Quantitative Proteomic Profiles of Androgen Receptor Signaling in the Liver of Fathead Minnows (Pimephales promelas)

Androgenic chemicals are present in the environment at concentrations that impair reproductive processes in fish. The objective of this experiment was to identify proteins and cell processes mediated through androgen receptor signaling using an androgen receptor agonist (17��-trenbolone) and antagonist (flutamide) in the liver. Female fathead minnows were exposed to nominal concentrations of either 17��-trenbolone (0.05, 0.5, or 5 ��g/L), flutamide (50, 150, or 500 ��g/L), or a mixture (500 ��g flutamide/L and 0.5 ��g 17��-trenbolone/L) for 48 h. The iTRAQ method was used to label peptides after protein extraction and trypsin-digestion from livers of untreated controls or from fish treated with 17��-trenbolone (5 ��g/L), flutamide (500 ��g/L), or a mixture of both compounds. Forty-five proteins were differentially altered by one or more treatments (p < 0.05). Many altered proteins were involved in cellular metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase), general and oxidative stress response (e.g., superoxide dismutase and heat shock proteins), and the regulation of translation (e.g., ribosomal proteins). Cellular pathway analysis identified additional signaling cascades activated or inhibited by flutamide that may not be androgen receptor mediated. We also compared changes in select proteins to changes in their mRNA levels and observed, in general, that proteins and mRNA changes did not correlate, suggesting complex regulation at the level of both the transcriptome and proteome. It is concluded that both transcriptomic and proteomic approaches offer unique and complementary insights into mechanisms of regulation. We demonstrate the utility of proteomic profiling for use on a model species with value to ecotoxicology but having limited genomic information.

环境中存在的雄激素类化学物质，其浓度可损害鱼类的生殖过程。本实验旨在利用雄激素受体激动剂17α-群勃龙（17α-trenbolone）与拮抗剂氟他胺（flutamide），在肝脏中筛选经雄激素受体信号通路介导的蛋白质及细胞过程。
将雌性黑头呆鱼暴露于标称浓度的17α-群勃龙（0.05、0.5或5 μg/L）、氟他胺（50、150或500 μg/L），或二者混合体系（500 μg/L氟他胺+0.5 μg/L 17α-群勃龙）中，暴露时长为48小时。本实验采用iTRAQ（同位素标记相对和绝对定量技术）法，对未经处理的对照组鱼肝脏，以及经17α-群勃龙（5 μg/L）、氟他胺（500 μg/L）或二者混合处理的鱼肝脏进行蛋白提取、胰蛋白酶消化后，对肽段进行标记。
经分析，共有45种蛋白质在至少一种处理组中出现显著表达差异（p < 0.05）。多数差异表达蛋白质参与细胞代谢过程（如甘油醛-3-磷酸脱氢酶、磷酸甘油酸变位酶）、基础应激与氧化应激应答（如超氧化物歧化酶与热休克蛋白），以及翻译调控过程（如核糖体蛋白）。细胞通路分析还发现，氟他胺可调控其他非雄激素受体介导的信号级联反应。
本研究还将部分差异蛋白质的表达变化与其mRNA水平变化进行了对比，整体发现蛋白质与mRNA的表达变化并无显著相关性，提示转录组与蛋白质组层面均存在复杂的调控机制。综上，转录组学与蛋白质组学方法均可为调控机制研究提供独特且互补的视角。本研究证实了蛋白质组谱分析在生态毒理学研究中有应用价值，同时也为基因组信息有限的模式物种研究提供了可行方案。