Article pubs.acs.org/crt
Screening for Modulatory Effects on Steroidogenesis Using the Human H295R Adrenocortical Cell Line: A Metabolomics Approach Jeroen C. W. Rijk,* Ad A. C. M. Peijnenburg, Marco H. Blokland, Arjen Lommen, Ron L. A. P. Hoogenboom, and Toine F. H. Bovee RIKILT, Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands S Supporting Information *
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.
1. INTRODUCTION Exposure to endocrine disrupting compounds (EDCs) via food or the environment can have major adverse effects on the endocrine and reproductive systems of humans and wildlife.1,2 In the body, EDCs are able to affect hormone homeostasis via a wide variety of mechanisms by acting on various targets. This includes activation and inhibition of endocrine signaling pathways by direct binding to receptors but also alteration of normal hormone levels by affecting hormone biosynthesis, transport, or metabolism. As a consequence, either animal experiments and/or implementation of a diverse set of in vitro test systems are needed to fully comprehend endocrine disruption in chemical safety testing. In this process of endocrine disruptor testing, a tiered approach starting with in vivo and in vitro screening assays allows prioritization of compounds for additional testing.3 Regarding in vitro assays, the emphasis is mainly focused on receptor mediated effects of EDCs as determined by assays based on receptor binding, proliferation, or transcription activation, making use of yeast or mammalian cells.4−9 Regarding the latter for instance, the stably transfected transactivation assays using the ERα-HeLa-9903 or BG1Luc4E2 © 2012 American Chemical Society
cell lines for the detection of estrogenic activity have been developed and OECD validated for such purposes.10,11 However, several EDCs exert their effects via nonreceptor mediated pathways, e.g., via alterations of common signal transduction pathways in steroid synthesis or through (non)competitive inhibition of enzymes involved in steroidogenesis (Figure 1). In time, various in vivo, ex vivo, and in vitro methodologies for measuring effects on steroidogenesis have been developed,12 of which the recently OECD validated H295R steroidogenesis assay is included in the US EPA Endocrine Disruptor Screening Program (EDSP).13 Compared to in vivo, the H295R steroidogenesis assay is one of the most complete and simple in vitro assays since it is based on the human H295R adrenocarcinoma cell line which expresses all key enzymes and steroids involved in adrenal and gonadal steroidogenesis. Moreover, regulation of steroidogenesis and hormone secretion patterns in H295R cells highly reflect those of freshly isolated adrenal cells.14−16 Although it is generally Received: April 20, 2012 Published: July 6, 2012 1720
dx.doi.org/10.1021/tx3001779 | Chem. Res. Toxicol. 2012, 25, 1720−1731
Chemical Research in Toxicology
Article
Figure 1. Overview of the human steroidogenic pathway, showing the major enzymes and steroids involved in mineralocorticosteroid, glucocorticosteroid, androgen, and estrogen biosynthesis.
Table 1. Class, Properties and Concentrations of the Compounds Tested on the H295R Cell Line test compound
class
properties
concentration tested (μM)
forskolin abiraterone prochloraz ketoconazole trilostane formestane aminoglutethimide fadrozole forskolin + fadrozole etomidate metyrapone
pharmaceutical pharmaceutical fungicide antifungal drug pharmaceutical pharmaceutical pharmaceutical pharmaceutical
adenylyl cyclase activator CYP17A1 inhibitor CYP17A1 inhibitor CYP17A1 inhibitor 3β-HSD inhibitor CYP19A1 inhibitor CYP11A1 and CYP19A1 inhibitor CYP19A1 inhibitor mixture CYP11B1 inhibitor CYP11B1 inhibitor
50 10 5 5 5 10 50 10 50 + 10 1 10
anesthetic agent pharmaceutical
in steroid profiles.29−31 Separation techniques such as ultraperformance liquid chromatography (UPLC)32 in combination with full scan high-resolution MS, such as time-of-flight (ToF) and Orbitrap MS, allow for acquisition of more complete chemical profiles of complex biological samples.33 In addition, the mass accuracy provided by ToF-MS and Orbitrap MS facilitates elemental composition assessment and structure elucidation.34 The main goal of the present study was to investigate the feasibility of (un)targeted metabolomics approaches to evaluate changes in the steroid profile upon exposure of H295R cells to known steroidogenic modulating compounds. The effects of 10 steroidogenic modulating compounds were investigated, i.e., the CYP17A1 inhibitors abiraterone, prochloraz, and ketoconazole, the 3β-hydroxysteroid dehydrogenase (3β-HSD) inhibitor trilostane, the aromatase (CYP19A1) inhibitors formestane, fadrozole, and aminoglutethimide, and the CYP11B1 inhibitors etomidate and metyrapone, while the adenylyl cyclase stimulator forskolin was chosen as an inducer of androgens and estrogens (Table 1). Medium extracts of exposed H295R cells were analyzed with UPLC-ToF-MS, and the obtained chemical profiles were preprocessed by MetAlign software for data reduction and alignment.35 The extracted steroid profiles were compared to the steroid profiles obtained by the quantitative targeted GC-MS/MS analysis of the same sample and to the
recognized that the H295R model allows investigation of effects of EDCs on mineral- and glucocorticosteroid production17 as well as investigation of effects at the genomic18,19 or enzyme level,20 current end points of the H295R assay are limited to testosterone and estradiol levels. The levels of these two hormones are in general determined by EIAs, RIAs,21,22 or targeted LC-MS/MS methods.23 While measuring changes in the testosterone and estradiol levels is useful for negative− positive screening of compounds in the H295R assay, evaluating multiple and various end points might be more informative and give insight into the underlying mechanisms of possible changes in steroid production.24 Within this scope, targeted profiling of the extended steroidogenesis pathway is an attractive alternative to provide additional and fundamental information about the effect of EDCs on the regulatory pathways in the H295R cells.16,25−27 Besides the main steroid biosynthesis pathways (Figure 1), H295R cells also display significant phase I and II metabolism, resulting in high levels of hydroxylated and sulphated steroid metabolites.28 Although the H295R model is well described, effects of steroidogenesis disrupting model compounds are in most cases only examined using a few steroids as an end point, while overall steroid profiles are lacking. As an alternative for targeted steroid profiling, recent developments in LC-MS and bioinformatics allow more comprehensive metabolomics approaches to evaluate changes 1721
dx.doi.org/10.1021/tx3001779 | Chem. Res. Toxicol. 2012, 25, 1720−1731
Chemical Research in Toxicology
Article
addition of 50 μL of substrate solution (ATPlite kit content). The plate was shaken again for 5 min at an orbital shaker (700 rpm) and after 10 min of incubation in the dark, the luminescence was measured at 590 nm using a Synergy HT multidetection microplate reader. The cell viability experiments were performed using assay-triplicates, and the final concentration of the DMSO solvent carrier was 0.1%. 2.4. Hormone Quantification Using EIAs. Extraction of free steroids was performed according to the procedure described by Hecker and Giesy.21 Aliquots of 450 μL of medium were mixed with 2.5 mL of diethylether and centrifuged at 2000g for 10 min. The supernatant was transferred into a fresh glass tube, and the extraction step was repeated. Next, the combined solvent fractions were evaporated under a gentle stream of nitrogen gas and reconstituted in 450 μL of EIA buffer. Progesterone, testosterone, and 17β-estradiol levels were determined by competitive EIA according to the manufacturer’s recommendations (Cayman Chemical Company, Ann Arbor, MI, USA). In a prior EIA analysis, extracts were diluted 1:100 for progesterone, 1:100 for testosterone, and 1:10 for 17β-estradiol. Relative changes in hormone production were expressed versus the DMSO solvent control (SC), and statistical analysis of data was conducted using R (v2.13.1).36 Differences between chemical treatments and SCs were evaluated using a one way ANOVA followed by a two-sided Dunnett’s test. Differences were considered significant at a p-value
