Transcriptional Induction of CYP1A1 by Oltipraz in Human Caco-2 Cells Is Aryl Hydrocarbon Receptor- and Calcium-dependent*

Oltipraz, a synthetic derivative of the cruciferous veg-etable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both cytochrome P-450 (CYP) and glutathione S -transferase expression and activities in rat tissues. Its effects, how-ever, are variable according to the enzyme, tissue, and species. We show here that, as previously found in rat lung and kidney, CYP1A1 is inducible by oltipraz in both rat intestine and Caco-2 cells, a cell line originated from a human colon adenocarcinoma. In these cells, a 50 (cid:1) M oltipraz treatment increased CYP1A1 mRNA ( (cid:1) 30-fold), protein and activity. mRNA level was augmented as early as 2 h after the beginning of treatment, suggesting a transcriptional activation, and was maximal between 8 and 12 h. Transient transfection of Caco-2 cells with constructs containing different sizes of the 5 (cid:1) -flanking region of the CYP1A1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in oltipraz-treated cells, which correlates with the presence of the xenobiotic I pGL3-XRE3. Caco-2 co-transfected the pRL-SV40 vector codes for Renilla luciferase (Promega) plus the p1A1-FL constructs. Similar experiments were performed with a “basic” control consisting of the promoterless pGL3-luciferase con- struct (pGL3-basic) and a pGL3-promoter plasmid containing the SV40 promoter upstream of the luciferase gene. Transient transfec- tion of Caco-2 cells was performed by the TransFast method (Pro-mega) according to manufacturer’s instructions. Briefly, 400 (cid:2) l of transfection media (Dulbecco’s modified Eagle’s medium without fetal bovine serum) containing 600 ng of luciferase reporter plasmid were added to the confluent Caco-2 cells along with 15 ng of the pRL-SV40 DNA and 4 (cid:2) l of TransFast. The plates were incubated for 2 h, and then 2 ml of Dulbecco’s modified Eagle’s medium were added and incubated for another 24 h, at which time the cells were treated for 16 h. Dual luciferase assays (firefly and Renilla ) were performed with a Promega kit as in the manufacturer’s protocol. Significant differences were evaluated with the Student’s t test. For the variations of intracellular calcium concentration, the Mann-Whitney U test was used.

Oltipraz, a synthetic derivative of 1,2-dithiole-3-thione, a constituent of cruciferous vegetables, is considered as one of the most promising chemopreventive agents in development, based on preclinical studies (1)(2)(3)(4)(5) and a recent Phase IIa clinical trial in China (6,7). This compound was first claimed to act as a chemopreventive agent by enhancing activities of phase II enzymes such as glutathione S-transferase, UDP-glucuronosyltransferases, NAD(P)H:quinone reductase, aflatoxin B 1 -aldehyde reductase, and epoxide hydrolase (for a review see Ref. 8).
Biochemical and genetic studies showed that induction of these detoxifying enzymes is primarily due to transcriptional activation of the genes and is regulated by an enhancer, called an antioxidant-responsive element (ARE) 1 or electrophile-responsive element (9 -11). NF-E2-related factor 2 (Nrf2) has been implicated as an essential component of an ARE-binding transcriptional complex (12)(13)(14), but the signal transduction pathways that relay the chemical signals to the ARE-protein complex remain to be elucidated.
More recently oltipraz was also reported to influence cytochrome P450 (CYP) expression and activities. It was found to be a potent inhibitor of both CYP1A and -2B in rat liver in vivo and in vitro (15) and of CYP1A2 and -3A4 in primary human hepatocyte cultures (16). Other studies demonstrated that oltipraz was also capable of increasing mRNA levels of CYP1A2 and CYP2B1/2 in rat liver following its transient inhibitory effect (15,17). A similar induction of CYP1A expression and activity was observed in lung and kidney, and CYP2B1 apoprotein was dramatically decreased via a proteasome-dependent pathway in rat lung after administration of oltipraz (18).
Several reports have implicated protein kinase C (PKC) in the XRE-inducible transcription of the CYP1A1 gene mediated through the AhR (20 -22). Recently, Huang et al. (13) demonstrated that regulation of the ARE also involves a PKC-mediated phosphorylation of Nrf2 and a mitogen-activated protein kinase (MAPK) pathway (14). Because MAPK and PKC targeting are regulated by intracellular calcium concentration ([Ca 2ϩ ] i ) (23)(24)(25), this ubiquitous second messenger might be one of the initial events involved in gene induction mediated by oltipraz or PAH. Interestingly, it has been shown that benzo-[a]pyrene and 7,12-dimethylbenz[a]anthracene, two known CYP1A1 inducers, increase [Ca 2ϩ ] i in the human mammary epithelial cell line MCF-10A (26).
Dithiolethiones and food contaminant PAH first accumulate in the gastrointestinal tract, which expresses some CYP1A metabolic capacity (27), suggesting that in the presence of oltipraz, activation of these carcinogens could be altered. In this study we confirmed that CYP1A is expressed in the rat intestine and is inducible after administration of oltipraz to rats. To further characterize the mechanism of induction of CYP1A1 by this compound, we used the Caco-2 cell line derived from a human colon carcinoma. These cells differentiate into enterocytes at confluency and exhibit a highly inducible CYP1A1 by benzimidazole or PAH derivatives (omeprazole and ␤-naphtoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively) (28,29). We brought evidence that CYP1A1 induction by oltipraz is dependent upon both [Ca 2ϩ ] i and the Ah receptor.
Animal Treatment-Male Wistar rats (ϳ180 g) were fed a diet (AO4; Centre d'Elevage Janvier, Le Genest, France) supplemented with oltipraz for 3 days at a final concentration of 0.075% (w/w). Three animals were used for each condition. Animals were killed by beheading, and the intestines were removed and washed with cold NaCl 0.15 M. The duodenum, jejunum, ileum, and colon were separated, and each was divided in two fragments. The first fragment was frozen in liquid nitrogen and stored at -80°C until mRNA isolation. Intestinal mucosa was obtained by scraping the second fragment and used for preparation of microsomes. All experimental procedures were done in compliance with French laws and regulation.
Cells-Caco-2 cells, originating from a human colorectal carcinoma were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, 1% non-essential amino acids, 1% L-glutamine, penicillin (100 units/ml), and streptomycin (10 g/ml). Experiments were carried out for 25 to 35 passages, and cells were allowed to grow and differentiate for up to 21 days after confluency. Because fetal calf serum has been shown to induce CYP1A1 (30), cells were maintained for 24 h without fetal calf serum prior to treatment with oltipraz.
RNA Isolation and Blot Analysis-Total RNA was extracted from rat intestinal samples by the method of Chirwing et al. (31) and from Caco-2 cells by the method of Chomczynski and Sacchi (32). Ten g of RNA samples were subjected to electrophoresis under denaturing conditions (6% (v/v) formaldehyde, 1.25% (w/v) agarose) and then transferred onto nylon filters (Amersham Biosciences) and fixed with a UV cross-linker. Evidence for equal RNA amount and integrity was obtained by methylene blue staining of the 18 S and 28 S ribosomal RNA species. Prehybridization and hybridization were performed according to Church and Gilbert (33). Membranes were washed with 3ϫ SSC, 0.1% SDS for 30 min and then twice with 1ϫ SSC, 0.1% SDS for 10 min at 65°C. The human CYP1A1-specific cDNA probe was a gift from Dr. de Waziers (INSERM U490, Paris, France). cDNA probes were 32 Plabeled by random priming using a Rediprime labeling kit (Amersham Biosciences). An 18 S ribosomal RNA oligonucleotide was 32 P-labeled and used as control.
Reporter Gene Constructs and Transient Transfection-The p1A1-FL construct containing the Ϫ1566, ϩ73 5Ј-region of the human CYP1A1 gene upstream of the firefly luciferase reporter gene (a gift from Prof. Barouki, INSERM U490, Paris, France) has been described previously (34). Successive deletions of the 5Ј-flanking region have been performed by PCR. The PCR products were subcloned into the luciferase reporter gene plasmid pGL3-basic (Promega) to yield the p1A1-FL(Ϫ1460), p1A1-FL(Ϫ1395), p1A1-FL(Ϫ1300), p1A1-FL(Ϫ-800), and p1A1-FL(Ϫ340) plasmids. A double-stranded oligonucleotide (CGACCTCAGGCTACGTGA-GAATAGTGCACTCAGGCTAGCGTGAGAAAGTGCACTCAGGCTA-GCGTGAGAATTGAGCT) containing three XRE sequences (underlined) was inserted between the KpnI and SacI sites of the pGL3promoter to give pGL3-XRE3. Caco-2 cells were co-transfected with the pRL-SV40 vector that codes for Renilla luciferase (Promega) plus the p1A1-FL constructs. Similar experiments were performed with a "basic" control consisting of the promoterless pGL3-luciferase construct (pGL3-basic) and a pGL3-promoter plasmid containing the SV40 promoter upstream of the luciferase gene. Transient transfection of Caco-2 cells was performed by the TransFast method (Promega) according to manufacturer's instructions. Briefly, 400 l of transfection media (Dulbecco's modified Eagle's medium without fetal bovine serum) containing 600 ng of luciferase reporter plasmid were added to the confluent Caco-2 cells along with 15 ng of the pRL-SV40 DNA and 4 l of TransFast. The plates were incubated for 2 h, and then 2 ml of Dulbecco's modified Eagle's medium were added and incubated for another 24 h, at which time the cells were treated for 16 h. Dual luciferase assays (firefly and Renilla) were performed with a Promega kit as described in the manufacturer's protocol.
Preparation of Microsomal Fractions-Intestinal mucosa or Caco-2 cells were homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA. After removal of nuclear and mitochondrial fractions by centrifugation at 3,000 ϫ g for 10 min and at 9,000 ϫ g for 20 min, microsomes were obtained by centrifugation at 100,000 ϫ g for 1 h. They were dissolved in 0.1 M phosphate buffer (pH 7.4) containing 10% glycerol and stored at -80°C until use. Protein concentration was determined according to the Bradford method (35).
Ethoxyresorufin O-Deethylase (EROD) Activity Assay-EROD activity, supported mainly by CYP1A, was measured on intestinal microsomes according to the method of Burke and Mayer (36). Measurement on living Caco-2 cells was performed as follows. In brief, the medium was removed after treatment, and cells were washed with 100 mM phosphate-buffered saline and then incubated with 50 M ethoxyresorufin (Sigma) and 1.5 mM salicylamide (Sigma) at 37°C. Kinetic reading with a spectrofluorometer was performed over a period of 30 min. Fluorescence values were converted to picomoles using a calibration curve of resorufin fluorescence, and results were expressed as pmol of resorufin/mg of total protein/min.
Intracellular Calcium Measurements-Caco-2 cells were cultured on 12-mm culture inserts (0.4 M pore size, polycarbonate, Transwell, Costar) and incubated with 2.5 M Fura-2-AM for 15 min at 37°C in Hepes-buffered medium (10 mM Hepes, 134.8 mM NaCl, 4.7 mM KCl, 1 mM MgCl 2 , 1.2 mM KH 2 PO 4 , 1 mM CaCl 2 , 10 mM glucose, pH 7.4, at 37°C) supplemented with 0.006% pluronic acid. Fura-2-loaded Caco-2 cells were placed in a continuously perfused recording chamber mounted on the stage of an epifluorescence microscope (Nikon Diaphot). The cells were irradiated alternately with 340 and 380 nm light, and fluorescence from the trapped dye was measured at 510 nm. The F340/ F380 ratio was used to estimate the intracellular calcium [Ca 2ϩ ] i (F340 and F380 is the fluorescence intensity at 340 and 380 nm, respectively). The monochromator and photometers, which produce and detect the fluorescence from ϳ10 to 15 cells in the field of view, were part of a Photon Technology International (PTI) DeltaRAM system, and the software systems to control the monochromator and to both acquire and process the data were also supplied by PTI.
Statistical Analysis-The results are presented as mean Ϯ S.D.
Significant differences were evaluated with the Student's t test. For the variations of intracellular calcium concentration, the Mann-Whitney U test was used.

Effect of Oltipraz on CYP1A1 Expression and Activity in Rat
Gut-In a first set of experiments we investigated the expression of CYP1A1 in rat intestine, one of the first tissues in contact with oltipraz after administration by oral route. To differentiate the different parts of the gastrointestinal tract, we measured CYP1A1 mRNA levels in the duodenum, jejunum, ileum, and colon of control and oltipraz-treated rats. After 72 h of treatment with oltipraz, CYP1A1 mRNA levels were increased mainly in the duodenum and jejunum when compared with control rats (Fig. 1A). CYP1A1 activity, measured by the deethylation of ethoxyresorufin (EROD), was also augmented in the duodenum and jejunum (Fig. 1B), although a significant increase was observed only in the jejunum (9-fold; p Ͻ 0.05).
Effect of Oltipraz on CYP1A1 Expression and Activity in Human Caco-2 Cells-To determine whether CYP1A1 is also induced in human cells by oltipraz, we used human Caco-2 cells, which are able to differentiate after ϳ21 days of confluency into cells expressing enterocyte characteristics. The differentiated state of the cells was confirmed by measuring the sucrase-isomaltase mRNA levels (data not shown). Twenty-one days after confluency, the cells were treated with 50 M oltipraz for 2, 4, 8, 12, 18, and 24 h, and CYP1A1 mRNA and protein levels and activity were determined by Northern blot, Western blot, and measurement of EROD activity, respectively ( Fig. 2A). In control cells, CYP1A1 transcripts and proteins were not detected. However, an increase of CYP1A1 mRNA level as early as 2 h after the beginning of oltipraz treatment was observed, whereas EROD activity and protein level were augmented after 4 h of treatment. The maximum of mRNA induction was obtained between 8 and 12 h of treatment. Treatment of Caco-2 cells with benzo[a]pyrene, a PAH, elicited the same pattern of CYP1A1 mRNA induction (Fig. 2B).
Effect of Oltipraz on CYP1A1 Transcriptional Activity in Human Caco-2 Cells-The rapid induction of CYP1A1 mRNA in oltipraz-treated cells suggested the involvement of a transcriptional mechanism. To test this hypothesis, we performed transient transfections with constructs containing the CYP1A1 gene 5Ј-flanking region upstream of the luciferase gene in order to measure the transcriptional activity of CYP1A1 in Caco-2 cells in the absence or presence of oltipraz. As shown in Fig. 3, neither pGL3-basic nor pGL3-promoter luciferase activities were affected after oltipraz treatment. In contrast, transfection of p1A1-FL(-1566) construct, which contains 1566 bp of the CYP1A1 gene 5Ј-flanking region upstream of the luciferase gene, resulted in a 5-fold increase of the luciferase activity in oltipraz-treated cells. CYP1A1 promoter activity was still induced by oltipraz when CYP1A1 deletion constructs (from Ϫ1566 to Ϫ1300 of the CYP1A1 promoter) were used. In contrast, there was no significant increase of luciferase activity when p1A1-FL(Ϫ800) or p1A1-FL(Ϫ340) constructs were transfected. Interestingly, five XREs, which are known to be involved in the CYP1A1 regulation by PAH, are present in the p1A1-FL(Ϫ1566), only one in the p1A1-FL(Ϫ800), and none in the p1A1-FL(Ϫ340) construct (19). Because oltipraz induction of CYP1A1 promoter activity seems to be correlated to the presence of XRE in the CYP1A1 DNA fragment subcloned upstream of the luciferase gene, Caco-2 cell transfection was performed with a construct containing a luciferase reporter gene driven by three XREs (pGL3-XRE3). Results from this experiment showed that the XRE-driven luciferase activity is inducible by oltipraz (2.5-fold, p Ͻ 0.05).
In cells treated by PAH or derivatives such as dioxin, the XRE binding sequences present in the 5Ј-flanking region of the CYP1A1 gene are able to interact specifically with a heterodimeric complex formed by the AhR and a protein partner, Arnt (37)(38)(39), thereby increasing CYP1A1 transcriptional activity. To determine whether induction of CYP1A1 by oltipraz is mediated by the AhR-XRE pathway, we co-treated Caco-2 cells with oltipraz and resveratrol, an AhR antagonist (40,41). As shown in Fig. 4A, in the presence of 50 and 100 M resveratrol the oltipraz-induced-CYP1A1 mRNA levels were partially and completely inhibited, respectively. Moreover, induction by oltipraz of luciferase activity measured after transient transfection of p1A1-Fl or pGL3-XRE3 was abolished in the presence of 25 M resveratrol (Fig. 4B). All of these results support the conclusion of the involvement of the AhR-XRE pathway in the oltipraz-mediated induction of CYP1A1 in Caco-2 cells.
Effects of Oltipraz on Intracellular Calcium Concentration-To further determine the mechanism involved in CYP1A1 induction by oltipraz, we studied upstream events that might occur at the cellular level and more particularly the [Ca 2ϩ ] i . In a first set of experiments we measured the [Ca 2ϩ ] i in Caco-2 cells in the absence or presence of oltipraz by using cell loaded-Fura-2-AM as a Ca 2ϩ -sensitive fluorescent probe. The intracellular Fura-2 fluorescence ratios recorded in the presence or absence of oltipraz were compared, knowing that any significant change in these ratios are suggestive of a significant change in [Ca 2ϩ ] i .
Following short-term incubations of Caco-2 cells with different concentrations of oltipraz, significant changes in the Fura-2 fluorescence ratio (F340/F380) were observed. F340/F380 ratio variations in the presence of oltipraz, representative of [Ca 2ϩ ] i variations, are illustrated by the recordings given in the left panel of Fig. 5A. An increase of the ratio was already observed with 5 M of oltipraz, which became more pronounced with increasing concentrations of oltipraz (25,50, and 100 M). The delta ratio, corresponding to the difference between the F340/ F380 ratios measured in cells prior to and following oltipraz treatment, respectively, showed a correlation between oltipraz concentration and [Ca 2ϩ ] i (Fig. 5A, right panel).
To determine whether the increase of [Ca 2ϩ ] i following oltipraz treatment was the consequence of an extracellular calcium entry, the cells were maintained in calcium-free medium supplemented with 10 mM EGTA (an extracellular calcium chelator) and treated with oltipraz. Under these conditions, the [Ca 2ϩ ] i increase was lowered dramatically in the calcium-free medium when compared with the [Ca 2ϩ ] i increase obtained with cells maintained in a calcium-containing medium (Fig. 5B).  when the calcium chelator was added 30, 60, and 240 min after oltipraz (Fig. 7B), thus indicating that the rapid, transient increase of [Ca 2ϩ ] i elicited upon addition of oltipraz (between 0 and 1 h) initiates the CYP1A1 induction mechanism. The oltipraz induction of luciferase activity measured after transient transfection of p1A1-Fl or pGL3-XRE3 was abolished in the presence of 50 M BAPTA-AM (Fig. 8). This finding also strongly supports the involvement of intracellular calcium in the oltipraz-mediated induction of CYP1A1 in Caco-2 cells.
To determine the mechanism involved in the increase of [Ca 2ϩ ] i following oltipraz treatment, we used verapamil and nifedipine, two inhibitors of voltage-dependent L-type calcium channels, and 2-APB, a blocker of store-operated Ca 2ϩ chan-nels. Treatment of Caco-2 cells with nifedipine or verapamil did not affect oltipraz-CYP1A1 induction (data not shown). On the other hand, 2-APB inhibited CYP1A1 induction in a dose-dependent manner (Fig. 9) and was found to prevent oltiprazinduced [Ca 2ϩ ] i increase (data not shown). These results strongly suggest that calcium entering through store-operated Ca 2ϩ channels is essential for CYP1A1 induction in Caco-2 cells treated with oltipraz.

DISCUSSION
The pleiotropic effects of oltipraz, a promising chemopreventive agent, are now well recognized, and recent evidence has been brought that this compound can induce both phase I and phase II enzymes either in liver or extrahepatic tissues. Although differences have been observed regarding the response of phase I and phase II enzymes between rat tissues, CYP1A appears to be among the most sensitive of the CYPs. CYP1A1 and CYP1A2, which are expressed, respectively, mostly in nonhepatic and hepatic tissues, are involved in the bioactivation of a number of carcinogens such as PAH as well as other toxicants. The results reported here show that oltipraz is also an inducer of CYP1A1 in the intestine and, using human Caco-2 cells, that its induction is calcium-and Ah receptor-dependent.
To our knowledge, this is the first demonstration that oltipraz dramatically increases CYP1A1 in both rat intestine and human Caco-2 cells. Moreover, the data presented here strongly indicate that this induction occurs through the XRE and that oltipraz and PAH inducers utilize the same AhRmediated signal transduction pathway to regulate the expression of CYP1A1. First, a comparative analysis of CYP1A1 mRNA levels shows the same pattern of induction in the presence of oltipraz or benzo[a]pyrene (starting after 2 h with a maximum induction between 8 and 12 h). Second, Caco-2 cells are known to possess a functional AhR (29). Third, the expression of CYP1A1 mRNA was superinduced when oltipraz was used in the presence of a protein synthesis inhibitor, cycloheximide (data not shown). Fourth, use of luciferase reporter constructs demonstrates that oltipraz is a potent inducer of both the XRE-containing 5Ј-flanking region of CYP1A1 gene and XRE-driven gene expressions. Fifth, resveratrol (an antagonist of AhR) inhibits the oltipraz increase of both CYP1A1 mRNA level and p1A1-Fl-or pGL3-XRE3-luciferase activities. Finally, oltipraz shares several structural features with CYP1A1/2 classical inducers such as 3-methylcholanthrene, TCDD, and ␤-naphtoflavone; it is hydrophobic, planar, and of polycyclic structure. Although its behavior is quite similar to that of PAH, it cannot be excluded that oltipraz (or one of its metabolites) is an activator of AhR without binding to it, as already shown for other compounds such as omeprazole (42,43).
Previous reports and the present study clearly indicate that oltipraz is able to increase transcriptional activity of phase II enzymes and CYP1A1 through the binding of transcriptional factors to the ARE and XRE sequences, respectively. However, early cellular events occurring upon cell exposure to oltipraz and leading to this transcriptional activation are still unknown and remain to be described. In an attempt to determine whether calcium, which is a well established transcriptional regulator, is involved in CYP1A1 induction, we looked for an effect of oltipraz on intracellular calcium level in Caco-2 cells. Our results clearly demonstrate that oltipraz induces a rapid and transient increase of [Ca 2ϩ ] i in these cells, which mediates oltipraz induction of CYP1A1. Increases in intracellular calcium in most cell types are mediated by either the release of calcium from the endoplasmic reticulum via inositol 1,4,5triphosphate or an influx via plasma membrane Ca 2ϩ channels (voltage-dependent calcium channels, receptor-operated calcium channels, and/or store-operated Ca 2ϩ channels) or by both. In the present case, the lack of [Ca 2ϩ ] i increase following the addition of oltipraz in a calcium-free medium (containing EGTA) strongly supports the involvement of plasma membrane calcium channels. This was confirmed by using 2-APB, an inhibitor of store-operated Ca 2ϩ channels, which inhibits oltipraz-CYP1A1 induction in a dose-dependent manner. Storeoperated Ca 2ϩ channels are defined as plasma membrane Ca 2ϩ channels, in which activation is mediated by a decrease in Ca 2ϩ in the endoplasmic reticulum (44). These channels are thought to be responsible for replenishing Ca 2ϩ lost from the endoplasmic reticulum via membrane Ca 2ϩ /Mg 2ϩ -ATPase. Thus, the addition of oltipraz in the cell culture medium rapidly increases [Ca 2ϩ ] i via an entry of this ion species through store-operated channels, which is likely to be activated following an effect on the endoplasmic reticulum. Our results also support the hypothesis that this rapid increase of [Ca 2ϩ ] i is the initial event leading to the induction of CYP1A1 by oltipraz. Interestingly, Marc et al. (45) have brought evidence for the involvement of Ca 2ϩ in the induction of mouse Cyp2b9/10 by phenobarbital, a compound that also induces CYP1A1 in rat (46) and CYP1A2 in rainbow trout hepatocytes (47). Moreover, TCDD, benzo-[a]pyrene, and 7,12-dimethylbenz[a]anthracene, three known CYP1A1 inducers, have been reported to increase [Ca 2ϩ ] i in the human mammary epithelial cell line MCF-10A (26,48). Therefore, intracellular Ca 2ϩ might be a key factor in the induction of CYP1A1 by various compounds. This hypothesis is reinforced by experiments carried out with Caco-2 cells treated with BAPTA-AM or 2-APB, which also partially inhibit CYP1A1 induction by TCDD, benzo[a]pyrene, or 3-methylcholanthrene (data not shown).
Several studies have demonstrated that changes in free intracellular Ca 2ϩ are rapidly transformed into changes in the activity of several kinases including protein kinase A, PKC, MAPKs (extracellular signal-regulated kinase and p38), Ca 2ϩ / calmodulin-dependent protein kinase (CaMK), and CaMKK (23,49). Previous investigations revealed that DNA binding by human and mouse AhR-Arnt heterodimers requires phosphorylation of both proteins, whereas formation of AhR-Arnt heterodimers requires phosphorylation of Arnt only (20,22). Moreover, Long et al. (21) showed that a PKC-mediated event is required for the AhR to form a functional transcriptional complex that leads to trans-activation. Thus, in the process of CYP1A1 induction by oltipraz, [Ca 2ϩ ] i might be a determinant step in the activation of PKC and hence the phosphorylation of AhR. Interestingly, recent findings indicate an important role for both PKC and MAPK pathways in the induction of AREdependent phase II detoxifying enzymes, with Nrf2 being phosphorylated by PKC (13,14). Consequently, it could be postulated that the increases in both CYP1A1 and phase II enzymes in the presence of oltipraz are dependent on a common initial signal, which is the oltipraz-induced entry of calcium inside the cells. This has yet to be established regarding phase II enzymes. Thus, the entry of extracellular calcium inside the cells through store-operated Ca 2ϩ channels might be the initial event leading to the activation of the AhR receptor and its subsequent binding to the XRE sequence.
Although oltipraz exhibits a chemopreventive effect during different stages of experimental colon carcinogenesis induced by azoxymethane in rats (4), compounds that induce CYP1A1 are regarded as potentially harmful because of the property of this CYP to activate carcinogenic PAH. Furthermore, rat intestinal conjugation enzymes have been demonstrated to be less susceptible to induction by oltipraz than their hepatic counterparts (50,51). In this context, the ability of oltipraz to induce CYP1A1 in intestinal cells raises the critical question of whether, in some cases, oltipraz might potentiate the deleterious effects of a particular compound to which an individual is also exposed.