Response of Human CYP1-Luciferase Plasmids to 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Polycyclic Aromatic Hydrocarbons

Postlind, Hans; Vu, Tien P.; Tukey, Robert H.; Quattrochi, Linda C.

doi:10.1006/taap.1993.1031

Cited by 138 publications

(90 citation statements)

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“…Our choice of Hep3B cells was based upon the observation that this hepatoma cell line displayed both the AHR and HIF1␣ signal transduction pathways. The use of the HepG2 derived CYP1A1 reporter line, 101L cells, was a matter of convenience and was used sparingly, primarily as a confirmation of our data generated in Hep3B cells (13).…”

Section: Methodsmentioning

confidence: 63%

“…To characterize the AHR/ ARNT mediated response, a time course was performed in cells transiently transfected with the dioxin inducible reporter PL1A1N (13). In keeping with the expected pharmacology of this reporter, luciferase activity was markedly up-regulated after 5, 10, and 23 h of dioxin exposure (14-, 16-, and 27-fold, respectively), but was unaffected by exposure to CoCl 2 (Fig.…”

Section: Resultsmentioning

confidence: 76%

“…Oligonucleotides-The underlined nucleotides define the core sequences of the DRE or HRE: OL73: 5Ј-TCGAGTAGATCACGCAAT-GGGCCCAGC; OL74: 5Ј-TCGAGCTGGGCCCATTGCGTGATCTAC; OL116: 5Ј-GGCCACATCCGGGACATCACAGA; OL117: 5Ј-TGGGGAT-GGTGAAGGGGACGAA; OL135: 5Ј-GAAGATCTTCCAGTGGTCCCA-GCCTACACC; OL200: 5Ј-GAAGATCTTCTTAGTGATGGTGATGGTG-ATGGAAGTCTAGTTTGTGTTTGGTTC; OL497: 5Ј-CGCCCCACCAC-GCCTCAT; OL498: 5Ј-AGCCCCCATCCTGTCTTCAT; OL523: 5Ј-GCC-CTACGTGCTGTCTCA; OL524: 5Ј-TGAGACAGCACGTAGGGC; OL1204: 5Ј-GGAGATCTGGTACCGGTGGCCCAGGGACTCTGCG; OL1205: 5Ј-GGAGATCTGATGCCCCCCAGGGGAGGTG. Materials-The Cyp1a1 enhancer-luciferase reporter plasmid, PL1A1N, and the HepG2-101L cells that stably express this reporter plasmid were a gift from Dr. Robert Tukey (13). The Hep3B cells were obtained from American Type Culture Collection (Mannassas, VA).…”

Section: Methodsmentioning

confidence: 99%

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Cross-talk between the Aryl Hydrocarbon Receptor and Hypoxia Inducible Factor Signaling Pathways

Chan¹,

Yao²,

Gu³

et al. 1999

Journal of Biological Chemistry

187

134

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The aryl hydrocarbon receptor (AHR) and the ␣-class hypoxia inducible factors (HIF1␣, HIF2␣, and HIF3␣) are basic helix-loop-helix PAS (bHLH-PAS) proteins that heterodimerize with ARNT. In response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, the AHR⅐ARNT complex binds to "dioxin responsive enhancers" (DREs) and activates genes involved in the metabolism of xenobiotics, e.g. cytochrome P4501A1 (Cyp1a1). The HIF1␣⅐ARNT complex binds to "hypoxia responsive enhancers" and activates the transcription of genes that regulate adaptation to low oxygen, e.g. erythropoietin (Epo). We postulated that activation of one pathway would inhibit the other due to competition for ARNT or other limiting cellular factors. Using pathway specific reporters in transient transfection assays, we observed that DRE driven transcription was markedly inhibited by hypoxia and that hypoxia responsive enhancer driven transcription was inhibited by AHR agonists. When we attempted to support this cross-talk model using endogenous loci, we observed that activation of the hypoxia pathway inhibited Cyp1a1 up-regulation, but that activation of the AHR actually enhanced the induction of Epo by hypoxia. To explain this unexpected additivity, we examined the Epo gene and found that its promoter harbors DREs immediately upstream of its transcriptional start site. These experiments outline conditions where inhibitory and additive cross-talk occur between the hypoxia and dioxin signal transduction pathways and identify Epo as an AHR-regulated gene. The AHR1 regulates a variety of biological responses to environmentally ubiquitous polycyclic aromatic hydrocarbons and dioxins (1,2). In what can be defined as an adaptive pathway, the AHR up-regulates a battery of XMEs that often metabolize many of these agonists to more soluble and excretable products. A classic example of this pathway is observed upon exposure to benzo [a]pyrene. This chemical binds to the AHR leading to the up-regulation of a battery of genes including Cyp1a1, Cyp1a2, and Cyp1b1 (3). The enzymes encoded by these loci have metabolic activity toward benzo[a]pyrene and thus play an important role in its elimination (4). At present, we understand many of the molecular events in what appears to be an adaptive response to polycyclic aromatic hydrocarbon exposure. In brief, the up-regulation of genes like Cyp1a1 are regulated by an agonist-induced heterodimerization between two bHLH-PAS proteins, the AHR and ARNT (5, 6). This heterodimeric pair interacts with DREs upstream of the regulated promoters leading to an increase in their transcription rate and a resultant increase in XME activity (7).Although we have developed models to describe how the AHR regulates the expression of XMEs, we still have very little knowledge about how this protein mediates the toxicity of potent agonists like dioxin. The molecular mechanisms of dioxin-induced effects like lymphoid involution, epithelial hyperplasia, tumor promotion, teratogenesis, or even death remain unclear. Moreover, although genetic studies indicate the ...

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Section: Methodsmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

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Cross-talk between the Aryl Hydrocarbon Receptor and Hypoxia Inducible Factor Signaling Pathways

Chan¹,

Yao²,

Gu³

et al. 1999

Journal of Biological Chemistry

187

134

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“…Therefore, to precisely detect AhR activators for humans, establishment of a convenient method with a human cell line is necessary. However, there are few cell lines established for such purpose (16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

Establishment of a Human Hepatoma Cell Line HepG2-A10 for a Reporter Gene Assay of Arylhydrocarbon Receptor Activators

Sekimoto

Kawamagari

Nakatani

et al. 2007

Genes and Environment

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To establish a human hepatic cell line for a convenient reporter gene assay of arylhydrocarbon receptor (AhR) activators, the chimera plasmid containing xenobiotic responsible element (XRE), minimal SV40 promoter, and luciferase reporter gene and the expression vector pRC/CMV containing a neomycin-resistant gene were co-transfected into a human hepatoma cell line, HepG2. Then, antibiotic (G418)-resistant HepG2 cells were selected and cloned. A cell clone, HepG2-A10, showed the highest responsibility to 3-methylcholanthrene (MC)-mediated induction of luciferase among the clones obtained. Expression levels of luciferase activity in HepG2-A10 cells were increased in a dose-and time-dependent manner by treatment with either MC, an AhR-ligand type activator, or omeprazole (OME), a non-AhR ligand type activator. In addition, expression levels of cytochrome P4501A subfamily genes (CYP1A1 and CYP1A2) were also increased in a dose-and time-dependent manner by treatment with MC. The presentˆndings demonstrate that a newly established human HepG2-A10 is a useful cell line for a convenient reporter gene assay of AhR activators.

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“…To obtain a cell with high sensitivity to dioxins, a HepG2 cell having a luciferase vector with a xenobiotic-responsive element (XRE) (Denison et al, 1988;Postlind et al, 1993) in the promoter region was prepared. HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, 20 U penicillin/ ml and 20 g streptomycin/ml at 37˚C in a humidified atmosphere of 5% CO 2 in air.…”

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confidence: 99%

Evaluation of Intestinal Dioxin Permeability Using Human Caco-2 Cell Monolayers

Natsume¹,

Satsu²,

Hatsugai³

et al. 2003

FSTR

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A system to evaluate the intestinal permeability for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was constructed with human intestinal Caco-2 cell monolayers. TCDD was added to the apical side of the cell monolayers and the basal TCDD concentration was estimated by using a HepG2 cell having a luciferase vector with a xenobiotic-responsive element in the promoter region. Addition of such food substances as chlorophyll to the apical side of the Caco-2 cell monolayers markedly decreased the apical-to-basal transport of TCDD. This system would be useful for use in searching for the materials having an inhibitory activity to intestinal TCDD absorption.

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Response of Human CYP1-Luciferase Plasmids to 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Polycyclic Aromatic Hydrocarbons

Cited by 138 publications

References 0 publications

Cross-talk between the Aryl Hydrocarbon Receptor and Hypoxia Inducible Factor Signaling Pathways

Cross-talk between the Aryl Hydrocarbon Receptor and Hypoxia Inducible Factor Signaling Pathways

Establishment of a Human Hepatoma Cell Line HepG2-A10 for a Reporter Gene Assay of Arylhydrocarbon Receptor Activators

Evaluation of Intestinal Dioxin Permeability Using Human Caco-2 Cell Monolayers

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