Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor

Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor

Available online at www.sciencedirect.com ABB Archives of Biochemistry and Biophysics 470 (2008) 187–199 www.elsevier.com/locate/yabbi Antagonistic ...

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Available online at www.sciencedirect.com

ABB Archives of Biochemistry and Biophysics 470 (2008) 187–199 www.elsevier.com/locate/yabbi

Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor Shin Nishiumi a, Norio Yamamoto b, Rie Kodoi a, Itsuko Fukuda c, Ken-ichi Yoshida a, Hitoshi Ashida a,c,* a

Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan b Research Section, Research & Development Department, House Wellness Foods Co., 3-20 Imoji, Itami, Hyogo 664-0011, Japan c Research Center for Food Safety and Security, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan Received 12 September 2007, and in revised form 6 November 2007 Available online 18 December 2007

Abstract Halogenated and polycyclic aromatic hydrocarbons, exogenous ligands of the aryl hydrocarbon receptor (AhR), cause various toxicological effects through the transformation of the AhR. In this study, we investigated the antagonistic effects of indigoids on the transformation in addition to their agonistic ones. In a cell-free system, indigoids induced the transformation dose-dependently, but suppressed the transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the binding of 3-methylcholanthrene to the AhR. In mouse hepatoma Hepa-1c1c7 cells, indigoids, especially indirubin, suppressed the transformation and expression of CYP1A1 by inhibiting the translocation of AhR into the nucleus. When orally administered to mice at 10 mg/kg BW/day for three successive days, indigoids did not induce AhR transformation and expression of the CYP1A subfamily in the liver, while indirubin and indigo upregulated quinone reductase activity. These results indicate that indigoids are able to bind to the AhR as ligands and exhibit antagonistic effects at lower concentrations in mammalian cells.  2007 Elsevier Inc. All rights reserved. Keywords: Aryl hydrocarbon receptor; Indigoid; Indirubin; Indigo; TCDD; Transformation; CYP1A1; Quinone reductase

The aryl hydrocarbon receptor (AhR),1 a basic helix– loop–helix (bHLH) protein belonging to the Per-ArntSim (PAS) family, is a ligand-activated transcription factor that is present in most cell and tissue types. A previous study using AhR-deficient mice suggested to be involved

* Corresponding author. Present address: Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan. Fax: +81 78 803 5878. E-mail address: [email protected] (H. Ashida). 1 Abbreviations used: AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; ARE, antioxidant response element; bHLH, basic helix–loop–helix; CYP, cytochrome P450; DMSO, dimethylsulfoxide; DRE, dioxin response element; ED50, 50% effective dose; ELISA, enzymelinked immunosorbent assay; EMSA, electrophoretic mobility shift assay; EROD, ethoxyresorufin O-deethylase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GST, glutathione S-transferease; HAH, halo-

0003-9861/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.11.021

in the development of the liver and immune systems [1]. The AhR is an orphan receptor whose endogenous ligands and biochemical functions have not been fully elucidated at the moment, although it is known to be stable in the cytosol where it forms a complex with a dimer of the 90kDa heat shock protein (hsp90) [2], an AhR-associated protein (ARA9, also termed XAP2 or AIP) [3] and p23 [4]. It has been found that halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs) including dioxins genated aromatic hydrocarbon; HAP, hydroxyapatite; hsp90, 90-kDa heat shock protein; IC50, 50% inhibitory concentration; MC, 3-methylcholanthrene; NF-E2, nuclear factor-erythroid 2p45; Nrf2, NF-E2-related factor; PAH, polycyclic aromatic hydrocarbon; PAS, Per-Arnt-Sim; QR, quinone reductase; SW-ELISA, southwestern ELISA; TCDF, 2,3,7,8-tetrachlorodibenzofuran; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UGT, UDP-glucuronosyltransferase.

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bind to the AhR as exogenous ligands [5]. Among these exogenous ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener and causes various toxicological effects through an AhR-dependent pathway. The binding of a ligand to the AhR results in its transformation to a DNA-binding form via the following process: A conformational change in the receptor protein, its translocation into the nucleus, the dissociation of hsp90, and heterodimerization with another bHLH/PAS family protein, an AhR nuclear translocator (Arnt) [6]. This heterodimer binds to a dioxin response element (DRE), a cis-acting element found in the 5 0 -regulatory regions of dioxinresponsive genes, and regulates the expression of a battery of genes, particularly genes encoding drug-metabolizing enzymes, such as cytochrome P450 (CYP) 1A subfamily [7], quinone reductase (QR) [8], glutathione S-transferase (GST), [9] and UDP-glucuronosyltransferase (UGT) [10]. Regarding the ligands of the AhR, indole derivatives [11], bilirubin [12], 7-ketocholesterol [13], flavonoids [14] and resveratrol [15] have been reported to interact with the AhR and induce or inhibit the transformation. Certain natural compounds are considered candidates for endogenous ligands of the AhR. Indirubin and indigo were detected in human urine and/or fetal bovine serum (FBS), and had AhR ligand activity in a yeast-based AhR reporter system [16] and caused AhR transformation in mammalian cells [17,18]. Indigo is produced by the fermentation of plant materials from Polygonum tinctorium, Isatis tinctoria and Indigofera tinctoria, and has been used as a dye for cloth of jeans and other fabrics. Indirubin, a pink pigment, is a by-product of indigo synthesis. These compounds are ingredients of the traditional Chinese medicine ‘Dang gui Long hui wang’ used against chronic myelogenous leukemia [19]. It has been also reported that they are formed from indole through oxidation by human CYPs and/or dimerization in the human body [20]. Therefore, we assumed that indigoids do not have the toxicity of HAHs and PAHs, although they were better inducers of AhR transformation than TCDD in a yeast-based AhR reporter system [16]. In this study, we evaluated the antagonistic effects of indigoids on the transformation of the AhR and its downstream events, in addition to confirming their agonistic effects. In a cell-free system using rat liver cytosol, 1 nM indirubin and indigo significantly suppressed the binding of 0.25 nM [3H] 3-methylcholanthrene (MC) to the AhR and the 1 nM TCDD-induced transformation, nevertheless indirubin and indigo induced the receptor’s transformation dose-dependently consistent with previous reports [17,18]. In mouse hepatoma Hepa-1c1c7 cells, 100 nM indirubin and indigo suppressed the transformation and the expression of the CYP1A subfamily caused by 0.5 nM TCDD, although indigoids themselves prompted these events at a higher concentration range. Moreover, the oral administration of indigoids did not cause transformation of the AhR or expression of the CYP1A subfamily, but indirubin and

indigo significantly upregulated QR activity in the liver. These results suggest that indigoids do not show HAH and PAH-like actions, but rather suppress the AhR-dependent toxicological actions of these environmental contaminants. Materials and methods Materials Indirubin, indigo and isoindigo were synthesized according to a previous report [21] and TCDD was obtained from AccuStandard (New Haven, CT) and dissolved in dimethylsulfoxide (DMSO). Their structures are shown in Fig. 1. Curcumin was purchased from Sigma Chemical Co. (St. Louis, MO). Corn oil and MC were obtained from Nacalai Tesque Inc. (Kyoto, Japan). For the electrophoretic mobility shift assay (EMSA), an oligonucleotide DRE probe containing an AhR-binding site, 5 0 -GAT CTG GCT CTT CTC ACG CAA CTC CG-3 0 and 5 0 -GAT CCG GAG TTG CGT GAG AAG AGC CA-3 0 were synthesized at Hokkaido System Science (Sapporo, Japan). For the AhR ligand-binding assay, [3H]MC (1.9 Ci/mmol) and hydroxyapatite (HAP) were obtained from Moravek Biochemicals Inc. (Brea, CA) and Bio-Rad Laboratories Inc. (Hercules, CA), respectively. For the Western blot analysis, anti-CYP1A1 goat IgG and anti-AhR mouse IgG antibodies were purchased from Daiichi Pure Chemicals (Tokyo, Japan) and Affinity BioReagents (Golden, CO), respectively, and anti-goat IgG and anti-mouse IgG antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

A cell-free incubation system All animal treatments in this study were approved by the Institutional Animal Care and Use Committee and carried out according to the Kobe University Animal Experimentation Regulations. Male Sprague–Dawley rats (6 weeks old) were purchased from Japan SLC (Shizuoka, Japan). Rat hepatic cytosol was prepared according to our previous report [14]. The cytosol (4 mg protein/mL) was incubated with various concentrations of indirubin, indigo and isoindigo with or without TCDD in HEDG buffer consisting of 25 mM Hepes, pH 7.4, 1.5 mM EDTA, 1.0 mM dithiothreitol (DTT) and 10% (v/v) glycerol at 20 C for 2 h in the dark. The control treatment was carried out with the same volume of DMSO (10 lL/mL) alone as vehicle. After the incubation, the reaction mixture was subjected to EMSA or southwestern enzyme-linked immunosorbent assay (SWELISA) for determination of AhR transformation.

O

O

NH

N H

H N

N H

O

O Indigo

Indirubin

H N Cl

O

Cl

Cl

O

Cl

O O N H Isoindigo

TCDD

Fig. 1. Structure of indigoids and TCDD.

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Cell culture and treatment Mouse hepatoma Hepa-1c1c7 cells and human hepatoma HepG2 cells were maintained in modified Eagle’s medium and Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co., Tokyo, Japan), respectively, supplemented with 10% (v/v) FBS (Biowest, Miami, FL), 4 mM L-glutamine, 100 U/mL penicillin and 100 lg/mL streptomycin at 37 C in a humidified 5% CO2 atmosphere. These cells on 60 mm-dishes (approximately 80% confluent at a density of 2.0 · 106 cells/dish) were treated with various concentrations of indigoids and/or TCDD for the periods specified in each figure. The control cells were treated with the same volume of DMSO (1 lL/mL) alone as a vehicle control. After the treatment, the cells were harvested with lysis buffer consisting of 10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 and 0.5 mM DTT containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 5 lg/mL leupeptin and 5 lg/mL aprotinin) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4) and stood on ice for 15 min with occasional mixing. The mixture was centrifuged at 1000g for 10 min at 4 C, and the supernatant obtained was used as a post-nuclear fraction. The precipitate was suspended in extraction buffer consisting of 20 mM Hepes, pH 7.6, 20% (v/v) glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1.0 mM DTT and 0.1% (v/v) NP-40 containing the same protease and phosphatase inhibitors, and rotated for 1 h at 4 C. This suspension was centrifuged at 15,000g for 20 min at 4 C, and the supernatant obtained was used as a nuclear extract.

Animal experiments Male C57BL/6 mice (7 weeks old) were purchased from Japan SLC. The mice were housed in a temperature-controlled (23–25 C) room at 60 ± 5% humidity under a 12-h light–dark cycle and acclimatized for seven days with access to a commercial chow and distilled water. They were divided at random into 6 groups of 5 mice each, and 5 groups were orally given indigo, indirubin, isoindigo, curcumin and MC, respectively, at 10 mg/kg body weight/day for three successive days after food was withheld for 12 h. The other group was given 0.2 mL of corn oil alone as a vehicle control for three successive days. On day 4, these mice were killed 24 h after the final administration. The liver was taken and homogenized with five times the volume of hypotonic buffer consisting of 10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 and 0.5 mM DTT containing protease inhibitors (1 mM PMSF, 5 lg/mL leupeptin and 5 lg/mL aprotinin) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). The homogenate was centrifuged at 1000g for 10 min at 4 C. The precipitate obtained was washed twice with hypotonic buffer containing 0.5% (v/v) Triton X-100 and once with hypotonic buffer alone, and was suspended in hypertonic buffer consisting of 10 mM Hepes, pH 7.6, 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 0.5 mM DTT containing the same protease and phosphatase inhibitors. This suspension was rotated for 1 h at 4 C and centrifuged at 15,000g for 20 min at 4 C, and the supernatant obtained was used as a nuclear extract. The supernatant from the 1000g centrifugation was recentrifuged at 10,500g for 20 min at 4 C, and the supernatant was further centrifuged at 105,000g for 70 min at 4 C. The supernatant obtained was used as a cytosolic fraction, while the pellet was suspended in 0.05 M phosphate buffer of pH 7.4 containing 20% (v/v) glycerol and 1 mM PMSF and referred to as a microsomal fraction. Determination of AhR transformation was performed by EMSA and SW-ELISA using the nuclear extract. The measurement of ethoxyresorufin O-deethylase (EROD) activity [22] and detection of the CYP1A subfamily by Western blotting analysis were carried out using the microsomal fraction. The activity of QR [23] and GST [24] was measured using the cytosolic fraction according to previous reports.

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tein) from the incubation described above and 250 ng of poly[dIdC] (Roche Diagnostics, Mannheim, Germany) in a final volume of 12 lL of HEDG buffer containing KCl (final concentration, 150 mM). For in vivo experiments, the reaction mixture was composed of nuclear extract (2.5 lg protein) and 250 ng of poly[dIdC] in a final volume of 12 lL of extraction buffer. After each reaction mixture was incubated for 15 min at room temperature, a [c-32P]-labeled DRE probe (ca. 25 kcpm, 10 fmol) was added and the mixture was incubated for a further 15 min at room temperature. AhR transformation was determined as previously described [14]. In the case of SW-ELISA, for the cell-free system, the reaction mixture was made up of 10 lL of HEDG buffer containing 750 mM KCl (final concentration, 150 mM) and 40 lL of the cytosolic protein from the incubation described above. For the cultured cell system and in vivo experiments, the reaction mixture comprised 37.5 lL of HEDG buffer and 12.5 lL of nuclear extract (20 lg protein). Each mixture (50 lL) containing the transformed AhR was plated into a DRE probe-bound 96-well microtiter plate, and the transformation was determined according to a previous report [25].

AhR ligand-binding assay The specific binding of [3H]MC to the AhR was determined by an AhR ligand-binding assay using HAP as previously described [26]. Briefly, the rat hepatic cytosol (4 mg protein) was incubated with [3H]MC dissolved in 0.25 lL/mL ethanol and indigoids in 0.25 lL/mL 1,4-dioxine in a total volume of 2 mL of HEDG buffer for 2 h at 20 C. Non-specific binding was determined by incubating with [3H]MC in the presence of 2,3,7,8tetrachlorodibenzofuran (TCDF) (200-fold molar excess of [3H]MC). An aliquot of 0.5 mL of the reaction mixture was incubated with 0.3 mL of HEDG buffer containing 50% HAP for 30 min at 4 C with occasional mixing. After the proteins exhibiting non-specific binding to HAP were washed out by HEDG containing 0.5% (v/v) Tween-80, the radioactivity of [3H]MC bound to HAP was measured using a liquid scintillation counter with a scintillation cocktail.

Measurement of the activity of EROD and QR in cultured hepatocytes Hepa-1c1c7 cells were seeded on a 96-well plate at a final density of 1 · 104 cells/well in 100 lL of medium and pre-cultured for 24 h at 37 C. The cells were treated with various concentrations of indigoids and/or TCDD dissolved in DMSO for further a 24 h at 37 C. For all treatments, the final concentration of DMSO in the medium was 1 lL/mL, and the control cells were treated with DMSO alone. For measurement of the EROD activity, the medium was changed to a fresh one containing 1 lM 7-ethoxyresorufin as a substrate and 10 lM dicumarol. After the cells were incubated for 1 h at 37 C, an aliquot of 75 lL of the medium was withdrawn from each well and transferred to another 96-well plate, and the EROD activity was measured as previously described [27]. The remaining cell monolayers on the 96-well plate were used for the measurement of cell density by staining with crystal violet. For the measurement of QR activity, the medium was removed, and the cells were then lysed with 50 lL of lysis solution consisting of 0.8% (w/v) digitonin and 2.0 mM EDTA. After the cells were incubated for 20 min at 37 C, 200 lL of a reaction mixture consisting of 25 mM Tris–HCl, pH 7.4, 0.07% (w/v) BSA, 0.01% (v/v) Tween-20, 5 lM FAD, 1 mM glucose-6-phosphate, 30 lM NADP+, 200 U/mL glucose-6-phosphate dehydrogenase, 720 lM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide and 50 lM menadione in acetonitrile was added to each well, and the QR activity was measured as previously described [28].

Determination of AhR transformation by EMSA and SW-ELISA

Detection of the CYP1A subfamily and AhR protein by Western blotting

The transformation of the AhR was determined by EMSA and SWELISA according to our previous reports [14,25]. In the case of EMSA, for the cell-free system, the reaction mixture consisted of cytosol (10 lg pro-

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% for the CYP1A subfamily and 7.5% for AhR). After electrophoresis, the proteins were transferred onto a PVDF

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membrane (GE Healthcare Bio-Science Co., Piscataway, NJ). This was followed by the blocking of the non-specific binding sites with 5% (w/v) skim milk in Tris buffered saline containing 0.05% (v/v) Tween-20 (TBST) for the CYP1A subfamily or with a casein-based blocking solution (Nacalai Tesque) for AhR for 1 h at room temperature. The membrane was washed with TBST six times for 5 min each and incubated with anti-CYP1A1 (1:5000) or anti-AhR antibody (1:1000) for 1 h at room temperature. After another wash with TBST under the same conditions, the membrane was incubated with the appropriate secondary antibody (1:30000) conjugated with horseradish peroxidase for 1 h at room temperature. Specific immune complexes were detected with an ECL plus Western Blotting Detection System (GE Healthcare UK Ltd., Buckinghamshire, England).

Statistical analysis All data are expressed as means ± SE of at least three independent determinations for each experiment. Statistical significance was analyzed using Student’s t-test, and a level of probability of 0.05 was used as the criterion of significance.

Results Agonistic and antagonistic effects of indigoids on AhR transformation in the cell-free system Since indirubin and indigo have agonistic effects on AhR transformation [16–18], we confirmed their ability to induce the transformation in the cell-free system using the rat hepatic cytosol by EMSA. Indirubin, indigo, isoindigo and TCDD induced the transformation in a dosedependent manner (Fig. 2A and B). To compare their ability, the intensity of the AhR/DRE complex was plotted against a log of the concentrations, and each 50% effective dose (ED50) value was determined (Fig. 2B). The ED50 values of indirubin, indigo, isoindigo and TCDD were 45, 2.3, 300 and 0.55 nM, respectively (Table 1). Since indigoids themselves induced AhR transformation, we investigated whether they are able to bind to the AhR by measuring the inhibition of the binding of [3H]MC, a known agonist of the AhR, to the receptor protein. As a result, 1 nM indirubin and indigo inhibited the binding of 0.25 nM [3H]MC to 36% and 35%, respectively (Fig. 2C). Indirubin inhibited the specific binding of [3H]MC to the AhR in a dose-dependent manner with a 50% inhibitory concentration (IC50) of 0.5 nM (Fig. 2D). Moreover, 1 nM indirubin almost completely inhibited the specific binding of 0.025 nM [3H]MC to the AhR, and the inhibitory effect was observed regardless of the [3H]MC concentration (Fig. 2E). Then, the antagonistic effect of indigoids on AhR transformation in addition to the agonistic effect was examined in the cell-free system by SW-ELISA (Fig. 2F). With respect to the agonistic effect (open bar), the results from the SW-ELISA were similar to those from the EMSA (Fig. 2A and B). In the case of the antagonistic effect on the 1 nM TCDD-induced transformation (closed bar), 1 nM indirubin, indigo and isoindigo decreased the transformation to 48%, 74% and 69%, respectively. At the higher concentration (100 nM), the suppressive effect

was weakened and indigo exhibited a significant synergistic effect. Agonistic effects of indigoids on AhR transformation, EROD activity and QR activity in cultured hepatocytes We, next, investigated the effects of indigoids and TCDD on AhR transformation and its downstream events in mouse hepatoma Hepa-1c1c7 cells. As shown in Fig. 3A, all compounds tested induced the transformation dose-dependently, but the ED50 value of each indigoid was higher than that of TCDD (23.5, 13.5, 31.5 and 0.1 nM for indirubin, indigo, isoindigo and TCDD, respectively; Table 1). TCDD has been reported to induce the expression of phases 1 and 2 drug-metabolizing enzymes, such as the CYP1A subfamily [7], QR [8], GST [9] and UGT [10], through the AhR-dependent pathway. Therefore, EROD activity, namely the enzymatic activity of the CYP1A subfamily, and QR activity were measured using Hepa-1c1c7 cells (Fig. 3B and C). Indirubin, indigo, isoindigo and TCDD induced EROD activity dose-dependently (Fig. 3B) with ED50 values of 383 nM, 244 nM, 97 nM and 9.6 pM, respectively (Table 1). They also induced QR activity (Fig. 3C) with ED50 values of 212 nM, 320 nM, 318 nM and 71.4 pM, respectively (Table 1). In the case of both EROD and QR activities, the ED50 value of each indigoid was higher than that of TCDD, and indigoids triggered these activities at more than 10 nM (Fig. 3B and C). From the result for QR activity, it was confirmed that the maximum induction ratio of indirubin and isoindigo was much higher than that of TCDD (Fig. 3C), suggesting that these compounds stimulate QR through not only the AhR-mediated pathway but also another pathway. When the time-dependent effect of indigoids and TCDD on AhR transformation was investigated using Hepa1c1c7 cells (Fig. 3D), the transformation was found to reach a maximum at 1 h for indirubin and isoindigo, 30 min for indigo and 2 h for TCDD. Indirubin and isoindigo had transient effects and maintained some transformation but with no significant effect by 24 h. The induction by indigo was also transient and recovered to the control level at 8 h, while TCDD maintained significant transformation for 24 h. Expression of CYP1A subfamily proteins by indigoids and TCDD in cultured hepatocytes We examined the dose-dependent effect of indigoids and TCDD on the expression of CYP1A subfamily proteins in Hepa-1c1c7 cells and human hepatoma HepG2 cells. In Hepa-1c1c7 cells (Fig. 4A), indirubin, indigo and isoindigo induced the expression of CYP1A1 and 1A2 at 1, 10 and 50 lM, respectively, while TCDD did so at only 0.01 nM. In HepG2 cells (Fig. 4B), indirubin and isoindigo also induced their expression at 10 and 50 lM, respectively, and indigo had no effect up to

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50 lM. TCDD induced the expression of CYP1A1 and 1A2 at 1 nM. These results suggest that the ability of indigoids to induce the expression of the CYP1A subfamily is weaker than that of TCDD consistent with the effects on AhR transformation and the activity of

191

EROD and QR (Fig. 3A–C). Moreover, it was also indicated that the reactivity of indigoids and TCDD to the human AhR is lower than that to the mouse AhR by a comparison between the results from Hepa-1c1c7 cells and HepG2 cells (Fig. 4A and B).

A TCDD 0.005 0

0.02 0.01

0.1 0.05

0.5 0.2

Indirubin 100 fM 100 pM

2 1

10

5 nM

10

1

0

100 nM

1

100 μM 10

1

AhR/DRE Indigo

Isoindigo

B

C

8

120

[3 H]MC specific bound (% of 0.25 nM [3H]MC)

AhR transformation (ratio to control)

7 6 5 4 3 2 1 0 -3 10

100 80 60

*

*

40 20 0

1 10 3 Concentration (nM)

10 6

1 nM Indigoids



Indirubin Indigo Isoindigo

Fig. 2. Agonistic and antagonistic effects of indigoids on AhR transformation in the cell-free system. (A and B) Rat hepatic cytosol was treated with the indicated concentrations of TCDD or indigoids for 2 h at 20 C. Control treatment was carried out by addition of DMSO alone as a vehicle. AhR transformation was determined by EMSA (A), and the AhR/DRE complex was quantified by a digital imaging analyzer as described in Materials and methods (B). m, Indirubin; j, indigo; , isoindigo; and d, TCDD. Data are plotted as a ratio to the DMSO-treatment against a log of concentrations, and values are represented as the means ± SE (n = 6). (C) The cytosol was treated with 1 nM indigoids and 0.25 nM [3H]MC for 2 h at 20 C. Non-specific binding was detected by incubation with 0.25 nM [3H]MC plus 50 nM TCDF. The inhibitory effect of indigoids against the binding of [3H]MC to the AhR was determined by an AhR ligand-binding assay using HAP as described in Materials and methods. Data are shown as a percentage of the 0.25 nM [3H]MC-binding, and values are represented as the means ± SE (n = 3). Asterisks indicate a significant difference from the value for the 0.25 nM [3H]MCtreatment, p < 0.05 by Student’s t-test. (D) The cytosol was treated with indirubin at the indicated concentrations and 0.25 nM [3H]MC, and the inhibitory effect of indirubin was determined as Fig. 2C. (E) The cytosol was treated with 1 nM indirubin and the indicated concentrations of [3H]MC. Non-specific binding was detected by incubation with [3H]MC and TCDF at a 200-fold molar excess of the concentration of [3H]MC, and the inhibitory effect of indirubin was determined as described in Fig. 2C. Closed circles represent the results from the treatment with [3H]MC alone, and closed triangles, those from the simultaneous treatment with [3H]MC and 1 nM indirubin. Data are shown as means ± SE (n = 3). (F) After the cytosol was treated with 1 or 100 nM indigoids and/or 1 nM TCDD and AhR transformation was determined by SW-ELISA as described in Materials and methods. DMSO alone was added to the cytosol as a vehicle control under the same conditions. Data are shown as a percentage of the 1 nM TCDD-induced transformation, and values are represented as means ± SE (n = 3–6). Asterisks and daggers indicate a significant difference from the value of corresponding controls (1 nM TCDD- and DMSO-treatment, respectively), p < 0.05 by Student’s t-test.

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E

120

1000

100 [ 3H]MC specific bound (dpm)

[3 H]MC specific bound (% of 0.25 nM [3 H]MC)

D

80 60 40 20 0 10 -4

1 10 -2 Indirubin (nM)

F

800 600 400 200 0 10 -2

10 2

1 10 -1 [3 H]MC (nM)

10 1

160 †

AhR transformation (% of 1 nM TCDD)

140

*

120



100 *

80





*

*

*

60 40



20 0 Indirubin (nM) –

1 100











1 100













1 100









Indigo (nM) –





1 1100 –

Isoindigo (nM) –









1 100 –









1

100















+

+

+

+

+

+

1 nM TCDD

+

Fig. 2 (continued )

Antagonistic effects on AhR transformation, translocation of AhR into the nucleus, expression of CYP1A subfamily proteins and induction of EROD activity by TCDD in cultured hepatocytes We, further, investigated antagonistic effects on the TCDD-induced transformation of the AhR, translocation of the AhR into the nucleus, expression of CYP1A subfamily proteins and induction of EROD activity in Hepa-1c1c7 cells. Indirubin and indigo at 100 nM significantly suppressed the transformation induced by 0.5 nM TCDD, but isoindigo had no effect (Fig. 5A). Indirubin exhibited dosedependent suppression of the transformation and a significant effect at 1 nM (Fig. 5A). In addition, 100 nM indirubin inhibited the TCDD-induced translocation of the AhR from the cytosol into the nucleus (Fig. 5B). Regarding the downstream event of the transformation, 100 nM indirubin and indigo decreased the expression level of CYP1A1 protein (Fig. 5C) and significantly suppressed the EROD activity (Fig. 5D). These results indicate that indigoids have not only

Table 1 ED50 values of indigoids and TCDD for AhR transformation and the activity of EROD and QR in the cell-free and cultured cell systems Compounds

Indirubin Indigo Isoindigo TCDD

Cell-free system

Cultured cell system

AhR transformation

AhR transformation

EROD activity

QR activity

45 nM 2.3 nM 300 nM 0.55 nM

23.5 nM 13.5 nM 31.5 nM 0.1 nM

383 nM 244 nM 97 nM 9.6 pM

212 nM 320 nM 318 nM 71.4 pM

Each ED50 value was calculated from Fig. 2B, Fig. 3A–C.

agonistic effects but also antagonistic effects on AhR transformation and its downstream events. Comparison of effects of indigoids and MC on AhR transformation and induction of drug-metabolizing enzymes in the liver of mice Finally, we compared the effects of indigoids and MC on AhR transformation and the expression of drug-

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A

193

0.6

AhR transformation (arbitrary unit)

0.5 0.4 0.3 0.2 0.1 0

B

10 -3

10 -1 10 1 10 3 Concentration (nM)

C

30

5

20

QR activity (ratio to control)

EROD activity (ratio to control)

25

6

15 10

4 3 2

5 1

0 -5

0 10 -3

1 10 3 Concentration (nM)

D

10 -3

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0.6 †

AhR transformation (arbitrary unit)

0.5



0.4 0.3

† † † †

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0.1 0 0 0.5 1

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4 Treatment time (h)

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Fig. 3. Induction of AhR transformation and the activity of EROD and QR by indigoids and TCDD in cultured hepatocytes. Hepa-1c1c7 cells were treated with the indicated concentrations of indirubin (closed triangles), indigo (closed squares), isoindigo (closed diamonds) and TCDD (closed circles), or DMSO alone as a vehicle control. (A) After a 1-h-treatment, nuclear extract was prepared and subjected to SW-ELISA to determine AhR transformation. Data are shown as means ± SE (n = 3–6). After a 24-h-treatment, the activity of EROD (B) and QR (C) was measured as described in Materials and methods. Data are shown as a ratio to the activity following DMSO-treatment, and values are represented as the means ± SE (n = 6). (D) Hepa-1c1c7 cells were treated with 10 lM indigoids, 1 nM TCDD and DMSO (open triangles) for the indicated times, and nuclear extract was prepared and subjected to SW-ELISA to determine AhR transformation. Data are shown as means ± SE (n = 3–6). Daggers indicate a significant difference from the respective values for the DMSO-treatment, p < 0.05 by Student’s t-test.

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(A) Hepa-1c1c7 cells Indirubin (nM) 10

100

Indigo (nM)

1,000 10,000 50,000

10

100 1,000 10,000 50,000

CYP 1A1 CYP 1A2 Isoindigo (nM) 10

TCDD (nM)

100 1,000 10,000 50,000

0 0.001 0.01 0.1

1

5

CYP 1A1 CYP 1A2

(B) HepG2 cells

Indigo (nM)

Indirubin (nM) 10

100 1,000 10,000 50,000

10

100 1,000 10,000 50,000

CYP 1A1 CYP 1A2 TCDD (nM)

Isoindigo (nM) 10

100 1,000 10,000 50,000

0 0.001 0.01 0.1

1

5

CYP 1A1 CYP 1A2 Fig. 4. Expression of CYP1A subfamily proteins induced by indigoids and TCDD in cultured hepatocytes. Hepa-1c1c7 cells (A) and HepG2 cells (B) were treated with the indicated concentrations of indigoids and TCDD for 24 h, or DMSO alone as a vehicle control. A post-nuclear fraction was prepared and subjected to Western blotting to detect the CYP1A subfamily proteins. Data are typical results from three independent experiments.

metabolizing enzymes in the liver of C57BL/6 mice. In this experiment, curcumin, a yellow pigment derived from Curcuma longa, was also used as a compound reported to induce the transformation in cultured cell systems [29,30]. An oral administration of indigoids and curcumin altered neither body weight nor tissue weights of the liver, thymus and spleen, although MC tended to increase liver weight and to decrease thymus weight compared to corn oil (data not shown) in agreement with previous reports using TCDD [31,32]. When AhR transformation in the liver was determined by EMSA (Fig. 6A), indirubin showed slight DNA-binding ability, while MC had a prominent effect. The same tendency was evident in the results of the SW-ELISA (Fig. 6B). Both the expression level of the CYP1A subfamily (Fig. 6C) and EROD activity (Table 2) remained unchanged after the administration of indigoids and curcumin, while MC caused a 5-fold increase. As to the phase 2 drug-metabolizing enzymes (Table 2), indirubin and indigo significantly increased QR activity, but they did not upregulate GST activity. MC potently enhanced both activities, while curcumin had no effect on either enzyme. These results indicate that the oral intake of indigoids does not induce AhR transformation in the liver, although it leads to a slight increase in DNA-binding ability and the significant increase in QR activity.

Discussion Previously, Adachi et al. identified indirubin and indigo to be endogenous ligands of the AhR based on experiments with a yeast-based AhR reporter system [16]. These indigoids were also reported to induce the transformation of the AhR or its downstream events in a cultured mammalian cell system and in vivo [17,18,33]. However, these compounds are ingredients of the traditional Chinese medicine ‘Dang gui Long hui wang’ [19], strongly suggesting that they do not trigger similar biochemical responses to HAHs and PAHs at concentrations found in the body. In the present study, the oral administration of indigoids at 10 mg/kg body weight/day for three successive days did not induce AhR transformation and its downstream events, although indirubin caused a slight induction of the transformation without significance, while MC, a well-known AhR agonist, exhibited significant effects at the same dosage (Fig. 6 and Table 2). In a previous study, the intraperitoneal injection of indirubin, indigo and indirubin 3 0 -oxime into rats caused the expression of CYP1A1 protein at 1.5–50 mg/kg body weight [18]. In an experiment using mice, the CYP1A subfamily was significantly induced to express by gavage of indirubin and indigo at 50 mg/kg body weight/day for three successive days [17]. From these results, post-oral administered

S. Nishiumi et al. / Archives of Biochemistry and Biophysics 470 (2008) 187–199

indigoids may be difficult to incorporate per se into the body, and almost of them may be metabolized to the inactive form(s) in the alimentary canal. However, a portion of indigoids would be incorporated into the body without metabolic conversion and caused a slight increase in the DNA-binding activity by indirubin (Fig. 6A and B) and a significant increase in QR activity (Table 2). Regarding the difference in the expression of CYP1A subfamily between our study (Fig. 6C) and previous one [18], it may be due to the difference in the dosages.

A AhR transformation (% of 0.5 nM TCDD)

120

B

100 80

*

*

*

*

60 40 20

0 Indirubin (nM)



1

10

100





Indigo (nM)









100



Isoindigo (nM)











100

Nuclear fraction AhR Post-nuclear fraction AhR 100 nM Indirubin





+

+

0.5 nM TCDD



+



+

C CYP1A1 100 nM indigoids





0.5 nM TCDD



+

D

Indirubin Indigo Isoindigo +

+

+

EROD activity (% of 0.5 nM TCDD)

120 100 *

80

*

60 40 20

0 100 nM indigoids



Indirubin Indigo Isoindigo

195

In this study, we demonstrated that indigoids has a poor ability to transform the AhR in the mammalian system: the order of ability to induce AhR transformation and its downstream events was TCDD >> indigo, indirubin and isoindigo in both the cell-free and cultured cell systems (Fig. 2A, B and F, Fig. 3A–C and Fig. 4). Previously, similar results regarding DRE-driven luciferase activity and EROD activity in a cultured cell system were reported [17,18]. Our results and previous findings are inconsistent with the results reported by Adachi et al. [16]: They showed that indirubin and indigo had more potent agonistic effects than TCDD by using a yeast-based AhR reporter system. This inconsistency would be due to the difference in experimental systems, namely the difference between mammalian cells and yeast cells. The reason why indigoids are poor inducers is as follows: transformation of the AhR and the downstream events of it require not only the binding of AhR agonists to the receptor and the formation of the AhR/Arnt heterodimer but also the phosphorylation of the AhR and Arnt [34,35]. Our recent report [36] showed that curcumin, a yellow pigment of C. longa (turmeric), bound to the AhR as a ligand and caused the translocation of the AhR into the nucleus, but it caused neither AhR transformation nor the phosphorylation of the AhR and Arnt, while TCDD did. Moreover, curcumin suppressed the TCDD-induced AhR transformation by inhibiting their phosphorylation. In this study, we confirmed that curcumin did not induce AhR transformation and the expression of CYP1A subfamily in vivo (Fig. 6). Therefore, we assume that indigoids also might exhibit the inhibitory effects on the phosphorylation of these proteins in mammalian cells. The discrepancy between mammalian cells and yeast cells also gives support to the notion that the factor acting to stabilize the

b Fig. 5. Suppressive effects of indigoids on AhR transformation, translocation of AhR into the nucleus, expression of CYP1A subfamily proteins and induction of the EROD activity induced by TCDD in cultured hepatocytes. (A) Hepa-1c1c7 cells were treated with indigoids at the indicated concentrations and 0.5 nM TCDD for 2 h, or DMSO alone as a vehicle control. Nuclear extract was prepared and subjected to SW-ELISA to determine the suppressive effect on AhR transformation. Data are shown as a percentage of the 0.5 nM TCDD-induced transformation, and values are represented as the means ± SE (n = 3). Asterisks indicate a significant difference from the value for the 0.5 nM TCDD-treatment, p < 0.05 by Student’s t-test. (B–D) Hepa-1c1c7 cells were treated with 100 nM indirubin and 0.5 nM TCDD, or DMSO alone as a vehicle control. (B) After a 2-h-treatment, the post-nuclear fraction and nuclear extract were subjected to Western blotting analysis to determine the nuclear translocation of the AhR. Data are typical results from three independent experiments. (C) After a 24-h-treatment, the post-nuclear fraction was subjected to Western blotting to determine the expression of CYP1A subfamily proteins. Data are typical results from three independent experiments. (D) After a 24-h-treatment, EROD activity was measured as described in Materials and methods. Data are shown as a percentage of the 0.5 nM TCDD-induced EROD activity, and values are represented as means ± SE (n = 3–6). Asterisks indicate a significant difference from the value for the 0.5 nM TCDD-treatment, p < 0.05 by Student’s t-test.

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A

B

Lane 1 2 3 4 5 6

0.14

AhR transformation (arbitrary unit)

0.12 AhR/DRE

*

0.10 0.08 0.06 0.04 0.02 0

o il in in go n o ndig irub indi cum I r d o Is In Cu

r Co

C

M

C Lane 1 2 3 4 5 6 7 8 9 10 11 12

CYP1A subfamily expression level (arbitrary unit)

CYP1As

200

*

150 100 50 0 rn Co

oil

igo

Ind

in

ub

I

ir nd

o

dig

in Iso

n mi

rcu Cu

C

M

Fig. 6. Effects of indigoids and MC on AhR transformation and expression of CYP1A subfamily proteins in the liver of mice. Male C57BL/6 mice were orally given indigo, indirubin, isoindigo, curcumin and MC at 10 mg/kg body weight/day, or corn oil as a vehicle control for three successive days. These mice were killed 24 h after the final administration. Each nuclear extract of the liver was subjected to EMSA (A) and SW-ELISA (B) to determine AhR transformation. (A) A typical result is shown from one of five mice. Lane 1, corn oil; lane 2, indigo; lane 3, indirubin; lane 4, isoindigo; lane 5, curcumin; and lane 6, MC. (B) Data are shown in arbitrary units, and the values are represented as means ± SE (n = 5). Asterisks indicate a significant difference from the control group, p < 0.05 by Student’s t-test. (C) The microsomal fraction from the liver was subjected to Western blotting to detect CYP1A subfamily proteins. The upper panel shows typical results from two of five mice in each group. Lanes 1 and 2, corn oil; lanes 3 and 4, indigo; lanes 5 and 6, indirubin; lanes 7 and 8, isoindigo; lanes 9 and 10, curcumin; and lanes 11 and 12, MC. The lower panel shows the band density of CYP1A subfamily proteins from the results for all mice. Data are shown as means ± SE (n = 5), and asterisks indicate a significant difference from the control group, p < 0.05 by Student’s t-test.

AhR is absent in yeast, while it is present in mammalian cells. In yeast, the binding of the ligands to the AhR directly correlates with the ability to induce the transformation. By contrast, in mammalian cells, the transformation is not caused by only binding of the ligands to the AhR due to the factor that stabilizes the receptor protein. In a yeast-based AhR reporter system, a-naphthoflavone, an antagonist of the AhR in mammalian cells, exhibited an agonistic effect on the transformation and had similar inducing effect to b-naphthoflavone [37]. These results suggest that indigoids have greater ability to bind to the AhR than TCDD, but might not be able to affect the factor(s)

in mammalian cells resulting in the poor ability to transform the AhR. Our results indicate that indigoids temporarily induce the transformation of the AhR (Fig. 3D). The transient expression of CYP1A1 and 1B1 induced by indirubin was reported in MCF-7 human breast cancer cells [33]. Moreover, bilirubin [38], lipoxin A4 [39] and 6-formylindolo[3,2-b]carbazole [40], candidates for endogenous AhR ligands, exhibited similar transient actions. The effects of these compounds have been suggested to be due to metabolism resulting in the disappearance of the ability as the AhR ligands and the following AhR-dependent responses.

S. Nishiumi et al. / Archives of Biochemistry and Biophysics 470 (2008) 187–199 Table 2 The activity of EROD, QR and GST after the oral administration of indigoids, curcumin and MC Administration of

Corn oil (vehicle control) Indirubin Indigo Isoindigo Curcumin MC

Activity (nmol/min/mg protein) EROD

QR

GST

120 ± 12 133 ± 6 141 ± 10 109 ± 8 101 ± 2 640 ± 76*

392 ± 15 580 ± 39* 456 ± 19* 458 ± 40 420 ± 25 634 ± 25*

136 ± 5 149 ± 12 134 ± 10 164 ± 16 149 ± 10 199 ± 16*

Male C57BL/6 mice were orally given indirubin, indigo, isoindigo, curcumin and MC at 10 mg/kg body weight/day for three successive days. Control mice received the same volume (0.2 mL) of corn oil as a vehicle control for three successive days. These mice were killed 24 h after the final administration. The activity of EROD, QR and GST was measured as described in Materials and methods. The data are shown as means ± SE (n = 5), and asterisks indicate a significant difference from the vehicle control group, p < 0.05 by Student’s t-test.

It was reported that indirubin and indigo are present at an average concentration of 0.2 nM in the urine of normal donors and that indirubin is present at 0.07 nM in FBS [16]. Data in the present study showed that the IC50 value for the inhibitory effect of indirubin against the binding of [3H]MC to the AhR was 0.5 nM (Fig. 2D). In addition, 1 nM indirubin exhibited a significant antagonistic effect on AhR transformation in the cell-free and cultured cell systems (Fig. 2F and Fig. 5A). On the other hand, known natural AhR antagonists, such as flavonoids [14], curcumin [29,36] and resveratrol [15], have an effect at the micromolar level. Although indigoids are poor inducers of AhR transformation, they would be able to bind to the receptor without inducting its transformation at lower concentrations. Indeed, 0.5 nM indirubin did not induce the transformation, but could bind to the AhR (Fig. 2A–E). Therefore, indigoids, especially indirubin, are more potent AhR antagonists than natural ones reported, and may exhibit effects at concentrations found in the body. We observed differences in the reactivity to the AhR among species (Table 1 and Fig. 4), and these results coincided with the previous ones [41–43]. In this study, we observed that the inducible ability for the expression of CYP1A subfamily by indigoids and TCDD in mouse hepatoma Hepa-1c1c7 cells is 10- to 100-fold higher than that in human hepatoma HepG2 cells (Fig. 4). The differences are explainable by the previous report that the ligand-binding affinity to mouse AhR was stronger than that to human AhR [41]. Moreover, the 50% lethal dosage of TCDD in guinea pig (the most sensitive species) is 1000-fold lower than that in hamster (the most resistant species) and 100-fold than that in mouse and rat [44]. These species-specific differences in response to TCDD are due to the differences in the ligand-binding affinity to the AhR [41], the DRE-binding ability of the AhR [45] and the transactivation [46,47]. Regarding the antagonistic effect, 2,5,2 0 ,5 0 -tetrachlorobiphenyl was reported to

197

be an antagonist of the mouse and rat AhR, but it did not exhibit the antagonistic effect in cultured cells derived from guinea pig and human, although it was able to bind to guinea pig AhR [43]. Therefore, species-specific characteristics of the AhR may influence the agonistic and antagonistic effects of indigoids. Indirubin and indigo are active constituents of the traditional Chinese medicine ‘Dang gui Long hui wang,’ which is used for chronic myelogenous leukemia. It was reported that these compounds inhibited cyclin-dependent kinases [19]. Recent report [48] demonstrated that the TCDD-induced AhR transformation was involved in the development of lymphoma in vivo. Moreover, AhR transformation inhibited apoptosis in several lymphoma cells, and 3 0 -methoxy-4 0 -nitroflavone, a potent AhR antagonist, suppressed this inhibitory effect [48]. Therefore, not only the inhibitory effect of indigoids on cyclin-dependent kinases but also the antagonistic effect on the transformation might be responsible for an anti-leukemia activity of the traditional Chinese medicine, because the actions of indirubin to cyclin-dependent kinases are independent on the AhR-dependent actions [49]. Another remarkable finding of this study is the expression of phase 2 drug-metabolizing enzymes induced by indigoids (Fig. 3C and Tables 1 and 2). Phase 2 drugmetabolizing enzymes, such as QR, GST and UGT, play an important role in detoxification and reduction in the toxicological responses of carcinogens, while phase 1 drug-metabolizing enzymes, for example the CYP1A subfamily, sometimes produce highly reactive compounds [50]. The expression of certain phase 2 drug-metabolizing enzymes is regulated by not only AhR transformation but also activation of nuclear factor-erythroid 2p45 (NFE2)-related factor (Nrf2), a redox-sensitive transcription factor [51]. Following exposure of oxidative stress or electrophiles to the cells, cytosolic Nrf2 is released from its repressor protein, Kelch-like ECH-associated protein 1, and translocates into the nucleus [52]. In the nucleus, Nrf2 binds to an antioxidant response element (ARE), which locates in the promoter region of genes for phase 2 drug-metabolizing enzymes, as a transcription factor, followed by the expression of these enzymes [53]. In this study, the oral administration of indirubin and indigo elevated the level of QR activity without significantly inducing EROD activity (Table 2). In contrast, MC induced EROD, QR and GST activities (Table 2). The maximum induction by indirubin and isoindigo of QR activity was greater than that by TCDD in Hepa-1c1c7 cells (Fig. 3C). These results suggest that MC induces the expression of both QR and GST through the AhR/ DRE-dependent pathway, while indigoids upregulate QR activity through the Nrf2/ARE-dependent pathway. It is known that there is a possible interaction between the AhR/DRE and Nrf2/ARE signaling pathways [reviewed in [54]], and it was proposed that the expression of NAD(P)H:quinone oxidoreductase 1 is regulated by this interaction [55]. From our results, it is suggested that

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indirubin may share the AhR- and Nrf2-dependent biological actions, and the Nrf2-dependent action may be superior to the AhR-dependent one, resulting in the induction of QR without the significant transformation of the AhR. In conclusion, indigoids are poor inducers of the transformation of the AhR and do not trigger AhR-dependent biological responses similar to HAHs and PAHs, although indigoids are able to bind to the AhR as ligands. Indigoids exhibit antagonistic rather than agonistic effects at concentrations found in the body. Moreover, indigoids have an ability to induce phase 2 drug-metabolizing enzymes probably through the Nrf2/ARE-dependent pathway. Taken together, these results demonstrate that indigoids, especially indirubin, may be useful for the prevention of dioxin-associated biological changes. Acknowledgment This work was supported by grants from Research Fellow of the Japan Society for the Promotion of Science (S.N.). References [1] P. Fernandez-Salguero, T. Pineau, D.M. Hilbert, T. McPhail, S.S. Lee, S. Kimura, D.W. Nebert, S. Rudikoff, J.M. Ward, F.J. Gonzalez, Science 268 (1995) 722–726. [2] H.S. Chen, G.H. Perdew, J. Biol. Chem. 269 (1994) 27554–27558. [3] B.K. Meyer, M.G. Pray-Grant, J.P. Vanden Heuvel, G.H. Perdew, Mol. Cell Biol. 18 (1998) 978–988. [4] V.S. Premnath, Y.B. Bhagyashree, K.C. William, Biochem. Pharmacol. 65 (2003) 941–948. [5] M. Van den Berg, L. Birnbaum, A.T. Bosveld, B. Brunstrom, P. Cook, M. Feeley, J.P. Giesy, A. Hanberg, R. Hasegawa, S.W. Kennedy, T. Kubiak, J.C. Larsen, F.X. van Leeuwen, A.K. Liem, C. Nolt, R.E. Peterson, L. Poellinger, S. Safe, D. Schrenk, D. Tillitt, M. Tysklind, M. Younes, F. Waern, T. Zacharewski, Environ. Health Perspect. 106 (1998) 775–792. [6] C.J. Elferink, T.A. Gasiewicz, J.P. Whitlock Jr., J. Biol. Chem. 265 (1990) 20708–20712. [7] K. Kawajiri, Y. Fujii-Kuriyama, Arch. Biochem. Biophys. 464 (2007) 207–212. [8] D. Brauze, M. Widerak, J. Cwykiel, K. Szyfter, W. Baer-Dubowska, Toxicol. Lett. 167 (2006) 212–220. [9] T.H. Rushmore, R.G. King, E.K. Paulson, C.B. Pickett, Proc. Natl. Acad. Sci. USA 87 (1990) 3826–3832. [10] T. Iyanagi, M. Haniu, K. Sogawa, Y. Fujii-Kuriyama, S. Watanabe, J.E. Shively, K.F. Anan, J. Biol. Chem. 261 (1986) 15607–15614. [11] E.C. Henry, J.C. Bemis, O. Henry, A.S. Kende, T.A. Gasiewicz, Arch. Biochem. Biophys. 450 (2006) 67–77. [12] D. Phelan, G.M. Winter, W.J. Rogers, J.C. Lam, M.S. Denison, Arch. Biochem. Biophys. 357 (1998) 155–163. [13] J.F. Savouret, M. Antenos, M. Quesne, J. Xu, E. Milgrom, R.F. Casper, J. Biol. Chem. 276 (2001) 3054–3059. [14] H. Ashida, I. Fukuda, T. Yamashita, K. Kanazawa, FEBS lett. 476 (2000) 213–217. [15] R.F. Casper, M. Quesne, I.M. Rogers, T. Shirota, A. Jolivet, E. Milgrom, J. Savouret, Mol. Pharmacol. 56 (1999) 784–790. [16] J. Adachi, Y. Mori, S. Matsui, H. Takigami, J. Fujino, H. Kitagawa, C.A. Miller 3rd, T. Kato, K. Saeki, T. Matsuda, J. Biol. Chem. 276 (2001) 31475–31478.

[17] K. Sugihara, S. Kitamura, T. Yamada, T. Okayama, S. Ohta, K. Yamashita, M. Yasuda, Y. Fujii-Kuriyama, K. Saeki, S. Matsui, T. Matsuda, Biochem. Biophys. Res. Commun. 318 (2004) 571–578. [18] F. Peter Guengerich, M.V. Martin, W.A. McCormick, L.P. Nguyen, E. Glover, C.A. Bradfield, Arch. Biochem. Biophys. 423 (2004) 309– 316. [19] R. Hoessel, S. Leclerc, J.A. Endicott, M.E. Nobel, A. Lawrie, P. Tunnah, Nat. Cell Biol. 1 (1999) 60–67. [20] E.M. Gillam, L.M. Notley, H. Cai, J.J. De Voss, F. Peter Guengerich, Biochemistry 39 (2000) 13817–13824. [21] S. Leclerc, M. Garnier, R. Hoessel, D. Marko, J.A. Bibb, G.L. Snyder, P. Greengard, J. Biernat, Y.Z. Wu, E.M. Mandelkow, G. Eisenbrand, L. Meijer, J. Biol. Chem. 276 (2001) 251–260. [22] B. Shiotani, Y. Nonaka, K. Kanazawa, G. Danno, H. Ashida, Biosci. Biotechnol. Biochem. 66 (2002) 356–362. [23] C. Lind, E. Cadenas, P. Hochstein, L. Ernster, Meth. Enzymol. 186 (1990) 287–301. [24] W.H. Habig, M.J. Pabst, W.B. Jakoby, J. Biol. Chem. 249 (1974) 7130–7139. [25] I. Fukuda, S. Nishiumi, Y. Yabushita, R. Mukai, R. Kodoi, K. Hashizume, M. Mizuno, Y. Hatanaka, H. Ashida, J. Immunol. Methods 287 (2004) 187–201. [26] L. Poellinger, J. Lund, E. Dahlberg, J.A. Gustafsson, Anal. Biochem. 144 (1985) 371–384. [27] M.T. Donato, M.J. Gomezlechon, J.V. Castell, Anal. Biochem. 213 (1993) 29–33. [28] H.J. Prochaska, P. Talalay, Cancer Res. 48 (1988) 4776–4782. [29] H.P. Ciolino, P.J. Daschner, T.T.Y. Wang, G.C. Yeh, Biochem. Pharmacol. 56 (1998) 197–206. [30] A.L. Rinaldi, M.A. Morse, H.W. Fields, D.A. Rothas, P. Pei, K.A. Rodrigo, R.J. Renner, S.R. Mallery, Cancer Res. 62 (2002) 5451– 5456. [31] F. Decloitre, M. Martin, J. Chauveau, Chem. Biol. Interact. 10 (1975) 229–238. [32] H. Yoshimura, S. Yoshihara, N. Ozawa, M. Miki, Proc. Natl. Acad. Sci. USA 320 (1979) 179–192. [33] B.C. Spink, M.M. Hussain, B.H. Katz, L. Eisele, D.C. Spink, Biochem. Pharmacol. 66 (2003) 2313–2321. [34] I. Pongratz, P.E. Stromstedt, G.F. Mason, L. Poellinger, J. Biol. Chem. 266 (1991) 16813–16817. [35] A. Berghard, K. Gradin, I. Pongratz, M. Whitelaw, L. Poellinger, Mol. Cell Biol. 13 (1993) 677–689. [36] S. Nishiumi, K. Yoshida, H. Ashida, Arch. Biochem. Biophys. 466 (2007) 267–273. [37] L.A. Carver, V. Jackiw, C.A. Bradfield, J. Biol. Chem. 269 (1994) 30109–30112. [38] C.J. Sinal, J.R. Bend, Mol. Pharmacol. 52 (1997) 590–599. [39] C.M. Schaldach, J. Riby, L.F. Bjeldanes, Biochemistry 38 (1999) 7594–7600. [40] M. Mukai, S.A. Tischkau, Toxicol. Sci. 95 (2007) 172–181. [41] M. Ema, N. Ohe, M. Suzuki, J. Mimura, K. Sogawa, S. Ikawa, Y. Fujii-Kuriyama, J. Biol. Chem. 269 (1994) 27337–27343. [42] J.T. Sanderson, G.D. Bellward, Toxicol. Appl. Pharmacol. 132 (1995) 131–145. [43] P.M. Garrison, K. Tullis, J.M. Aarts, A. Brouwer, J.P. Giesy, M.S. Denison, Fundam. Appl. Toxicol. 30 (1996) 194–203. [44] L.S. Birnbaum, Environ. Health Perspect. 102 (1994) 157–167. [45] P.A. Bank, E.F. Yao, C.L. Phelps, P.A. Harper, M.S. Denison, Eur. J. Pharmacol. 228 (1992) 85–94. [46] C.C. Abnet, R.L. Tanguay, W. Heideman, R.E. Peterson, Toxicol. Appl. Pharmacol. 159 (1999) 41–51. [47] M. Korkalainen, J. Tuomisto, R. Pohjanvirta, Biochem. Biophys. Res. Commun. 273 (2000) 272–281. [48] C.F. Vogel, W. Li, E. Sciullo, J. Newman, B. Hammock, J.R. Reader, J. Tuscano, F. Matsumura, Am. J. Pathol. in press. [49] M. Knockaert, M. Blondel, S. Bach, M. Leost, C. Elbi, G.L. Hager, S.R. Nagy, D. Han, M. Denison, M. Ffrench, X.P. Ryan, P.

S. Nishiumi et al. / Archives of Biochemistry and Biophysics 470 (2008) 187–199 Magiatis, P. Polychronopoulos, P. Greengard, L. Skaltsounis, L. Meijer, Oncogene 23 (2004) 4400–4412. [50] P. Talalay, BioFactors 12 (2000) 5–11. [51] M.K. Kwak, K. Itoh, M. Yamamoto, T.R. Sutter, T.W. Kensler, Mol. Med. 7 (2001) 135–145. [52] N. Wakabayashi, A.T. Dinkova-Kostova, W.D. Holtzclaw, M.I. Kang, A. Kobayashi, M. Yamamoto, T.W. Kensler, P. Talalay, Proc. Natl. Acad. Sci. USA 101 (2004) 2040–2045.

199

[53] K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh, I. Hatayama, M. Yamamoto, Y. Nabeshima, Biochem. Biophys. Res. Commun. 236 (1997) 313–322. [54] C. Ko¨hle, K.W. Bock, Biochem. Pharmacol. 73 (2007) 1853–1862. [55] G. Ma, K. Kinneer, Y. Bi, J.Y. Chan, Y.W. Kan, Biochem. J. 377 (2004) 204–213.