Abstract
To investigate possible species-specificity of aryl hydrocarbon receptor (AhR)-mediated signal transduction pathways, activities of 2,3,7,8-tetrochlorodibenzo-p-dioxin (TCDD) and six synthetic flavonoids were evaluated in mouse hepatoma and guinea pig adenocarcinoma cells transfected with an AhR-responsive luciferase reporter. Rank order potency in these two cell lines was similar for the ability of these flavonoids to antagonize TCDD-induced reporter gene expression. However, in the presence of flavone alone, a species-specific difference in agonist activity was observed. In guinea pig cells, several flavonoids demonstrated agonist activity up to 50% of the maximum TCDD response. In mouse cells, however, no significant agonist activity was observed at the same concentrations based on luciferase enzyme activity, protein expression, and mRNA analysis. Moreover, competitive ligand-binding assays, using [3H]TCDD in cytosolic fractions, demonstrated that 3′-methoxy-4′-nitroflavone had a similar IC50 in both recombinant cell lines, suggesting that the flavone has similar binding affinity to receptors from both species. However, electrophoretic mobility shift assay using the cytosolic fractions demonstrated that this flavone elicited binding to the DRE by guinea pig but not mouse AhR complex. The dependence of the AhR in this differential interaction was further demonstrated using in vitro synthesized guinea pig and mouse Ah receptors and mouse Arnt. Together, these data suggest that the differential agonist/antagonist activity of these flavone derivatives is caused by the efficacy of these flavonoids in eliciting an AhR conformation that recognizes regulatory response elements in a species-specific manner.
Footnotes
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↵1 Litron Laboratory Ltd., Rochester, New York.
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The research was funded in part by National Institute of Environmental Health Sciences grants ES09702 and ES01247 and a grant from the American Institute for Cancer Research.
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The data were presented in part at the 40th Annual Meeting of Society of Toxicology, March 2001, San Francisco, California (Toxicol Sci60:439).
- Abbreviations:
- AhR
- aryl hydrocarbon receptor
- Arnt
- Ah receptor nuclear translocation partner
- TCDD
- 2,3,7,8-tetrochlorodibenzo-p-dioxin
- DRE
- dioxin responsive elements
- 3′AAF
- 3′-acetamideflavone
- 3′5′MF
- 3′,5′-methoxyflavone
- 4′N7,8BF
- 4′-nitro-7,8-benzoflavone
- 3′M4′NF
- 3′-methoxy-4′-nitroflavone
- 3′DMAF
- 3′-dimethylaminoflavone
- ANF
- 7,8-benzoflavone
- DMSO
- dimethyl sulfoxide
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- EMSA
- electrophoretic mobility shift assay
- HEDG
- HEPES/EGTA/dithiothreitol/glycerol
- Received October 31, 2002.
- Accepted January 15, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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