U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561, USA.
Aquat Toxicol. 2011 May;103(1-2):71-8. doi: 10.1016/j.aquatox.2011.02.008. Epub 2011 Feb 18.
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.
蛋白质表达的变化可用于检测生物标志物，这些标志物可用于诊断性筛选具有内分泌干扰活性的化学物质。在这项研究中，表面增强激光解吸/电离飞行时间质谱（SELDI-TOF-MS）与短期鱼类试验相结合，用于研究血浆蛋白表达的变化，作为筛选具有雄激素活性的化学物质的一种方法。成年妊娠雌性鲷鱼（Cyprinodon variegatus）被放置在单独的水族箱中，分别用于海水对照、乙醇溶剂对照以及以下雄激素激动剂处理：二氢睾酮（DHT）、甲基二氢睾酮（MDHT）、睾酮（T）、甲基睾酮（MT）和群勃龙（TB）。0.6μg/L 硫丹和 40μg/L 毒死蜱（CP）的处理作为非雄激素性应激对照。使用间歇式流动通过给药装置以 20L/h 的速度供应暴露水来维持测试浓度。在第 7 天对鱼进行采样，将血浆稀释，在弱阳离子交换 CM10 蛋白芯片上处理，并进行分析。光谱处理产生了 249 个单独的 m/z 峰簇，用于雄激素暴露的鱼类。偏最小二乘法判别分析用于建立一个雄激素反应模型，使用 DHT 暴露和未暴露溶剂对照鱼的样本光谱作为训练集。非芳香化雄激素的雄激素分类模型具有≥79%的特异性（真阴性率）和≥70%的敏感性（真阳性率）。T 和 MT 等芳香化雄激素被分类为雄激素，特异性分别为 42%和 79%。T 的敏感性降低被认为是由于其被芳香酶代谢转化为雌激素。这些研究的结果表明，使用高通量质谱和多变量方法的组合，诊断性血浆蛋白表达模型可以正确地根据化学物质的雄激素活性对其进行分类。
