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Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada
(Received September 17, 2004; accepted December 15, 2004)
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; Ema et al., 1992). The AHR exists as cytoplasmic aggregates bound to two 90-kDa heat-shock proteins (HSP90), the cochaperone p23 and a 43-kDa protein termed hepatitis B virus X-associated protein (Ma and Whitlock, 1996; Carver and Bradfield, 1997; Meyer et al., 1998). Upon ligand binding, the AHR dissociates from HSP90, and the ligand-receptor complex translocates to the nucleus. Then, the activated AHR dimerizes with the AHR nuclear translocator protein (ARNT) and binds to a class of promoter DNA sequences, called xenobiotic response elements (XREs), of the target genes to activate their transcription (Nebert et al., 1993; Whitlock, 1999). The AHR-regulated genes consist of four phase I enzymes, cytochrome P450 1A1 (CYP1A1), CYP1A2, CYP1B1, and CYP2S1, and several phase II enzymes that are involved in the metabolism of xenobiotics and endogenous compounds (Nebert et al., 2000; Rivera et al., 2002).
Among the AHR-regulated genes, CYP1A1 is the most capable in producing polar, toxic, or even carcinogenic metabolites from various AHR ligands including aromatic and halogenated hydrocarbons (Schrenk, 1998). These metabolites have been shown to be involved in the mediation of a broad range of distinct toxic responses such as immune suppression, endocrine disruption, birth defects, and carcinogenesis (Poland and Knuston, 1982).
tert-Butylhydroquinone (tBHQ) is a major metabolite of 3-tert-butyl-hydroxyanisole (BHA) in vivo in dogs, rats, and humans (Nakamura et al., 2003). BHA has been widely used for many years as an antioxidant to preserve and stabilize the freshness, nutritive value, flavor, and color of foods and animal food products (Williams et al., 1999; Li et al., 2002). Both tBHQ and BHA have received a lot of attention due to their ability to induce phase II detoxification enzymes, including glutathione S-transferase and NADPH-quinone oxidoreductase, and, thus, their potential role in cancer prevention (Li et al., 2002). In contrast to the beneficial effects of BHA, a number of studies have shown that chronic exposure to high concentrations of BHA in diet induces neoplastic lesions in the forestomach, urinary bladder, and esophagus of rats, mice, hamsters, and pigs (Li et al., 2002).
Although the carcinogenicity of BHA in animals, particularly at high concentrations, has been well documented, the relevance of these results to the low exposure of humans to BHA has been questioned (Li et al., 2002). Recently, a study on the carcinogenicity of BHA, at low concentrations, alone or in combination with other phenolic chemicals, including caffeic acid, sesamol, 4-methoxyphenol and catechol, in rats has shown that the above phenolic compounds exerted synergistic effects on rat forestomach carcinogenesis (Li et al., 2002). Assessing the risk from human consumption of BHA and tBHQ is complicated by the fact that the precise mechanisms of their carcinogenicity are not well understood. The metabolic formation of tBHQ has been suggested to, at least in part, contribute to the carcinogenicity effect of BHA.
In an attempt to investigate the mechanisms by which tBHQ induces toxicity or carcinogenicity, we examined the effect of tBHQ on Cyp1a1 mRNA, protein, and enzyme activity in murine hepatoma Hepa 1c1c7 cells. The involvement of the AHR-dependent signaling pathway was also investigated by using an AHR antagonist and gel electrophoretic mobility shift assay (EMSA). Here, we provide the first direct evidence for an AHR-dependent induction of Cyp1a1 gene expression and enzyme activity by the tBHQ, which has a single unsaturated phenolic ring (Fig. 1).
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Materials and Methods |
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-32P]dCTP and [
-32P]ATP (3000 Ci/mmol) were supplied by DNA Core Services Laboratory, University of Alberta. Bromphenol blue, ß-mercaptoethanol, glycine, acrylamide, N'N'-bis-methylene-acrylamide, ammonium persulfate, nitrocellulose membrane (0.45 µm), and N,N,N',N'-tetramethylethylenediamine were purchased from Bio-Rad (Hercules, CA). Cyp1a1 and ARNT goat anti-mouse polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Skim milk was obtained from Difco (Detroit, MI). All other chemicals were purchased from Fisher Scientific Co. (Toronto, ON, Canada). Cell Culture and Treatments. Murine hepatoma Hepa 1c1c7 (generously provided by Dr. O. Hankinson, University of California, Los Angeles, CA) and HepG2 cells (obtained from the American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium, without phenol red, supplemented with 10% fetal bovine serum, 20 µM L-glutamine, 50 µg/ml gentamicin sulfate, 100 IU/ml penicillin, 10 µg/ml streptomycin, and 25 ng/ml amphotericin B, 0.1 mM nonessential amino acids, and vitamin supplement solution. Cells were grown in 75-cm2 tissue culture flasks at 37°C in a 5% CO2 humidified incubator.
tBHQ and resveratrol were dissolved in dimethyl sulfoxide. Actinomycin D (Act-D) and cycloheximide (CHX) were dissolved in 75% ethanol and sterile distilled water, respectively. For all experiments, control groups for each of the treatments were administered the appropriate solvent. For analysis of mRNA and protein expression levels, 
1 x 106 cells were added to a six-well tissue culture plate in 2 ml of culture media. For analysis of 7-ethoxyresorufin O-deethylase (EROD) activity, 1 x 105 cells were added to each well of a 96-well tissue culture plate in 200 µl of culture media. On 60 to 80% confluence (1–2 days), appropriate stock solutions of the test chemicals were directly added to the culture media. For experiments involving Act-D, resveratrol, and CHX, the first two chemicals were added 2 h before, whereas CHX was added 0.5 h before treatment of cells with tBHQ.
Effect of tBHQ on Cell Viability. The effect of tBHQ on cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (Vakharia et al., 2001). The assay measures the conversion of MTT to formazan crystal by enzymes in the mitochondria of metabolically active cells. Briefly, Hepa 1c1c7 cells were seeded into 96-well microtiter cell culture plates and incubated for 24 h at 37°C in a 5% CO2 humidified incubator. Cells were treated with various concentrations of tBHQ (0, 1, 10, 50, 100, and 500 µM). After a 24-h incubation, the medium was removed and replaced with cell culture medium containing 1.2 mM MTT dissolved in PBS (pH 7.4). After 2 h of incubation, the formed crystals were dissolved with isopropanol. The intensity of the color in each well was measured at a wavelength of 550 nm using the Bio-Tek EL 312e microplate reader (Bio-Tek Instruments, Winooski, VT).
Determination of Cyp1a1 Activity. The Cyp1a1-dependent EROD activity was performed on intact, living cells as described previously (Elbekai and El-Kadi, 2004; Korashy and El-Kadi, 2004). Enzymatic activity was normalized for cellular protein content, which was determined using a modified fluorescent assay (Elbekai and El-Kadi, 2004; Korashy and El-Kadi, 2004).
RNA Extraction and Northern Blot Analysis. After incubation with the test compounds for the specified time periods, total cellular RNA was isolated using TRIzol reagent, according to manufacturer's instructions (Invitrogen), and quantified by measuring the absorbance at 260 nm. Northern blot analysis of total RNA was performed as described elsewhere (Sambrook et al., 1989). Briefly, aliquots of 20 µg of RNA were separated in a denaturing (2.2 M formaldehyde) agarose (1.1%) gel and transferred to Hybond-N nylon membranes. The RNA was cross-linked to the membranes using the UV Stratalinker 2400, followed by baking at 65°C for 2 h. Prehybridization of the membranes was carried out in a solution containing 6x SSPE (0.9 M NaCl, 0.2 M NaH2PO4·H2O, 0.02 M EDTA), 50% deionized formamide, 5x Denhardt's reagent (0.1% polyvinylpyrrolidone, 0.1% Ficoll, 0.1% bovine serum albumin), 0.5% SDS, and 100 µg/ml sheared salmon sperm DNA, for at least 4 h at 42°C. Hybridization with the 32P-labeled cDNA probes was carried out in the same solution, minus Denhardt's reagent, for 16 to 24 h at 42°C. The membranes were then washed twice at room temperature in a solution containing 2x SSPE and 0.5% SDS for 5 min for the first wash and 15 min for the second wash. This was followed by a 30-min wash in 0.1x SSPE, 0.5% SDS at 42°C and a final 30-min wash in 0.1x SSPE, 0.5% SDS at a temperature of 62°C for 30 min. The washed membranes were rinsed in a 0.1x SSPE solution, dried, sealed in plastic wrap, and visualized by exposure to Kodak Biomax MS film (Eastman Kodak, Rochester, NY). Hybridization signals were quantified relative to the signals obtained for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
The cDNA probes for mouse Cyp1a1 and GAPDH mRNA were generously provided by Dr. John R. Bend (University of Western Ontario, London, ON, Canada). All probes were 32P-labeled by the random primer method according to the manufacturer's (Invitrogen) instructions.
Western Blot Analysis. Cells treated with tBHQ for 24 h were collected in lysis buffer containing 50 mM HEPES, 0.5 M sodium chloride, 1.5 mM magnesium chloride, 1 mM EDTA, 10% (v/v) glycerol, 1% Triton X-100, and 5 µl/ml protease inhibitor cocktail. The cytosolic fractions were obtained by incubating the cell lysates on ice for 1 h, with intermittent vortexing every 10 min, followed by centrifugation at 12,000g for 10 min at 4°C. Proteins (100 µg) were resolved by denaturing electrophoresis, as described previously (Sambrook et al., 1989; Elbekai and El-Kadi, 2004; Korashy and El-Kadi, 2004). Briefly, the cytosol supernatants were dissolved in 1x sample buffer, boiled for 5 min, separated by 7.5% SDS-polyacrylamide gel electrophoresis, and electrophoretically transferred to a nitrocellulose membrane. Protein blots were blocked for 24 h at 4°C in blocking buffer containing 5% skim milk powder, 2% bovine serum albumin, and 0.05% (v/v) Tween 20 in Tris-buffered saline solution (0.15 M sodium chloride, 3 mM potassium chloride, 25 mM Tris-base). After blocking, the blots were incubated with a primary polyclonal goat anti-mouse Cyp1a1 antibody for 2 h at room temperature in Tris-buffered saline solution containing 0.05% (v/v) Tween 20 and 0.02% sodium azide. Incubation with a peroxidase-conjugated rabbit anti-goat IgG secondary antibody was carried out in blocking buffer for 1 h at room temperature. The bands were visualized with the enhanced chemiluminescence method according to the manufacturer's instructions (Amersham Biosciences Inc., Piscataway, NJ).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis of mRNA. RT was performed using murine leukemia virus reverse transcriptase (Invitrogen) as described previously (Li et al., 1998). Purified total RNA (2.5 µg) was used in each reaction. PCR was performed using AmpliTAQ DNA polymerase (PerkinElmer Life and Analytical Sciences, Boston, MA) according to the manufacturer's instructions. RT reaction mixture (2.5 µl) was used for each PCR. Agarose gel electrophoresis was used to separate PCR-amplified products. Ethidium bromide was added to the gel before solidification. PCR products were visualized under a UV transluminator and digitally recorded. Band intensity was quantitated using ImageQuant software (Amersham Biosciences Inc.). Primer sequences used were: CYP1A1 forward, 5'-GACCTGAATGAGAAGTTCTACAGC-3'; reverse, 5'-CGGAAGGTCTCCAGGATGAAG-3'; and ß-actin forward, 5'-CTACAATGAGCTGCGTGTGG-3'; reverse, 5'-TAGCTCTTCTCCAGGGAGGA-3', as described previously (Li et al., 1998; Roblin et al., 2004).
Nuclear Extracts and EMSA. Nuclear extracts were prepared as described previously (Rogers and Denison, 2002). A complementary pair of synthetic oligonucleotides containing the XRE3 binding site for the transformed AHR/ARNT complex (5'-GATCTGGCTCTTCTCACGCAACTCCG-3' and 5'-GATCCGGAGTTGCGTGAGAAGAGCCA-3') were synthesized, purified, annealed, and radiolabeled with [-32P]ATP as described previously (Denison et al., 1988). For EMSA analysis, 10 µg of nuclear extract proteins was incubated for 30 min in a reaction mixture containing 2 µg of poly(dI/dC), 1 ng (100,000 cpm) [32P]XRE, 25 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol (v/v), and 80 mM KCl. After incubation, the reaction mixture was then separated by electrophoresis through a 4% nondenaturing polyacrylamide gel, and the results were recorded by autoradiography. For competition experiments, nuclear extracts were preincubated at room temperature for 30 min with a 100-fold molar excess of unlabeled XRE or 1 µg of anti-ARNT antibody (Santa Cruz Biotechnology, Inc.) before the addition of the labeled XRE. Preincubation with antibody made to the C-terminal region of ARNT is known to inhibit AHR/ARNT heterodimer binding to XRE (Santiago-Josefat et al., 2001).
Statistical Analysis. All results are presented as mean ± S.D. The comparison of the results from the various experimental groups and their corresponding controls was carried out by a one-way analysis of variance followed by Student Newman-Keuls post hoc tests. The differences were considered significant when P < 0.05.
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Increase in Cyp1a1 Expression after tBHQ Treatment. Northern blot analysis was performed to examine the time-dependent effect of tBHQ on Cyp1a1 mRNA. At a concentration of 100 µM, tBHQ caused a time-dependent increase in Cyp1a1 mRNA level (Fig. 3). The onset of induction was achieved only 3 h after the addition of tBHQ, but Cyp1a1 mRNA levels remained elevated for at least 12 h after tBHQ treatment.
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To examine the concentration-dependent effect of tBHQ on Cyp1a1 mRNA, the cells were treated with various concentrations of tBHQ (1–100 µM). Figure 4 illustrates that tBHQ caused a concentration-dependent increase in Cyp1a1 mRNA levels already apparent at a concentration of 10 µM, whereas maximum induction was obtained at about 100 µM.
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To assess whether the increase in Cyp1a1 mRNA level was accompanied by an increase in protein expression, quantitative measurement of Cyp1a1 protein using Western blot analysis was carried out. Our results clearly show that only the highest concentrations tested, 50 and 100 µM tBHQ, caused significant induction in Cyp1a1 protein levels (Fig. 5).
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Furthermore, to assess the functional implication of exposure to tBHQ, Cyp1a1-dependent EROD activity was also measured in Hepa 1c1c7 cells (Fig. 6). Significant increase in EROD activity was measured at 10, 50, and 100 µM tBHQ (1.6-, 3.3-, and 3.0-fold increase compared with control, respectively), which correlates with the mRNA data.
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Effect of tBHQ on CYP1A1 in HepG2 Cells. To examine whether tBHQ causes similar effects in human cells, human hepatoma HepG2 cells were treated with various concentrations of tBHQ (0–100 µM). Similar to the results obtained with Hepa 1c1c7 cells, tBHQ caused a concentration-dependent increase in CYP1A1 at the mRNA and activity levels in HepG2 cells (Fig. 7, A and B). However, at the highest concentration tested, 100 µM, the induction in CYP1A1 mRNA and activity was less than that obtained with the 50 µM concentration, probably due to reduced cell viability (about 25% decrease in the cell viability; data not shown).
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Effect of RNA and Protein Synthesis Inhibitors. To determine whether the increase in Cyp1a1 expression in response to tBHQ was a result of de novo RNA synthesis or a post-transcriptional effect, Hepa 1c1c7 cells were cotreated with the RNA polymerase inhibitor Act-D. Treatment with 5 µg/ml Act-D 2 h before exposure to 100 µM tBHQ completely abolished the increase in Cyp1a1 mRNA (Fig. 8A). Act-D also completely blocked the increase in Cyp1a1-dependent EROD activity in response to 100 µM tBHQ (Fig. 8B).
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We also determined whether the increased Cyp1a1 mRNA levels, due to tBHQ exposure, were dependent on the action of a highly labile or de novo synthesized protein. Hepa 1c1c7 cells were treated with the protein synthesis inhibitor, CHX (1 µg/ml) in the presence or absence of tBHQ (100 µM) for 6 h. Our results clearly demonstrated that in the absence of tBHQ, CHX alone significantly increased the Cyp1a1 mRNA level. In addition, cotreatment of cells with CHX and tBHQ superinduced the increase in Cyp1a1 mRNA levels by CHX (Fig. 9A). With respect to EROD activity, CHX significantly inhibited the Cyp1a1 induction in response to tBHQ treatment (Fig. 9B), indicating a requirement for de novo protein synthesis for increased EROD activity.
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AHR-Dependent Induction of Cyp1a1. To further investigate the role of AHR in the induction of Cyp1a1 by tBHQ, Hepa 1c1c7 cells were incubated with an AHR antagonist, resveratrol (100 µM), 2 h before treatment with tBHQ (100 µM). Our results demonstrated that resveratrol significantly inhibited the increase in Cyp1a1-dependent EROD activity in response to tBHQ (Fig. 10).
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Ligand-dependent activation of the AHR/ARNT heterodimer to a DNA-binding form (XRE), so-called transformation, can be monitored by incubation of nuclear proteins with the ligand in vitro, followed by EMSA. EMSA results (Fig. 11) demonstrate that tBHQ significantly induces the formation of a heterodimer/32P-XRE complex that comigrates with that induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of tBHQ-induced AHR/ARNT heterodimer binding to XRE was confirmed by competition experiments in the presence of 100-fold molar excess of unlabeled XRE or by the addition of anti-ARNT antibody.
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). Most of them, the "classical" ligands, including polycyclic aromatic hydrocarbons and halogenated aryl hydrocarbons, share the structural features of being planar, aromatic, and hydrophobic (Denison and Nagy, 2003). Recently, a relatively large number of AHR ligands whose structures and physiochemical characteristics differ from classical ligands have been identified (Denison and Nagy, 2003). The majority of these "nonclassical" AHR ligands have a low affinity to the AHR and are relatively weak inducers of Cyp1a1, compared with TCDD. A wide range of structural diversity in AHR ligands indicates that a greater spectrum of chemicals can interact with and activate this receptor than previously thought (Denison and Nagy, 2003). Here, our data provide strong evidence that tBHQ, which has a single unsaturated phenolic ring, is a weak ligand of AHR compared with TCDD, and can directly induce Cyp1a1 gene expression and enzymatic activity in an AHR-dependent manner.
Humans consume appreciable amounts of phenolic antioxidants including BHA and its active metabolite, tBHQ, as food additives from dietary sources (Ma and Kinneer, 2002). tBHQ has been found to be a prototype inducer of a non-receptor-signaling pathway that selectively induces phase II enzymes in an AHR-independent manner and exhibits anticarcinogenic effects (Li et al., 2002; Munzel et al., 2003). Induction of phase II enzymes by tBHQ requires an antioxidant response element located in the enhancers of genes and is mediated through a Nrf2-dependent signal transduction (Ma and Kinneer, 2002). Nrf2 is a redox-sensitive member of the CNC bzip (cap `n' collar basic leucine zipper) family of transcription factors that forms a cytoplasmic complex with Keap1 (Ma and Kinneer, 2002). In the presence of tBHQ, Nrf2 dissociates from Keap1 and translocates into the nucleus, followed by dimerization with musculoaponeurotic fibrosarcoma protein, binding to antioxidant response element, and subsequently inducing transcription of genes (Ma and Kinneer, 2002). Studies on Nrf2-null mice revealed that lack of expression of Nrf2 markedly enhances the susceptibility of the mice to cancer by benzo-[a]pyrene. Thus, induction of phase II enzymes through Nrf2 can account for chemoprotection by tBHQ against certain carcinogens (Ma and Kinneer, 2002).
In contrast, evidence is being provided that the bioactivation of tBHQ through a Cu(II)/Cu(I) redox cycle mechanism results in the formation of 2-tert-butyl(1,4)paraquinone, semiquinone anion radical, and reactive oxygen species. The reactive oxygen species may be involved in oxidative DNA damage and hence may contribute to the carcinogenicity of BHA (Li et al., 2002). However, other mechanisms may be involved.
Despite the extensive interest in the effect of tBHQ on human health, little is known about the physiologically relevant concentration attainable in human plasma and tissue, but it has been estimated that the daily consumption of tBHQ may reach 28 to 42 mg/day (European Food and Safety Authority, 2004). Moreover, it has been reported that when a single dose of 125 mg of tBHQ was given orally to adult male volunteers, the plasma level of tBHQ was 31 to 37 mg/l, which is equivalent to 187 to 223 µM (European Food and Safety Authority, 2004). Considering that the daily consumption of tBHQ is about 28 to 42 mg, we estimate the plasma concentration will range from 50 to 75 µM.
To further understand the mechanisms involved in BHA carcinogenesis, we hypothesized that tBHQ acts as an AHR ligand and induces Cyp1a1. The reason is that the conversion of AHR ligands into electrophilic compounds by Cyp1a1 results in the formation of covalent adducts, which can react directly with intracellular nucleophiles, including DNA, and initiate the cancer process (Spink et al., 2002). To test this hypothesis, we investigated the effect of tBHQ on Cyp1a1 mRNA expression, protein, and EROD activity in Hepa 1c1c7 cells. Our data clearly show that treatment of Hepa 1c1c7 cells with tBHQ (10–100 µM) causes concentration-dependent induction in Cyp1a1 mRNA and EROD activity. However, at the protein level, only the highest concentrations tested, 50 and 100 µM, caused significant induction in Cyp1a1 protein levels. Similarly, in human HepG2 cells, tBHQ caused significant induction in CYP1A1 at the mRNA and activity levels. In agreement with our study, Sugatani et al. (2004) have shown that treatment of HepG2 cells with tBHQ at 80 µM for 24 h produced a significant induction of CYP1A1 mRNA, whereas other studies have reported that treatment of Hepa 1c1c7 cells with 25 µM or 500 µM tBHQ had no effect on Cyp1a1 at the mRNA or activity levels (Liu et al., 1994; Vasiliou et al., 1995; Lamb and Franklin, 2002). We have found that tBHQ does not induce Cyp1a1 expression at 500 µM due to increased cytotoxicity at that concentration (Fig. 2).
Other aspects of our data are also worth noting. The time course study for Cyp1a1 mRNA induction indicates that the increase in response to tBHQ was somewhat delayed, with the increase in Cyp1a1 mRNA levels being clearly achieved 3 h after tBHQ treatment. However, the onset of Cyp1a1 mRNA increase was rapid and readily apparent by 1 h for TCDD (data not shown). In addition, induction of EROD activity 24 h after treatment with 100 µM tBHQ was only 40% of that observed for TCDD (data not shown). Taken together, these data suggest that tBHQ induces Cyp1a1, albeit with lower affinity than TCDD.
Further evidence for the involvement of the AHR comes from the result of the EMSA. The presence of a nuclear AHR complex is dependent on ligand binding by the cytosolic receptor, nuclear translocation of the liganded AHR, its heteromerization with ARNT, and subsequent specific and high-affinity DNA binding of the heteromeric transformed AHR complex within the nucleus (Phelan et al., 1998). Our results not only suggest that tBHQ can induce AHR transformation, nuclear accumulation, and DNA binding, similar to that observed with other AHR ligands such as TCDD, but also support a role for the AHR in the induction of Cyp1a1. To support our conclusion, the EROD activity induced by tBHQ was significantly prevented by the AHR antagonist, resveratrol, suggesting a direct contribution of an AHR-dependent mechanism.
We also attribute the changes in mRNA expression to a direct effect on transcription. Inhibition of tBHQ-mediated induction of Cyp1a1 mRNA in Hepa 1c1c7 cells treated with the RNA synthesis inhibitor Act-D demonstrates a requirement for de novo mRNA synthesis, consistent with increased gene transcription mediated by the ligand-bound AHR complex. It has been previously reported that in Hepa 1c1c7 cells, superinduction of Cyp1a1 mRNA is a transcriptional event involving a liganded AHR (Israel et al., 1985) and is considered to reflect the existence of a labile repressor protein that inhibits the response of the receptor-enhancer system through protein-protein interactions (Lusska et al., 1992). Our results demonstrate that CHX alone significantly increased the Cyp1a1 mRNA, which was superinduced in the presence of CHX and tBHQ, indicating that the superinduction requires the activation of AHR by an agonist. Therefore, tBHQ increases the Cyp1a1 gene expression through an AHR-dependent mechanism. The Cyp1a1-mediated induction by CHX alone, in the absence of an additional exogenous inducing agent, has been noted in various cell types (Giachelli et al., 1991). In addition, inhibition of tBHQ-mediated induction of Cyp1a1 activity by CHX indicates that de novo Cyp1a1 protein synthesis is required.
Taken all together, the results provided here present us with the first evidence that tBHQ, a phenolic antioxidant, can directly modulate the expression of Cyp1a1 through an AHR-dependent pathway by acting as an AHR ligand. In addition, induction of Cyp1a1 by tBHQ may be directly or indirectly involved in BHA carcinogenesis.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: AHR, aryl hydrocarbon receptor; Act-D, actinomycin D; ARNT, aryl hydrocarbon receptor nuclear translocator; BHA, 3-tert-butyl-hydroxyanisole; tBHQ, tert-butylhydroquinone; CHX, cycloheximide; CYP1A1 or Cyp1a1, cytochrome P4501A1; EMSA, gel electrophoretic mobility shift assay; EROD, 7-ethoxyresorufin O-deethylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSP90, 90-kDa heat-shock protein; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Nrf2, nuclear factor erythroid 2-related factor 2; PCR, polymerase chain reaction; RT, reverse transcription; SSPE, sodium chloride/sodium phosphate/EDTA; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element.
Address correspondence to: Dr. Ayman O. S. El-Kadi, Faculty of Pharmacy and Pharmaceutical Sciences, 3118 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, AB, Canada T6G 2N8. E-mail: aelkadi{at}pharmacy.ualberta.ca
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