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EFFECTS OF TRENBOLONE ON EXPRESSION OF ESTROGEN-RESPONSIVE PLASMA PROTEINS IN ADULT SHEEPSHEAD MINNOW, (CYPRINODON VARIEGATUS)
Citation:
HEMMER, M. J., K. SALINAS, BECKY L. HEMMER, L. R. GOODMAN, C. C. WALKER, AND G. M. CRIPE. EFFECTS OF TRENBOLONE ON EXPRESSION OF ESTROGEN-RESPONSIVE PLASMA PROTEINS IN ADULT SHEEPSHEAD MINNOW, (CYPRINODON VARIEGATUS). Presented at 46th Annual meeting of the Society of Toxicology, Charlotte, NC, March 25 - 29, 2007.
Description:
Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies using sheepshead minnows (SHM), mass spectral analysis detected four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/z) in the plasma of estrogen-treated male fish and gravid females as opposed to unexposed males. The 3025.5 m/z peptide was positively identified as SHM zona radiata protein fragment 2, an egg membrane protein, further supporting the four peptides as suitable estrogen-responsive biomarkers. In this study, a 21-day aqueous exposure of adult male and female SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17ß-trenbolone on expression of the four estrogen-responsive peptides and on expression of plasma vitellogenin (VTG). Fish were continuously exposed to 0.005, 0.05 and 5.0 µg trenbolone/L, a vehicle control (triethylene glycol, TEG), and a seawater (SW) control using an intermittent flow-through dosing system. Plasma was analyzed for 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 measurable levels of VTG and absence of the four peptide biomarkers. The estrogen-responsive peptides were present in females from the SW, TEG, 0.005, and 0.05 µg/L exposures, but all four peptide biomarkers were significantly reduced in the highest treatment of 5.0 µg/L (p<0.0002 to p< 0.005). Mean plasma VTG ranged from 11.8 to 4.5 mg/ml in the SW control and 5.0 µg/L treatment, respectively. No significant difference in VTG was found between the trenbolone and control treatments (¿=0.05) which can be explained by VTG¿s high accumulation and slow clearance from the bloodstream. These results demonstrate trenbolone can inhibit expression of estrogen-responsive proteins and may be capable of causing adverse reproductive effects.Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies using sheepshead minnows (SHM), mass spectral analysis detected four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/z) in the plasma of estrogen-treated male fish and gravid females as opposed to unexposed males. The 3025.5 m/z peptide was positively identified as SHM zona radiata protein fragment 2, an egg membrane protein, further supporting the four peptides as suitable estrogen-responsive biomarkers. In this study, a 21-day aqueous exposure of adult male and female SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17ß-trenbolone on expression of the four estrogen-responsive peptides and on expression of plasma vitellogenin (VTG). Fish were continuously exposed to 0.005, 0.05 and 5.0 µg trenbolone/L, a vehicle control (triethylene glycol, TEG), and a seawater (SW) control using an intermittent flow-through dosing system. Plasma was analyzed for 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 measurable levels of VTG and absence of the four peptide biomarkers. The estrogen-responsive peptides were present in females from the SW, TEG, 0.005, and 0.05 µg/L exposures, but all four peptide biomarkers were significantly reduced in the highest treatment of 5.0 µg/L (p<0.0002 to p< 0.005). Mean plasma VTG ranged from 11.8 to 4.5 mg/ml in the SW control and 5.0 µg/L treatment, respectively. No significant difference in VTG was found between the trenbolone and control treatments (¿=0.05) which can be explained by VTG¿s high accumulation and slow clearance from the bloodstream. These results demonstrate trenbolone can inhibit expression of estrogen-responsive proteins and may be capable of causing adverse reproductive effects.