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ToxSci Advance Access published online on September 29, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh292
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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Received July 30, 2004
Accepted September 7, 2004

Endocrine Toxicology

A Novel Nonradioactive Method for Measuring Aromatase Activity Using a Human Ovarian Granulosa-Like Tumor Cell Line and an Estrone ELISA

Ken Ohno 1, Naohiro Araki 1, Toshihiko Yanase 2, Hajime Nawata 2, and Mitsuru Iida 1*

1 EcoScreen R & D section, Endocrine Disrupting Chemical Analysis Center, Otsuka Life Science Initiative, Otsuka Pharmaceutical Co., Ltd. 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima, 771-0195, Japan
2 Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medicine Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

* To whom correspondence should be addressed. E-mail: iidam{at}hq.otsuka.co.jp.


  Abstract

Aromatase is a key enzyme in steroidogenesis and plays an important role in sexual differentiation, fertility, and carcinogenesis. Importantly, a variety of chemicals in the environment may influence its activity and thereby disrupt endocrine function. In the current studies, we developed a novel nonradioactive method for measuring aromatase activity that uses a specific ELISA for estrone along with KGN human ovary granulose-like carcinoma cells. This cell line has relatively high aromatase activity, and, because it lacks 17{alpha}-hydroxylase, it secretes little or no androstenedione, 17{beta}-estradiol, or estrone. Therefore, aromatase activity can be assayed simply by measuring the production of estrone in the culture medium after addition of the substrate, androstenedione. Furthermore, by making a slight change in the commercial ELISA kit and optimizing the experimental conditions, we developed a sensitive aromatase assay that could measure a wide range of estrone concentrations with very low interference by androgens. We used this assay to investigate the effects of 23 chemicals that have been previously reported to affect aromatase activity in vitro. We confirmed that 17 of 23 test chemicals had inhibitory or inducible effects, although the specific effects of some were different than previously reported. In conclusion, we have developed a simple, sensitive, and nonradioactive assay that can be used for large-scale screening of compounds that can disrupt endocrine function by influencing aromatase activity.

Keywords: aromatase; endocrine disrupter; screening assay; KGN cell line; benomyl.
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