The Synergistic Upregulation of Phase II Detoxification Enzymes by Glucosinolate Breakdown Products in Cruciferous Vegetables

The Synergistic Upregulation of Phase II Detoxification Enzymes by Glucosinolate Breakdown Products in Cruciferous Vegetables

Toxicology and Applied Pharmacology 174, 146 –152 (2001) doi:10.1006/taap.2001.9207, available online at http://www.idealibrary.com on The Synergisti...

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Toxicology and Applied Pharmacology 174, 146 –152 (2001) doi:10.1006/taap.2001.9207, available online at http://www.idealibrary.com on

The Synergistic Upregulation of Phase II Detoxification Enzymes by Glucosinolate Breakdown Products in Cruciferous Vegetables Chu Won Nho and Elizabeth Jeffery 1 Division of Nutritional Sciences, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801 Received January 25, 2001; accepted April 20, 2001

The Synergistic Upregulation of Phase II Detoxification Enzymes by Glucosinolate Breakdown Products in Cruciferous Vegetables. Nho, C. W., and Jeffery, E. (2001). Toxicol. Appl. Pharmacol. 174, 146 –152. Cruciferous vegetables contain secondary metabolites termed glucosinolates that break down to products that upregulate hepatic detoxification enzymes. We have previously shown that a mixture of four major glucosinolate breakdown products from Brussels sprouts interact to produce synergistic induction of phase II detoxification enzymes. Here we tested the hypothesis that this synergism is at the level of transcription and is due to the interaction between the oral bifunctional inducer, indole-3-carbinol (I3C), and monofunctional inducer, crambene (1-cyano 2-hydroxy 3-butene). Adult male rats were treated by gavage with either corn oil (vehicle); crambene (50 mg/kg), I3C (56 mg/kg), or a mix of crambene and I3C at the doses shown. Given orally, I3C alone and crambene with I3C caused significant induction of CYP1A activity and CYP1A1 mRNA levels, whereas crambene alone had no significant effect on CYP1A activity or mRNA levels. Crambene and I3C individually caused induction of glutathione S-transferase (GST) and quinone reductase (QR) activity. The mixture of crambene and I3C caused induction of GST and QR that was significantly greater than the sum of the induction by individual treatments. Upregulation of total GST activity was not as great as that of QR, possibly because some subunits did not show this effect. GST Ya2 mRNA showed a synergistic upregulation by crambene and I3C, while Yc1 and Yc2 showed only an additive response. We speculate that this different regulation is partly due to differences in gene sequences within the antioxidant response element and xenobiotic response element in the regulatory region of GST Ya2 compared to those within the regulatory region of the Yc1/Yc2 subunits. © 2001 Academic Press Key Words: detoxification enzymes; glucosinolates; crambene; indole-3-carbinol; synergism; quinone reductase; glutathione Stransferases.

Numerous epidemiological studies have shown that cruciferous vegetables have a role in dietary prevention of cancers 1 To whom correspondence should be addressed at Division of Nutritional Sciences, National Soybean Research Center, 1101 W. Peabody Dr., Urbana, Illinois 61801. Fax: (217) 333-8046; E-mail: [email protected]

0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

(Steinmetz and Potter, 1991). This protective effect may be due to their glucosinolate content. Brassicas, including all types of cabbages, broccoli, cauliflower, and Brussels sprouts, are a genus of the family Crucifeacea and have relatively high glucosinolate content. Glucosinolates can be hydrolyzed by the enzyme myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1; Verhoeven et al., 1997). Myrosinase is present in plant cells in a separate compartment from glucosinolates. When the plant cells are damaged, e.g., by cutting or chewing, the myrosinase comes into contact with the glucosinolates and hydrolysis occurs. Upon hydrolysis of glucosinolates, three major classes of breakdown products can be formed: isothiocyanates, nitriles, and thiocyanates. Many of these products are bioactive, causing upregulation of detoxification enzymes. The bioactive agents have been divided into two groups, termed mono- and bifunctional inducers. Monofunctional inducers upregulate a number of phase II detoxification enzymes, including quinone reductase (QR) and glutathione S-transferases (GSTs). Bifunctional inducers upregulate a similar array of phase II enzymes, but in addition they upregulate a few phase I enzymes, including CYP1A1. Because CYP1A1 is known to bioactivate polycyclic hydrocarbon carcinogens, bifunctional inducers are considered less closely associated with cancer prevention than are monofunctional inducers (Prochaska and Talalay, 1988). In addition, some monofunctional inducers have been found to act as competitive inhibitors of cytochrome P450 activation of carcinogens (Stresser et al., 1995; Teel and Huynh, 1998). GSTs are a major group of phase II detoxification enzymes, mediating the conjugation between GSH and a variety of xenobiotics. The cytosolic GSTs consist of at least five classes, including ␣⫺, ␮⫺, ␲⫺, ␴⫺, and ␶⫺GST. Each class contains a number of subunits and active GST consists of homo- and heterodimers within the class. In liver, many of the GST isozymes are expressed constitutively; many more are inducible (Hayes and Pulford, 1995). The mechanism of induction of ␣⫺GST subunit Ya2 has been studied extensively; GST Ya2 is induced by many compounds, including monofunctional inducers via a DNA-regulatory region termed the antioxidant response element (ARE) (Nguyen and Pickett, 1992). The ␣⫺GST subunit Yc1 is reported to be both constitutively expressed in rat liver and inducible when rats are fed mono-

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functional and bifunctional inducers. The GST subunit Yc2 is only found constitutively in the liver of the rat fetus and young rats but, like Yc1, is induced by monofunctional and bifunctional inducers in the adult liver (Hayes and Pulford, 1995). Crambene (1-cyano-2-hydroxy-3-butene), formed through the hydrolysis of progoitrin, is an aliphatic nitrile occurring naturally in cruciferous vegetables including Brussels sprouts, broccoli, and cauliflower. Seeds from the plant Crambe abyssinica are the richest source of crambene (Fenwick et al., 1983). Crambene is known to upregulate GST synthesis in liver and other organs (March et al., 1998). Indole-3-carbinol (I3C) is formed through the hydrolysis of glucobrassicin. I3C and its acid condensation products formed in the stomach following ingestion are known to have anticarcinogenic properties (Takahashi et al., 1995b). The mechanism may be by induction of phase I P450-dependent and/or phase II detoxification enzymes (Wortelboer et al., 1992; Chen et al., 1996) and is thought to involve a direct effect of I3C acid condensates as ligand bound to the Ah receptor interacting with a sequence in the regulatory region of these detoxification genes, the xenobiotic response element (XRE) (Jellinck et al., 1993). Recently, the individual and collective effects of specific glucosinolate breakdown products on induction of detoxification enzymes were examined (Staack et al., 1998). Crambene, I3C, iberin, and phenethylisothiocyanate (PEITC), a mixture formulated in the relative proportions found in Brussels sprouts, or the four individual compounds, were fed to rats for 7 days. At the doses used, only crambene and I3C caused significant induction of the phase II enzymes QR and GST when given individually. The group receiving the mix showed significantly greater activity than the sum of the individual treatments. Since only crambene and I3C caused significant induction individually, we hypothesized that these two compounds were responsible for the synergistic induction. The dose of crambene was chosen based on previous work showing that 30 –100 mg/kg caused a dose-related response and that GST was stable at 7 days (Wallig et al., 1992; March et al., 1998). Here we report that a mix of I3C and crambene, at the same doses as used in the previous study (Staack et al., 1998), caused a synergistic increase in activity for both QR and GST. When upregulation of GST was studied in detail, we found that, while induction of all three subunits studied was at the transcriptional level, transcription was only synergistically upregulated for one of three subunits evaluated. MATERIALS AND METHODS Chemicals. Crambene was isolated and purified from the seeds of Crambe abyssinica (Wallig et al., 1992). I3C was purchased from Sigma (St. Louis, MO). Animals, diet, and treatments. Adult male CDF 344 (crl/BR) rats (Charles River Laboratories, Wilmington, MA), weighing 150 –200 g, were used in all

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experiments. Animals were acclimated for a week on a regular chow diet and for 3 days on a AIN76A powdered diet purchased from Teklad (Madison, WI), which was continued through the experiment. Rats (four animals/group) received either crambene (50 mg/kg), I3C (56 mg/kg), or a mixture of crambene and I3C at these same doses, given by gavage daily for 7 days. Two groups received vehicle (corn oil) alone: a control group that was fed ad libitum, and a second group that was pair fed to the group receiving the crambene ⫹ I3C mixture. Preparation of hepatic cytosol and microsomes. Rats were killed by carbon dioxide asphyxiation followed by cervical dislocation. The livers were perfused with 1.15% KCl and homogenized in 4 vol of buffer (0.15 M KCl, 0.25 M K 2HPO 4/KH 2O 4, pH 7.25). Homogenates were centrifuged at 10,000g for 20 min at 4°C. The supernatant was centrifuged at 105,000g for 1 h at 4°C to separate microsomal and cytosolic fractions. Microsomal fractions were resuspended in 0.1 M freezing buffer (Na 2HPO 4/NaH 2PO 4 buffer, pH 7.4) containing 1.0 mM dithiothreitol and 0.25 M sucrose. Aliquots of cytosol and microsomes were snap frozen in liquid nitrogen and stored at ⫺80°C until needed. Measurement of enzyme activities. Ethoxyresorufin O-deethylase (EROD) activity was determined essentially as published (Pohl and Fouts, 1980). A 0.2-ml microsomal suspension (2 mg of protein/ml) was incubated with 10 ␮l ethoxyresorufin (4.2 ␮M in methanol) for 4 min and the reaction was stopped by adding 2 ml methanol. Resorufin formation was measured by fluorimetry using an excitation wavelength of 550 nm and an emission wavelength of 580 nm. Resorufin produced was compared to a standard curve of known concentration of resorufin (Sigma). Total GST activity was measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (Habig et al., 1974). Cytosolic samples were diluted 1:100, which gave rates in the linear range, and were measured in triplicate. The reaction was started by adding CDNB, and initial velocity was measured at 340 nm for 90 s. Units of specific activity are reported as nmol CDNB conjugate formed per mg cytosolic protein per min. QR activity was measured spectrophotometrically as the dicumorol-sensitive reduction of 2,6-dichlorophenolindophenol (DCPP) (Benson et al., 1980). The reaction was initiated by adding 10 ␮l of DCPP (12 mM) to 300 ␮l of cytosol in 2.65 ml reaction buffer. The initial velocity of the reaction was measured at 600 nm for 90 s. Assays were performed in triplicate with and without dicumorol. The units of specific activity are reported as nmol DCPP reduced/mg cytosolic protein/min. All assays were performed at room temperature. Northern blotting. Total RNA was extracted from frozen livers according to the manufacture’s instructions using Trizol reagent (Life Technologies). The concentration and purity of RNA were determined spectrophotometrically at 260 nm and by the 260/280 nm ratio, respectively. Fifteen or 20 ␮g of total RNA was separated on a 1.5% agarose/formaldehyde gel, blotted onto a nitrocellulose membrane, and hybridized with a 32P-random-labeled cDNA probe at 65°C for 2 h in a hybridization oven. After hybridization, blots were washed twice at room temperature with wash buffer [2⫻ SSC and 0.1% sodium dodecyl sulfate (SDS)] for 5 min each. The blots were exposed to X-ray films overnight at ⫺80°C. Autoradiographs were scanned and quantitatively analyzed by densitometry. The cDNA probe for rat GST Ya2 was a gift from Dr. Cecil Pickett, Schering–Plough Research Institute (Kenilworth, NJ) and the cDNA probe for human CYP1A1 was a gift from Dr. Roland Wolf, University of Dundee (Dundee, UK). Western blot analysis. Cytosolic proteins (20 ␮g) were separated on a 12% SDS–polyacrylamide gel and transferred onto polyvinylidene difluoride membranes for 1 h at room temperature with 0.25– 0.3 A. After blocking with 5% bovine serum albumin (BSA), the membrane was incubated with primary antibodies (monoclonal Yc1 or Yc2 antibody) for 1 h at room temperature in buffer containing 1% BSA. Goat anti-rabbit alkaline phophatase-conjugated IgG was used as secondary antibody (Sigma). The blots were developed using a color substrate mix (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium; Sigma) for alkaline phosphatase. The monoclonal anti-rat GST Yc1 and Yc2 antibodies (Buetler et al., 1995) were generous gifts from Dr. J.Hayes, Ninewells Hospital (Dundee, UK).

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Statistical analysis. Data were analyzed for treatment effects across groups using one-way analysis of variance (ANOVA). Where a significant effect was found, Fisher’s least significant difference test for multiple comparisons was used to identify differences between groups. p ⬍ 0.05 was considered to be significant. To determine synergism, we used the method of contrasts, which compares treatments 2 and 3 vs treatment 4, where crambene alone was treatment 2, I3C alone was treatment 3, and the mixed treatment crambene ⫹ I3C was treatment 4. The method of contrasts was run from a SAS program, version 8.0, using different variances and a p ⬍ 0.05 was considered to be significant.

RESULTS

Induction of Detoxification Enzyme Activities in Vivo Hepatic GST enzyme activity was induced 1.4- and 1.5-fold by crambene and I3C, respectively. The mixture of crambene ⫹ I3C caused a 2.1-fold induction in GST activity, which was significantly greater than additive (p ⬍ 0.05; Fig. 1A). Hepatic QR enzyme activity was induced 1.8- and 2.1-fold by crambene and I3C, respectively. The mixture of crambene ⫹ I3C caused a 4-fold induction in QR activity, which was significantly greater than additive (p ⬍ 0.05; Fig. 1B). Hepatic EROD activity was not significantly increased by crambene but was significantly increased 8-fold by I3C. The mixture of crambene ⫹ I3C also caused an 8-fold increase in EROD activity, which was not significantly greater than I3C alone (Fig. 1C). In agreement with the measurement of activity, CYP1A1 mRNA was not significantly increased by crambene alone. In contrast, I3C alone and the mixture of crambene ⫹ I3C caused 32-fold and 30-fold increases, respectively, in the level of mRNA encoding for CYP1A1 (Fig. 2). Induction of mRNAs Encoding GST-␣ Class Subunits The mRNA encoding GST subunit Ya2 was induced 2-fold both by crambene alone and by I3C alone. The mixture of crambene ⫹ I3C caused a 5-fold induction in mRNA encoding GST subunit Ya2, which was significantly greater than additive (p ⬍ 0.05; Fig. 3). Crambene, I3C, and the crambene ⫹ I3C mix induced GST Yc1 mRNA approximately 3-, 3-, and 5.5-fold, respectively (Fig. 4). Similarly, the GST Yc2 mRNA was induced 2.5-, 2.4, and 4.5-fold by crambene, I3C, and crambene ⫹ I3C, respectively (Fig. 5). There was no synergistic induction found for either GST Yc1 or Yc2 subunit, unlike GST Ya2. Induction of GST Yc Proteins Since the nucleotide sequence of the cDNAs encoding GST Yc1 and Yc2 shows approximately 93% homology (Hayes et al., 1994), cross-reaction may occur between Yc1 and Yc2 cDNA probes and the corresponding mRNAs. In order to confirm the mRNA induction for each subunit, a Western blot was performed using rat GST Yc1 and Yc2 monoclonal antibodies. The induction pattern of GST Yc1 and Yc2 proteins by different treatments closely matched the mRNA induction profiles (Fig. 6).

FIG. 1. Determination of hepatic phase I and phase II enzyme activities in rats. Effect of crambene and indole-3-carbinol on rat hepatic GST activity (A), QR activity (B), and EROD activity (C). Rats were treated daily for 7 days with crambene (CB, 50 mg/kg), I3C (56 mg/kg), or both together (MIX) and killed on the 8th day. Control animals received corn oil and were fed ad libitum (CO) or pair fed to the group receiving the mix (PF). GST and QR activity was measured in the hepatic cytosolic fraction and EROD activity in the hepatic microsomal fraction as described under Materials and Methods. Values are means ⫾ SD (n ⫽ 4); values bearing different superscripts differ significantly (p ⬍ 0.05). Actual mean value is shown above each bar; a dashed line across the graph identifies the control, baseline value.

DISCUSSION

In this study, we have shown that there is a synergistic induction of phase II enzymes, including GST and QR activ-

SYNERGISTIC UPREGULATION OF DETOXIFICATION ENZYMES

FIG. 2. Effects of crambene and I3C on the expression of mRNA encoding CYP1A1. (A) mRNA levels were measured by Northern blot, as described under Materials and Methods. Fifteen micrograms of total RNA was loaded per well and hybridized with a human CYP1A1 cDNA probe. Each lane represents RNA of one individual animal. (B) Densitometry readings of P450 1A1 mRNA were normalized to 28S ribosomal RNA and expressed as percentage of control mRNA. Rats were treated daily for 7 days with crambene (CB, 50 mg/kg), I3C (56 mg/kg), or both together (MIX) and killed on the 8th day. Control animals received corn oil and were fed ad libitum (CO) or pair fed to the group receiving the mix (PF). Values are means ⫾ SD (n ⫽ 4); values bearing different superscripts differ significantly (p ⬍ 0.05). Actual mean value is shown above each bar; a dashed line across the graph identifies the control, baseline value.

ities, in the presence of two components found in cruciferous vegetables, crambene and I3C. In contrast, there was no synergistic induction observed in the phase I enzyme measured. These results confirm the previous study in which there was a synergistic phase II enzyme induction by a mixture of four different glucosinolate breakdown products (Staack et al., 1998). Furthermore, these results show that crambene and I3C alone can produce the synergism. In this study, the oral bifunctional inducer I3C showed significant induction of P4501A activity and P4501A1 mRNA levels in agreement with previous findings (Takahashi et al., 1995a). I3C has been reported as a protective agent against AFB 1 carcinogenecity in long-term studies in vivo. Increased P4501A levels may be the reason for this, since P4501A is responsible for metabolism of AFB 1 to less toxic and less carcinogenic metabolites (Takahashi et al., 1995b). Such a mechanism, however, cannot be extended to explain protection from other carcinogens, such as polyarylhydrocarbons, which are bioactivated by P4501A1 (Whitlock, 1999). No induction was found by crambene alone for P4501A activity or mRNA levels. While some glucosinolate hydrolysis products have been reported to inhibit cytochrome P450 activity (Stresser et

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al., 1995; Teel and Huynh, 1998), no inhibition was observed under these dosing conditions. In the group that received both I3C and crambene, induction of P4501A activity and P4501A1 mRNA levels was no greater than that seen with I3C alone. This suggests that P4501A induction was solely due to I3C and that crambene was not able to have a synergistic or enhancing effect on P4501A induction. While QR and GST activates both exhibited synergism in the rats receiving both crambene and I3C, the increase was far greater for QR than for GST. With respect to induction of GST, the fold increase in GST activity was much less than the fold increase in GST Ya2 mRNA level in rats receiving either I3C or crambene. Similarly, the synergistic increase was far greater for GST Ya2 mRNA than for total GST activity. This may be explained by the complex make-up of cytosolic GST, which consists of 13 subunits across five different families including ␣⫺, ␮⫺, ␲⫺, ␴⫺, and ␶⫺GST (Hayes and Pulford, 1995). Since all 13 subunits do not respond to the same extent, when measuring total GST activity, a synergistic response in synthesis of one subunit may be dampened by activity of subunits where synthesis is not synergistically induced. Among these GST subunits, rat GST Ya2 has been studied in the greatest detail and is known to be one of the major players in induction of GST activity. Thus, the exaggerated visualization of the synergistic effect when induction of GST Ya2 mRNA is eval-

FIG. 3. Induction of mRNA encoding GST Ya2 subunit by crambene, I3C, or both. (A) mRNA levels were measured by Northern blot, as described under Materials and Methods. Fifteen micrograms of total RNA was loaded per well and hybridized with a rat GST Ya2 cDNA probe. Each lane represents RNA of one individual animal. (B) Densitometry readings of GST Ya2 mRNA were normalized to 28S ribosomal RNA and expressed as percentage of control mRNA. Rats were treated daily for 7 days with crambene (CB, 50 mg/kg), I3C (56 mg/kg), or both together (MIX) and killed on the 8th day. Control animals received corn oil and were fed ad libitum (CO) or pair fed to the group receiving the mix (PF). Values are means ⫾ SD (n ⫽ 4); values bearing different superscripts differ significantly (p ⬍ 0.05). Actual mean value is shown above each bar; a dashed line across the graph identifies the control, baseline value.

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FIG. 4. Induction of mRNA expression of GST Yc1 by crambene, I3C, or both. (A) mRNA levels were measured by Northern blot, as described under Materials and Methods. Fifteen micrograms of total RNA was loaded per well and hybridized with a rat GST Yc1 specific 400-bp cDNA probe. Each lane represents RNA of one individual animal. (B) Densitometry readings of GST Yc1 mRNA were normalized to 28S ribosomal RNA and expressed as percentage of control mRNA. Rats were treated daily for 7 days with crambene (CB, 50 mg/kg), I3C (56 mg/kg), or both together (MIX) and killed on the 8th day. Control animals received corn oil and were fed ad libitum (CO) or pair fed to the group receiving the mix (PF). The shaded portion of each bar represents control levels of enzyme activity and the open portion of each bar represents the enzyme levels induced over control by each treatment group. Values are means ⫾ SD (n ⫽ 4); values bearing different superscripts differ significantly (p ⬍ 0.05). Actual mean value is shown above each bar; a dashed line across the graph identifies the control, baseline value.

uated alone suggests that the GSTYa2 subunit is responsible for a substantial portion of the increase in total GST activity. We also examined induction of GST Yc1 and Yc2 subunits by Northern and Western blots. Since GST Yc1 and Yc2 show about 90% homology in gene sequences, we needed to confirm the mRNA induction by evaluating protein levels using specific monoclonal antibodies. In adult rat liver GST Yc1 is constitutively expressed, while GST Yc2 is expressed constitutively only in young rats up to 8 weeks of age (Hayes et al., 1994). Both GSTs are inducible in young and old rats. Since the animals used in our study were 7 weeks old, we observed notable basal levels of both GST Yc1 and Yc2 mRNA and protein. We also confirmed that I3C was able to significantly induce GST Yc2 protein, as reported in previous studies (Manson et al. 1997, 1998). Among the three different subunits examined, the only synergistic response that we observed was in transcription of the GST Ya2 subunit. Although GST Yc1 and Yc2 are in the ␣⫺GST class with Ya2, neither Northern nor Western blot

FIG. 5. Induction of expression of GST Yc2 mRNA by crambene, I3C, or both. (A) mRNA levels were measured by Northern blot, as described under Materials and Methods. Fifteen micrograms of total RNA was loaded per well and hybridized with a rat GST Yc2 specific 430-bp cDNA probe. Each lane represents RNA of one individual animal. (B) Densitometry readings of GST Yc2 mRNA were normalized to 28S ribosomal RNA and expressed as percentage of control mRNA. Rats were treated daily for 7 days with crambene (CB, 50 mg/kg), I3C (56 mg/kg), or both together (MIX) and killed on the 8th day. Control animals received corn oil and were fed ad libitum (CO) or pair fed to the group receiving the mix (PF). The shaded portion of each bar represents control levels of enzyme activity and the open portion of each bar represents the enzyme levels induced over control by each treatment group. Values are means ⫾ SD (n ⫽ 4); values bearing different superscripts differ significantly (p ⬍ 0.05). Actual mean value is shown above each bar; a dashed line across the graph identifies the control, baseline value.

showed synergism in the rats receiving I3C and crambene together. This further suggests that the subunit Ya2 may be responsible for synergism in GST activity by crambene and

FIG. 6. Western blot analysis of cytosolic proteins of livers from rats treated with crambene, I3C, or both. Rats were treated daily for 7 days with crambene (CB, 50 mg/kg), I3C (56 mg/kg), or both together (MIX) and killed on the 8th day. Control animals received corn oil and were fed ad libitum (CO) or pair fed to the group receiving the mix (PF). Twenty-five micrograms of cytosolic proteins was loaded in each lane. Each lane represents one cytosolic sample from each group. The Yc1 and Yc2 proteins were detected using monoclonal anti-rat GST Yc1 or Yc2 antibodies. Samples were loaded onto SDS–PAGE as follows: lane 1, 25 ␮g of control liver; lane 2, 25 ␮g of crambene-treated liver; lane 3, 25 ␮g of I3C-treated liver; lane 4, 25 ␮g of crambene ⫹ I3C-treated liver. The molecular weights of GST Yc1 and Yc2 proteins are 2.8 and 2.5 kDa, respectively.

SYNERGISTIC UPREGULATION OF DETOXIFICATION ENZYMES

I3C. Further investigation is needed across a large number of GST subunits to confirm this. The presence of an ARE is obligatory in the regulatory region of detoxification enzymes for a response to monofunctional inducers to occur (Rushmore and Pickett, 1990). The ARE has been found in the regulatory region for ␥-glutamyl cysteinyl synthase and several phase II detoxification enzymes, including several GSTs, QR, epoxide hydrolase, and UDPglucuronosyltransferase. This battery of enzymes is thought to protect cells against the toxic and neoplastic action of carcinogens. We suggest that crambene upregulates phase II enzymes via an ARE, since crambene effects are consistent with those of a monofunctional inducer. I3C has been classified as a bifunctional inducer of phase I and phase II detoxification enzymes (Loub et al., 1975; Wattenberg, 1975). Induction of these enzymes has been shown to depend on activation of the XRE found in the regulatory region of the gene sequence of these enzymes (Chen et al., 1996; Jellinck et al., 1993). Our data are consistent with the possibility that the synergism observed at the transcriptional level is due to concurrent stimulation at both the ARE and XRE by crambene and I3C, respectively. For example, the stimulation of both might prolong binding at the ARE, resulting in cooperativity. Furthermore, that the synergistic induction occurred for the GST Ya2 subunit, but not in the GST Yc1/Yc2 subunit further supports this hypothesis. Although GST Ya2 and Yc1/Yc2 contain AREs and XREs in common, the XRE motifs of GST Yc1/Yc2 are not in the 5⬘-regulatory region as they are in GST Ya2 (Pulford and Hayes, 1996). In conclusion, this study shows that a mixture of crambene and I3C cause synergistic induction of phase II detoxification enzymes, including GST and QR enzyme activity. Of the GST subunits tested, only GST Ya2 showed a synergistic induction by this mixture of I3C ⫹ crambene. The induction could be traced to the level of transcription. The molecular mechanisms of synergistic induction of phase II enzymes by an I3C and crambene mixture are under investigation. ACKNOWLEDGMENTS This research was supported by grants from the USDA (National Research Initiative 99 –35503-7010) and Standard Process, Inc., Palmyra, Wisconsin.

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