Léger Thibaut, Le Guével Rémy, Solhi Hélène, Evrard Bertrand, Darde Thomas, Desdoits-Lethimonier Christèle, Glorennec Philippe, Bonvallot Nathalie, Chalmel Frédéric, David Arthur
Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, F-35000, Rennes, France; Toxicology of Contaminants Unit, Fougères Laboratory, French Agency for Food, Environmental and Occupational Health & Safety (ANSES), 35306 Fougères CEDEX, France.
ImPACcell-Biosit SFR UMS CNRS 3480 - INSERM 018, France.
Anal Chim Acta. 2025 Feb 8;1338:343594. doi: 10.1016/j.aca.2024.343594. Epub 2024 Dec 28.
Considering the large diversity of chemicals present in the environment and the need to study their effects (alone or as mixtures), the development of high-throughput in vitro assays in line with the Replacement, Reduction, Refinement (3R) strategy is essential for chemical risk assessments.
We developed a robust analytical workflow based on both low resolution tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HRMS) to quantify 13 steroids in NCI-H295R cell culture medium, human plasma and serum. The workflow was validated by screening media from the NCI-H295R cell line exposed in dose-response experiments to 5 endocrine disruptors (EDs) such as bisphenol A, prochloraz, ketoconazole, atrazine and forskolin. Absolute quantifications of the 13 steroids performed on a triple quadrupole (QqQ) MS/MS demonstrated that the performances obtained were in line with OECD recommendations. HRMS (MS1-HRMS) provided measurements nearly as sensitive and as reproducible as those obtained using multiple reaction monitoring (MRM) and ELISA. A bioinformatics workflow, using HRMS, was implemented to detect and annotate disrupted metabolites. HRMS allowed to detect disruptions in pathways associated to fatty acids, purines and amino acids metabolisms after exposure to the EDs tested, in addition to that linked to steroidogenesis.
We developed a robust MS1-HRMS workflow, from sample preparation to compound quantification or annotation, compatible with absolute steroid quantification, to screen NCI-H295R cell media exposed to potential EDs. Using only 200 μL of medium, the method integrates MS/MS and HRMS analyses, 96-well plate solid-phase extraction for high throughput, and automated pre-annotation for cost efficiency. This optimized workflow identifies EDs in cell assays by detecting disruptions in steroidogenesis and other biological pathways.
鉴于环境中存在的化学物质种类繁多，且需要研究它们（单独或作为混合物）的影响，开发符合替代、减少、优化（3R）策略的高通量体外检测方法对于化学风险评估至关重要。
我们开发了一种基于低分辨率串联质谱（MS/MS）和高分辨率质谱（HRMS）的强大分析工作流程，用于定量NCI-H295R细胞培养基、人血浆和血清中的13种类固醇。通过筛选在剂量反应实验中暴露于5种内分泌干扰物（EDs）（如双酚A、咪鲜胺、酮康唑、阿特拉津和福司可林）的NCI-H295R细胞系的培养基，对该工作流程进行了验证。在三重四极杆（QqQ）MS/MS上对13种类固醇进行的绝对定量表明，所获得的性能符合经合组织的建议。HRMS（MS1-HRMS）提供的测量结果几乎与使用多反应监测（MRM）和酶联免疫吸附测定（ELISA）获得的结果一样灵敏且可重复。实施了一种使用HRMS的生物信息学工作流程来检测和注释受干扰的代谢物。除了与类固醇生成相关的途径外，HRMS还能够检测到暴露于测试的EDs后与脂肪酸、嘌呤和氨基酸代谢相关的途径中的干扰。
我们开发了一种强大的MS1-HRMS工作流程，从样品制备到化合物定量或注释，与绝对类固醇定量兼容，用于筛选暴露于潜在EDs的NCI-H295R细胞培养基。该方法仅使用200μL培养基，整合了MS/MS和HRMS分析、用于高通量的96孔板固相萃取以及用于成本效益的自动预注释。这种优化的工作流程通过检测类固醇生成和其他生物途径中的干扰来识别细胞检测中的EDs。
