3-Methoxy-4-nitroflavone, a reported aryl hydrocarbon receptor antagonist, enhances Cyp1a1 transcription by a dioxin responsive element-dependent mechanism

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Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, regulating expression of a group of specific genes including cytochrome P450 1A1 (Cyp1a1). Stably transfected luciferase with dioxin responsive elements (DRE) in its promoter region has been commonly used as a reporter gene to study the mechanism of AhR signaling and compare potencies of TCDD and related compounds. However, how these two genes might respond to structurally diverse AhR ligands was unknown. This study investigates their expression in the same cells in response to TCDD, the most potent agonist, and 3M4NF, a reported potent antagonist. Our data suggest that these two compounds appear to play different roles in regulating these genes. While TCDD enhanced transcription of both genes, 3M4NF induced the endogenous Cyp1a1, but not the reporter gene. Mechanistic studies indicated that the increase in induction of CYP1A1 protein by 3M4NF was mediated by AhR-dependent transcriptional activation. Further analysis of the Cyp1a1 promoter sequence did not reveal any 3M4NF-specific responsive elements other than DREs. Rather, the interaction between the 3M4NF-bound receptor complex and DREs was confirmed by the observation that a single nucleotide mutation in DRE core sequences obliterated AhR enhancer activity in response to both TCDD and 3M4NF. Together these data suggest that 3M4NF, a weak AhR agonist, activates the AhR to recognize and interact with the same DREs as TCDD. However, depending on its concentration as well as the promoter context of a particular gene, the ability of 3M4NF to act as an AhR antagonist or agonist may appear different for various genes.

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Chemicals

Seven flavonoid compounds, 3,4-dimethoxyflavone (3,4MF), 3-acetamideflavone (3AAF), 3,5-dimethoxyflavone (3,5MF), 4-nitro-7,8-benzoflavone (4N7,8BF), 3-dimethylaminoflavone (3DMAF), 7,8-benzoflavone (ANF), and 3-methoxy-4-nitroflavone (3M4NF), were synthesized by the procedure of Cunningham et al. (1992) as previously described [21]. TCDD was purchased from Cambridge Isotopes (Cambridge, MA); benzo(a)pyrene (BaP), actinomycin D, and cycloheximide were from Sigma Chemicals (St.

Differential activity of 3M4NF on Cyp1a1 and p2DLuc genes

Initial experiments were designed to investigate if a stably transfected luciferase reporter gene has responsiveness that is similar to an endogenous gene following AhR activation by structurally diverse ligands. The effects of 3M4NF and TCDD on expression of CYP1A1 and reporter luciferase proteins were examined in Hepa.2DLuc.3A4 cells by Western blot analysis (Fig. 1A). As expected, TCDD produced a concentration-dependent increase in both the endogenous and reporter proteins. Unlike TCDD, 3

Discussion

This study investigated AhR-mediated endogenous Cyp1a1 and luciferase reporter gene expression within the same cell system in response to TCDD and 3M4NF. Taken together, data on mRNA, protein, and luciferase enzymatic activity indicated that TCDD and 3M4NF played different roles in regulating these genes. While TCDD enhanced expression of both reporter and endogenous genes, 3M4NF acted as an agonist for Cyp1a1, but an apparent antagonist for luciferase.

Immunoprecipitation and

Acknowledgements

We thank Dr. J.P. Whitlock, Jr. (Stanford University) for the kind gift of Cyp1a1 promoter deletion constructs D16, D17, D8, and individual DRE-driven CAT reporter constructs. We thank Dr. A.S. Kende (University of Rochester) for the synthesis of substituted flavones. We also thank the members of our laboratory for the critical review of the manuscript.

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    The research was funded in part by NIEHS Grant 09702, Center Grant ES01247, and a grant from the American Institute for Cancer Research. The data were presented in part at the 41st Annual Meeting of Society of Toxicology, March 2002, Nashville, Tennessee (Toxicol. Sci. 66 (1-S) 255).

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