Research article

Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen

Natàlia Garcia-Reyero^1,8 , Kevin J Kroll¹ , Li Liu² , Edward F Orlando³ , Karen H Watanabe⁴ , María S Sepúlveda⁵ , Daniel L Villeneuve⁶ , Edward J Perkins⁷ , Gerald T Ankley⁶ and Nancy D Denslow¹

¹Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA

²ICBR, University of Florida, Gainesville, FL 32611, USA

³Department of Animal & Avian Sciences, University of Maryland, College Park, MD 20742, USA

⁴Division of Environmental and Biomolecular Systems, Oregon Health & Science University, West Campus, Beaverton, OR, 97006, USA

⁵Department of Forestry & Natural Resources, Purdue University, Lafayette, IN, 47907, USA

⁶U.S. Environmental Protection Agency, ORD, NHEERL, MED, Duluth, MN, 55804, USA

⁷Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS, 39180, USA

⁸Current address: Department of Chemistry, Jackson State University, Jackson, MS 39217, USA

author email corresponding author email

BMC Genomics 2009, 10:308doi:10.1186/1471-2164-10-308

Published:	13 July 2009

Abstract

Background

Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17α-ethinylestradiol (EE₂)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE₂/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels.

Results

Steroidogenesis was down-regulated by EE₂ as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE₂/L or with the mixture of 5 ng EE₂/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE₂ were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern.

Conclusion

Our results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.