Comparison of Gene Expression Patterns between 2,3,7,8-Tetrachlorodibenzo-p-dioxin and a Natural Arylhydrocarbon Receptor Ligand, Indirubin

Jun Adachi*, Yoshitomo Mori{dagger}, Saburo Matsui* and Tomonari Matsuda*,1

* Department of Technology and Ecology, Graduate School of Global Environmental Studies, Kyoto University, Kyoto, Japan, and {dagger} Japanese Ministry of the Environment, Tokyo, Japan

Received December 26, 2003; accepted March 10, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Indirubin is a natural arylhydrocarbon receptor (AhR) ligand isolated from human urine. We previously reported that it was more potent than the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a yeast assay system. Here we compared gene expression changes in HepG2 cells exposed to 10 nM of indirubin or TCDD using nylon-membrane-based cDNA arrays with 1176 genes to elucidate the toxic differences at the transcriptional level. The gene expression profiles for TCDD and indirubin were very similar. The number of up-regulated genes (fold change ≥2.0) was 11 and 4 and the number of down-regulated genes (fold change ≤0.5) was 17 and 21 in TCDD-treated and indirubin-treated cells, respectively. Cytochrome P450 (CYP) 1A1, 1A2, 19A1, insulin-like growth factor binding protein 1 (IGFBP1), and IGFBP10 were confirmed to be up-regulated using real-time reverse transcription polymerase chain reaction. CYP1A1 and CYP1A2 mRNAs were induced by as little as 1 pM of indirubin, whereas they were not induced by 10 pM of TCDD. In the time-course experiment, CYP1A1 mRNA was induced by indirubin transiently. Indirubin was also metabolized by CYP1A1 and lost its ligand activity. Indirubin would appear to be a good substrate of CYP1A1 given its low dissociation constant. Our results suggest that indirubin rapidly activates its own metabolism via AhR-mediated induction of CYP1A1 and this characteristic is consistent with the notion that indirubin is a physiological ligand of AhR.

Key Words: indirubin; AhR; TCDD; CYP1A1; metabolism; gene expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor that is present in most cells and tissue types of the body (Pohjanvirta and Tuomisto, 1994Go) and mediates most, if not all, toxic and biological effects of dioxins in various species and tissues (Fernandez-Salguero et al., 1995Go, 1996Go; Lucier et al., 1993Go; Mimura et al., 1997Go). The ligand-bound AhR forms a heterodimer with Ah receptor nuclear translocator (ARNT) and activates the transcription of the Ah gene battery which includes cytochrome P450 1A1 (CYP1A1), 1A2 (CYP1A2), glutathione S-transferase Ya subunit (GSTA1, Ya), NAD(P)H: quinone oxidoreductase (NQO1), UDP-glucuronosyltransferase 1A6 (UGT1A6), and aldehyde-3-dehydrogenase 3 (ALDH3A1) genes (Nebert et al., 2000Go). In addition, the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been reported to modulate the expression of genes for protein kinase C (PKC) family (Moore et al., 1993Go; Puga et al., 2000Go), estrogen receptor (Lu et al., 1994Go), several cytokines such as interleukin-1ß (IL-1ß; Gaido and Maness, 1994Go), IL-2 (Jeon and Esser, 2000Go), tumor growth factor-ß2 (TGF-ß2; Vogel and Abel, 1995Go) and tumor necrosis factor-{alpha} (TNF-{alpha}; Vogel and Abel, 1995Go), cyclooxygenase isoenzymes COX-1 and COX-2 (Puga et al., 1997Go; Wolfle et al., 2000Go), the immediately-early proto-oncogenes c-fos and c-jun (Puga et al., 1992Go), and tumor suppressor p27 (Kolluri et al., 1999Go).

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., 2001Go). 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., 1992Go). 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., 1999Go). It also inhibits inflammation in delayed-type hypersensitivity reactions (Kunikata et al., 2000Go). 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., 2000Go). 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., 2001Go). 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.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 1. Molecular structures of indirubin, TCDD, and B[a]P.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Analytical grade chemicals were purchased from Wako (Osaka, Japan). Microsomes from baculovirus-infected insect cells coexpressing NADPH-cytochrome P450 reductase and human CYP1A1 and control insect cell microsomes were purchased from BD Gentest (Wobum, MA). Indirubin was synthesized as described previously (Hoessel et al., 1999Go) and kindly provided by Dr. Saeki (Nagoya City University, Nagoya, Japan). The purity of indirubin was confirmed by HPLC.

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 (70–80% 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.


View this table:
[in this window]
[in a new window]
 
TABLE 1 RT-PCR Primers

 
Competitive RT-PCR. Total RNA was isolated as mentioned above. Quantitative competitive RT-PCR for CYP1A1, CYP1A2, CYP2C, CYP2D6, and CYP2E1 mRNA was carried out using a human cytochrome P450 competitive RT-PCR set and RNA PCR Kit (AMV) Ver. 2.1 (Takara) as instructed. With this method, target mRNA and appropriate copies of competitor RNA are mixed and reverse transcribed. Target cDNA and competitor cDNA share the same primer to be amplified and are separated by electrophoresis because the amplified genes differ in length. The ratio of target band to competitor band is equal to the ratio of target mRNA copies to competitor RNA copies, thus one can know the number of target mRNA copies from the known number of competitor RNA copies. The competitor RNA is designed to contain competitive primers' sequences to amplify all target genes (CYP1A1, 1A2, 2C, 2D6, 2E1, and GAPDH). The sequences of the primers and competitor RNA are not available for proprietary reasons. The PCR reaction mixtures were heated to 94°C for 2 min and immediately cycled through 30 s of denaturing at 94°C, 60 s of annealing at 60°C and 60 s of extension step 72°C. The reaction comprised 33 cycles for the CYP1A1 gene, 38 cycles for the CYP1A2 gene, 40 cycles for the CYP2A6, 2C, 2D6, and 2E1 genes, and 28 cycles for the GAPDH gene. After the amplification, PCR products (5 µl) were separated on a 2.5% agarose gel and visualized by ethidium bromide staining. Images were captured digitally using a Kodak Electrophoresis Documentation and Analysis System (EDAS) 290LE (Eastman Kodak, Rochester, NY) and the bands were quantified using NIH image program. All signals were normalized to GAPDH mRNA as a control.

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., 2001Go; Miller, 1999Go). 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., 2001Go), 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., 2001Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Indirubin and TCDD-Responsive Genes
Changes of gene expression were analyzed using Atlas human 1.2-toxicology microarray (CLONTECH) with 1176 genes per array in order to characterize the effect of indirubin and TCDD on human hepatocarcinoma HepG2 cells. Table 2 shows the differential gene expression profiles of TCDD-treated and indirubin-treated cells. The number of up-regulated genes (fold change ≥ 2.0) in TCDD-treated and indirubin-treated cells were 11 and 4, respectively. The CYP1A1 gene was prominently up-regulated, 446-fold in TCDD-treated cells and 88 fold in indirubin-treated cells. Other up-regulated genes were IGFBP10, GSTP1, CYP19A1, IGFBP1, NPC1, CYP1A2, SOX9, EGR1, IGF2, and CDKN1A. The number of down-regulated genes (fold change ≤0.5) in TCDD-treated and indirubin-treated cells was 17 and 21, respectively. We further analyzed the mRNA levels of CYP1A1, CYP1A2, CYP19A1, IGFBP1, and IGFBP10 by real-time RT-PCR to confirm the finding of the cDNA microarray analysis (Table 2). As shown in Figure 2, CYP19A1, IGFBP1, and IGFBP10 mRNA were induced by indirubin dose-dependently.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Differential Gene Expression Profiles of TCDD-Treated Cells and Indirubin-Treated Cells

 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 2. Effect of indirubin on CYP19A1, IGFBP1, and IGFBP10 mRNA levels in HepG2 cells. HepG2 cells (4 x 106) were seeded into 10-cm dishes, and 24 h later a stock solution of indirubin or DMSO (control) was added to the medium. After 8 h, total RNA was extracted and CYP19A1, IGFBP1, and IGFBP10 mRNA expression was quantified by real-time RT-PCR. Each dot represents the average obtained from two separate experiments.

 
Dose-Response and Time Course Changes of CYP1A mRNA Induced by Indirubin, TCDD, and B[a]P
We examined the expression of CYP1A1 and 1A2 mRNA induced by AhR ligands, indirubin, TCDD and benzo[a]pyrene (B[a]P) in HepG2 cells. Since fetal bovine serum is known to contain endogenous AhR ligands (Guigal et al., 2001Go), we measured the CYP1A1 mRNA level without adding any test chemicals. At 8 h after the change of medium, CYP1A1 mRNA level was 2550 copies/ng total RNA. But at 32 h, the level had dropped to 100 copies/ng total RNA. Therefore, we allowed a 24 h period between cell seeding and chemical exposure to lower the background level. This operation made it possible to observe CYP1A mRNA induction at very low concentrations.

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 ~1000–2000 copies CYP1A1 mRNA/ng total RNA (10–20 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.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 3. Effect of indirubin, TCDD, and B[a]P on CYP1A1 and CYP1A2 mRNA levels in HepG2 cells. (A) Induction of the CYP1A1 gene by 1 pM of indirubin. HepG2 cells were treated as described in Figure 2. CYP1A1 mRNA expression was quantified by competitive RT-PCR. (B) and (C) Dose-response curves of indirubin, TCDD and B[a]P for CYP1A1 (B) or CYP1A2 (C) mRNA induction. In Figure 3B, the lower panel shows part of the upper panel enlarged. Cells were treated with indirubin, TCDD, or B[a]P in the same conditions described in Figure 2. Error bars represent standard deviations obtained from three separate experiments. (D) Kinetics of CYP1A1 mRNA induction by indirubin, TCDD, and B[a]P. HepG2 cells were seeded into 10-cm dishes, and 24 h later a stock solution of indirubin, TCDD, or B[a]P in DMSO was added to the medium to yield a final concentration of 10 nM, 200 pM, or 5 µM, respectively. HepG2 cells were incubated from 0 to 24 h prior to total RNA isolation. Error bars represent standard deviations obtained from three separate experiments.

 
As shown in Figure 3C, indirubin induced CYP1A2 mRNA as well as CYP1A1 mRNA expression. The background level of CYP1A2 mRNA was 10–16 copies/ng total RNA (0.1–0.16 copies/cell). At 1 pM, indirubin significantly induced the expression (0.67 copies/cell) (p < 0.01), and the level increased up to 1 nM (1.6 copies/cell). TCDD and B[a]P also induced CYP1A2 mRNA expression in a dose-dependent manner, but they were less potent than indirubin.

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., 2000Go), 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., 2001Go). 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.



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 4. Indirubin metabolism by human recombinant CYP1A1. Four types of reaction mixtures with or without indirubin, CYP1A1, control microsome, and NADPH were made as indicated at the bottom of the figure. Each reaction mixture (100 µl) was incubated at 37°C for 3 h. After the incubation, 1 µl of the mixture was subjected to a yeast assay and the equivalent concentration of indirubin was calculated from the dose-response curve of indirubin. The results obtained from three separate experiments were expressed as a histogram with standard deviations. **Significantly different from control (p < 0.01).

 
Dissociation Constants of Indirubin, TCDD, and B[a]P for CYP1A1
Since we had not yet identified the indirubin metabolite, we could not determine the Km and Vmax values of this metabolic reaction. Alternatively, we measured the dissociation constants of indirubin, B[a]P, and TCDD for CYP1A1 to examine the interaction between the human CYP1A1 enzyme and these AhR ligands. To measure the dissociation constant, the degree of inhibition of 7-ethoxyresorufin O-deethylation (EROD) by each AhR ligand was determined. As shown in Figure 5, Lineweaver-Burk plots indicated that indirubin inhibited EROD activity by a mixed type inhibition as well as the typical CYP1A1 substrate B[a]P. The dissociation constant of indirubin and B[a]P represented by a Ki value was 2.9 and 7.4 nM, respectively. We also examined the inhibitory effect of TCDD, but no effect was observed at 775 nM of TCDD (data not shown).



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 5. Lineweaver-Burk plot for the inhibition of CYP1A1 enzyme activity by indirubin and B[a]P. The 7-ethoxyresorufin O-deethylation (EROD) activity of recombinant human CYP1A1 was inhibited by indirubin (A) and B[a]P (B). The Lineweaver-Burk plots suggest that indirubin and B[a]P are mixed-type inhibitors of EROD activity with a calculated Ki of 2.9 and 7.4 nM respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gene expression profile of TCDD in some cell lines and experimental animals has been reported (Frueh et al., 2000Go; Hurst et al., 2001Go; Kurachi et al., 2002Go; Martinez et al., 2002Go; Puga et al., 2000Go; Zeytun et al., 2002Go). In this study, we compared the gene expression profile of TCDD with that of the natural AhR ligand, indirubin. Genes whose expression was regulated by indirubin or TCDD were involved in xenobiotic metabolism (CYP1A1, CYP1A2, GSTP1), cytokine and cell signal transduction (IGFBP1, IGFBP10, IGF2, IL1R1, ILF2), steroid hormone and sex differentiation (TRIP15, CYP19A1, SOX9), cell cycle (CDKN1A, RBBP3, CDK1), cell adhesion (ITGB1, CDH2), transcriptional activation (EGR1, TCF12, BTF2p34), DNA replication and repair (TOP2A, TOP1, MSH2), and cholesterol and glucose transportation (NPC1, SLC2A2). The expression of CYP19A1, IGFBP1, and IGFBP10 mRNA was confirmed by real-time RT-PCR. There is a XRE-like sequence (5'-GCGTG-3', –3139 ~–3135) in the promoter region of CYP19A1. Moreover, another AhR ligand, diindolylmethane (DIM), was reported to induce CYP19A1 expression in human adrenocortical carcinoma cells (Sanderson et al., 2001Go). CYP19A1 is produced in hepatoma cells and responsible for the conversion of androgens to estrogens (Castagnetta et al., 2003Go). These results suggest that CYP19A1 is an AhR-regulated gene. Further study is needed to elucidate whether the induction of CYP19A1 contributes to the estrogenic effect of TCDD. The mRNA expression of IGFBP1, 10 is a novel response to AhR ligands. IGFBP1 has an XRE sequence (5'-TNGCGTG-3', –87 ~ –81) and an XRE-like sequence (–914 ~ –910) in the promoter region. IGFBP10 also has three XRE sequences (–3116 ~ –3110, –2360 ~ –2354, –2233 ~ –2227) and one XRE-like sequence (–392 ~ –388). IGFBP1 is the smallest IGFBP and controls IGF-I's access to cell surface receptors. Excess levels of IGFBP1 may contribute to growth failure in intrauterine growth restriction and in pediatric chronic renal failure (Lee et al., 1997Go). The binding affinity of IGFBP10 (CYR61/CCN1) for IGFs is two or three orders of magnitude less than that of IGFBP1 (Brigstock, 2003Go). IGFBP10 binds to cell surface integrins and induces intracellular signaling events that include kinase activation and gene transcription (Brigstock, 2003Go). IGFBP10 is estrogen-inducible and overexpressed in steroid-dependent breast tumors and promotes breast cancer. It also plays an essential role in placental angiogenesis during embryogenesis. These mRNA inductions in vitro require further study in animal models in vivo. AhR was implicated in cell cycle arrest at G1 (Ma and Whitlock, 1996Go; Weiss et al., 1996Go) through the down-regulation of cyclins and cyclin-dependent kinases, and/or by induction of cyclin-dependent kinase inhibitors such as p21 (CDKN1A) (Cover et al., 1999Go; Kolluri et al., 1999Go; Rininger et al., 1997Go; Yoon et al., 2002Go). In our microarray analysis, CDKN1A was up-regulated and RBBP3 (E2F1) and CDK1 (Cdc2) were down-regulated. RBBP3 is inactive when bound to retinoblastoma (RB) protein (Nevins, 2001Go). When RB is phosphorylated by CDK4, RBBP3 uncouples from RB and works as a transcription factor to move the cell cycle from G1 to S. CDK1 is a member of the Ser/Thr protein kinase family and a catalytic subunit of the highly conserved protein kinase complex known as M-phase promoting factor (MPF), which is essential for the G2/M transition in the eukaryotic cell cycle (Doree and Hunt, 2002Go). So, indirubin and TCDD-mediated activation of AhR may produce an arrest in G1 and G2 of the cell cycle via the transcriptional regulation of CDKN1A, RBBP3, and CDK1. Interestingly, indirubin also inhibits several CDKs and other kinases directly (Hoessel et al., 1999Go). Thus, indirubin may control the cell cycle via AhR-dependent and independent pathways. As shown in Table 2, the pattern of gene expression was almost the same between indirubin and TCDD. Technical developments that offer increased sensitivity when examining large numbers of genes for a given organism should make it possible to evaluate changes in RNA expression comprehensively and identify indirubin or TCDD-specific genes sometime soon.

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., 2001Go). 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., 2001Go), (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., 2001Go). 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., 1995Go). 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., 1999Go). 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., 2000Go). 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., 2001Go; Schaldach et al., 1999Go). 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, 1999Go). 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., 2003Go). 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, 2000Go).

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., 1999Go; Kolluri et al., 1999Go; Ma and Whitlock, 1996Go; Rininger et al., 1997Go; Weiss et al., 1996Go; Yoon et al., 2002Go), cell differentiation (Phillips et al., 1995Go), the development of organs (Fernandez-Salguero et al., 1995Go, 1996Go; Schmidt et al., 1996Go), and the modification of hormone signalling by cross-talk with hormone receptors (Ohtake et al., 2003Go). 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., 2002Go). 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., 1999Go). An indirubin derivative was also reported to inhibit CDK2 via direct interaction with the kinase's ATP binding site (Hoessel et al., 1999Go). 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.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. Charles A. Miller III (Environmental Health Sciences Department and Tulane-Xavier Center for Bioenvironmental Research, Tulane University, New Orleans, LA) for providing the yeast strain YCM3 and for helpful suggestions and Dr. Ken-ichi Saeki (Faculty of Pharmaceutical Sciences, Nagoya City University) for providing indirubin. This work was supported in part by the Japanese Ministry of the Environment, the Japanese Ministry of Health, Labour and Welfare and Grants-in-aid for Scientific Research 13027245 and 12055101 from the Japanese Ministry of Education, Science, Sports and Culture, and New England and Industrial Technology Development Organization (NEDO). J.A. was supported in part by a J.S.P.S. Research Fellowship for Young Scientists.


    NOTES
 
A portion of this article was previously published in the 22nd International Symposium on Halogenated Environmental Organic Pollutants and POPs (2002) 56, 13–15 and the 2nd Pacific Conference on Reproductive Biology and Environmental Sciences (2002) 35–37.

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adachi, J., Mori, Y., Matsui, S., Takigami, H., Fujino, J., Kitagawa, H., Miller, C. A. 3rd, Kato, T., Saeki, K., and Matsuda, T. (2001). Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. J. Biol. Chem. 276, 31475–31478.[Abstract/Free Full Text]

Brigstock, D. R. (2003). The CCN family: A new stimulus package. J. Endocrinol. 178, 169–175.[Abstract/Free Full Text]

Burczynski, M. E., and Penning, T. M. (2000). Genotoxic polycyclic aromatic hydrocarbon ortho-quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor. Cancer Res. 60, 908–915.[Abstract/Free Full Text]

Castagnetta, L. A., Agostara, B., Montalto, G., Polito, L., Campisi, I., Saetta, A., Itoh, T., Yu, B., Chen, S., and Carruba G. (2003). Local estrogen formation by nontumoral, cirrhotic, and malignant human liver tissues and cells. Cancer Res. 63, 5041–5045.[Abstract/Free Full Text]

Chang, T. K., Chen, J., and Lee, W. B. (2001). Differential inhibition and inactivation of human CYP1 enzymes by trans-resveratrol: Evidence for mechanism-based inactivation of CYP1A2. J. Pharmacol. Exp. Ther. 299, 874–882.[Abstract/Free Full Text]

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, 22548–22555.[Abstract/Free Full Text]

Cover, C. M., Hsieh, S. J., Cram, E. J., Hong, C., Riby, J. E., Bjeldanes, L. F., and Firestone, G. L. (1999). Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells. Cancer Res. 273, 1244–1251.

Denison, M. S., Pandini, A., Nagy, S. R., Baldwin, E. P., and Bonati, L. (2002). Ligand binding and activation of the Ah receptor. Chem. Biol. Interact. 141, 3–24.[CrossRef][ISI][Medline]

Doree, M., and Hunt, T. (2002). From Cdc2 to Cdk1: When did the cell cycle kinase join its cyclin partner? J. Cell Sci. 15, 2461–2464.

Fernandez-Salguero, P. M., Hilbert, D. M., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1996). Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol. Appl. Pharmacol. 140, 173–179.[CrossRef][ISI][Medline]

Fernandez-Salguero, P. M., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 5, 722–726.

Frueh, F. W., Hayashibara, K. C., Brown, P. O., and Whitlock Jr., J. P. (2000). Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression. Toxicol. Lett. 45, 189–203.

Gaido, K. W., and Maness, S. C. (1994). Regulation of gene expression and acceleration of differentiation in human keratinocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 127, 199–208.[CrossRef][ISI][Medline]

Gillam, E. M. J., Notley, L. M., Cai, H., De Voss, J. J., and Guengerich, F. P. (2000). Oxidation of indole by cytochrome P450 enzymes. Biochemistry 39, 13817–13824.[CrossRef][ISI][Medline]

Guigal, N., Seree, E., Nguyen, Q. B., Charvet, B., Desobry, A., and Barra, Y. (2001). Serum induces a transcriptional activation of CYP1A1 gene in HepG2 independently of the AhR pathway. Life Sci. 68, 2141–2150.[CrossRef][ISI][Medline]

Hoessel, R., Leclerc, S., Endicott, J. A., Nobel, M. E. M., Lawrie, A., Tunnah, P., Leost, M., Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W., Eisenbrand, G., and Meijer, L. (1999). Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat. Cell Biol. 1, 60–67.[CrossRef][ISI][Medline]

Hurst, C. H., Abbott, B., Schmid, J. E., and Birnbaum, L. S. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) disrupts early morphogenetic events that form the lower reproductive tract in female rat fetuses. Toxicol. Sci. 508, 87–98.

Jeon, M. S., and Esser, C. (2000). The murine IL-2 promoter contains distal regulatory elements responsive to the Ah receptor, a member of the evolutionarily conserved bHLH-PAS transcription factor family. J. Immunol. 165, 6975–6983.[Abstract/Free Full Text]

Kolluri, S. K., Weiss, C., Koff, A., and Gottlicher, M. (1999). p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev. 13, 1742–1753.[Abstract/Free Full Text]

Kunikata, T., Tatefuji, T., Aga, H., Iwaki, K., Ikeda, M., and Kurimoto, M. (2000). Indirubin inhibits inflammatory reactions in delayed-type hypersensitivity. Eur. J. Pharmacol. 410, 93–100.[CrossRef][ISI][Medline]

Kurachi, M., Hashimoto, S., Obata, A., Nagai, S., Nagahata, T., Inadera, H., Sone, H., Tohyama, C., Kaneko, S., Kobayashi, K., and Matsushima, K. (2002). Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive genes in mouse liver by serial analysis of gene expression. Biochem. Biophys. Res. Commun. 65, 368–377.

Lee, P. D., Giudice, L. C., Conover, C. A., and Powell, D. R. (1997) Insulin-like growth factor binding protein-1: Recent findings and new directions. Proc. Soc. Exp. Biol. Med. 216, 319–357.[Abstract]

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, 311–318.[CrossRef][ISI][Medline]

Lu, Y., Wang, X., and Safe, S. (1994). Interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoic acid in MCF-7 human breast cancer cells. Toxicol. Appl. Pharmacol. 127, 1–8.[CrossRef][ISI][Medline]

Lucier, G. W., Portier, C. J., and Gallo, M. A. (1993). Receptor mechanisms and dose-response models for the effects of dioxins. Environ. Health Perspect. 22, 36–44.

Ma, Q., and Whitlock Jr., J. P. (1996). The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Mol. Cell. Biol. 271, 2144–2150.

Martinez, J. M., Afshari, C. A., Bushel, P. R., Masuda, A., Takahashi, T., and Walker, N. J. (2002). Differential toxicogenomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in malignant and nonmalignant human airway epithelial cells. Toxicol. Sci. 141, 409–423.[CrossRef]

Miller, C. A. 3rd. (1999). A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures. Tox. Appl. Pharmacol. 160, 297–303.[CrossRef][ISI][Medline]

Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p–dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes to Cells 2, 645–654.[Abstract/Free Full Text]

Moore, M., Narasimhan, T. R., Steinberg, M. A., Wang, X., and Safe, S. (1993). Potentiation of CYP1A1 gene expression in MCF-7 human breast cancer cells cotreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin and 12-O-tetradecanoylphorbol-13-acetate. Arch. Biochem. Biophys. 305, 483–488.[CrossRef][ISI][Medline]

Nebert, D. W., Roe, A. L., Dieter, M. Z., Solis, W. A., Yang, Y., and Dalton, T. P. (2000). Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem. Pharmacol. 59, 65–85.[CrossRef][ISI][Medline]

Nevins, J. R. (2001). The Rb/E2F pathway and cancer. Hum. Mol. Genet. 10, 699–703.[Abstract/Free Full Text]

Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y., and Kato, S. (2003). Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 423, 545–550.[CrossRef][ISI][Medline]

Petrulis, J. R., and Bunce, N. J. (1999). Competitive inhibition by inducer as a confounding factor in the use of the ethoxyresorufin-O-deethylase (EROD) assay to estimate exposure to dioxin-like compounds. Toxicol. Lett. 105, 251–260.[CrossRef][ISI][Medline]

Phillips, M., Enan, E., Liu, P. C., and Matsumura, F. (1995). Inhibition of 3T3-L1 adipose differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Cell Sci. 108, 395–402.[Abstract/Free Full Text]

Pohjanvirta, R., and Tuomisto, J. (1994). Short-term toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals: Effects, mechanisms, and animal models. Pharmacol. Rev. 46, 483–549.[ISI][Medline]

Puga, A., Nebert, D. W., and Carrier, F. (1992). Dioxin induces expression of c-fos and c-jun proto-oncogenes and a large increase in transcription factor AP-1. DNA Cell Biol. 11, 269–281.[ISI][Medline]

Puga, A., Hoffer, A., Zhou, S., Bohm, J. M., Leikauf, G. D., and Shertzer, H. G. (1997). Sustained increase in intracellular free calcium and activation of cyclooxygenase-2 expression in mouse hepatoma cells treated with dioxin. Biochem. Pharmacol. 54, 1287–1296.[CrossRef][ISI][Medline]

Puga, A., Maier, A., and Medvedovic, M. (2000). The transcriptional signature of dioxin in human hepatoma HepG2 cells. Biochem. Pharmacol. 60, 1129–1142.[CrossRef][ISI][Medline]

Rannug, U., Bramstedt, H., and Nilsson, U. (1992). The presence of genotoxic and bioactive components in indigo dyed fabrics—a possible health risk? Mutat. Res. 282, 219–25.[CrossRef][ISI][Medline]

Rininger, J. A., Stoffregen, D. A., Babish, J. G. (1997). Murine hepatic p53, RB, and CDK inhibitory protein expression following acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Chemosphere 51, 1557–1568.[CrossRef]

Sanderson, J. T., Slobbe, L., Lansbergen, G. W., Safe, S., and van den Berg, M. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 61, 40–48.[Abstract/Free Full Text]

Schaldach, C. M., Riby, J., and Bjeldanes, L. F. (1999). Lipoxin A4: A new class of ligand for the Ah receptor. Biochemistry 38, 7594–7600.[CrossRef][ISI][Medline]

Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996). Characterization of a murine Ahr null allele: Involvement of the Ah receptor in hepatic growth and development. Proc. Natl. Acad. Sci. U.S.A. 93, 6731–6736.[Abstract/Free Full Text]

Spink, B. C., Hussain, M. M., Katz, B. H., Eisele, L., and Spink, D. C. (2003). Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin. Biochem. Pharmacol. 66, 2313–2321.[CrossRef][ISI][Medline]

Vogel, C., and Abel, J. (1995). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on growth factor expression in the human breast cancer cell line MCF-7. Arch. Toxicol. 69, 259–265.[CrossRef][ISI][Medline]

Weiss, C., Kolluri, S. K., Kiefer, F., and Gottlicher, M. (1996). Complementation of Ah receptor deficiency in hepatoma cells: Negative feedback regulation and cell cycle control by the Ah receptor. Exp. Cell. Res. 226, 154–163.[CrossRef][ISI][Medline]

Wolfle, D., Marotzki, S., Dartsch, D., Schafer, W., and Marquardt, H. (2000). Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Carcinogenesis 21, 15–21.[Abstract/Free Full Text]

Yoon, B. I., Hirabayashi, Y., Kawasaki, Y., Kodama, Y., Kaneko, T., Kanno, J., Kim, D. Y., Fujii-Kuriyama, Y., and Inoue, T. (2002). Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity. Toxicol. Sci. 70, 150–156.[Abstract/Free Full Text]

Zeytun, A., McKallip, R. J., Fisher, M., Camacho, I., Nagarkatti, M., and Nagarkatti, P. S. (2002). Analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced gene expression profile in vivo using pathway-specific cDNA arrays. Toxicology 23, 241–260.[CrossRef]