed (inset of Figure 4B). Similar results were obtained with P450 2A6 with both BITC and PEITC (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Both BITC and PEITC are potent chemopreventive agents in animals treated with a number of different chemical carcinogens including a variety of nitrosamines (17). A number of studies have correlated the consumption of vegetables with a reduced risk of cancer (41–45), suggesting that compounds such as the isothiocyanates present in vegetables may have chemopreventive properties in humans. The data presented here support the hypothesis that BITC and PEITC, which are present at varying levels in a number of cruciferous vegetables such as watercress, gardencress, cauliflower and cabbage, could potentially be effective chemopreventive agents in humans. We have investigated the effects of BITC and PEITC on the catalytic activity of the human P450 2A enzymes, P450 2A6 and 2A13.

P450 2A6 is a hepatic enzyme that plays a major role in the initial metabolism of nicotine, the addictive component of cigarettes, to its inactive metabolite cotinine (12–14). Inhibition of P450 2A6-mediated metabolism of nicotine would lead to slower elimination of nicotine from the bodies of smokers and therefore would be expected to decrease the number of cigarettes needed to maintain a constant level of nicotine in the body (46). Overall, this decrease in the number of cigarettes smoked per day would decrease the exposure to carcinogenic nitrosamines. Both BITC and PEITC were potent inhibitors of P450 2A6-mediated nicotine metabolism. At a nicotine concentration of 200 µM, which is slightly higher than the reported Km for P450 2A6-mediated nicotine 5'-oxidation in the absence of cytosol (16), BITC (25 µM) and PEITC (3 µM) inhibited nicotine metabolism by 84 and 75%, respectively (Table II).

P450 2A13, an extrahepatic P450 expressed in high levels in the respiratory system, is an efficient catalyst of NNK bioactivation, with a reported Km of 11 µM (5). The efficient metabolism of NNK by P450 2A13 and its presence in the lung, the target tissue for NNK-mediated carcinogenesis, have lead to the suggestion that P450 2A13 is the primary enzyme responsible for the metabolic activation of NNK in smokers in vivo (3,5). Since NNK is one of the most potent pulmonary carcinogens in cigarette smoke, inhibition of P450 2A13-mediated metabolism of NNK would be expected to lower the risk of lung cancer in smokers. P450 2A13 efficiently catalyzes both {alpha}-methylene and {alpha}-methyl hydroxylation of NNK (3,5). Both these hydroxylations give rise to reactive intermediates that can bind to DNA (1). BITC and PEITC inhibited the formation of both OPBA and HPB, the products of {alpha}-methylene hydroxylation and {alpha}-methyl hydroxylation, respectively (Figure 3). Both pathways were inhibited to the same extent. An aliquot of 1 µM BITC inhibited the 2A13-catalyzed metabolism of 10 µM NNK by 40% while 0.3 µM PEITC inhibited NNK metabolism by 32% (Table II).

As shown here, both BITC and PEITC inhibited P450 2A6-mediated nicotine metabolism and P450 2A13-mediated NNK metabolism. In order to determine the relative potencies of BITC and PEITC as inhibitors of P450s 2A6 and 2A13 we used coumarin as the probe substrate. Members of the P450 2A subfamily, including P450s 2A6 and 2A13, are efficient and specific coumarin 7-hydroxylases and the conversion of coumarin to 7-hydroxycoumarin is often used as a probe of P450 2A activity in vivo. PEITC was found to be a better inhibitor than BITC for both P450 2A6- and 2A13-mediated coumarin 7-hydroxylation. The KI for the inhibition of coumarin 7-hydroxylation by PEITC was 10-fold lower for P450 2A13 than for P450 2A6 (Table I). The difference in the KIs for BITC-mediated inhibition was not as great as the difference with PEITC, 3-fold versus 10-fold. The KI for the non-competitive inhibition of P450 2A6-mediated coumarin 7-hydroxylation was 0.3 µM. This KI was 60-fold lower than that previously reported for P450 2A6 co-expressed with reductase in baculovirus microsomes (28). Differences between this study and the previous study are as follows: (i) we used the purified reconstituted enzyme system rather than a microsomal preparation; (ii) the source of the reductase and the ratios of P450 to reductase are different; and (iii) we did not use cytochrome b5 in our experiments. All of these variables may have a significant effect on the inhibition observed. The apparent KI for the inhibition of P450 2A13-mediated coumarin 7-hydroxylation by PEITC was 30 nM. However, since this KI is close to the concentration of enzyme in this reaction (20 nM), a rather large error (±10 nM) was observed. A KI in the nanomolar range makes PEITC a very promising chemopreventive agent that could potentially decrease the risk of lung cancer in smokers.

Both BITC and PEITC have been reported to be mechanism-based inactivators of a number of P450 enzymes (28–32). In these cases the mechanism of inactivation appears to be through binding of a reactive intermediate to the apoprotein. The ability of BITC and PEITC to act as mechanism-based inactivators of the P450 2A enzymes has not been studied extensively. In these studies we have shown that BITC and PEITC inactivated both P450 2A6 and 2A13 in a time-, concentration- and NADPH-dependent manner. The kinetic parameters for the inactivation of P450 2A6 by BITC were determined (Figure 4A). The inactivation was fairly potent with an apparent KI of 28 µM. The kinact. was 0.055 min–1 and the t1/2 was 12.6 min. The KI for the inactivation of P450 2A6 by BITC (28 µM) was similar to that reported for P450s 2B1 (13 µM) and 2E1 (2.7 µM) (30,31). However, owing to the very potent inhibition of P450 2A6 by PEITC and P450 2A13 by both BITC and PEITC the kinetic parameters for the inactivation could not be determined. The inactivation of P450 2A13 by BITC appears to be very fast (Figure 4B). At low concentrations of BITC the inactivation seemed to be complete at the first time-point. Shorter time-points were not physically possible to perform. At higher BITC concentrations the inactivation was linear for a longer period of time; however, at the higher concentrations the inhibition of the enzyme in the secondary reaction became a major problem. Owing to the potency of PEITC as an inhibitor in this reaction we were not able to determine either time- or concentration-dependent activity loss using the classical approaches to investigate mechanism-based inactivation. Inhibition of catalytic activity by PEITC was observed at dilutions as high as 1:1000 in the secondary reaction making an accurate determination of the KI, kinact and t1/2 values impossible. To be able to investigate whether inactivation of P450s 2A6 and 2A13 by PEITC was occurring, spin-column gel filtration had to be used to eliminate the inhibition of catalytic activity by PEITC in the secondary reaction. Table III clearly shows NADPH-dependent inactivation since the loss in activity in the samples incubated with NADPH is greater than in the –NADPH controls when BITC was incubated with P450s 2A6 and 2A13 and PEITC with P450 2A13. We were also able to show that the loss in activity of both P450s 2A6 and 2A13 with PEITC is time-dependent (Table IV).

The mechanism of inactivation of P450s 2A6 and 2A13 by the two isothiocyanates was investigated. No significant losses in native heme were observed with either isothiocyanate nor did we observe any adducts to the heme moiety, consistent with our earlier studies on the inactivation of the P450 2E and 2B enzymes by BITC and PEITC (30–32). LC/MS analysis of the inactivated proteins indicated that the mode of inactivation appears to be predominantly through the formation of adducts to the P450 apoprotein (Figure 5); however, the data are not conclusive. Both PEITC and BITC are reactive without activation and can bind non-enzymatically to proteins. Although we observed some non-specific binding of BITC and PEITC to both enzymes, there was a significant difference between the control and inactive samples with respect to the extent of adduct formation and the masses of the adducted proteins (Figure 5). The mass of the single adduct in the inactive sample was ~15–24 mass units greater than the mass of the single adduct in the exposed control sample, consistent with the incorporation of one oxygen atom through metabolic activation in the inactivated sample.

In summary, we have demonstrated that BITC and PEITC are very potent inhibitors of P450 2A6 and 2A13-mediated coumarin 7-hydroxylation. Both compounds were also able to significantly inhibit the metabolism of nicotine by P450 2A6 and the metabolic activation of NNK by P450 2A13. In addition, both BITC- and PEITC-inactivated P450s 2A6 and 2A13 in an NADPH-dependent manner, possibly through the formation of apoprotein adducts. Owing to the potent inhibition of both P450 2A6 and 2A13 by BITC and PEITC, we were not able to characterize fully these two isothiocyanates as mechanism-based inactivators, although we did show NADPH-, time- and concentration-dependent inactivation which suggests that the inactivation is mechanism-based. The high potency of PEITC and BITC as inhibitors of the human P450 2A enzymes together with the low toxicity of these compounds in humans makes these two naturally occurring isothiocyanates excellent candidates for consideration as chemopreventive agents in smokers.


   Notes
 
{ddagger} Present address: Cancer Center, University of Minnesota, Minneapolis, MN, USA Back


   Acknowledgments
 
These studies were supported in part by a postdoctoral fellowship to L.v.W from the Philip Morris External Research Program and by research grant CA 16954 from the National Institutes of Health.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 21, 2005; revised July 29, 2005; accepted December 6, 2005.


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L. Mi, Z. Xiao, B. L. Hood, S. Dakshanamurthy, X. Wang, S. Govind, T. P. Conrads, T. D. Veenstra, and F.-L. Chung
Covalent Binding to Tubulin by Isothiocyanates: A MECHANISM OF CELL GROWTH ARREST AND APOPTOSIS
J. Biol. Chem., August 8, 2008; 283(32): 22136 - 22146.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
G. D. Stoner, A. A. Dombkowski, R. K. Reen, D. Cukovic, S. Salagrama, L.-S. Wang, and J. F. Lechner
Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate
Cancer Res., August 1, 2008; 68(15): 6460 - 6467.
[Abstract] [Full Text] [PDF]

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 Drug Metab. Dispos.Home page
H. H. Yoo, M. W. Lee, Y. C. Kim, C.-H. Yun, and D.-H. Kim
Mechanism-Based Inactivation of Cytochrome P450 2A6 by Decursinol Angelate Isolated from Angelica Gigas
Drug Metab. Dispos., October 1, 2007; 35(10): 1759 - 1765.
[Abstract] [Full Text] [PDF]

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