1. Influence of cAMP on reporter bioassays for dioxin and dioxin-like compounds.
- Author
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Kasai A, Yao J, Yamauchi K, Hiramatsu N, Hayakawa K, Meng Y, Maeda S, and Kitamura M
- Subjects
- Animals, Base Sequence, Cell Line, Tumor, Colforsin pharmacology, Consensus Sequence genetics, Cytochrome P-450 CYP1A1 genetics, Dioxins analysis, Dose-Response Relationship, Drug, Environmental Pollutants analysis, Enzyme Activators pharmacology, False Positive Reactions, Hepatocytes, Mammary Tumor Virus, Mouse drug effects, Mammary Tumor Virus, Mouse genetics, Mice, Molecular Sequence Data, Receptors, Aryl Hydrocarbon drug effects, Response Elements drug effects, Response Elements genetics, Terminal Repeat Sequences drug effects, Terminal Repeat Sequences genetics, Transfection, Biological Assay methods, Cyclic AMP pharmacology, Gene Expression Regulation drug effects, Genes, Reporter drug effects, Promoter Regions, Genetic drug effects
- Abstract
In reporter assays for detection of dioxins, the dioxin-responsive element (DRE) is generally used as a sensor sequence. In several systems, the CYP1A1 promoter containing DREs (DRE(cyp)) is inserted into a part of the long terminal repeat of mouse mammary tumor virus (LTR(MMTV)) to improve sensitivity of assays. We found that DRE(cyp)-LTR(MMTV) responds not only to dioxins and dioxin-like compounds but also to forskolin, a cAMP-elevating agent. This effect was dose-dependent and reproduced by other cAMP-elevating agents including 8-bromo-cAMP and 3-isobutyl-methylxanthine. The cAMP response element (CRE) and CRE-like sequences were absent in DRE(cyp)-LTR(MMTV) and not involved in this process. In contrast to the effect of dioxin, the activation of DRE(cyp)-LTR(MMTV) by cAMP was independent of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor for DRE. Furthermore, neither DRE(cyp), LTR(MMTV) nor the consensus sequence of DRE alone was activated in response to cAMP. These data elucidated for the first time that the combination of DRE(cyp) with LTR(MMTV) causes a peculiar response to cAMP and suggested that use of AhR antagonists is essential to exclude false-positive responses of DRE(cyp)-LTR(MMTV)-based bioassays for detection and quantification of dioxins and dioxin-like compounds.
- Published
- 2006
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