Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan
Received October 23, 2002; accepted December 9, 2002
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor repressor; aryl hydrocarbon receptor; aryl hydrocarbon receptor nuclear translocator; cytochrome P450; induction; TCDD.
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INTRODUCTION |
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The expression of AhRR in tissues has been determined in mice (Mimura et al., 1998) and Atlantic killifish (Karchner et al., 2002
). Recently, Fujita et al.(2002)
reported that AhRR mRNA is expressed in the lung, kidney, spleen, and thymus of the human fetus, but not in brain, liver, heart, and muscle. However, the constitutive expression of AhRR in human adult tissues and its inducibility is unknown. In the present study, the constitutive expression levels of AhRR mRNA in normal adult human tissues and in various human tissuederived cell lines were investigated. To investigate the inducibility of human AhRR gene, nine human tissuederived cell lines were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene (3-MC). In our previous study (Iwanari et al., 2002
), we found that nitro-substituted PAHs (NPAHs) can induce the human CYP 1 family in a chemical-specific manner. Therefore, the induction potency of AhRR by various PAH and NPAH compounds was also compared in HepG2 and OMC-3 cells.
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MATERIALS AND METHODS |
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RNA samples from normal human tissues.
Total RNA samples of human liver, breast, colon, kidney, bladder, uterus, and ovary were obtained from Stratagene (La Jolla, CA). Total RNA samples of human testis and adrenal gland were obtained from Clontech (Palo Alto, CA), and the total RNA sample of human lung was from Cell Applications Inc. (San Diego, CA). The liver sample was obtained at the normal margin to trabecular carcinoma from a single donor, a 45-year-old male. The breast sample was pooled tissues from two female donors, 39- and 49-years-old. The colon sample was pooled tissues from two female donors, 62- and 67-years-old. The kidney, lung, and ovary samples were each a single 56-year-old male, a 40-year-old male, and a 73-year-old female, respectively. The bladder sample was pooled tissues from two female donors, 24- and 42-years-old. The uterus sample was pooled tissues from three female donors, 54-, 68-, and 76-years-old. The testis sample was pooled tissues from 45 Caucasians, ages 1964. The adrenal gland sample was pooled tissues from 62 male/female Caucasians, ages 1561. Information concerning smoking or medication of the donors was not available.
Cell lines and cell culture.
The human cell lines HepG2 (hepatocellular carcinoma), A549 (lung carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma) were obtained from Riken Gene Bank (Tsukuba, Japan). MCF-7 (breast carcinoma), LS-180 (colon carcinoma), ACHN (renal carcinoma), and HT-1197 (bladder carcinoma) were from American Type Culture Collection (Rockville, MD). Cells were plated on 100-mm diameter dishes. HepG2, A549, and HeLa cells were cultured in Dulbeccos Modified Eagle Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) with 10% fetal bovine serum (FBS; Invitrogen, Melbourne, Australia). MCF-7, LS-180, ACHN, and HT-1197 cells were cultured in DMEM with 0.1 mM nonessential amino acid (Invitrogen) and 10% FBS (Invitrogen). NEC14 cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical) with 10% FBS (Invitrogen). OMC-3 cells were cultured in Hams F12 medium (Nissui Pharmaceutical) with 10% FBS (Bio Whittaker, Walkersville, MD). These cells were cultured in an atmosphere of 5% CO2/95% air at 37°C.
Treatment of cells and isolation of total RNA.
To determine the concentration-dependent induction of AhRR mRNA, HepG2 cells were treated with 0.1 pM10 nM TCDD for 24 h or 0.1 nM10 µM 3-MC for 6 h. To determine the time-dependent induction of AhRR mRNA, HepG2 cells were treated with 10 nM TCDD or 10 µM 3-MC for 0.5, 1, 2, 3, 6, 12, 24, 48, and 72 h. To determine the AhRR inducibility in various human tissuederived cell lines, cells were treated with 10 nM TCDD or 10 µM 3-MC for 24 h. To determine the chemical-dependent induction of AhRR, HepG2 and OMC-3 cells were treated with 1 µM of pyrenes (Py, 1-NP, and 1-AP), 1 µM of dinitropyrenes (1,3-, 1,6-, and 1,8-DNPs), 10 µM of fluoranthenes (Flu, 2-NF, and 3-NF), 10 µM of chrysenes (Chry, 6-NC, and 6-AC), 5 µM of benzo[a]pyrenes (B[a]P and 6-NB[a]P), 10 µM of benz[a]anthracenes (B[a]A and 7-NB[a]A), 10 nM of TCDD, or 10 µM of 3-MC for 24 h. The concentrations of various chemicals were determined in our previous study (Iwanari et al., 2002). It was confirmed that the concentrations of these chemicals did not affect the cell growth or viability. All chemicals were dissolved in DMSO and the final concentration of the solvent in the culture medium was 0.1%. Control cells were treated with 0.1% DMSO. Total RNA was isolated from the cells using ISOGEN according to the protocol supplied by the manufacturer. The RNA concentration and its purity were determined spectrometrically.
Reverse transcriptase-polymerase chain reaction (RT-PCR).
A 1-µl portion of the RT mixture was added to a PCR mixture containing 0.4 µM of each primer, 0.2 mM dNTPs, 1.0 µCi [-32P]dCTP, 1.0 U Taq DNA polymerase, 1.5 mM MgCl2, 67 mM TrisHCl buffer (pH 8.8), 16.6 mM (NH4) 2SO4, 0.45% Triton X-100, and 0.2 mg/ml gelatin in a final volume of 25 µl. PCR reactions were performed with a DNA Thermal Cycler (Takara). RT-PCR analysis of human AhRR was performed as follows: DNA was denatured at 94°C for 3 min and cycled immediately for 30 cycles (cell lines) and 28 cycles (human tissues): denaturing at 94°C for 45 s, annealing at 56°C for 45 s, and extension at 72°C for 1 min. The primers were AhRR-S: 5-AGACTCCAGGACCCACAA-3 and AhRR-AS: 5-CAGCGTCGGACCACACA-3. RT-PCR analyses of human CYP1A1, CYP1A2, CYP1B1, and ß-actin were also performed as we described previously (Iwanari et al., 2002
). A 20-µl portion of the PCR reaction mixture was electrophoresed on a 10% polyacrylamide gel that was subsequently dried. The data were analyzed with a Fujix Bio-Imaging Analyzer BAS 1000 (Fuji Film, Tokyo, Japan). To normalize RNA loading and PCR variations, the signals of targets were corrected with the signals of ß-actin mRNA as the internal standard.
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RESULTS |
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Concentration-Dependent Induction of AhRR in HepG2 Cells by TCDD and 3-MC
To determine the concentration-dependent induction of AhRR mRNA, HepG2 cells were treated with various concentrations of TCDD for 24 h, and 3-MC for 6 h. To normalize RNA loading and PCR variations, the expression levels were corrected with the expression level of ß-actin mRNA as the internal standard. We confirmed that the chemicals at the indicated concentrations did not significantly influence the ß-actin mRNA levels. As shown in Figure 2, AhRR mRNA was induced by TCDD and 3-MC in a concentration-dependent manner. Treatment with 0.1 nM of TCDD induced AhRR mRNA by three-fold, and treatment with 1 nM and 10 nM TCDD induced AhRR mRNA by seven- and eight-fold, respectively. Treatment with 1 µM of 3-MC resulted in the induction of AhRR mRNA by five-fold, and the maximum induction (nine-fold) was observed by 10 µM of 3-MC.
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DISCUSSION |
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The expression level of AhRR is high in testis but not in the testis embryonal carcinoma cell line NEC14. In addition, the expression levels of AhRR is high in uterus adenocarcinoma cell line HeLa but not in the uterus. Thus, there was no apparent relationship between human tissues and cell lines concerning the AhRR expression level. In order to determine the inducibility of human AhRR, various human tissue derived cell lines were treated with TCDD or 3-MC. AhRR in HepG2 cells was induced by TCDD or 3-MC in concentration- and time-dependent manners. These responses of AhRR were similar to those of the CYP1 family demonstrated in our previous study (Iwanari et al., 2002). These results support the notion that AhRR is induced via binding of the AhR/ARNT heterodimer to the XREs in the 5-flanking region of the AhRR gene. The differences in the induction levels of AhRR and CYP1A1 might be partly due to differences in the binding affinity of the AhR/ARNT heterodimer to XREs, since it has been reported that its binding affinity to XREs on the AhRR gene was lower than that to the XRE on the CYP1A1 gene (Baba et al., 2001
). The liganded AhR and ARNT heterodimer activates the expression of the AhRR gene, and the expressed AhRR, in turn, inhibits the function of AhR. Accordingly, it has been suggested that AhR and AhRR constitute a regulatory loop of xenobiotic signal transduction (Mimura et al., 1998
). However, these phenomena could not be demonstrated, since the inductions of AhRR were parallel with those of the CYP1 family. It has been reported that proteins corresponding to the AhRR/ARNT heterodimer in human fibroblasts bind to XRE (Gradin et al., 1999
, 2002
). It was considered that the expression of AhRR resulted in no inducibility of CYP1B1 mRNA in human fibroblasts. In contrast to human fibroblasts, the binding of the proteins corresponding to the AhRR/ARNT heterodimer to XRE in HepG2 cells was not observed (Gradin et al., 1999
). Therefore, it was suggested that the expression level of AhRR protein in HepG2 cells might not be high enough, although the AhRR mRNA was induced by TCDD or 3-MC.
In our previous study (Iwanari et al., 2002), we demonstrated that the CYP1 family is inducible in HepG2, MCF-7, LS-180, and OMC-3 cells, but not in ACHN, A549, HT-1197, HeLa, and NEC14 cells. These results were confirmed in the present study (Fig. 4
). The inducibility of AhRR in various cells was similar to those of the CYP1 family. We first demonstrated that in HeLa cells, the constitutive expression level of AhRR is remarkably high. In HeLa cells, the CYP1 family was also not induced. It has been reported that the high constitutive expression of AhRR would repress the induction of CYP1A1 in normal human skin fibroblasts (Gradin et al., 1999
). Therefore, it is suggested that the high expression level of AhRR might work as a negative factor in the low induction of the CYP1 family in HeLa cells. In other noninducible cell lines, ACHN, A549, HT-1197, and NEC14 cells, the expression levels of AhRR mRNA were not so high. Another downregulation mechanism might be involved in the noninducibility of the CYP1 family in these cells.
In our previous study (Iwanari et al., 2002), we also found that human CYP1A1, CYP1A2, and CYP1B1 were induced by NPAHs as well as their parent PAHs in chemical- and CYP isoform-specific manners. In the present study, the inducibility of AhRR by NPAHs and PAHs was compared with those of the CYP1 family in HepG2 and OMC-3 cells. The induction profile of AhRR by PAHs and NPAHs was similar to those of the CYP1 family (Iwanari et al., 2002
), although the extent of induction was different between the AhRR and CYP1 isoforms.
In conclusion, we demonstrated that AhRR is constitutively expressed in almost human tissues, and that AhRR mRNA was induced in a ligand concentration- and treatment time-dependent, and chemical- and cell-specific manner. Further characterization of the AhRR function in humans may contribute to understand the mechanisms responsible for differences in response to PAH exposure among tissues or cell types.
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ACKNOWLEDGMENTS |
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NOTES |
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