Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
1 To whom correspondence should be addressed at Faculty of Pharmacy & Pharmaceutical Sciences, 3126 Dentistry / Pharmacy Centre, University of Alberta, Edmonton, AB, Canada T6G 2N8. Fax: 7804921217. E-mail: aelkadi{at}pharmacy.ualberta.ca.
Received May 25, 2005; accepted August 4, 2005
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor; Cyp1a1; heavy metals; transcriptional; posttranscriptional.
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INTRODUCTION |
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AhR is a ligand-activated basic helix-loop-helix transcriptional factor located in the cytoplasm and is bound to two 90-kDa heat-shock proteins (HSP90) and an AhR interacting protein (AIP) (Heid et al., 2000; Ma and Whitlock, 1997
; Pollenz, 2002
). Activation of the AhR upon binding with a ligand, such as TCDD, causes dissociation of HSP90 and AIP. Subsequently, the ligandreceptor complex translocates to the nucleus, where it heterodimerizes with the transcriptional factor protein, aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR/ARNT complex then binds to a specific DNA sequence, called the xenobiotic-responsive element (XRE) located in the promoter region of the Cyp1a1 gene. This results in the initiation of the mRNA transcription process. Newly synthesized mRNA is then transported out of the nucleus where it is translated into protein (Pollenz, 2002
; Whitlock, 1999
).
The frequent persistent occurrence and accumulation of heavy metals in the environment, even in traces, and the potential to human exposure from numerous sources, including contaminated air, water, soil, and food, makes heavy metals some of the most hazardous and toxic substances in the environment (ATSDR, 2003). Cocontamination with complex mixtures of heavy metals and HAHs is a common environmental problem with multiple biological consequences, particularly to the metabolizing enzyme systems in the body (Korashy and El-Kadi, 2004
). Recently, we have examined in murine hepatoma Hepa 1c1c7 cells the potential effects of three prominent environmental heavy metals, mercury (Hg2+), lead (Pb2+), and copper (Cu2+), on the constitutive and inducible expression of Cyp1a1 gene mediated by four potential aromatic hydrocarbon contaminants, including TCDD, 3-methylcholanthrene, ß-naphthoflavone, and benzo[a]pyrene (Korashy and El-Kadi, 2004
). At the constitutive level, we have shown that all three metals induced Cyp1a1 mRNA in a concentration-dependent manner, but did not affect its catalytic activity. At the inducible level, Hg2+ and Pb2+ further potentiated, while Cu2+ inhibited the HAH-mediated induction of Cyp1a1 mRNA, whereas the catalytic activity was inhibited by the three metals. The detailed sequence of cellular events occurring between the increase of Cyp1a1 mRNA and the decrease of its catalytic activity is still not known.
Although the regulation of the CYP1A1 by classical AhR ligands, such as TCDD, is known to be regulated at the transcriptional level (Pasco et al., 1988; Whitlock, 1999
), the current available studies on the relative contribution of posttranscriptional mechanisms in the regulation of CYP1A1 by AhR ligands are controversial. Regardless, very little information is known about the regulation of this gene by other CYP1A1 modifiers, including the heavy metals.
In the current work, we have explored the molecular mechanisms by which heavy metals affect the regulation of Cyp1a1 gene expression in Hepa 1c1c7 cells. We provide here the first evidence implicating the involvement of transcriptional and posttranslational mechanisms in the induction of Cyp1a1 by the heavy metals. Importantly, we have shown that the induction of Cyp1a1 mRNA by heavy metals is an AhR dependent, and that inhibition of AhR degradation further enhanced the induction of Cyp1a1 mRNA observed. Moreover, at the posttranslational level, heavy metals decreased the degradation rate of the Cyp1a1 protein. Furthermore, all metals significantly inhibited the TCDD-mediated induction of Cyp1a1 activity, which coincided with an increase in heme oxygenase-1 (HO-1) and a decrease in cellular heme content.
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METHODS AND MATERIALS |
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Cell culture and treatments.
Murine hepatoma Hepa 1c1c7 cells (generously provided by Dr. O Hankinson, University of California, Los Angeles) were maintained in DMEM, without phenol red, supplemented with 10% fetal bovine serum, 20 µM L-glutamine, 50 µg/ml gentamicin sulfate, 100 IU/ml penicillin G, 10 µg/ml streptomycin, 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 under a 5% CO2 humidified environment.
For enzyme activity assay, Hepa 1c1c7 cells were seeded in 96-well cell culture plates at a cell density of 7.5 x 104 cells/well in DMEM culture media, and for RNA and protein assays at a cell density of 11.5 x 106 cells/well in six-well cell culture plates. The cells were treated in serum-free media with TCDD dissolved in dimethylsulfoxide (DMSO) and/or metals dissolved in double deionized water, in the presence and absence of the chemical inhibitors actinomycin D (Act-D) dissolved in 75% ethanol, CHX dissolved in double deionized water, MG-132 dissolved in DMSO, or the combination of CHX and MG-132. The various metals were added 30 min prior to TCDD treatment or after the addition of chemical inhibitors, whenever applicable.
RNA extraction and Northern blot analysis.
After incubation with the test compounds for the indicated time periods, total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to manufacturer's instructions. Northern blot analysis was performed as described previously (Korashy and El-Kadi, 2004; Sambrook et al., 1989
). Briefly, total RNA (20 µg) was electrophoresed on a 1.1% formaldehyde-agarose denaturing gel, transferred to Hybond-N-nylon membranes, and hybridized with a 32P-labeled cDNA probe specific for mouse Cyp1a1 and HO-1 (generously provided by Dr. J. R. Bend, University of Western Ontario, London). The nylon membrane blots were subsequently stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA probe, which was used as a loading control. The intensity of Cyp1a1 mRNA was quantified, relative to the densitometric signals obtained for Gapdh mRNA (TA Desnitometer, model TBX, Tobias Associates, Inc., Ivyland, PA).
Protein extraction and Western blot analysis.
After incubation with the test compounds for the indicated time periods, Hepa 1c1c7 cells were collected in lysis buffer (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 of protease inhibitor cocktail). The total cellular proteins were obtained by incubating the cell lysates on ice for 1 h, with intermittent vortexing every 10 min, followed by centrifugation at 12,000 x g for 10 min at 4°C. The supernatant fractions were collected for determination of protein concentration using bovine serum albumin as a standard by the Lowry method (Lowry et al., 1951). Western blot analysis was performed using a previously described method (Korashy and El-Kadi, 2004
; Sambrook et al., 1989
). Briefly, 25 µg of protein from each treatment group was separated by 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then electrophoretically transferred to nitrocellulose membrane. Protein blots were then blocked overnight at 4°C in blocking solution containing 0.15 M sodium chloride, 3 mM potassium chloride, 25 mM Tris-base (TBS), 5% skim milk powder, 2% bovine serum albumin, and 0.5% Tween-20. After blocking, the blots were incubated with a primary polyclonal goat anti-mouse Cyp1a1 antibody for 2 h at room temperature in TBS 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 solution for 1 h at room temperature. The bands were visualized using the enhanced chemiluminescence method according to the manufacturer's instructions (Amersham Biosciences Inc., Piscataway, NJ). The blots were subsequently stripped in a solution containing 62.5 mM Tris-HCl pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 30 min at 50°C and thereafter reprobed with actin goat anti-human primary antibody, which was used as loading control, as described above.
Determination of Cyp1a1 mRNA and protein half-lives.
Two independent experiments, Act-D- and CHX-chase experiments, were performed in order to determine the half-lives of the Cyp1a1 mRNA and protein, respectively. Hepa 1c1c7 cells were grown to 70% confluence in six-well cell culture plates and then preincubated with 1 nM TCDD for 12 or 24 h, to induce mRNA or protein, respectively. After the induction period, cells were then washed three times with fresh media to remove TCDD.
For the Act-D-chase experiment, the cells were incubated in fresh serum-free media containing 5 µg/ml Act-D, a transcription inhibitor, to block any further synthesis of RNA. Thirty min later, 5 µM Hg2+, 25 µM Pb2+, or 10 µM Cu2+ was added. Total RNA was isolated at 0, 1, 3, 6, 12, and 24 h after the addition of Act-D, followed by Northern blot quantification analysis.
For the CHX-chase experiment, the cells were incubated in fresh serum-free media containing 10 µg/ml CHX, a translation inhibitor, to block any further synthesis of protein. Thirty min later, 5 µM Hg2+, 25 µM Pb2+, or 10 µM Cu2+ was added. The cell protein was then extracted at 0, 1, 3, 6, 12, and 24 h after the addition of CHX, followed by Western blot quantification analysis.
In both experiments, Northern and Western blot analyses were quantified by densitometry. Cyp1a1 mRNA and protein half-lives were calculated from the slope of the semi-logarithmically transformed best fit line. The decay curves were analyzed individually using linear regression of mRNA or protein amount, expressed as percentage of mRNA or protein remaining versus time. The half-lives obtained from three separate experiments were then used to calculate the mean half-life (mean ± SEM, n = 3).
Nuclear and cytosolic protein preparation.
Hepa 1c1c7 cells, plated at 1 x 106 cells/100 mm dish, were grown to 90% confluence and harvested after treatment with 5 µM Hg2+, 25 µM Pb2+, 10 µM Cu2+, or 10 nM TCDD in serum-free media for 3 h. The nuclear proteins were then extracted and prepared as described previously (Rogers and Denison, 2002). Hepatic cytosol of untreated guinea pig (generously provided by Dr. M. S. Denison, University of California, Davis) was prepared as described previously (Brown et al., 2004
) and then was incubated in vitro with 5 µM Hg2+, 25 µM Pb2+, 10 µM Cu2+, or 20 nM TCDD for 3 h.
Gel electrophoretic mobility shift assay (EMSA).
XRE complementary oligonucleotides, 5-GGAGTTGCGTGAGAAGAGCC-3 and 5-GGCTCTTCTCACGCAACTCC-3, were synthesized, then annealed by heating to 70°C for 7 min, then allowed to cool to room temperature. The double-stranded XRE was then labeled with -32P-ATP at the 5-end using T4 polynucleotide kinase (Invitrogen), according to the manufacturer's instructions, and used as a probe for EMSA reactions. EMSA was performed as described previously (Gharavi and El-Kadi, 2005
; Rogers and Denison, 2002
). Briefly, aliquots of the nuclear extract (10 µg) or cytosolic protein (80 µg) were incubated for 30 min at room temperature in a reaction mixture (30 µl) containing 25 mM HEPES, pH 7.9, 80 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol (v/v), 1 µg salmon sperm DNA, and 5 µg poly(dI.dC). Thereafter,
1 ng (100,000 cpm) 32P-labeled XRE was incubated with the mixture for another 30 min before being separated through a 4% nondenaturing polyacrylamide gel. For the competition assay, nuclear or cytosolic proteins were preincubated at room temperature for 30 min with a 100-fold molar excess of unlabeled XRE before the addition of the 32P-labeled XRE. The gel was dried at 80°C for 1h, and then visualized by autoradiography.
Determination of Cyp1a1 enzymatic activity.
Cyp1a1-dependent 7-ethoxyresorufin O-deethylase (EROD) activity was performed on intact, living Hepa 1c1c7 cells, using 7-ethoxyresorufin as a substrate, as previously described (Kennedy et al., 1993; Korashy and El-Kadi, 2004
). Enzymatic activity was normalized for cellular protein content, which was determined using a modified fluorescent assay (Korashy and El-Kadi, 2004
; Lorenzen and Kennedy, 1993
).
Measurement of cellular heme content.
Cellular heme content was determined fluorimetrically as described previously (Ward et al., 1984). Briefly, Hepa 1c1c7 cells were plated in 100-mm Petri dishes for 48 h. The cells were then incubated in fresh serum-free media with 1 nM TCDD in the presence and absence of 5 µM Hg2+, 25 µM Pb2+, or 10 µM Cu2+ for 24 h. Pelleted cells were boiled in 2 M oxalic acid (100 µl) for 30 min and then rapidly resuspended in cold PBS (0.9 ml) followed by centrifugation at 14,000 x g for 15 min. The supernatant was assayed for protoporphyrin IX using Eclipse fluorescence spectrophotometer (Varian Australia PTY LTD., Australia) with excitation and emission wavelengths of 405 and 600 nm, respectively. Cellular heme content was normalized for cellular protein which was determined using Lowry method (Lowry et al., 1951
).
Statistical analysis.
All results are presented as mean ± SEM. The comparison of the results from the various experimental groups with their corresponding controls was carried out by a one-way analysis of variance (ANOVA) followed by StudentNewmanKeul's test to assess significant differences from the control group. The differences were considered significant when p < 0.05.
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RESULTS |
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Effect of Heavy Metals on the TCDD Concentration-Dependent Induction of Cyp1a1 mRNA
To further examine the effect of heavy metals on the kinetics of Cyp1a1 mRNA induced by different concentrations of TCDD, Hepa 1c1c7 cells were treated with 5 µM Hg2+, 25 µM Pb2+, or 10 µM Cu2+ in the presence or absence of increasing concentrations of TCDD (0.1, 1, and 10 nM). Thereafter, Cyp1a1 mRNA was measured using Northern blot analysis.
TCDD alone caused a concentration-dependent increase in Cyp1a1 mRNA levels (Fig. 2). However, at the highest concentration tested, 10 nM, the induction of Cyp1a1 mRNA was less than that obtained with 1 nM concentration (Fig. 2). Hg2+ or Pb2+ further increased the Cyp1a1 mRNA mediated by TCDD at all concentrations of TCDD tested. Cu2+, on the other hand, decreased the Cyp1a1 mRNA induction mediated by TCDD (Fig. 2).
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Inhibition of metal-mediated Cyp1a1 mRNA induction by a RNA synthesis inhibitor.
To investigate whether heavy metals are able to increase the de novo Cyp1a1 RNA synthesis, Hepa 1c1c7 cells were treated with 5 µM Hg2+, 25 µM Pb2+, 10 µM Cu2+, or 1 nM TCDD in the presence or absence of 5 µg/ml Act-D, a RNA synthesis inhibitor, for 6 h. Total RNA was then isolated and quantified by Northern blot analysis. If metals increase the amount of Cyp1a1 mRNA through increasing its de novo RNA synthesis, we would expect to observe a decrease in the content of Cyp1a1 mRNA after the inhibition of its RNA synthesis.
Figure 3 shows that in untreated cells, as expected, the amount of Cyp1a1 mRNA was barely detectable (lane 1). However, pretreatment of the cells with Act-D for 6 h completely inhibited the constitutive expression of Cyp1a1 mRNA (lane 2). In metal-treated cells, metals alone significantly increased the Cyp1a1 mRNA level (lanes 3, 5, and 7); however, pretreatment of the cells with Act-D completely inhibited the induction of Cyp1a1 mRNA in response to metals (lanes 4, 6, and 8). Furthermore, the induction of Cyp1a1 mRNA by TCDD (lane 9), which is known to be a transcriptional event, was completely prevented by Act-D (lane 10). These results suggest that heavy metals increase the Cyp1a1 mRNA level by increasing its de novo RNA synthesis, in a manner similar to that observed with TCDD.
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As shown in Figure 4, CHX alone caused a significant accumulation of Cyp1a1 mRNA 6-fold greater than DMSO-treated cells (lane 2). However, cotreatment of the cells with CHX and metals further increased the accumulation of Cyp1a1 mRNA by CHX (lanes 4, 6, and 8), a phenomenon known as "superinduction" (Israel et al., 1985; Joiakim et al., 2004
; Ma et al., 2000
). Densitometric scan of the autoradiogram indicates that the rate of Cyp1a1 mRNA synthesis induced by Hg2+, Pb2+, or Cu2+, plus CHX were 2-, 3-, or 4-fold greater than those induced by each metal alone, respectively, and Cyp1a1 mRNA synthesis was about 25%, 37%, or 38% greater than those induced by CHX alone (p < 0.05). Furthermore, the cotreatment of the cells with CHX and TCDD superinduced the increase in Cyp1a1 mRNA level by CHX (lane 10). It should be noted here that, in superinduction studies, the weak TCDD-induced Cyp1a1 mRNA signals observed in Fig. 4 and 5 reflect shortened X-ray film exposure times. The exposure period were reduced by half of the standard exposure time (2436 h) to maintain an appropriate perspective of the magnitude of the superinduction.
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Superinduction of metal-mediated Cyp1a1 mRNA induction by a 26S proteasome inhibitor.
Several studies have demonstrated that inhibition of proteasomal-dependent degradation of the AhR protein prolongs its half-life, thereby increasing the amount of functional AhR/ARNT complex and, hence, the transcription of Cyp1a1 in response to TCDD (Joiakim et al., 2004; Ma and Baldwin, 2000
). The results of these studies suggest the involvement of proteasome-dependent pathway in the transcriptional regulation of Cyp1a1. To test the possibility that inhibition of proteasomal-dependent degradation of AhR protein would increase Cyp1a1 transcription in response to metals, Hepa 1c1c7 cells were pretreated with 25 µM MG-132, a potent 26S proteasome inhibitor (Ma and Baldwin, 2000
; Ma et al., 2000
), with or without 10 µg/ml CHX, both in the presence and absence of Hg2+, Pb2+, Cu2+, or TCDD.
Treatment of Hepa 1c1c7 cells with either CHX or MG-132 alone for 6 h (Fig. 5), caused a 5- and 3-fold increase in the Cyp1a1 mRNA levels (lanes 2 and 3), respectively. Cotreatment of CHX and MG-132, however, did not further increase the accumulation of Cyp1a1 mRNA beyond what was observed with each inhibitor alone (lane 4). On the other hand, treatment of the cells with CHX and metals or MG-132 and metals, superinduced Cyp1a1 mRNA expression. Contrastingly, when the cells were pretreated with CHX and MG-132 in the presence of metals, the superinduction of the Cyp1a1 mRNA was greater than the sum of the increases of Cyp1a1 mRNA that was observed with exposure to each inhibitor alone (lanes 68, 1012, and 1416). Similar results were observed when the cells were pretreated with CHX and/or MG-132 in the presence of TCDD (lanes 1820).
Taken together, these results suggest that the increased accumulation of Cyp1a1 mRNA in response to metals is an AhR-dependent pathway.
Activation of AhR/XRE binding by heavy metals.
The effect of metals on AhR translocation to the nucleus and the binding to the XRE of the Cyp1a1 was measured by EMSA. Hepa 1c1c7 cells were treated with metals for 3 h, and their nuclear extracts were subjected to EMSA. Extracts from TCDD-treated cells were used as a positive control for AhR transformation. Figure 6 shows that Hg2+ or Pb2+, but not Cu2+, increased the DNA-binding capacity of the nuclear AhR, as shown by the intensity of the bands. The specificity of metal-induced AhR/ARNT heterodimer binding to XRE was confirmed by competition assay in the presence of 100-fold molar excess of unlabeled XRE.
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TCDD alone caused a concentration-dependent increase in Cyp1a1 activity levels (Fig. 10A). However, at the highest concentration tested, 10 nM, the induction of Cyp1a1 activity was less than that obtained with 1 nM concentration (Fig. 10A). Although metals alone, in the absence of TCDD, did not significantly affect the Cyp1a1 activity, it was noted that the three metals significantly inhibited the induction of Cyp1a1 catalytic activity mediated by TCDD at concentrations 0.1 and 1 nM. However, at the highest concentration tested of TCDD, 10 nM, only Cu2+ decreased the Cyp1a1 catalytic activity (Fig. 10A).
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Taken together, these results suggest that the decreased cellular heme content, at least in part, plays a role in reduced Cyp1a1 catalytic activity observed in heavy metals treated cells.
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DISCUSSION |
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Knowledge of the regulation of Cyp1a1 gene expression shows that the activation of a cytosolic transcriptional factor, AhR, is the first step in a series of molecular events promoting Cyp1a1 transcription and translation processes. To examine whether the regulation of Cyp1a1 gene expression by heavy metals is a transcriptional and/or posttranscriptional event, a series of experiments were carried out. Initially, we have found that all tested metals significantly increased the basal Cyp1a1 mRNA levels in a time-dependent manner. Interestingly, the induction pattern of Cyp1a1 mRNA by heavy metals was similar to those obtained with TCDD, in which heavy metals caused a rapid increase in Cyp1a1 mRNA, which was evident after 3 h of treatment, reaching steady-state after 12 h. Although heavy metal-mediated induction of Cyp1a1 mRNA was 10-fold lower than those induced by TCDD, the level of induction was 15-fold higher than control. On the other hand, the TCDD-mediated induction of Cyp1a1 mRNA was further increased by Hg2+ and Pb2+, but was decreased by Cu2+ cotreatment. The alteration in Cyp1a1 mRNA steady-state levels in response to metals can therefore be attributed to transcriptional and/or posttranscriptional mechanisms.
In the current study, the transcriptional regulation of Cyp1a1 gene expression by heavy metals was demonstrated through different approaches. First, the inhibition of the RNA transcription, using Act-D, completely abolished the induction of Cyp1a1 mRNA in response to heavy metals, implying that metals increase the de novo Cyp1a1 RNA synthesis, in a manner similar to that obtained with TCDD. Second, the coadministration of metals with CHX, a protein translation inhibitor, and/or MG-132, a 26S proteasome inhibitor, superinduced the Cyp1a1 mRNA in response to metals. Superinduction of Cyp1a1 mRNA by CHX or MG-132 has been previously reported in Hepa 1c1c7 (Gharavi and El-Kadi, 2005; Ma et al., 2000
; Sindhu and Kikkawa, 1999
) and in other cell lines (Daujat et al., 1991
; Joiakim et al., 2004
; Lamb and Franklin, 2002
). Recent studies have demonstrated that the superinduction of Cyp1a1 gene by CHX or MG-132 is a transcriptional mechanism and reflects a change in the synthesis, rather than stabilization, of Cyp1a1 mRNA (Joiakim et al., 2004
; Ma and Baldwin, 2000
, 2002
).
Interestingly, it has been previously reported that superinduction of the transcription of AhR-regulated genes by CHX or MG-132 is a gene-specific effect. For example, it has been shown that Cyp1a1 and TCDD-inducible poly(ADP-ribose) polymerase (TiPARP), which are regulated by AhR, are superinduced by CHX or MG-132 (Ma, 2002), whereas the induction of phase II AhR-regulated genes, NAD(P)H: quinone oxido reductase (Nqo1) and glutathione S-transferase Ya subunit (Gst ya), by TCDD requires both AhR and nuclear factor erythroid 2-related factor 2 (Nrf2) (Ma et al., 2004
). This is because Nrf2 is a labile protein, and therefore, the induction is susceptible to CHX but not MG-132. Although the exact mechanism of superinduction is not clear, several proposed mechanisms have been reported. The most acceptable mechanism suggests the presence of a labile transcriptional protein that negatively regulates the expression of Cyp1a1. This is supported by the observations that inhibition of protein synthesis, using CHX, decreases the concentration of repressor proteins, such as I
Ba (Ma et al., 2000
), resulting in an increase in the rate of Cyp1a1 transcription (Israel et al., 1985
; Lamb and Franklin, 2002
; Lusska et al., 1992
). Moreover, it has been hypothesized that inhibition of the turnover of AhR protein by a protein synthesis inhibitor (Ma and Baldwin, 2002
; Ma et al., 2000
) or proteasome inhibitor (Ma and Baldwin, 2000
; Pollenz, 2002
) accounts for the superinduction of Cyp1a1, based on the fact that AhR activation is an integral step for the induction of Cyp1a1 gene expression (Ma et al., 1995
). Taken together, these results suggest that the superinduction of Cyp1a1 in response to metals in the presence of CHX or MG-132 is both a transcriptional and an AhR-dependent pathway; in a manner similar to that obtained with TCDD (Ma and Baldwin, 2002
; Sindhu and Kikkawa, 1999
).
The direct evidence for the involvement of AhR in the transcriptional regulation of Cyp1a1 by heavy metals is supported by our previous observation that heavy metals dose-dependently induced Cyp1a1 mRNA in wild-type, but not in mutant AhR-deficient Hepa 1c1c7 cells (Korashy and El-Kadi, 2004). Further evidence comes from the result of the EMSA. The presence of a nuclear AhR complex is dependent on ligand binding to the cytosolic receptor, nuclear translocation of the liganded AhR, its heterodimerization with ARNT, and subsequent specific and high-affinity DNA binding (Phelan et al., 1998
). Our results not only suggest that metals can induce AhR transformation, nuclear accumulation, and DNA binding, similar to that which is observed with classical AhR ligands such as TCDD, but also support a role for the AhR in the induction of Cyp1a1 by heavy metals. Surprisingly, unlike Hg2+ and Pb2+, Cu2+ fails to induce the nuclear AhR/ARNT/XRE complex formation. At present, we have no mechanistic explanation for the differential effects of Cu2+ in vitro and in intact cells. However these results indicate the involvement of different molecular processes.
The mechanism by which heavy metals directly activate the in vitro AhR translocation is still unknown. However, several mechanisms of activation of the AhR by nonclassical ligands were proposed. In this regard, it has been recently demonstrated that the divalent metal arsenite induced AhR nuclear translocation and binding to the Cyp1a1 gene promoter in Hepa 1c1c7 cells, possibly by disrupting the molecular interaction between AhR and its associated protein, HSP90 (Kann et al., 2005). Based on these observations, we postulate that heavy metals may bind to the AhR-associated proteins, or to a non-TCDD binding site on the receptor, resulting in AhR conformational changes which lead to activation of the receptor and its translocation to the nucleus (Werlinder et al., 2001
).
To test the hypothesis that heavy metals may modulate Cyp1a1 mRNA at the posttranscriptional level, we assessed the turnover rate of Cyp1a1 mRNA using an Act-D chase experiment. Our results showed that the Cyp1a1 mRNA induced by TCDD is short-lived, with an estimated half-life of 3.8 h. Our results are in agreement with previous reports that the half-life of Cyp1a1 mRNA induced by TCDD in Hepa 1c1c7 cells ranges from 34.5 h (Chen et al., 1995; Miller et al., 1983
). Previous studies have reported that the half-life for Cyp1a1 mRNA induced by a different AhR ligand such as dimethybenzanthracene in MCF7 cells was about 7 h (Ciolino and Yeh, 1999
), whereas in human hepatoma HepG2 cells the half-life was about 2.5 h (Lekas et al., 2000
). The differences in the half-lives could be attributed to different cell lines, Cyp1a1 inducers, and/or the type of RNA synthesis inhibitor used. In our study, the Cyp1a1 mRNA half-life in Hepa 1c1c7 cells treated with metals was not statistically different from those of TCDD-induced Cyp1a1 mRNA, indicating that metals did not alter the stability of Cyp1a1 mRNA. In agreement with our results, but with a different metal, it has been recently reported that arsenite did not affect the Cyp1a1 mRNA stability in HepG2 cells (Bessette et al., 2005
).
The inhibitory effects of heavy metals on the induction of Cyp1a1 activity by TCDD suggest a posttranslational mechanism, though the posttranslational regulation of Cyp1a1 is not well studied. This is the first report, to our knowledge, that showed that Hepa 1c1c7 Cyp1a1 protein is short-lived, with a half-life of about 2.7 h. Similarly, in V79 cells, the half-life of Cyp1a1 protein was 2.8 h (Werlinder et al., 2001). In the present study, all three metals significantly decreased the degradation rate of Cyp1a1 protein, providing the first evidence that metals are capable of regulating Cyp1a1 gene expression through a posttranslational mechanism.
Interestingly, the stabilization of Cyp1a1 protein by heavy metals was in contrast with the inhibitory effects of metals on the induction of Cyp1a1 activity-mediated by TCDD. Although it is unclear how heavy metals decreased the induction of Cyp1a1 activity, it has been previously demonstrated that the decrease in heme availability, the prosthetic group of CYP450, causes a reduction in the Cyp1a1 activity (Meyer et al., 2002). This is supported by our current observations that heavy metals induced HO-1 mRNA expression and decreased cellular heme content. Another potential mediator that may be involved in the inhibition of TCDD-mediated induction of Cyp1a1 activity by heavy metals is reactive oxygen species (ROS). We have recently demonstrated that increased ROS production by the AhR ligands, benzo[a]pyrene, 3-methylcholanthrene, and ß-naphthoflavone, at high concentrations were accompanied by a decrease in the Cyp1a1 catalytic activity, but not the mRNA or protein expression levels, which were significantly induced in a concentration-dependent manner (Elbekai et al., 2004
). The AhR-mediated decrease in Cyp1a1 activity was reversed by the antioxidant N-acetylcysteine, suggesting a direct involvement of ROS in the inhibition of Cyp1a1 activity. Furthermore, recent data from our laboratory showed that all metals used in the current study are able to generate ROS (data not shown).
In conclusion, data presented here clearly demonstrate that Hg2+, Pb2+ and Cu2+ can directly activate the AhR and modulate the Cyp1a1 gene expression at both transcriptional and posttranslational levels. Furthermore, the inhibitory effects of heavy metals on the TCDD-inducible Cyp1a1 activity are attributed, at least in part, to decreased heme availability.
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NOTES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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Bessette, E. E., Fasco, M. J., Pentecost, B. T., and Kaminsky, L. S. (2005). Mechanisms of arsenite-mediated decreases in benzo[k]fluoranthene-induced human cytochrome P4501A1 levels in HepG2 cells. Drug Metab. Dispos. 33, 312320.
Brown, D. J., Van Overmeire, I., Goeyens, L., Denison, M. S., De Vito, M. J., and Clark, G. C. (2004). Analysis of Ah receptor pathway activation by brominated flame retardants. Chemosphere 55, 15091518.[CrossRef][ISI][Medline]
Chen, Y. H., Riby, J., Srivastava, P., Bartholomew, J., Denison, M., and Bjeldanes, L. (1995). Regulation of CYP1A1 by indolo[3,2-b]carbazole in murine hepatoma cells. J. Biol. Chem. 270, 2254822555.
Ciolino, H. P., and Yeh, G. C. (1999). The steroid hormone dehydroepiandrosterone inhibits CYP1A1 expression in vitro by a post-transcriptional mechanism. J. Biol. Chem. 274, 3518635190.
Daujat, M., Clair, P., Astier, C., Fabre, I., Pineau, T., Yerle, M., Gellin, J., and Maurel, P. (1991). Induction, regulation and messenger half-life of cytochromes P450 IA1, IA2 and IIIA6 in primary cultures of rabbit hepatocytes. CYP 1A1, 1A2 and 3A6 chromosome location in the rabbit and evidence that post-transcriptional control of gene IA2 does not involve mRNA stabilization. Eur. J. Biochem. 200, 501510.[Abstract]
Elbekai, R. H., Korashy, H. M., Wills, K., Gharavi, N., and El-Kadi, A. O. (2004). Benzo[a]pyrene, 3-methylcholanthrene and beta-naphthoflavone induce oxidative stress in hepatoma hepa 1c1c7 cells by an AHR-dependent pathway. Free Radic. Res. 38, 11911200.[CrossRef][ISI][Medline]
Gharavi, N., and El-Kadi, A. O. (2005). Tert-butylhydroquinone is a novel aryl hydrocarbon receptor ligand. Drug Metab. Dispos. 33, 365372.
Guengerich, F. P. (2004). Cytochrome P450: What have we learned and what are the future issues? Drug Metab. Rev. 36, 159197.[CrossRef][Medline]
Heid, S. E., Pollenz, R. S., and Swanson, H. I. (2000). Role of heat shock protein 90 dissociation in mediating agonist-induced activation of the aryl hydrocarbon receptor. Mol. Pharmacol. 57, 8292.
Israel, D. I., Estolano, M. G., Galeazzi, D. R., and Whitlock, J. P., Jr. (1985). Superinduction of cytochrome P1450 gene transcription by inhibition of protein synthesis in wild type and variant mouse hepatoma cells. J. Biol. Chem. 260, 56485653.[Abstract]
Joiakim, A., Mathieu, P. A., Elliott, A. A., and Reiners, J. J., Jr. (2004). Superinduction of CYP1A1 in MCF10A cultures by cycloheximide, anisomycin, and puromycin: A process independent of effects on protein translation and unrelated to suppression of aryl hydrocarbon receptor proteolysis by the proteasome. Mol. Pharmacol. 66, 936947.
Kann, S., Huang, M. Y., Estes, C., Reichard, J. F., Sartor, M. A., Xia, Y., and Puga, A. (2005). Arsenite-induced aryl hydrocarbon receptor nuclear translocation results in additive induction of phase I and synergistic induction of phase II genes. Mol. Pharmacol. 68, 336346.
Kennedy, S. W., Lorenzen, A., James, C. A., and Collins, B. T. (1993). Ethoxyresorufin-O-deethylase and porphyrin analysis in chicken embryo hepatocyte cultures with a fluorescence multiwell plate reader. Anal. Biochem. 211, 102112.[CrossRef][ISI][Medline]
Korashy, H. M., and El-Kadi, A. O. (2004). Differential effects of mercury, lead and copper on the constitutive and inducible expression of aryl hydrocarbon receptor (AHR)-regulated genes in cultured hepatoma hepa 1c1c7 cells. Toxicology 201, 153172.[CrossRef][ISI][Medline]
Lamb, J. G., and Franklin, M. R. (2002). Cell-based studies reveal differences in glutathione S-transferase induction between oltipraz and tert-butylhydroquinone. J. Biochem. Mol. Toxicol. 16, 154161.[CrossRef][ISI][Medline]
Lekas, P., Tin, K. L., Lee, C., and Prokipcak, R. D. (2000). The human cytochrome P450 1A1 mRNA is rapidly degraded in HepG2 cells. Arch. Biochem. Biophys. 384, 311318.[CrossRef][ISI][Medline]
Lorenzen, A., and Kennedy, S. W. (1993). A fluorescence-based protein assay for use with a microplate reader. Anal. Biochem. 214, 346348.[CrossRef][ISI][Medline]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265275.
Lusska, A., Wu, L., and Whitlock, J. P., Jr. (1992). Superinduction of CYP1A1 transcription by cycloheximide role of the DNA binding site for the liganded ah receptor. J. Biol. Chem. 267, 1514615151.
Ma, Q. (2002). Induction and superinduction of 2,3,7,8-tetrachlorodibenzo-rho-dioxin-inducible poly(ADP-ribose) polymerase: Role of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator transcription activation domains and a labile transcription repressor. Arch. Biochem. Biophys. 404, 309316.[CrossRef][ISI][Medline]
Ma, Q., and Baldwin, K. T. (2000). 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced degradation of aryl hydrocarbon receptor (AhR) by the ubiquitin-proteasome pathway. Role of the transcription activaton and DNA binding of AhR. J. Biol. Chem. 275, 84328438.
Ma, Q., and Baldwin, K. T. (2002). A cycloheximide-sensitive factor regulates TCDD-induced degradation of the aryl hydrocarbon receptor. Chemosphere 46, 14911500.[CrossRef][ISI][Medline]
Ma, Q., Dong, L., and Whitlock, J. P., Jr. (1995). Transcriptional activation by the mouse Ah receptor. Interplay between multiple stimulatory and inhibitory functions. J. Biol. Chem. 270, 1269712703.
Ma, Q., Kinneer, K., Bi, Y., Chan, J. Y., and Kan, Y. W. (2004). Induction of murine NAD(P)H:Quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap n collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): Cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction. Biochem. J. 377, 205213.[CrossRef][ISI][Medline]
Ma, Q., Renzelli, A. J., Baldwin, K. T., and Antonini, J. M. (2000). Superinduction of CYP1A1 gene expression. Regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced degradation of ah receptor by cycloheximide. J. Biol. Chem. 275, 1267612683.
Ma, Q., and Whitlock, J. P., Jr. (1997). A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 272, 88788884.
Meyer, R. P., Podvinec, M., and Meyer, U. A. (2002). Cytochrome P450 CYP1A1 Accumulates in the Cytosol of Kidney and Brain and Is Activated by Heme. Mol. Pharmacol. 62, 10611067.
Miller, A. G., Israel, D., and Whitlock, J. P., Jr. (1983). Biochemical and genetic analysis of variant mouse hepatoma cells defective in the induction of benzo(a)pyrene-metabolizing enzyme activity. J. Biol. Chem. 258, 35233527.
Pasco, D. S., Boyum, K. W., Merchant, S. N., Chalberg, S. C., and Fagan, J. B. (1988). Transcriptional and post-transcriptional regulation of the genes encoding cytochromes P-450c and P-450d in vivo and in primary hepatocyte cultures. J. Biol. Chem. 263, 86718676.
Phelan, D., Winter, G. M., Rogers, W. J., Lam, J. C., and Denison, M. S. (1998). Activation of the ah receptor signal transduction pathway by bilirubin and biliverdin. Arch. Biochem. Biophys. 357, 155163.[CrossRef][ISI][Medline]
Pollenz, R. S. (2002). The mechanism of AH receptor protein down-regulation (degradation) and its impact on AH receptor-mediated gene regulation. Chem. Biol. Interact. 141, 4161.[CrossRef][ISI][Medline]
Rogers, J. M., and Denison, M. S. (2002). Analysis of the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in human ovarian carcinoma BG-1 cells. Mol. Pharmacol. 61, 13931403.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Shimada, T., and Fujii-Kuriyama, Y. (2004). Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Cancer Sci. 95, 16.[ISI][Medline]
Sindhu, R. K., and Kikkawa, Y. (1999). Superinduction of oxidized tryptophan-inducible cytochrome P450 1A1 by cycloheximide in hepa lclc7 cells. In Vitro. Mol. Toxicol. 12, 149162.[ISI][Medline]
Ward, J. H., Jordan, I., Kushner, J. P., and Kaplan, J. (1984). Heme regulation of HeLa cell transferrin receptor number. J. Biol. Chem. 21, 1323513240.
Werlinder, V., Backlund, M., Zhukov, A., and Ingelman-Sundberg, M. (2001). Transcriptional and post-translational regulation of CYP1A1 by primaquine. J. Pharmacol. Exp. Ther. 297, 206214.
Whitlock, J. P., Jr. (1999). Induction of cytochrome P450 1A1. Annu. Rev. Pharmacol. Toxicol. 39, 103125.[CrossRef][ISI][Medline]