21 research outputs found
Recombinant cell bioassays for the detection of (gluco)corticosteroids and endocrine-disrupting potencies of several environmental PCB contaminants
Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide >> corticosterone > dexamethasone > cortisol = betamethasone > prednisolone > aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity
Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor
Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67μM, 60μM, and 22nM for GR, and 39μM, 20μM, and 982nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestagens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular Δ4 steroidogenic pathway. Therefore, adding an in vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable
Screening for Modulatory Effects on Steroidogenesis Using the Human H295R Adrenocortical Cell Line: A Metabolomics Approach
The recently OECD validated H295R steroidogenesis assay
provides
an <i>in vitro</i> 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 <i>m</i>/<i>z</i>-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
Structural Changes of 6a-Hydroxy-Pterocarpans Upon Heating Modulate Their Estrogenicity
The isoflavonoid composition of an
ethanolic extract of fungus-treated
soybean sprouts was strongly altered by a combined acid/heat treatment.
UHPLC-MS analysis showed that 6a-hydroxy-pterocarpans were completely
converted to their respective, more stable, 6a,11a-pterocarpenes,
whereas other isoflavonoids, from the isoflavone and coumestan subclasses,
were affected to a much lesser extent (loss of ∼15%). Subsequently,
mixtures enriched in prenylated 6a-hydroxy-pterocarpans (pools of
glyceollin I/II/III and glyceollin IV/VI) or prenylated 6a,11a-pterocarpenes
(pools of dehydroglyceollin I/II/III and dehydroglyceollin IV/VI)
were purified, and tested for activity on both human estrogen receptors
(ERα and ERβ). In particular, the response toward ERα
changed, from agonistic for glyceollins to antagonistic for dehydroglyceollins.
Toward ERβ a decrease in agonistic activity was observed. These
results indicate that the introduction of a double bond with the concomitant
loss of a hydroxyl group in 6a-hydroxy-pterocarpans extensively modulates
their estrogenic activity