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ToxSci Advance Access published online on August 17, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfl079
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© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received May 25, 2006
Accepted August 8, 2006

Endocrine Toxicology

A Proteomic (SELDI-TOF-MS) Approach to Estrogen Agonist Screening

Calvin C. Walker 1 *, Kimberly A. Salinas 1, Peggy S. Harris 1, Sherry S. Wilkinson 1, James D. Watts 2, and Michael J. Hemmer 1

1 US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, Gulf Breeze, FL, USA
2 Senior Environmental Employee, Gulf Ecology Division, Gulf Breeze, FL, USA

* To whom correspondence should be addressed.
Calvin C. Walker, E-mail: walker.calvin{at}epa.gov


  Abstract

A small fish model and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria for seawater control, solvent control, and treatments of 17{beta}-estradiol (E2), methoxychlor, bisphenol-A, 4-tert-pentylphenol, endosulfan and chlorpyriphos. Fish plasma was applied to weak cation exchange (CM10) ProteinChip® arrays, processed and analyzed. The array produced approximately 42 peaks for E2 plasma and 30 peaks for solvent control plasma. Estrogen responsive mass spectral biomarker peaks were identified by comparison of E2 treated and control plasma spectra. Thirteen potential protein biomarkers with a range from 1-13 kDa were up- or down regulated in E2 treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist and non-agonist stressor treated males and normal female fish plasma. One of the biomarkers, m/z 3025.5, was identified by MS/MS as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens methoxychlor, bisphenol-A and 4-tert-pentylphenol elicited protein expression profiles consistent with the estrogen expression model. Estrogen responsive peaks were not detected in plasma from fish in the seawater, vehicle, endosulfan or chlorpyriphos treatments. No difference was found between plasma protein expression of seawater control and solvent control fish. We show that water exposure of fish to estrogen agonists produces distinct plasma protein biomarkers that can be reproducibly detected at low levels using protein chips and mass spectrometry.

Keywords: EDC; protein profiling; SELDI; fish; sheepshead minnow; estrogen.
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