Endocrine disrupting chemicals have been reported to exert effects directly on enzymes involved in steroid biosynthesis. Here, we present a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for profiling the steroid metabolome of H295R human adrenocarcinoma cells. Our method can simultaneously analyse 19 precursors, intermediates and end-products, representing the adrenal steroid biosynthesis pathway. In order to obtain better insights into the processes of steroidogenesis, we investigated the dose-response relationship of forskolin, an activator of adenylate cyclase, on steroid production in H295R cells. We observed that 1.5 μM forskolin stimulated steroid production at approximately 50% of the maximum rate for most steroids. Hence, we studied the time course for steroid synthesis over 72 h in H295R cells that were stimulated with forskolin. At 24 h, we observed a peak in steroid levels for the intermediate metabolites, such as progesterone and pregnenolone, while end-products such as testosterone and cortisol continued to increase until 72 h. Finally, we show how global data provide a unique basis to develop a comprehensive, dynamic model for steroidogenesis using first order kinetics. The timeline data made it possible to estimate all reaction rate constants of the network. We propose this method as a unique and sensitive screening tool to identify effects on adrenal steroidogenesis by endocrine disrupting compounds.
Keywords: Dynamical model; H295R human adrenocarcinoma cells; LC-MS/MS method; Steroidogenesis.
Copyright © 2018. Published by Elsevier Ltd.