Ovulation is Inhibited by an Environmentally Relevant Phthalate Mixture in Mouse Antral Follicles in Vitro

Abstract Phthalates are solvents and plasticizers found in consumer products including cosmetics, food/beverage containers, housing materials, etc. Phthalates are known endocrine-disrupting chemicals that can directly target the ovary, potentially causing defects in ovulation and fertility. Women are exposed to multiple different phthalates daily, therefore this study investigated the effects of an environmentally relevant phthalate mixture (PHTmix) on ovulation. Ovulation is initiated by the luteinizing hormone (LH) surge, which induces prostaglandin (PG) production, progesterone (P4)/progesterone receptor (PGR) signaling, and extra-cellular matrix (ECM) remodeling. We hypothesized that the PHTmix would directly inhibit ovulation by altering the levels of PGs, P4/PGR, and enzymes involved in ECM remodeling. Antral follicles from CD-1 mice were treated with vehicle control alone (dimethylsulfoxide, DMSO), hCG alone (LH analog), and hCG+PHTmix (1-500μg/ml), and samples were collected across the ovulatory period. The PHTmix decreased ovulation rates at all doses tested in a dose dependent manner when compared to hCG. PG levels were decreased by the PHTmix when compared to hCG, which was potentially mediated by altered levels of PG synthesis (Ptgs2) and transport (Slco2a1) genes. The PHTmix altered P4 and Pgr levels when compared to hCG, leading to decreases in downstream PGR-mediated genes (Edn2, Il6, Adamts1). ECM remodeling was potentially dysregulated by altered levels of ovulatory mediators belonging to the matrix metalloproteases and plasminogen activator families. These data suggest that phthalate exposure inhibits ovulation by altering PG levels, P4/PGR action, and ECM remodeling.

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219 EFFECT OF THE ENDOCRINE DISRUPTING CHEMICALS ON PLACENTAL TRANSPORT IN BeWo CELLS AS AN IN VITRO MODEL

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab219 ◽

2015 ◽

Vol 27 (1) ◽

pp. 199

Author(s):

J.-H. Lee ◽

E.-B. Jeung

Keyword(s):

In Vitro Model ◽

Endocrine Disrupting Chemicals ◽

Reproductive Hormones ◽

Dependent Manner ◽

Endocrine Disrupting ◽

Calcium Cations ◽

Vitro Model ◽

Dose Dependent ◽

Iron And Calcium

The placenta exchanges vital factors, including oxygen, carbon dioxide, copper, iron, calcium cations, and glucose, which are essential to fetal growth. Each molecule is transferred by specific receptors that are located at the cell membrane or in the cytoplasm. Copper, iron, calcium cations, and glucose transfer genes are regulated by estrogens, vitamin D, and human placental lactogen. Regulations of these receptors depend on pregnancy time length and maternal and fetal nutrient environment with various pathways. Some synthetic plastics known as endocrine disrupting chemicals (EDC) have a similar structure to reproductive hormones such as estrogens. Thus, these substances have a potential effect on the expression of genes which are regulated by estrogens or progesterone by interfering their pathways. Having an estrogenic property, EDC interact with oestrogen receptors and elevate or decrease the expression of target genes which are responsible for transporting essential molecules such as copper, iron, and calcium. To examine the effects of EDC exposure during pregnancy, we conducted an in vitro model study using the BeWo human trophoblast cell line. The BeWo cell was treated with well-known EDC, octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA) in a dose-dependent manner (10–7, 10–6, and 10–5 M) for 24 h. The expression of copper (CTR1, ATP7A), iron (IREG1, HEPH), and calcium transporting genes (PMCA1, TRPV6), were measured by real-time RT–PCR and Western blot. The expression of copper, iron, and calcium transporting genes were elevated in a dose-dependent manner by all well-known EDC, including OP, NP, and BPA, as well as E2. To unveil the mechanism of these elevations of ionic transporting genes, an ERE promoter study will be needed. Taken together, essential cation transporting genes in placenta are modulated by EDC.

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Scientific discrepancies in European regulatory proposals on endocrine disruptors—REACH regulation quo vadis?

Archives of Toxicology ◽

10.1007/s00204-021-03152-7 ◽

2021 ◽

Author(s):

Andreas Natsch

Keyword(s):

Low Dose ◽

Endocrine Disrupting Chemicals ◽

Test Methods ◽

Consumer Products ◽

In Vitro Tests ◽

In Vitro Test ◽

Animal Testing ◽

European Regulatory ◽

Quo Vadis

AbstractThe EU chemical strategy for sustainability places a high focus on endocrine-disrupting chemicals (ED), the importance of their identification with increased testing and a ban in consumer products by a generic approach. It is assumed that for ED no threshold and hence no safe dose exists, leading to this generic approach. This view appears to be linked to the claim that for ED ‘low-dose non-monotonic dose response’ (low-dose NMDR) effects are observed. Without this hypothesis, there are no scientific reasons why classical risk assessment cannot be applied to the ED mode-of-action. Thus, whether for ED low-dose NMDR effects are considered a reproducible scientific fact by European authorities is Gretchen’s question in this politicized field. Recent documents by the SCCS, EFSA and ECHA reviewed herein illustrate the diverging views within European scientific bodies on this issue. Furthermore, ED researchers never replicated findings on low-dose NMDR in blinded inter-laboratory experiments and the CLARITY-BPA core studies could not find evidence for reproducible NMDR for BPA. ECHA proposes a battery of in vitro tests to test all chemicals for ED properties. However, these tests were never validated for relevance and their high positivity rate could lead to increased follow-up animal testing. Based on (i) lack of reproducibility data for low-dose NMDR, (ii) diverging views within European authorities on NMDR and (iii) lack of fully validated in vitro test methods it might be premature to fast-track the wide-ranging changes in the regulatory landscape proposed by the authorities ultimately leading to drastically increased animal testing.

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Characterization of Estrogenic and Androgenic Activities for Bisphenol A-like Chemicals (BPs): In Vitro Estrogen and Androgen Receptors Transcriptional Activation, Gene Regulation, and Binding Profiles

Toxicological Sciences ◽

10.1093/toxsci/kfz173 ◽

2019 ◽

Vol 172 (1) ◽

pp. 23-37 ◽

Cited By ~ 11

Author(s):

Katherine E Pelch ◽

Yin Li ◽

Lalith Perera ◽

Kristina A Thayer ◽

Kenneth S Korach

Keyword(s):

Bisphenol A ◽

Transcriptional Activation ◽

Food Packaging ◽

Endocrine Disrupting Chemicals ◽

Consumer Products ◽

Production Volume ◽

Bisphenol S ◽

Androgenic Activity ◽

Data Gap

Abstract Bisphenol A (BPA) is a high production volume chemical widely used in plastics, food packaging, and many other products. It is well known that endocrine-disrupting chemicals might be harmful to human health due to interference with normal hormone actions. Recent studies report widespread usage and exposure to many BPA-like chemicals (BPs) that are structurally or functionally similar to BPA. However, the biological actions and toxicity of those BPs are still relatively unknown. To address this data gap, we used in vitro cell models to evaluate the ability of 22 BPs to induce or inhibit estrogenic and androgenic activity. BPA, Bisphenol AF (BPAF), bisphenol Z (BPZ), bisphenol C (BPC), tetramethyl bisphenol A (TMBPA), bisphenol S (BPS), bisphenol E (BPE), 4,4-bisphenol F (4,4-BPF), bisphenol AP (BPAP), bisphenol B (BPB), tetrachlorobisphenol A (TCBPA), and benzylparaben (PHBB) induced estrogen receptor (ER)α and/or ERβ-mediated activity. With the exception of BPS, TCBPA, and PHBB, these same BPs were also androgen receptor (AR) antagonists. Only 3 BPs were found to be ER antagonists. Bisphenol P (BPP) selectively inhibited ERβ-mediated activity and 4-(4-phenylmethoxyphenyl)sulfonylphenol (BPS-MPE) and 2,4-bisphenol S (2,4-BPS) selectively inhibited ERα-mediated activity. None of the BPs induced AR-mediated activity. In addition, we identify that the BPs can bind to ER or AR with varying degrees by a molecular modeling analysis. Taken together, these findings help us to understand the molecular mechanism of BPs and further consideration of their usage in consumer products.

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Matrix Metalloproteases as Influencers of the Cells’ Social Media

International Journal of Molecular Sciences ◽

10.3390/ijms20163847 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3847 ◽

Cited By ~ 17

Author(s):

Daniel Young ◽

Nabangshu Das ◽

Anthonia Anowai ◽

Antoine Dufour

Keyword(s):

Inflammatory Diseases ◽

Matrix Metalloproteases ◽

Phase Iii ◽

Biological Functions ◽

Mmp Inhibitors ◽

Ecm Remodeling ◽

Surface Receptors ◽

Phase Iii Clinical Trials ◽

The Matrix ◽

The Impact

Matrix metalloproteinases (MMPs) have been studied in the context of cancer due to their ability to increase cell invasion, and were initially thought to facilitate metastasis solely through the degradation of the extracellular matrix (ECM). MMPs have also been investigated in the context of their ECM remodeling activity in several acute and chronic inflammatory diseases. However, after several MMP inhibitors failed in phase III clinical trials, a global reassessment of their biological functions was undertaken, which has revealed multiple unanticipated functions including the processing of chemokines, cytokines, and cell surface receptors. Despite what their name suggests, the matrix aspect of MMPs could contribute to a lesser part of their physiological functions in inflammatory diseases, as originally anticipated. Here, we present examples of MMP substrates implicated in cell signaling, independent of their ECM functions, and discuss the impact for the use of MMP inhibitors.

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2-Photon Characterization of Optical Proteolytic Beacons for Imaging Changes in Matrix-Metalloprotease Activity in a Mouse Model of Aneurysm

Microscopy and Microanalysis ◽

10.1017/s1431927616000088 ◽

2016 ◽

Vol 22 (2) ◽

pp. 349-360

Author(s):

Darren G. Haskett ◽

David Maestas ◽

Stephen J. Howerton ◽

Tyler Smith ◽

D. Catalina Ardila ◽

...

Keyword(s):

Mouse Model ◽

Ex Vivo ◽

Disease Process ◽

Developed Countries ◽

Proteolytic Cleavage ◽

Matrix Metalloproteases ◽

Heterogeneous Distribution ◽

Ecm Remodeling ◽

Photon Spectra

AbstractAbdominal aortic aneurysm is a multifactorial disease that is a leading cause of death in developed countries. Matrix-metalloproteases (MMPs) are part of the disease process, however, assessing their role in disease initiation and progression has been difficult and animal models have become essential. Combining Förster resonance energy transfer (FRET) proteolytic beacons activated in the presence of MMPs with 2-photon microscopy allows for a novel method of evaluating MMP activity within the extracellular matrix (ECM). Single and 2-photon spectra for proteolytic beacons were determined in vitro. Ex vivo experiments using the apolipoprotein E knockout angiotensin II-infused mouse model of aneurysm imaged ECM architecture simultaneously with the MMP-activated FRET beacons. 2-photon spectra of the two-color proteolytic beacons showed peaks for the individual fluorophores that enable imaging of MMP activity through proteolytic cleavage. Ex vivo imaging of the beacons within the ECM revealed both microstructure and MMP activity. 2-photon imaging of the beacons in aneurysmal tissue showed an increase in proteolytic cleavage within the ECM (p<0.001), thus indicating an increase in MMP activity. Our data suggest that FRET-based proteolytic beacons show promise in assessing MMP activity within the ECM and will therefore allow future studies to identify the heterogeneous distribution of simultaneous ECM remodeling and protease activity in aneurysmal disease.

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Antimicrobial Agent Triclosan Inhibits Acetylcholinesterase Activity in Vitro in a Dose-Dependent Manner

10.1101/2021.06.11.448059 ◽

2021 ◽

Author(s):

Narasimha Pullaguri ◽

Andrea Kagoo ◽

Anamika Bhargava

Keyword(s):

Antimicrobial Agent ◽

Ache Activity ◽

Acetylcholinesterase Activity ◽

Consumer Products ◽

In Vivo Studies ◽

Dependent Manner ◽

Human Health Effects ◽

Dose Dependent

The antimicrobial agent, Triclosan, is widely used in many consumer products. It has been designated as a "contaminant of emerging concern (CEC)" because its exposure is known to cause adverse ecological and human health effects. Triclosan is not labelled as GRAS/GRAE (generally recognized as safe and effective), but its use is still prevailing. In vivo studies have revealed that exposure to triclosan results in a decreased acetylcholinesterase (AChE) activity. However mechanistic insights into AChE inhibition by triclosan are missing. Using in vitro AChE activity assay with purified AChE, we show that triclosan acts as a direct inhibitor of AChE and inhibits AChE activity in a dose-dependent manner. Given the function of AChE, any alteration in its activity can be neurotoxic. Our results provide important mechanistic insights into triclosan induced neurotoxicity with AChE as a target.

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The Tetramethylpyrazine Derivative Statmp-151: A Novel Small Molecule Stat3 Inhibitor With Promising Activity Against Breast Cancer

Frontiers in Pharmacology ◽

10.3389/fphar.2021.651976 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chen Fan ◽

Yijie Wang ◽

Hui Huang ◽

Wenzhen Li ◽

Jialin Ma ◽

...

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Potential Candidate ◽

Dependent Manner ◽

The Matrix

Breast cancer is the most common malignancy in women and is a molecularly heterogeneous disease. Signal transducer and activator of transcription 3 (Stat3) is overexpressed and hyperactivated in a variety of human tumours, including breast cancer, thus representing a promising target for breast cancer treatment. In the present study, we evaluated the activities of a novel Stat3 inhibitor named Statmp-151 in the human breast cancer cell lines MCF-7 and MDA-MB-231 and the murine mammary carcinoma cell line 4T1. The in vitro results showed that Statmp-151 inhibited the proliferation of breast cancer cell lines in a dose- and time-dependent manner and suppressed the phosphorylation of Stat3 in a dose-dependent manner. Flow cytometry (FCM) assays revealed that Statmp-151 affected mitochondrial membrane potential and reactive oxygen species (ROS) production. Furthermore, Statmp-151 inhibited cell migration, as shown by analysis of the matrix metalloproteinases MMP2 and MMP9. Finally, in a 4T1 tumour-bearing mouse model, intraperitoneal injection of 30 mg/kg/day Statmp-151 significantly suppressed the growth of tumours without obvious toxicity. These results indicated that Statmp-151 might be a potential candidate for the treatment of breast cancer.

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GTP Is Required for Iron-Sulfur Cluster Biogenesis in Mitochondria

Journal of Biological Chemistry ◽

10.1074/jbc.m706808200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1362-1371 ◽

Cited By ~ 30

Author(s):

Boominathan Amutha ◽

Donna M. Gordon ◽

Yajuan Gu ◽

Elise R. Lyver ◽

Andrew Dancis ◽

...

Keyword(s):

Atp Hydrolysis ◽

Dependent Manner ◽

Gtp Hydrolysis ◽

Iron Sulfur Cluster ◽

Isolated Mitochondria ◽

Iron Sulfur ◽

The Matrix

Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.

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ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner

Journal of Cell Science ◽

10.1242/jcs.105.3.807 ◽

1993 ◽

Vol 105 (3) ◽

pp. 807-818

Author(s):

D.C. Mah ◽

P.A. Dijkwel ◽

A. Todd ◽

V. Klein ◽

G.B. Price ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Matrix ◽

Pcr Amplification ◽

S Phase ◽

Dependent Manner ◽

The Matrix ◽

S Period ◽

Cycle Dependent

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

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Radiolabeled r-Hirudin as a Measure of Thrombin Activity at, or within, the Rabbit Aorta Wall In Vitro and In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642467 ◽

1994 ◽

Vol 71 (04) ◽

pp. 499-506 ◽

Cited By ~ 10

Author(s):

Mark W C Hatton ◽

Bonnie Ross-Ouellet

Keyword(s):

Rabbit Aorta ◽

Balloon Injury ◽

Recombinant Hirudin ◽

Aorta Wall ◽

Thrombin Activity ◽

Before And After ◽

The Matrix

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

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