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

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 (17β-trenbolone) and antagonist (flutamide) in the liver. Female fathead minnows were exposed to nominal concentrations of either 17β-trenbolone (0.05, 0.5, or 5 μg/L), flutamide (50, 150, or 500 μg/L), or a mixture (500 μg flutamide/L and 0.5 μg 17β-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 17β-trenbolone (5 μg/L), flutamide (500 μg/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，其暴露浓度可对鱼类的生殖过程造成损伤。本实验旨在利用雄激素受体（androgen receptor）激动剂17β-群勃龙（17β-trenbolone）与拮抗剂氟他胺（flutamide），在鱼类肝脏中筛选经雄激素受体信号通路介导的蛋白质与细胞过程。 将雌性黑头软口鲦（fathead minnow）暴露于以下标称浓度的处理液中，暴露时长为48小时：17β-群勃龙（0.05、0.5或5 μg/L）、氟他胺（50、150或500 μg/L），以及二者的混合液（500 μg/L氟他胺与0.5 μg/L 17β-群勃龙）。本实验从未处理对照组以及经17β-群勃龙（5 μg/L）、氟他胺（500 μg/L）或二者混合液处理的鱼类肝脏中提取蛋白质，经胰蛋白酶消化后，采用同位素相对标记与绝对定量（iTRAQ）法对肽段进行标记。 经统计检验（p < 0.05），共有45种蛋白质在至少一种处理组中发生了差异表达。差异表达的蛋白质主要参与细胞代谢（如甘油醛-3-磷酸脱氢酶（glyceraldehyde 3-phosphate dehydrogenase）、磷酸甘油酸变位酶（phosphoglycerate mutase））、基础应激与氧化应激应答（如超氧化物歧化酶（superoxide dismutase）与热休克蛋白（heat shock proteins）），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。 本研究还将筛选出的蛋白质表达变化与其mRNA水平变化进行了对比，总体发现蛋白质与mRNA的表达变化并无显著相关性，这表明转录组（transcriptome）与蛋白质组（proteome）层面均存在复杂的调控机制。综上可知，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可用于兼具生态毒理学（ecotoxicology）研究价值但基因组信息有限的模式物种。