Comparison of estrogen-responsive plasma protein biomarkers and reproductive endpoints in sheepshead minnows exposed to 17β-trenbolone

Abstract

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/ z) in the plasma of estrogen agonist-treated male and gravid female sheepshead minnows ( Cyprinodon variegatus, SHM), which served as distinct estrogenic biomarkers. In this study, a 21-day reproductive assay with adult SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17β-trenbolone, on expression of these four estrogen-responsive peptides. In addition, the response of the peptide biomarkers were compared to traditional reproductive endpoints of fecundity, histopathology, secondary sex characteristics, length, weight, hepatosomatic index, female gonadosomatic index and plasma vitellogenin (VTG) levels. Fish were continuously exposed to 0.005, 0.05, and 5.0 μg/l, a solvent control (triethylene glycol, TEG), and a seawater control (SW) using an intermittent flow-through dosing system. Plasma was analyzed for the presence of the four peptide biomarkers by MALDI-TOF MS and VTG protein by quantitative ELISA. Male fish from the trenbolone treatments and controls showed no expression of the four peptide biomarkers or measurable levels of VTG. The estrogen-responsive biomarkers and plasma VTG were constitutively expressed in females from the SW, TEG, 0.005 and 0.05 μg/l exposures. All four peptide biomarkers were significantly reduced ( p < 0.0002 to p < 0.005) at the 5.0 μg/l treatment level which corresponded with significant reductions in fecundity and changes in ovarian morphology. A distinct but non-significant reduction in VTG was also observed in female fish from the 5.0 μg/l treatment. Results of this study suggest application of these estrogen-responsive protein biomarkers may be a cost effective alternative to fecundity measures which are labor intensive and expensive to conduct.