* Department of Technology and Ecology, Graduate School of Global Environmental Studies, Kyoto University, Kyoto, Japan, and Japanese Ministry of the Environment, Tokyo, Japan
Received December 26, 2003; accepted March 10, 2004
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ABSTRACT |
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Key Words: indirubin; AhR; TCDD; CYP1A1; metabolism; gene expression.
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INTRODUCTION |
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We previously reported that indirubin was more potent than TCDD in inducing transactivation of a reporter gene in yeast that expressed human AhR and ARNT proteins (Adachi et al., 2001). Indirubin was also proved to be a high (Kd = 12.2 nM) affinity AhR ligand by competitive binding assay using rat liver cytosol and [3H]TCDD (Rannug et al., 1992
). Indirubin is a pink colored pigment and synthesized as a by-product of indigo (Fig. 1). Indirubin's bioactivity has been studied because it is also an active component of the Chinese traditional medicine, Danggui Longhui Wan, which is used to cure chronic myelocytic leukemia (CML). Indirubin is a potent inhibitor of cyclin-dependent kinases (CDKs) and of glycogen synthase kinase-3ß (GSK-3ß), which may play an important role in the development of Alzheimer's disease (Hoessel et al., 1999
). It also inhibits inflammation in delayed-type hypersensitivity reactions (Kunikata et al., 2000
). Furthermore, indirubin was confirmed to be a product of the human cytochrome P450-catalyzed metabolism of indole, a product of the tryptophan catabolite (Gillam et al., 2000
). We have previously detected indirubin in human urine of healthy individuals and in fetal bovine serum at average concentrations of 0.2 and 0.07 nM, respectively (Adachi et al., 2001
). Both indirubin and TCDD are potent AhR ligands. But TCDD has pleiotropic toxicity while indirubin seems to be nontoxic since it is excreted into our urine everyday. To elucidate this toxic difference, the present study was undertaken to compare the gene expression profiles and metabolism of the potent natural AhR ligand, indirubin, with those of the prototypical AhR ligand, TCDD.
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MATERIALS AND METHODS |
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Cell culture and treatments. The human hepatocarcinoma cell line HepG2 was obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan. The cells were grown at 37°C in air supplemented with 5% CO2. Cells cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum were seeded into 10-cm dishes (4 x 106 cells/dish) containing fresh medium and 24 h later (7080% confluence) exposed to the test chemicals. The chemicals were dissolved in DMSO and added to the medium directly. The final DMSO concentration was 1.0% (v/v).
Gene expression arrays. HepG2 cells were exposed to 10 nM of indirubin or TCDD for 8 h and subsequently total RNA was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality and quantity were assessed using agarose gel electrophoresis and spectrophotometric absorbency at 260/280 nm. Using 50 µg of total RNA, poly (A)+ RNA enrichment and radiolabeled cDNA probe synthesis were carried out using an Atlas Pure Total RNA Labeling System (Clontech, Palo Alto, CA). The 33P-labeled cDNA probes were separated from unincorporated nucleotides and small cDNA fragments by using a spin column (Atlas Nucleo Spin Extraction Kit, Clontech). We used Clontech Atlas human 1.2-toxicology microarrays with 1176 genes per array. Clontech Atlas arrays are nylon-membrane-based cDNA arrays used for broadscale differential gene expression profiling. The human 1.2-toxicology array contains genes associated with xenobiotic metabolism, drug resistance, stress response, apoptosis, cell cycle, cell surface antigens, transcriptional activation, oncogenes, cytokines, signal transduction, cytoskeleton, energy metabolism, and DNA metabolism.
Purified 33P-labeled cDNA probes were hybridized with the array and the array was washed according to the manufacturer's instructions. Arrays were placed on an imaging plate (Fuji, Tokyo, Japan) for one week and visualized using a bioimaging analyzer (FLA-2000, Fuji). Image files were imported into AtlasImage 2.01 (CLONTECH) for quantification. Each data point was normalized to the total intensity of the array filter (the sum of signal values over the background for all genes on the filter) by using the "global normalization" feature in AtlasImage 2.01. An intensity ratio (treated/control) threshold value of 2 for up-regulation and 0.5 for down-regulation was used in an attempt to detect significant changes in expression.
Real-time RT-PCR. To confirm the results of the microarray analysis, the expression of some genes was analyzed by real-time RT-PCR. Total RNA was extracted as described previously. cDNA was synthesized from 100 ng of total RNA using RNA PCR Kit (AMV) Ver.2.1(Takara, Shiga, Japan) as the manufacturer instructed. Synthesized cDNA (2 µl) was amplified in a total volume of 25 µl containing 0.3 mM dNTP, 50 mM KCl, 3.4 mM MgCl2, 1 µl of SYBR Green (1000 x diluted, Cambrex, Rockland, ME), 1.25 U of TaKaRa Ex Taq R-PCR version (Takara), and 0.2 µM of each primer. Primer sequences are listed in Table 1. PCR was performed using a Smart Cycler (Cepheid, Sunnyvale, CA). After 30 s denaturation at 94°C, PCR was carried out for 40 cycles with denaturation at 94°C for 3 s, annealing and extension at 60°C for 30 s. All signals were normalized against actin beta mRNA as a control.
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Indirubin metabolism by CYP1A1. Four types of reaction mixtures were made. Reaction mixture 1 contained 15 pmol of recombinant CYP1A1 enzyme, 4 mM NADPH, and 3.3 mM MgCl2 in 0.1 M sodium-potassium phosphate buffer (pH 7.4). Reaction mixture 2 was the same except that it contained 15 pmol of control microsomes instead of CYP1A1 and had 100 nM indirubin. Reaction mixture 3 was equivalent to mixture 1 except that it had 100 nM indirubin and lacked NADPH. Reaction mixture 4 was the same as mixture 1 but it included 100 nM indirubin. Each reaction mixture (100 µl) was incubated at 37°C for 3 h. Then 1 µl of the mixture was subjected to a yeast assay. The yeast assay was performed essentially as described previously (Adachi et al., 2001; Miller, 1999
). In the yeast strain YCM3, human AhR and ARNT genes are integrated into chromosome III. AhR and ARNT are expressed from the galactose-regulated GAL 1, 10 promoter. Ligand-dependent activation of AhR leads to formation of the AhR/ARNT heterodimer. Expression of the lacZ reporter plasmid is directed by the AhR/ARNT complex binding to five xenobiotic response elements (XREs) in the promoter region. Thus, AhR ligand activity can be detected and quantified by measuring ß-galactosidase activity.
Measuring the dissociation constant of AhR ligands. The 7-ethoxyresorufin O-deethylation activity of the recombinant human CYP1A1 was determined by a continuous spectrofluorometric method as described previously (Chang et al., 2001), but with minor modifications. Briefly, the general reaction mixture, prepared in a spectrofluorimetric cuvette, contained 2 ml of sodium-potassium phosphate buffer (0.1 M, pH 7.4), 7-ethoxyresorufin (ranging from 0.1 µM to 1.0 µM), 50 µM NADPH, 2.5 pmol human recombinant CYP1A1 enzyme and the inhibitor at the concentration indicated in each figure. The reaction was run at 30°C. A baseline of fluorescence was recorded at an excitation wavelength of 510 nm and an emission wavelength of 586 nm. Calibration curves were constructed with a resorufin standard and a linear regression analysis was used to calculate the amount of resorufin formed in each incubation sample. The Ki values were determined according to a previously described procedure (Chang et al., 2001
).
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RESULTS |
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Figure 3A shows that 1 pM of indirubin significantly induced CYP1A1 mRNA expression (9.4-fold) after 8 h exposure. The expression was induced by indirubin, TCDD, and B[a]P in a dose-dependent manner (Fig. 3B). The indirubin dose-response curve was different from the TCDD dose-response curve in that it was not sigmoid in shape. The mRNA expression was relatively constant for the treatments with 1 pM to 1 nM of indirubin. These doses of indirubin were associated with 10002000 copies CYP1A1 mRNA/ng total RNA (1020 copies/cell). These levels were significantly higher than the control level (1 copy/cell) (p < 0.01). When cells were exposed to 100 nM of indirubin, we observed
7900 copies of CYP1A1 mRNA/ng RNA (
79 copies/cell). In contrast, TCDD did not induce CYP1A1 mRNA expression significantly at a concentration of 10 pM. But the expression level elevated sharply at a concentration over 100 pM. One nM of TCDD induced
32,000 copies of CYP1A1 mRNA/ng total RNA (
320 copies/cell). Another AhR ligand, B[a]P, induced CYP1A1 mRNA expression at a concentration over 100 nM.
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The time course of CYP1A1 mRNA induction by indirubin, TCDD, and B[a]P, is shown in Fig. 3D. CYP1A1 mRNA levels increased to a maximum at 8 h after exposure to indirubin and then declined, whereas CYP1A1 mRNA levels increased for up to 24 h after exposure to TCDD or B[a]P.
CYP2C, CYP2D6, CYP2E1, and CYP2A6 were confirmed to catalyze the production of indirubin from isatin in vitro (Gillam et al., 2000), so we examined the mRNA expression levels of these enzymes. But we did not observe a significant induction of CYP2C, CYP2D6, CYP2E1, or CYP2A6 mRNA by indirubin (data not shown).
Metabolism of Indirubin by CYP1A1
To determine the metabolism of indirubin by CYP1A1, 100 nM of indirubin was mixed with recombinant human CYP1A1 and NADPH at 37°C for 3 h, and subsequently the AhR ligand activity was measured by assaying the AhR-responsive reporter gene activity of the yeast strain YCM3 (Adachi et al., 2001). When indirubin was incubated with CYP1A1 and NADPH, the AhR ligand activity was dramatically decreased in the yeast assay in comparison with the control which did not contain CYP1A1 or NADPH (Fig. 4). These results indicate that indirubin is a substrate of the human CYP1A1 enzyme and that metabolism by CYP1A1 degrades indirubin's AhR binding activity.
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DISCUSSION |
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We observed that CYP1A1 mRNA levels transiently increased to 2550 copies/ng total RNA at 8 h and dropped to 100 copies/ng total RNA at 32 h after the change of medium without adding any AhR ligands in HepG2 cells. It was also reported that CYP1A1 mRNA expression was transiently induced by FBS in HepG2 cells (Guigal et al., 2001). We speculate that indirubin in FBS primarily contributes to the induction. This is because (1) indirubin accounted for half of the total AhR activity of FBS in our previous study (Adachi et al., 2001
), (2) both indirubin and FBS induce CYP1A1 mRNA expression transiently, and (3) the concentration of indirubin in FBS is sufficient (0.07 nM) to induce CYP1A1 mRNA expression.
The lowest observed effective concentration (LOEC) for CYP1A1 mRNA induction by indirubin and TCDD was 1 pM and 100 pM, respectively. The LOEC for CYP1A2 mRNA induction was the same as for CYP1A1 mRNA induction. As far as we know, no other AhR ligand has been reported to induce CYP1A mRNA expression at 1 pM in human cells. However, the maximum induction level of CYP1A1 differed between TCDD and indirubin, whereas that of CYP1A2 was the same for the two chemicals. This differential induction of CYP1A1 vs. CYP1A2 by TCDD compared to indirubin may due to differences in the location and number of XRE, transcription factors other than AhR, mRNA stability and AhR degradation by a ubiquitination-dependent mechanism. Moreover the dominant induction of CYP1A1 compared to CYP1A2 is important information with regard to the metabolism of AhR ligands. The data on the potency of indirubin and TCDD obtained from our study using human cells agrees with that obtained in the yeast AhR signalling assay (Adachi et al., 2001). We found that indirubin is able to induce CYP1A1 and CYP1A2 mRNA expression in human cells at the physiological concentration. Therefore, it seems reasonable to suppose that indirubin is an endogenous AhR ligand activating AhR-mediated signaling mechanisms in human cells.
As shown in the time-course experiment (Fig. 3D), 10 nM of indirubin induced CYP1A1 mRNA expression transiently, whereas 200 pM of TCDD induced expression throughout 24 h. It was reported that 1 nM of TCDD also induced CYP1A1 enzyme activity (as detected with the EROD assay) steadily over a 24 h period in HepG2 cells (Chen et al., 1995). In contrast to TCDD, another putative endogenous AhR ligand, lipoxin A4, was reported to induce CYP1A1 mRNA expression transiently (maximum level at 8 h) in a similar manner to indirubin (Schaldach et al., 1999
). A transient CYP1A1 mRNA induction is consistent with the finding data that CYP1A1 mRNA was rapidly degraded in HepG2 cells (t0.5 = 2.4 h) (Lekas et al., 2000
). Thus we hypothesized that the transient CYP1A1 mRNA induction was due to the metabolism of indirubin by the induced enzyme (CYP1A1). The indirubin metabolism experiment (Fig. 4) clearly supported this hypothesis. We measured the dissociation constant between indirubin and CYP1A1 to supply this metabolic response with a quantitative underpinning. The dissociation constant of indirubin and B[a]P represented by Ki was 2.9 and 7.4 nM, respectively. Ki values of other AhR ligands, lipoxin A4 and resveratrol, were reported to be 1100 and 1200 nM respectively under the similar experimental conditions (Chang et al., 2001
; Schaldach et al., 1999
). To our knowledge, the dissociation constant of indirubin-CYP1A1 is the lowest value reported among CYP1A1 substrates. TCDD did not inhibit the EROD activity in our experiment using human CYP1A1 and it was reported that TCDD competitively inhibited the EROD activity (Ki value, 200 nM) using rat hepatic microsomes (Petrulis and Bunce, 1999
). Our results do not directly show but strongly suggest, that indirubin will efficiently bind the substrate-binding site of CYP1A1 and be rapidly metabolized, whereas TCDD will not be metabolized by CYP1A1 effectively. In MCF-7 human breast cancer cells, indirubin induced CYP1A1 and 1B1 expression transiently and the potency of indirubin increased when cells were treated with the CYP inhibitor, ellipticine (Spink et al., 2003
). The Ki of a well known CYP1A1 substrate, B[a]P, was as low as that of indirubin. However, the long-term induction of CYP1A1 mRNA expression by B[a]P (Fig. 3D) was observed. One of its metabolites benzo[a]pyrene-7,8-dione (BPQ) may contribute to this induction, because it was found to be a potent and rapid inducer of CYP1A1 mRNA, with an EC50 value identical to that of the parent B[a]P in HepG2 cells (Burczynski and Penning, 2000
).
Inducing the expression of drug-metabolizing enzymes is a well known function of AhR. But AhR also plays roles in cell-cycle regulation (Cover et al., 1999; Kolluri et al., 1999
; Ma and Whitlock, 1996
; Rininger et al., 1997
; Weiss et al., 1996
; Yoon et al., 2002
), cell differentiation (Phillips et al., 1995
), the development of organs (Fernandez-Salguero et al., 1995
, 1996
; Schmidt et al., 1996
), and the modification of hormone signalling by cross-talk with hormone receptors (Ohtake et al., 2003
). Which is the primary role of AhR? One view is that the primary role of AhR is the latter and such physiological roles are triggered by a "true" AhR ligand like hormones or vitamins. CYP1A1 may have been developed to control the level of the ligand in cells. Another view is that the primary role of AhR is just to detoxify toxic AhR ligands. The majority of natural AhR ligands identified to date are dietary or related to dietary plant products (Denison et al., 2002
). Among these ligands, indirubin was reported to inhibit the activity of many kinases, such as GSK-3ß, CDKs, extracellular-signal-regulated kinase 2 (Erk2), casein kinase 1, and c-Src tyrosine kinase (Hoessel et al., 1999
). An indirubin derivative was also reported to inhibit CDK2 via direct interaction with the kinase's ATP binding site (Hoessel et al., 1999
). Therefore, it may be reasonable to conclude that if indirubin is not be metabolized, it may accumulate in some organs because of its hydrophobic nature and cause toxicity via direct inhibition of kinases. The other functions like cell-cycle arrest via AhR may have developed to transiently stop the cell cycle when the drug-metabolizing enzymes are running at full capacity.
Since, as far as we know, indirubin is the most potent AhR ligand, and shows strongest affinity for CYP1A1, it is reasonable to speculate that a natural AhR ligand like indirubin, rather than environmental AhR ligands such as dioxins and PAHs, promoted the evolution of the AhR-CYP1A1 system.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Technology and Ecology, Graduate School of Global Environmental Studies, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Fax: + 81-75-753-5171. E-mail: matsuda{at}eden.env.kyoto-u.ac.jp.
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