Tonoli David, Fürstenberger Cornelia, Boccard Julien, Hochstrasser Denis, Jeanneret Fabienne, Odermatt Alex, Rudaz Serge
†School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Geneva, Switzerland.
‡Department of Human Protein Sciences, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
Chem Res Toxicol. 2015 May 18;28(5):955-66. doi: 10.1021/tx5005369. Epub 2015 Apr 17.
The screening of endocrine disrupting chemicals (EDCs) that may alter steroidogenesis represents a highly important field mainly due to the numerous pathologies, such as cancer, diabetes, obesity, osteoporosis, and infertility that have been related to impaired steroid-mediated regulation. The adrenal H295R cell model has been validated to study steroidogenesis by the Organization for Economic Co-operation and Development (OECD) guideline. However, this guideline focuses solely on testosterone and estradiol monitoring, hormones not typically produced by the adrenals, hence limiting possible in-depth mechanistic investigations. The present work proposes an untargeted steroidomic footprinting workflow based on ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution MS for the screening and mechanistic investigations of EDCs in H295R cell supernatants. A suspected EDC, triclocarban (TCC), used in detergents, cosmetics, and personal care products, was selected to demonstrate the efficiency of the reported methodology, allowing the simultaneous assessment of a steroidomic footprint and quantification of a selected subset of steroids in a single analysis. The effects of exposure to increasing TCC concentrations were assessed, and the selection of features with database matching followed by multivariate analysis has led to the selection of the most salient affected steroids. Using correlation analysis, 11 steroids were associated with a high, 18 with a medium, and 8 with a relatively low sensitivity behavior to TCC. Among the candidates, 13 identified steroids were simultaneously quantified, leading to the evaluation and localization of the disruption of steroidogenesis caused by TCC upstream of the formation of pregnenolone. The remaining candidates could be associated with a specific steroid class (progestogens and corticosteroids, or androgens) and represent a specific footprint of steroidogenesis disruption by TCC. This strategy was devised to be compatible with medium/high-throughput screening and could be useful for the mechanistic elucidation of EDCs.
对可能改变类固醇生成的内分泌干扰化学物质（EDCs）进行筛选是一个极为重要的领域，这主要是因为众多疾病，如癌症、糖尿病、肥胖症、骨质疏松症和不孕症等，都与类固醇介导的调节受损有关。肾上腺H295R细胞模型已通过经济合作与发展组织（OECD）的指南验证，用于研究类固醇生成。然而，该指南仅侧重于睾酮和雌二醇的监测，而这两种激素并非肾上腺通常产生的，因此限制了可能的深入机制研究。本研究提出了一种基于超高压液相色谱（UHPLC）与高分辨率质谱联用的非靶向类固醇组学足迹分析工作流程，用于筛选和研究H295R细胞上清液中的EDCs及其作用机制。选择了一种用于洗涤剂、化妆品和个人护理产品中的疑似EDC——三氯生（TCC），以证明所报道方法的有效性，该方法能够在单次分析中同时评估类固醇组学足迹并对选定的类固醇子集进行定量。评估了暴露于不同浓度TCC的影响，并通过数据库匹配选择特征，随后进行多变量分析，从而选出受影响最显著的类固醇。通过相关性分析，确定有11种类固醇对TCC具有高敏感性，18种具有中等敏感性，8种具有相对低敏感性。在这些候选物中，对13种已鉴定的类固醇进行了同时定量，从而评估和定位了TCC在孕烯醇酮形成上游导致的类固醇生成紊乱。其余候选物可能与特定的类固醇类别（孕激素和皮质类固醇，或雄激素）相关，并代表了TCC对类固醇生成干扰的特定足迹。该策略设计为与中/高通量筛选兼容，可能有助于对EDCs进行机制阐释。
