scholarly journals The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay With Metabolic Competence

2020 ◽  
Vol 178 (2) ◽  
pp. 281-301
Author(s):  
Chad Deisenroth ◽  
Danica E DeGroot ◽  
Todd Zurlinden ◽  
Andrew Eicher ◽  
James McCord ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Biological Effects ◽  
Parent Compound ◽  
False Negative ◽  
Metabolic Enzymes ◽  
Vitro Assay ◽  
Transactivation Assay ◽  
Alginate Immobilization ◽  
Active Substances ◽  
Receptor Transactivation

Abstract The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances. A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative endocrine active substances, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as estrogen receptor active or inactive. The results demonstrate the utility of applying the AIME method for identification of false-positive and false-negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.

Bioactivation of Quinolines in a Recombinant Estrogen Receptor Transactivation Assay Is Catalyzed by N-Methyltransferases

2019 ◽  
Vol 32 (4) ◽  
pp. 698-707
Author(s):  
Markus Brinkmann ◽  
Bogdan Barz ◽  
Danielle Carrière ◽  
Mirna Velki ◽  
Kilian Smith ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transactivation Assay ◽  
Receptor Transactivation

Role of metabolism in the endocrine-disrupting effects of chemicals in aquatic and terrestrial systems

2003 ◽  
Vol 75 (11-12) ◽  
pp. 1917-1932 ◽  
Author(s):  
M. van den Berg ◽  
T. Sanderson ◽  
N. Kurihara ◽  
A. Katayama
Keyword(s):  
Risk Assessment ◽  
Estrogen Receptor ◽  
Parent Compound ◽  
Water Soluble ◽  
Assessment Process ◽  
Endocrine Disrupting ◽  
Pcb Metabolites ◽  
Hydroxy Groups ◽  
Active Substances

This review describes the role of metabolism with endocrine active substances. Many modern synthetic compounds are readily metabolized to more polar forms that often contain hydroxy groups. This presence of polar groups and aromatic moieties in the parent compound or metabolite can play an important role in the mechanism of endocrine disruption. In addition, phase II metabolism (e.g., glucuronidation) can also lead to deactivation of the endocrine properties. In the case of bisphenol A and alkylphenols, metabolism can be considered as a detoxification mechanism as glucuronides decrease of inhibit binding to the estrogen receptors. In the case of phthalate esters, the primary metabolites, the monoesters, and further degraded metabolites do not interact with the estrogen receptor either. In contrast, the demethylation of methoxychlor in fish and other vertebrate species leads to metabolites with an increased affinity for the estrogen receptor. Certain PCB metabolites with hydroxy groups on the para position without vicinal chlorines have estrogenic activity, but these metabolites are not relevant for the environment. PCB metabolites with methylsulfonyl groups are commonly found in environmental biota and have been associated with several endocrine, developmental, and reproductive effects. Some DDT metabolites bind weakly to the estrogen receptor, but the major biotransformation product p,p-DDE is an androgen receptor (AR) antagonist. Vinclozolin is an anti-androgen and this effect appears to caused by two of its more water-soluble metabolites. The chloro-s-triazines exhibit an in vitro induction of aromatase, but their dealkylated metabolites show a decrease or lack of this effect. It is recognized that common metabolic processes can differ strongly among species that complicates ecotoxicological risk assessment of endocrine active substances. In conclusion, the testing of metabolites for endocrine-disrupting properties should be encouraged in the future to establish a better risk assessment process. An appendix containing levels and half-lives of various endocrine-disrupting chemicals in the environment and in wildlife is included at the end of this article.

Estrogenic activity of bovine milk high or low in equol using immature mouse uterotrophic responses and an estrogen receptor transactivation assay

2009 ◽  
Vol 33 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Tina S. Nielsen ◽  
Jan V. Nørgaard ◽  
Stig Purup ◽  
Xavier C. Fretté ◽  
Eva C. Bonefeld-Jørgensen
Keyword(s):  
Estrogen Receptor ◽  
Estrogenic Activity ◽  
Bovine Milk ◽  
Transactivation Assay ◽  
Immature Mouse ◽  
Receptor Transactivation

scholarly journals Bioactive Compounds, Antioxidant Activity, and Biological Effects of European Cranberry (Vaccinium oxycoccos)

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 24 ◽  
Author(s):  
Tunde Jurikova ◽  
Sona Skrovankova ◽  
Jiri Mlcek ◽  
Stefan Balla ◽  
Lukas Snopek
Keyword(s):  
Antioxidant Activity ◽  
Biological Effects ◽  
Folk Medicine ◽  
Meat Products ◽  
Biologically Active ◽  
Anticancer Activities ◽  
Fruit Species ◽  
American Cranberry ◽  
Antibacterial And Antifungal ◽  
Active Substances

Lesser known fruits or underutilized fruit species are recently of great research interest due to the presence of phytochemicals that manifest many biological effects. European cranberry, Vaccinium oxycoccos fruit, as an important representative of this group, is a valuable source of antioxidants and other biologically active substances, similar to American cranberry (V. macrocarpon) which is well known and studied. European cranberry fruit is rich especially in polyphenolic compounds anthocyanins (12.4–207.3 mg/100 g fw), proanthocyanins (1.5–5.3 mg/100 g fw), and flavonols, especially quercetin (0.52–15.4 mg/100 g fw), which mostly contribute to the antioxidant activity of the fruit. Small cranberry is also important due to its various biological effects such as urinary tract protection (proanthocyanidins), antibacterial and antifungal properties (quercetin, proanthocyanidins, anthocyanins), cardioprotective (proanthocyanidins) and anticancer activities (proanthocyanidins), and utilization in food (juice drinks, jams, jellies, sauces, additive to meat products) and pharmacological industries, and in folk medicine.

scholarly journals Short Communication: Identification and evaluation of bioactivity in forest plants used for medicinal purposes by the Kutai community of East Kalimantan, Indonesia

2018 ◽  
Vol 19 (1) ◽  
pp. 253-259 ◽  
Author(s):  
ABDUL RASYID ZARTA ◽  
WIWIN SUWINARTI ◽  
FARIDA ARIYANI ◽  
IRAWAN WIJAYA KUSUMA ◽  
ENOS TANGKE ARUNG
Keyword(s):  
Antioxidant Activity ◽  
Secondary Metabolites ◽  
Plant Species ◽  
Short Communication ◽  
Biological Effects ◽  
Bacterial Activity ◽  
Plant Materials ◽  
Vitro Assay ◽  
East Kalimantan ◽  
Forest Plants

Zarta AR, Ariyani F, Suwinarti W, Kusuma IW, Arung ET. 2018. Short Communication: Identification and evaluation of bioactivity in forest plants used for medicinal purposes by the Kutai community of East Kalimantan, Indonesia. Biodiversitas 19: 253- 259. The Indonesian forest is one of the most species-rich ecosystems in the world. Within such forests are plant species with secondary metabolites that have novel molecular structure and diverse biological activity with excellent potential to be used medicinally in prevention and cure of various diseases afflicting humans. Plant materials often contain various forms of antioxidants. Phenolic compounds found in plants have many biological effects. Flavonoids and other phenolics play a protective role against metabolic damage caused by disease and environmental stressors. The communities of Kutai Kartanegara in East Kalimantan Indonesia are representative of many traditional peoples who have evolved ways of treating human ailment and disease by use of specific plants sourced from their forests. The purpose of the research described in this paper was to identify significant medicinal plant species used by the Kutai ethnic community and to prepare extracts from these plants, mainly from the leaves, and to evaluate the extracts for bioactivity; namely by general identification of secondary metabolites, and by estimation of their antioxidant activity, toxicity, and antibacterial activity. Samples of ten plant species, used medicinally by the Kutai community, were extracted using ethanol solvent. Assay of antioxidant activity was carried out by the spectrophotometric method using DPPH (1,1-diphenyl-2-picrylhydrazyl radical) as the control. The degree of toxicity of the extracts was determined by the BSLT (Brine Shrimp Lethality Test) while anti-bacterial activity was evaluated using an in vitro assay of growth inhibition of cultures of the bacterium Escherichia coli. The result showed that nine of the plant species had strong antioxidant activity (IC50); extracts of two of the species were very toxic, while one other was toxic; and at least eight of the species had extracts that exhibited anti-bacterial activity. The phytochemical compounds identified in several of the ten species included flavonoids, tannins, saponins, steroids, triterpenoids, and alkaloids.

Abstract P130: Estrogen Receptor β Mediates the Rescue of Cardiac Function in Advanced Heart Failure by Promoting Neoangiogenesis and Reducing Fibrosis

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Andrea Iorga ◽  
Rod Partow-Navid ◽  
Humann Matori ◽  
Jingyuan Li ◽  
Soban Umar ◽  
...  
Keyword(s):  
Heart Failure ◽  
Estrogen Receptor ◽  
Estrogen Receptor Alpha ◽  
Cardiac Fibrosis ◽  
Biological Effects ◽  
Estrogen Receptor Beta ◽  
Pressure Overload ◽  
Decompensated Heart Failure ◽  
Beta Agonist ◽  
Receptor Beta

Estrogen can act via the estrogen receptor alpha (ERa) or estrogen receptor beta (ERb) to exert its biological effects, and both of these receptors are present in the heart. We have previously shown that short-term estrogen (E2) treatment can rescue pressure overload-induced decompensated heart failure (HF) in mice, and that this rescue is achieved mainly through the ERb. Furthermore, E2 has been shown to regulate angiogenesis in different tissues. Because HF has been associated with decreased angiogenesis and increased fibrosis, here we investigated whether the E2-induced rescue of HF by the selective ERb agonist DPN can regulate cardiac fibrosis and neoangiogenesis. We used transaortic constriction to induce HF, and once the ejection fraction (EF) reached ∼30%, one group of animals was sacrificed (HF group), and the other three groups received either 17b-estradiol via a subcutaneous pellet implant (0.012mg/pellet, n=16), selective ERa agonist (PPT, 0.625mg/kg/day), or selective ERb agonist (DPN, 0.625mg/kg/day) for 10 days. Serial echocardiography was performed to monitor cardiac structure and function. As expected, E2 rescued HF by restoring EF from 33.17±1.12% to 53.05±1.29%. Mice treated with DPN had a significant EF improvement from 33.17±1.12% to 45.25±2.1% (n=7), while the EF of PPT-treated mice did not improve (31.09±2.3%, n=6). Similarly, only the fractional shortening of DPN-treated mice improved from 15.7±0.58% in HF to 21.95±1.65% with DPN treatment vs. 14.72±1.24% with PPT. Next, we examined whether promotion of cardiac neoangiogenesis and suppression of fibrosis by the selective ERb agonist are possible mechanisms in the rescue action of HF by DPN. DPN treatment was able to reverse the interstitial and perivascular fibrosis observed in HF, while PPT had no effect. The selective ERb agonist also stimulated neoangiogenesis, as the capillary density was increased from 0.46±0.04 microvessels/cardiomyocyte in HF to 0.67±0.07 with DPN treatment, whereas PPT treatment had no effect (0.43±0.03). Our data strongly suggests that upregulation of cardiac neoangiogenesis and reversal of fibrosis are pivotal mechanisms in rescuing advanced HF by the estrogen receptor beta agonist DPN.

scholarly journals The Effects of Mono-(2-Ethylhexyl) Phthalate (MEHP) on Human Estrogen Receptor (hER) and Androgen Receptor (hAR) by YES/YAS In Vitro Assay

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1558 ◽  
Author(s):  
Kim ◽  
Park ◽  
Kim ◽  
Kim
Keyword(s):  
Estrogen Receptor ◽  
Androgen Receptor ◽  
Endocrine System ◽  
Yeast Cells ◽  
Signal Recovery ◽  
Vitro Assay ◽  
Recovery Test ◽  
Human Estrogen Receptor ◽  
Ethylhexyl Phthalate

Endocrine active compounds with structural similarities to natural hormones such as 17β-estradiol (E2) and androgen are suspected to affect the human endocrine system by inducing hormone-dependent effects. This study aimed to detect the (anti-)estrogenic and (anti-)androgenic activities of mono-(2-ethylhexyl) phthalate (MEHP) by yeast estrogen/androgen bioassay (YES/YAS). In addition, the mechanism and uptake of MEHP to receptors during agonistic and antagonistic activities were investigated through the activation signal recovery test and chromatographic analysis using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Estrogenic and androgenic activities of MEHP were not observed. However, MEHP exhibited anti-estrogenic (IC50 = 125 μM) and anti-androgenic effects (IC50 = 736 μM). It was confirmed that these inhibitory effects of MEHP were caused by receptor-mediated activity of the estrogen receptor and non-receptor-mediated activity of the androgen receptor in an activation signal recovery test. When IC50 concentrations of anti-estrogenic and androgenic activity of MEHP were exposed to yeast cells, the uptake concentration observed was 0.0562 ± 0.0252 μM and 0.143 ± 0.0486 μM by LC-MS/MS analysis.

scholarly journals Identification and Characterization of a Novel Functional Estrogen Receptor on Human Sperm Membrane That Interferes with Progesterone Effects

1999 ◽  
Vol 84 (5) ◽  
pp. 1670-1678 ◽  
Author(s):  
Michaela Luconi ◽  
Monica Muratori ◽  
Gianni Forti ◽  
Elisabetta Baldi
Keyword(s):  
Estrogen Receptor ◽  
Tyrosine Phosphorylation ◽  
Biological Effects ◽  
Human Sperm ◽  
Protein Band ◽  
Human Spermatozoa ◽  
Functional Surface ◽  
Nongenomic Action ◽  
17Β Estradiol ◽  
Experimental Approaches

The presence of a novel functional estrogen receptor on the human sperm surface has been demonstrated by using different experimental approaches. Ligand blot analysis of sperm lysates, using peroxidase-conjugated estradiol as probe, identified a specific estradiol-binding protein of approximately 29-kDa apparent molecular mass. The same protein band was also revealed by using αH222 antibody, which is directed against the steroid binding domain of the genomic estrogen receptor. The biological effects of estrogen receptor were investigated by analyzing calcium fluxes, tyrosine phosphorylation, and acrosome reaction (AR) in response to 17β-estradiol (17βE2) and by measuring the steroid influence on calcium and AR in responses to progesterone (P), a well-known physiological stimulus for human spermatozoa. Our results demonstrate that 17βE2 induces a rapid and sustained increase of intracellular calcium concentrations ([Ca2+]i). This effect is totally dependent on the presence of extracellular calcium, because it is completely abolished in a calcium-depleted medium. The dose-response curve for calcium increase to 17βE2 is biphasic with a first component in the nanomolar range (effective concentration 50 = 0.60 ± 0.12 nmol/L) and a second component in the micromolar range (EC50 = 3.80 ± 0.26 μmol/L). 17βE2 stimulates tyrosine phosphorylation of several sperm proteins, including the 29-kDa protein band, and determines a reduction of calcium response to P, finally resulting in inhibition of P-stimulated sperm AR. Conversely, no direct effect of 17βE2 is observed on AR. 17βE2 effects on calcium are clearly mediated by a membrane receptor, because they are reproduced by the membrane-impermeable conjugate of the hormone BSA-E2 and reduced by sperm preincubation with αH222 antibody. Taken together, our results clearly show the presence of a functional surface estrogen receptor, of 29 kDa, on human spermatozoa. This receptor may play a role in the modulation of nongenomic action of P in these cells during the process of fertilization.

scholarly journals Standardization of Estrogen Receptor Measurement in Breast Cancer Suggests False-Negative Results Are a Function of Threshold Intensity Rather Than Percentage of Positive Cells

2011 ◽  
Vol 29 (22) ◽  
pp. 2978-2984 ◽  
Author(s):  
Allison W. Welsh ◽  
Christopher B. Moeder ◽  
Sudha Kumar ◽  
Peter Gershkovich ◽  
Elaine T. Alarid ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
False Negative ◽  
Negative Results ◽  
False Negative Results ◽  
Threshold Assessment ◽  
Quantitative Immunofluorescence ◽  
Er Positive ◽  
Quantitative Immunoblotting ◽  
Positive Results

Purpose Recent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance. Methods An assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods. Results Two methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/μg immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHC-negative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays. Conclusion Assessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.

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