Steroid metabolomics profiling of compound exposure in H295R cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
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PROVIDER: S-DIXA-075 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.
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Project description:Honey consumption is attributed to potentially advantageous effects on human health due to its antioxidant capacity as well as anti-inflammatory and antimicrobial activity, which are mainly related to phenolic compound content. Phenolic compounds are secondary metabolites of plants, and their content in honey is primarily affected by the botanical and geographical origin. In this study, a high-resolution mass spectrometry (HRMS) method was applied to determine the phenolic profile of various honey matrices and investigate authenticity markers. A fruitful sample set was collected, including honey from 10 different botanical sources (n = 51) originating from Greece and Poland. Generic liquid-liquid extraction using ethyl acetate as the extractant was used to apply targeted and non-targeted workflows simultaneously. The method was fully validated according to the Eurachem guidelines, and it demonstrated high accuracy, precision, and sensitivity resulting in the detection of 11 target analytes in the samples. Suspect screening identified 16 bioactive compounds in at least one sample, with abscisic acid isomers being the most abundant in arbutus honey. Importantly, 10 markers related to honey geographical origin were revealed through non-targeted screening and the application of advanced chemometric tools. In conclusion, authenticity markers and discrimination patterns were emerged using targeted and non-targeted workflows, indicating the impact of this study on food authenticity and metabolomic fields.
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Project description:BackgroundInflammation is integral to early disease progression in children with CF. The effect of modifiable environmental factors on infection and inflammation in persons with CF is poorly understood. Our prior studies determined that secondhand smoke exposure (SHSe) is highly prevalent in young children with CF. SHSe is associated with increased inflammation, heightened bacterial burden, and worsened clinical outcomes. However, the specific metabolite and signaling pathways that regulate responses to SHSe in CF are relatively unknown.MethodsHigh-resolution metabolomics was performed on plasma samples from infants (n = 25) and children (n = 40) with CF compared to non-CF controls (n = 15). CF groups were stratified according to infant or child age and SHSe status.ResultsGlobal metabolomic profiles segregated by age and SHSe status. SHSe in CF was associated with changes in pathways related to steroid biosynthesis, fatty acid metabolism, cysteine metabolism, and oxidative stress. CF infants with SHSe demonstrated enrichment for altered metabolite localization to the small intestine, liver, and striatum. CF children with SHSe demonstrated metabolite enrichment for organs/tissues associated with oxidative stress including mitochondria, peroxisomes, and the endoplasmic reticulum. In a confirmatory analysis, SHSe was associated with changes in biomarkers of oxidative stress and cellular adhesion including MMP-9, MPO, and ICAM-1.ConclusionsSHSe in young children and infants with CF is associated with altered global metabolomics profiles and specific biochemical pathways, including enhanced oxidative stress. SHSe remains an important but understudied modifiable variable in early CF disease.
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Project description:Increasing evidence suggests that welding fume exposure is associated with systemic inflammation. Although celluar metabolites may be associated with inflammation, there is limited information on metabolomic changes during welding fume exposure. Such changes may play an important role in the occurrence, development, and prevention of metal-associated diseases. We aim to investigate human metabolomics changes pre- and post-welding fume exposure.This study included 52 boilermakers totally. We collected plasma samples pre- and post-shift welding fume exposure and prepared samples using the automated MicroLab STAR® system. Metabolite concentrations were measured using ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) methods. Two-way analysis of variance was used to test the significance of metabolite changes with false discovery rate correction.Analysis detected several metabolic changes after welding fume exposure, mainly involved in the lipid pathway [glucocorticoid class (cortisol, corticosterone, and cortisone), acylcarnitine class, and DiHOME species (9,10-DiHOME and 12,13-DiHOME)], amino acid utilization (isoleucine, proline and phenylalanine), and S-(3-hydroxypropyl) mercapturic acid (3-HPMA). These compounds are all associated with inflammation according to previous studies. Further, additive interaction effects linked smoking and 3-HPMA levels. In the metabolite set enrichment analysis for diseases, the top two disease-associated metabolite pathways were systemic inflammation-related diseases including rheumatoid arthritis and systemic lupus erythematosus.This global metabolomics study shows evidence that metabolite changes during welding fume exposure are closely associated with systemic inflammation. The altered metabolites detected may be potential health monitoring biomarkers for boilermakers, especially for inflammation-related disease prevention.
| S-EPMC6106842 | biostudies-other
Project description:Major rooibos flavonoids--dihydrochalcones, aspalathin and nothofagin, flavones--orientin and vitexin, and a flavonol, rutin, were investigated to determine their influence on the activity of adrenal steroidogenic enzymes, 3?-hydroxysteroid dehydrogenase (3?HSD2) and cytochrome P450 (P450) enzymes, P450 17?-hydroxylase/17,20-lyase (CYP17A1), P450 21-hydroxylase (CYP21A2) and P450 11?-hydroxylase (CYP11B1). All the flavonoids inhibited 3?HSD2 and CYP17A1 significantly, while the inhibition of downstream enzymes, CYP21A2 and CYP11B1, was both substrate and flavonoid specific. The dihydrochalcones inhibited the activity of CYP21A2, but not that of CYP11B1. Although rutin, orientin and vitexin inhibited deoxycortisol conversion by CYP11B1 significantly, inhibition of deoxycorticosterone was <20%. These three flavonoids were unable to inhibit CYP21A2, with negligible inhibition of deoxycortisol biosynthesis only. Rooibos inhibited substrate conversion by CYP17A1 and CYP21A2, while the inhibition of other enzyme activities was <20%. In H295R cells, rutin had the greatest inhibitory effect on steroid production upon forskolin stimulation, reducing total steroid output 2.3-fold, while no effect was detected under basal conditions. Nothofagin and vitexin had a greater inhibitory effect on overall steroid production compared to aspalathin and orientin, respectively. The latter compounds contain two hydroxyl groups on the B ring, while nothofagin and vitexin contain a single hydroxyl group. In addition, all of the flavonoids are glycosylated, albeit at different positions--dihydrochalcones at C3' and flavones at C8 on ring A, while rutin, a larger molecule, has a rutinosyl moiety at C3 on ring C. Structural differences regarding the number and position of hydroxyl and glucose moieties as well as structural flexibility could indicate different mechanisms by which these flavonoids influence the activity of adrenal steroidogenic enzymes.
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Project description:Rehmanniae Radix Preparata (RR), the dry rhizome of Rehmannia glutinosa Libosch., is a traditional herbal medicine for improving the liver and kidney function. Ample clinical and pharmacological experiments show that RR can prevent post-menopausal osteoporosis and senile osteoporosis. In the present study, in vivo and in vitro experiments, as well as a UHPLC-Q/TOF-MS-based metabolomics study, were used to explore the preventing effect of RR on glucocorticoid-induced osteoporosis (GIOP) and its underlying mechanisms. As a result, RR significantly enhanced bone mineral density (BMD), improved the micro-architecture of trabecular bone, and intervened in biochemical markers of bone metabolism in dexamethasone (DEX)-treated rats. For the in vitro experiment, RR increased the cell proliferation and alkaline phosphatase (ALP) activity, enhanced the extracellular matrix mineralization level, and improved the expression of runt-related transcription factor 2 (RUNX2) and osteopontin (OPN) in DEX-injured osteoblasts. For the metabolomics study, a total of 27 differential metabolites were detected in the DEX group vs. the control group, of which 10 were significantly reversed after RR treatment. These metabolites were majorly involved in steroid hormone biosynthesis, sex steroids regulation, and amino acid metabolism. By metabolic pathway and Western blotting analysis, it was further ascertained that RR protected against DEX-induced bone loss, mainly via interfering steroid hormone biosynthesis, as evidenced by the up-regulation of cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1), and the down-regulation of 11β-hydroxysteroid dehydrogenase (HSD11B1). Collectively, these results indicated that RR had a notable preventing effect on GIOP, and the action mechanism might be related to steroid hormone biosynthesis.
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