Development of a Novel Non Radioactive Aromatase Assay Using Primary Human Skin Cells

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-368
Hanieh Sadegh Pour Saleh*1, Nadja Zoeller1, Manuel Butting1, Roland Lauster2, Roland Kaufmann1, Stefan Kippenberger1 and August Bernd1
1University Hospital Frankfurt, Germany, 2Technical University of Berlin, Germany
Aromatase is a member of the P450 enzyme family. It is expressed in a variety of tissues and cell species e.g. in liver, skin and adipose tissue. The aromatase is located in the membrane of the smooth endoplasmatic reticulum and catalyzes two reactions in the process of estrogen biosynthesis from androgens. Specifically, the aromatase converts androstendione to estrone and testosterone to estradiol.

In our experimental setup primary human skin fibroblasts were incubated for 1 to 14 days with different concentrations of testosterone (0.1 - 20 µM) as substrate for the aromatase. The estradiol concentration in the cell free culture medium was measured as analytical parameter of aromatase activity using the Roche Elecsys 2010 system. It could be shown that the measured estradiol concentration increases in human skin cells in a time- and concentration-dependent manner.

To determine a suitable aromatase inhibitor, the skin fibroblasts were treated for 3 days with 20 µM testosterone and different concentrations (1 - 50 µM) of apigenin, 7-OH-flavone and chrysin, respectively. In addition to the estradiol measurement the cell proliferation was determined by BrdU incorporation and the cytotoxicity by LDH assay. Our results reveal that both apigenin and chrysin are toxic to human skin cells at higher concentrations whereas 7-OH-flavone inhibits aromatase without toxic effects.

To study the stimulating effect on the aromatase activity, the dermal cells were treated for 3 days with 20 µM testosterone and different concentrations of the glucocorticoid dexamethasone (0.001 - 1 µM). Our data demonstrate that the aromatase activity of skin fibroblasts is stimulated 70-fold by dexamethasone at concentrations above 0.05 µM.

From these results we conclude that the described non radioactive assay is suitable to monitor aromatase activity in primary human skin cells.

Nothing to Disclose: HS, NZ, MB, RL, RK, SK, AB

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