3′-Methoxy-4′-nitroflavone, a reported aryl hydrocarbon receptor antagonist, enhances Cyp1a1 transcription by a dioxin responsive element-dependent mechanism☆
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Chemicals
Seven flavonoid compounds, 3′,4′-dimethoxyflavone (3′,4′MF), 3′-acetamideflavone (3′AAF), 3′,5′-dimethoxyflavone (3′,5′MF), 4′-nitro-7,8-benzoflavone (4′N7,8BF), 3′-dimethylaminoflavone (3′DMAF), 7,8-benzoflavone (ANF), and 3′-methoxy-4′-nitroflavone (3′M4′NF), 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 3′M4′NF 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 3′M4′NF 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 3′M4′NF. Taken together, data on mRNA, protein, and luciferase enzymatic activity indicated that TCDD and 3′M4′NF played different roles in regulating these genes. While TCDD enhanced expression of both reporter and endogenous genes, 3′M4′NF 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).