Advanced glycation end-products induce endoplasmic reticulum stress in human aortic endothelial cells

Background: Advanced glycation end products (AGEs), the final products of the Maillard reaction, have been shown to impair endothelial proliferation and function, thus contributing to endothelial cell injury present in diabetes, inflammatory and cardiovascular diseases. Endoplasmic reticulum (ER) stress triggered under hyperglycemic, hypoxic and oxidative conditions has been implicated in endothelial dysfunction through activation of the unfolded protein response (UPR). The present study investigates the role of AGEs in ER stress induction in human aortic endothelial cells exposed to variable AGE treatments. Methods: Human aortic endothelial cells (HAEC) were treated with increasing concentrations (100, 200 μg/mL) of AGE-bovine serum albumin (AGE-BSA) at different time-points (24, 48, 72 h). The induction of ER stress and the involved UPR components were investigated on mRNA and protein levels. Apoptosis was quantitatively determined by flow cytometry detecting propidium iodide expression and annexin V binding simultaneously. Results: AGEs administration significantly reduced HAEC proliferation in a time- and dose-dependent manner. An immediate induction of the ER chaperones GRP78, GRP94 and the transcriptional activator, XBP-1 was observed at 24 h and 48 h. A later induction of the phospho-lF2α and proapoptotic transcription factor CHOP was observed at 48 h and 72 h, being correlated with elevated early apoptotic cell numbers at the same time-points. Conclusions: The present study demonstrates that AGEs directly induce ER stress in human aortic endothelial cells, playing an important role in endothelial cell apoptosis. Targeting AGEs signaling pathways in order to alleviate ER stress may prove of therapeutic potential to endothelial dysfunction-related disorders

Lack of Association Between Estrogen Receptor-Alpha Single-Nucleotide Polymorphism (Codon 594 G-->A) and Postmenopausal Osteoporosis: A Pilot Study

Background: Recent studies have suggested that the estrogen receptor alpha (ERα) gene is implicated in reduced bone mineral density (BMD). Objective: In the present study, we investigated the relationship between genetic polymorphism in ΕRα 2014G-->A (T594T) (codon 594 G-->A) and osteoporosis in postmenopausal women. Methods: A total of 59 postmenopausal women were included in the study (21 normal, 24 osteoporotic, 14 osteopenic). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the ΕRα 2014G->A polymorphism. Results: The three groups were found to be in genetic equilibrium. Also, there were no allele (p=0.578) and/or genotype frequencies (p=0.59) among the groups, i.e. the three groups could be treated as a genetically uniform population. As expected, normal women exhibited the highest BMD values (1.11 g/cm2) followed by the osteopenic (0.91 g/cm2) and the osteoporotic women (0.73 g/cm2). There was significant difference (p<0.05) in age only between the osteoporotic group (mean age 48.7 years) and either the normal (mean age 46 years) or the osteopenic group (mean age 45 years). No allele and/or genotype effect on BMD or age was detected. Conclusion: Our results support the lack of association between ERα codon 594 and BMD in postmenopausal women, since we found no allele and/or genotype effect on BMD or age. Further studies in a larger sample of postmenopausal women are needed to confirm our results

Estrogen Receptor Subtypes Elicit a Distinct Gene Expression Profile of Endothelial-Derived Factors Implicated in Atherosclerotic Plaque Vulnerability

In the presence of established atherosclerosis, estrogens are potentially harmful. MMP-2 and MMP-9, their inhibitors (TIMP-2 and TIMP-1), RANK, RANKL, OPG, MCP-1, lysyl oxidase (LOX), PDGF-β, and ADAMTS-4 play critical roles in plaque instability/rupture. We aimed to investigate (i) the effect of estradiol on the expression of the abovementioned molecules in endothelial cells, (ii) which type(s) of estrogen receptors mediate these effects, and (iii) the role of p21 in the estrogen-mediated regulation of the aforementioned factors. Human aortic endothelial cells (HAECs) were cultured with estradiol in the presence or absence of TNF-α. The expression of the aforementioned molecules was assessed by qRT-PCR and ELISA. Zymography was also performed. The experiments were repeated in either ERα- or ERβ-transfected HAECs and after silencing p21. HAECs expressed only the GPR-30 estrogen receptor. Estradiol, at low concentrations, decreased MMP-2 activity by 15-fold, increased LOX expression by 2-fold via GPR-30, and reduced MCP-1 expression by 3.5-fold via ERβ. The overexpression of ERα increased MCP-1 mRNA expression by 2.5-fold. In a low-grade inflammation state, lower concentrations of estradiol induced the mRNA expression of MCP-1 (3.4-fold) and MMP-9 (7.5-fold) and increased the activity of MMP-2 (1.7-fold) via GPR-30. Moreover, p21 silencing resulted in equivocal effects on the expression of the abovementioned molecules. Estradiol induced different effects regarding atherogenic plaque instability through different ERs. The balance of the expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.The European Partnership for the Assessment of Risks from Chemicals has received funding from the European Union’s Horizon Europe research and innovation program under Grant Agreement No 101057014 and has received co-funding of the authors’ institutions. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.info:eu-repo/semantics/publishedVersio

Ιnvestigation of the mechanism of action of spruce lignans in breast cancer cells and in endothelial cells

The aim of the present work was to investigate the biological effects of plant lignans, in endothelial and breast cancer cells. Lignans are plant secondary products with several types of biological activity. The study is divided in two parts. In the first part, plant lignans were investigated regarding their possible anti-inflammatory/antiatherogenic properties in human aortic endothelial cells (HAEC). Our findings suggest that the lignans7-hydroxymatairesinol and 7-hydroxymatairesinol 2, exhibit strong anti-inflammatory properties in endothelial cells, at least in part, through attenuation of NF-κΒ and ERK phosphorylation. We also investigated the possible estrogenic/antiestrogenic activities of the plant lignans with the use of the estrogen dependent cell line MCF-7 (ERα positive, ERβ negative). According to our resultes lignans modulate MCF-7 cell proliferation but none of the tested compounds exerts estrogenic or antiestrogenic activities through the estrogen response elements (EREs).Η παρούσα διατριβή αφορά τη διερεύνηση των βιολογικών δράσεων φυτικών λιγνανίων σε ενδοθηλιακά κύτταρα και κύτταρα καρκίνου μαστού. Τα λιγνάνια είναι δευτερογενείς μεταβολίτες των φυτών, ανήκουν στην τάξη των φυτοοιστρογόνων και εμφανίζουν πληθώρα βιολογικών δράσεων. Το πρώτο μέρος της μελέτης, αφορά τη διερεύνηση της επίδρασης απομονωμένων λιγνανίων σε ενδοθηλιακά κύτταρα αορτής. Με τον τρόπο αυτό διερευνήθηκαν πιθανές αντιαθηρωματικές/αντιφλεγμονώδεις δράσεις των υπό εξέταση ενώσεων. Σύμφωνα με τα πειραματικά αποτελέσματα τα βασικά λιγνάνια της διατροφής 7-υδρόξυματαιρεσινόλη και το απομονωμένο κύριο στερεοϊσομερές της 7-υδρόξυματαιρεσινόλη 2, παρουσιάζουν ισχυρές αντιφλεγμονώδεις δράσεις οι οποίες πραγματοποιούνται εν μέρει, μέσω αναστολής του μεταγραφικού παράγοντα ΝF-κΒ και της φωσφορυλίωσης της κινάσης ERK. Οι πυρηνικοί υποδοχείς ER και PPARγ δεν φαίνεται να ενέχονται στο μηχανισμό αντιφλεγμονώδους δράσης των ενώεσων αυτών στη διαδικασία της αθηρωμάτωσης. Στο δεύτερο μέρος της διατριβής διερευνήθηκε η οιστρογονική/αντιοιστρογονική δράση των υπό εξέταση ενώσεων σε κύτταρα καρκίνου μαστού. Για το σκοπό αυτό χρησιμοποιήθηκε η οιστρογονοεξαρτώμενη κυτταρική σειρά MCF-7 που εκφράζει κυρίως τον υποδοχέα οιστρογόνων ERα. Τα υπό εξέταση λιγνάνια ρυθμίζουν τον πολλαπλασιασμό κυττάρων οιστρογονοεξαρτώμενου καρκίνου μαστού, δεν ασκούν ωστόσο οιστρογονικές ή αντιοιστρογονικές δράσεις μέσω οιστρογονοαποκρινόμενων στοιχείων EREs

Applicability of in silico tools for the prediction of dermal absorption for pesticides

Based on the “Human in vitro dermal absorption datasets” published as supporting information to the revised EFSA Guidance on Dermal Absorption, in silico models for prediction of absorption across the skin have been evaluated. For this evaluation, a systematic literature search and review was performed, identifying 288 publications describing mathematical models for prediction of dermal absorption. Eleven models potentially relevant to the regulatory assessment of pesticides and which cover a range of approaches were selected for in depth evaluation. This included three mixture models taking into account physicochemical properties of the co‐formulants such as polar surface area, hydrogen bonding or octanol‐water partition coefficients. Additional data on the pesticidal active substances and information on the composition of some of the formulations covered in the dermal absorption dataset were gathered, as these were required as input parameters for the selected models. The models were implemented with settings reflecting as much as possible realistic exposure scenarios and the experimental conditions under which the measured data were obtained. As the majority of the models predicted either maximum flux or the permeation coefficient, further combination with a model achieving translation into percentage absorption was required. This was done with and without consideration of the lag time. Only one model directly predicted percentage dermal absorption, which was a spreadsheet‐based single substance model taking into consideration several skin parameters, experimental conditions, various physicochemical properties of the active substance and the type of vehicle. Statistical analysis of model predictions revealed overall low concordance with measured values, thereby limiting regulatory acceptance. Additional analysis was performed on the results of two mixture models and the above mentioned complex single substance model which showed moderate correlation between predicted and measured data. Options to improve model performance were discussed and Bayesian random effects modelling was explored to adjust predicted percentage dermal absorption to measured data as was model combination. When taking into account observed uncertainties of predictions, one of the models may provide a Tier 2 tool to estimate dermal absorption value in the absence of adequate experimental data when the predicted values are in the range of 10 to 70%. However, further work is needed to better understand the effect of co‐formulants on dermal absorption of pesticides and to improve model predicitivity

Phenolic Acid Composition, Antiatherogenic and Anticancer Potential of Honeys Derived from Various Regions in Greece

<div><p>The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.</p></div

Floral source and regions of honey collection.

a<p>Indicating pollen grains of <i>Thymus capitatus.</i></p

Correlation study between the results of biological tests and phenolic acid content^a^,^b^,^c.

a<p>PCA, protochatechuic acid, p-HBA, p-hydroxybenzoic acid, VA, vanillic acid, CA, caffeic acid, p-COUA, p-coumaric acid, TP, total phenolic content.</p>b<p>Values represent Pearson’s correlation coefficient (r).</p>c<p>The minimum level of significance was set at p<0.05 (*), p<0.01 (**), p<0.001 (***).</p