Screening for Modulatory Effects on Steroidogenesis Using the Human H295R Adrenocortical Cell Line: A Metabolomics Approach

Abstract

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and estradiol levels as end points, measured with specific enzyme immunoassays (EIAs) or targeted liquid chromatography (LC) and gas chromatography (GC)-mass spectrometry (MS) methods. However, recent developments in LC-MS and bioinformatics allow for more comprehensive approaches to evaluate changes in steroid profiles. In the current work, the H295R cell model was combined with a metabolomics approach to monitor changes in metabolite profiles in both a targeted and untargeted way. H295R cells were exposed for 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz, ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole, etomidate, and metyrapone, known to affect steroidogenesis. After exposure, the levels of 9 natural steroids were determined by a quantitative targeted GC-MS/MS method and compared to a metabolomics method using Ultra Performance Liquid Chromatography-Time-of-Flight-Mass Spectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suited for negative-positive screening, but the MS methods also generated specific fingerprints, allowing chemical class prediction of the compound under investigation. Although the targeted GC-MS/MS was more sensitive, which was an advantage regarding analysis of the estrogens 17β-estradiol and estrone, the untargeted UPLC-ToF-MS was able to evaluate effects on the synthesis of the corticosteroids. Moreover, untargeted comparison of the aligned chemical profiles allowed identification of all m/z-values that are differential between exposed and nonexposed H295R cells. In conclusion, application of a comprehensive metabolite profiling methodology not only provides a tool to screen compounds for steroidogenic modulating properties, but also allows chemical class prediction. As such, steroid profiling methodologies in conjunction with the H295R assay can contribute to the prioritization of chemicals for additional safety testing.

Role of GABA-active neurosteroids in the efficacy of metyrapone against cocaine addiction

“…While typically regarded as a relatively selective 11beta-hydroxylase inhibitor, the consequences of this inhibition are only recently being appreciated [36]. The effects of metyrapone on the steroid metabolome includes a simultaneous large decrease in corticosterone/cortisol along with an accompanying large increase in 11-deoxysteroid metabolites including DHDOC and THDOC [93,94]. Evidence that metyrapone administration can also increase progesterone, DHP or allopregnanolone has also been reported [37,38,41].…”

Adrenocortical endocrine disruption

“…The use of gas chromatography tandem mass spectrometry analysis of H295R media has been developed, and used to evaluate steroid profiles, and compared with metabolomics methodology using ultra performance liquid chromatography-time-of-flight-mass-spectrometry (UPLC-ToF-MS) to identify specific "fingerprints" of endocrine disrupting chemicals (see [52,53]). It has also been proposed to use this cell line, along with other assays, in an integrated testing strategy for oestrogenicity to broaden sensitivity in identification of endocrine disruption through this mechanism [54].…”

“…It has also been proposed to use this cell line, along with other assays, in an integrated testing strategy for oestrogenicity to broaden sensitivity in identification of endocrine disruption through this mechanism [54]. Although the endpoints reported in these studies [52][53][54] pertain largely to sex steroids, they can also be applied to develop fingerprints using cortisol, aldosterone and other steroids specific to adrenocortical function. Such a development to the OECD Test Guideline on H295R in endocrine disruption evaluation [55], which currently only considers production of 17b-oestradiol and testosterone as endpoints, and validation of cortisol and aldosterone as endpoints, would be a major step in recognizing the unique role of the adrenal cortex and the extent to which glucocorticoid and/or mineralocorticoid producing adrenocortical cells may be a target for endocrine disruption.…”

Towards a non-animal risk assessment for anti-androgenic effects in humans

“…For example, in Lab 1, the LOEC for prochloraz was 0.0001 μg/ml, whereas for Lab 2 the LOEC was 0.1 μg/ml, indicating that if this assay were to be used to provide dose response data some further optimization may be required. Refinements already proposed include the addition of metabolomic assessment (Rijk et al, 2012) and the use of a co-culture system to mimic the fetoplacental unit (Thibeault et al, 2014). Overall, since this human cell-based assay is capable of covering a number of modes of action including LH receptor-mediated effects as well as effects mediated through interaction with steroidogenic enzymes it is likely to be useful as part of an integrated in vitro approach to risk assessment.…”