Benzo[a]pyrene paradoxically inhibits transcription of a transiently transfected aryl hydrocarbon receptor (AhR)-responsive reporter gene in EA.hy926 cells

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 338-354-Physiological Impacts of Endocrine Disrupting Chemicals
Basic/Translational
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-350
Maiko Numakura*, Toshio Ishikawa, Satoshi Takahashi, Yamato Mashimo, Nakayuki Yoshimura, Yuko Fujimaki, Makoto Kinoshita and Tamio Teramoto
Teikyo Univ Schl of Med
Background: Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by xenobiotics such as dioxin. The agonist-activated AhR binds to dioxin response elements (DREs) and induces transcription of target genes. Benzo[a]pyrene (B[a]P), a toxic component of tobacco smoke, is well known as an activator of AhR and is reported to be possibly involved in smoking-induced atherosclerosis and carcinogenesis. In order to investigate the role of B[a]P in development of atherosclerosis, we tried to test its effects on a human endothelial cell line. In the preliminary experiment, we happened to find paradoxical effects of B[a]P on AhR-mediated transcription. Thus, we decided to probe it further. 

Methods: EA.hy926 cells (human endothelial cell line) and HepG2 cells (human hepatoma cell line) were maintained in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum and antibiotics. They were transiently transfected with an AhR-responsive firefly luciferase reporter gene carrying four tandem DREs (or the parental reporter gene with no DREs) and an internal control Renilla luciferase gene using the transfection reagent FuGENE HD, and treated with B[a]P or other AhR agonists (indole-3-carbinol (I3C) and 3-methyl-2-thiohydantoin (MTH)). Four hours later, normalized firefly luciferase activity (ratio of firefly/Renillaluciferase luminescence) was measured.

Results: In HepG2 cells, B[a]P, I3C and MTH all enhanced AhR-mediated transcription, as expected. In EA.hy926 cells, however, AhR-mediated transcription was elicited by I3C and MTH, but clearly repressed by B[a]P dose-dependently. Furthermore, the AhR-mediated transcription induced by I3C or MTH was inhibited by addition of B[a]P in EA.hy926 cells, but not in HepG2 cells. Even after co-transfection of an AhR-overexpressing plasmid, B[a]P still inhibited AhR-mediated transcription in EA.hy926 cells.

Discussion and Conclusions: B[a]P, a well-recognized AhR activator, exhibits an AhR antagonist activity in EA.hy926 cells transiently transfected with an AhR-responsive reporter gene. Our findings demonstrate that a compound regarded as an AhR activator may actually act as an AhR inactivator, at least in a certain experimental setting, and suggest a possibility that an AhR agonist might inhibit AhR activity in vivo, depending on cell types and conditions.

Nothing to Disclose: MN, TI, ST, YM, NY, YF, MK, TT

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm