Endocrine Disruption and Steroid Hormone Action

An increasing number of chemicals have been suspected of mimicking hormonal effects and altering steroid hormone regulation. Endocrine-disrupting chemicals (EDCs) may contribute to the development and progression of major diseases, including developmental disorders, immune diseases, various forms of cancer, and cardio-metabolic diseases such as diabetes. But the mechanisms underlying chemical-induced disruption of endocrine functions are often unknown. Thus, there is a need to identify key events of disrupted steroid homeostasis as well as to develop suitable tools – in vitro and in silico – for early detection of chemical-induced disturbances of steroid homeostasis.

This project focusses on generating experimental data that can serve as a basis to improve regulatory test systems for the characterisation of EDCs affecting steroidogenesis and peripheral steroid action. Current regulatory tests for EDCs focus on effects on oestrogen, androgen, and thyroid receptor signalling and on sex steroid production, but do not adequately assess mineralocorticoids, glucocorticoids, and adrenal androgens. The project aims to refine the use of the established regulatory cell-based test system (the human adrenal cancer cell line H295R) to characterise effects on these other steroids. Furthermore, effects of EDCs on peripheral steroid action will be addressed through a broad range of methods, computer-based models, biological testing systems and using targeted and untargeted steroid profiling.

Regulatory authorities are participating in large-scale projects on chemical safety focusing on substances that disrupt endocrine functions. However, the currently available methods and potential targets under investigation are still highly limited and additional testing strategies are required. The tools that will be developed in this project should help in the identification of potentially hazardous chemicals and the steroidomic profiling should facilitate the investigation of their modes of action and subsequently their potential hazard to humans.

WP1 - Chemicals disrupting adrenal steroidogenesis and/or peripheral steroid action

Rationale for the project

The current OECD H295R assay (Test Guideline 496) has considerable potential for improvement. Parameters such as serum, incubation time, use of basal and stimulated cell state, and extended steroidomic analyses using LC-MS/MS combined with gene expression analyses will be addressed. Several pathways cannot be studied using H295R cells and modified cells as well as alternative systems will be considered. Moreover, targeted investigation of enzymes and receptors involved in peripheral steroid action will be performed by parallel screening using pharmacophore models of various steroid targets and in combination with a panel of bioassays. The combination of in silicoanalyses and biological testing should also provide mechanistic information.

Objectives

The objectives of this project are to generate a list of high priority chemicals to be tested for endocrine activities; to establish extended readouts from adrenal H295R cells to identify and characterise chemicals interfering with steroid synthesis; to establish modified models for assessment of steroidotoxic compounds; to validate a method for serum and urinary steroid metabolite quantification; and to establish new in silico and in vitro tools for the identification of EDC key events and for read-across patterns of in silico activity from known EDCs to poorly characterised high priority chemicals.

Partners: Serge Rudaz (University of Geneva), Daniela Schuster (University of Innsbruck)

WP2 - Steroidomic profiling in adrenal cell models

Rationale for the project

Disruption of steroid hormone action by endocrine disrupting chemicals (EDCs) may be associated with developmental defects, reproductive diseases, cardio-metabolic diseases and various forms of cancer. EDCs can affect steroid homeostasis by perturbing steroid synthesis, metabolism and receptor function due to direct effects or altered gene expression. The OECD Health Effects test guideline 456 (H295R Steroidogenesis Assay) was developed and standardised as a screen for chemical effects on steroidogenesis, and is likely to be widely used for regulatory screening and prioritisation of EDCs. This projects aims to provide improved readouts for the OECD H295R test system with targeted steroid analyses as well as extended steroid profiling of the test system.

Objectives

Steroids in the H295R test system will be measured by targeted and untargeted analytical strategies with the aim of identifying EDC biomarkers of effect for human biomonitoring studies. The proof-of-concept of the untargeted strategy in H295R cells has already been demonstrated using triclocarban exposure.

The proposed extended steroid profile will have to deal with a reduced number of unambiguously identified steroids for both statistical and biological interpretations. For this purpose, a user-friendly dynamic steroid database containing mass spectrometry and chromatographic data will be used. A data mining strategy combining multivariate and correlation analysis will be used as a relevant approach to extract and highlight marker candidates.  The developed method will constitute a potent approach to screen and classify potential or confirmed EDCs affecting adrenal steroidogenesis in a straightforward manner.

Partner: Prof. Alex Odermatt (University of Basel)