Application of protein expression profiling to screen chemicals for androgenic activity.

Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) coupled with a short term fish assay was used to investigate changes in plasma protein expression as a means to screen chemicals for androgenic activity. Adult gravid female sheepshead minnows (Cyprinodon variegatus) were placed into separate aquaria for seawater control, ethanol solvent control, and the following androgen agonist treatments at 5.0µg/L: dihydrotestosterone (DHT), methyldihydrotestosterone (MDHT), testosterone (T), methyltestosterone (MT) and trenbolone (TB). Treatments of 0.6µg/L endosulfan and 40µg/L chlorpyrifos (CP) served as non-androgenic negative stressor controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus supplying exposure water at 20L/h. Fish were sampled at 7 days, the plasma diluted, processed on weak cation exchange CM10 ProteinChip arrays and analyzed. Spectral processing resulted in 249 individual m/z peak clusters for the androgen exposed fish. Partial least squares-discriminant analysis was used to develop an androgen-responsive model using sample spectra from exposures with DHT and unexposed solvent control fish as the training set. The androgen classification model performed with ≥79% specificity (% true negative) and ≥70% sensitivity (% true positive) for non-aromatizable androgens. The aromatizable androgens T and MT were classified as androgenic with specificities of 42 and 79%, respectively. The reduction in sensitivity observed with T is thought to be caused by its metabolic conversion to an estrogen by aromatase. The results of these studies show diagnostic plasma protein expression models can correctly classify chemicals by their androgenic activity using a combination of high throughput mass spectrometry and multivariate approaches.

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

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, 3025.5m/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, 17beta-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 microg/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 microg/l exposures. All four peptide biomarkers were significantly reduced (p<0.0002 to p<0.005) at the 5.0 microg/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 microg/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.

A simple and rapid matrix-assisted laser desorption/ionization time of flight mass spectrometry method to screen fish plasma samples for estrogen-responsive biomarkers.

In the present study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry coupled with a short-term fish assay. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria consisting of vehicle control and the following estrogen agonist treatments: 17beta-estradiol (0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.5, and 1.0 microg/L, 4-tert-pentylphenol (100 microg/L), methoxychlor (6 and 12 microg/L), and bisphenol A (100 and 1,000 microg/L). Treatments with chlorpyrifos (80 microg/L) and endosulfan (0.6 microg/L) served as nonestrogenic negative controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus. Plasma was obtained from individuals, diluted and applied to an inert surface, and analyzed by MALDI. Multiple protein peaks, ranging from 2.9 to 12.9 kDa, were identified as markers of estrogenic effects when comparing estrogen-treated and control fish using interpercentile reference values. A binary classification tree model was constructed from plasma protein profiles of the vehicle control and the 0.2 microg/L of 17beta-estradiol treatments and then used to evaluate all samples. Treatments with the estrogen agonists 17beta-estradiol, 4-tert-pentylphenol, methoxychlor, and bisphenol-A generated reproducible diagnostic biomarkers based on the presence of specific estrogen-responsive plasma proteins. The controls and nonestrogenic compounds chlorpyrifos and endosulfan did not produce this estrogen-responsive protein profile. A no-observed-effect level for 17beta-estradiol at 0.025 microg/L was estimated from concentration-response exposures. The MALDI method described here provides a straightforward, sensitive, and specific tool to screen chemicals for estrogenic activity.

A proteomic (SELDI-TOF-MS) approach to estrogen agonist screening.

A small fish model and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry 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 17beta-estradiol (E2), methoxychlor (MXC), bisphenol-A (BPA), 4-tert-pentylphenol (TPP), endosulfan (ES), and chlorpyriphos (CP). 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 to 13 kDa were up- or downregulated in E2-treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist, and nonagonist stressor-treated males and normal female fish plasma. One of the biomarkers, mass-to-charge ratio 3025.5, was identified by high-resolution tandem mass spectrometry as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens MXC, BPA, and TPP elicited protein expression profiles consistent with the estrogen expression model. Estrogen-responsive peaks were not detected in plasma from fish in the seawater, vehicle, ES, or CP 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.