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Medicinal plants used in European folk medicine attached to Lamiales, Gentianales or Asterales orders are used to treat inflammatory disorders. Many targets have been identified but to date, implication of purinergic receptor P2X7 activation has not yet been investigated. We managed to evaluate the protective effect on P2X7 activation by plant extracts used as anti-inflammatory in European folk medicine by the YO-PRO-1 uptake dye inâ vitro bioassay. Results revealed that among our selected plants, species from Scrophularia and Plantago genus were able to decrease significantly P2X7 activation (>50 % at 0.1 and 1â µg/mL). UPLC/MS, dereplication and metabolomic analysis of Scrophularia extracts, allowed us to identify the cinnamoyl-iridoid harpagoside as putative inhibitor of P2X7 activation. These results open a new research field regarding the anti-inflammatory mechanism of cinnamoyl-iridoids bearing plants, which may involve the P2X7 receptor.
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Plantas Medicinais , Scrophularia , Receptores Purinérgicos P2X7 , Iridoides/farmacologia , Anti-Inflamatórios/farmacologia , Extratos Vegetais/farmacologiaRESUMO
Background: It is unclear whether delays in care affect prognosis of patients with lung cancer. The primary objective of this study was to describe the care pathway of patients diagnosed with lung cancer in a French region. Secondary objectives were to identify markers associated with 1) time from imaging to treatment and 2) with 1-year survival. Methods: In a retrospective cohort study, clinical data from multidisciplinary team meetings for all incident lung cancer cases discussed in 2018 in one French region were matched with medico-administrative data from the National Health Insurance Database. Care pathway time intervals were estimated for small cell lung cancer (SCLC), resected nonsmall cell lung cancer (NSCLC) and unresected NSCLC. Factors associated with delay in the care pathway were identified using linear regression; 1-year survival was analysed using Cox modelling. Results: A total of 685 patients were included. Median time between imaging and treatment was 49â days (interquartile range: 33-73), and was lower in cases of metastatic disease, SCLC and private care. At 1â year, 48% had died (resected NSCLC 12%). In unresected NSCLC, time from diagnostic imaging to first treatment <49â days was associated with a higher risk of death. Time intervals were similar in patients with squamous cell carcinoma versus adenocarcinoma or undifferentiated carcinoma. Discussion: Time intervals in the care pathways of lung cancer were similar to previous reports, confirming the robustness of retrospective databases. In unresectable NSCLC, rapid care was not associated with better survival.
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Chlorpyrifos is a pesticide that is toxic to human health and has been banned for the past decade. Due to its persistent and bioaccumulative properties, chlorpyrifos is still present in soil. Pregnant women can be exposed to chlorpyrifos through drinking water and herbal products, such as essential oils (EOs), resulting in adverse effects to the mother and fetus. Our objective was to evaluate and compare the potential endocrine disrupting effects of chlorpyrifos "free" or in contaminated lavender EO. We studied the release of four hormones and the activation of the P2X7 cell death receptor in human placental JEG-Tox cells as key biomarkers of endocrine toxicity for pregnant women (hPlacentox assay). We observed that "free" chlorpyrifos disrupted placental hormones and activated the P2X7 receptor, whereas chlorpyrifos in lavender EO disrupted only the placental hormones. We confirm that chlorpyrifos can be classified as an endocrine disrupting chemical (EDC) for pregnant women and point out that its endocrine disrupting effect may not be apparent when present in lavender EOs. Our results reveal the existence of specific reverse cocktail effects that may have protective properties against EDCs.
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Clorpirifos , Água Potável , Disruptores Endócrinos , Lavandula , Óleos Voláteis , Praguicidas , Clorpirifos/toxicidade , Disruptores Endócrinos/toxicidade , Feminino , Hormônios , Humanos , Placenta , Hormônios Placentários , Gravidez , Receptores de Morte Celular , Receptores Purinérgicos P2X7 , SoloRESUMO
Pregnant women may use EOs in case of morning sickness, nausea, stress management, etc. Little is known about the potential danger that EOs represent for the placenta and therefore for the pregnancy. Our aim was to explore and compare the placental toxicity and potential endocrine disrupting effects of niaouli, orange, tea tree, wintergreen and ylang-ylang EOs, and their key compounds: 4-terpineol, 1,8-cineol, limonene, methyl salicylate and benzyl salicylate. We studied the release of four hormones and the activation of P2X7 receptor in JEG-Tox human placental cells as key biomarkers for endocrine toxicity. We observed that niaouli, orange, tea tree, wintergreen and ylang-ylang EOs and their key components disrupted at least one of the studied hormones but none of them activated the P2X7 cell death receptor. The tested EOs appear then to be more hormonal modulators rather than EDCs in human placental cells. The hormonal effects observed with the key components were very different from those observed with the EOs. EOs are very complex mixtures, and it is essential to study whole EOs rather than their components individually in safety assessment.
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Forskolin, used in folk medicine since ancient times, is now available as a dietary supplement, with an indication as a fat burner and appetite suppressant. However, the safety of forskolin is poorly documented especially for pregnant women. The question that we raised is what about the safety of forskolin in pregnant women? As the placenta, an endocrine organ, is the key organ of pregnancy, we evaluated the in vitro placental toxicity of forskolin. We focused first on the activation of a P2X7 degenerative receptor as a key biomarker for placental toxicity, and second on steroid and peptide hormonal secretion. We observed that forskolin activated P2X7 receptors and disturbed estradiol, progesterone, hPL and hyperglycosylated hCG secretion in human placental JEG-Tox cells. To the best of our knowledge, we highlighted, for the first time, that forskolin induced endocrine disturbance in placental cells. Forskolin does not appear to be a safe product for pregnant women and restrictions should be taken.
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In pregnant women, the lungs, skin and placenta are exposed daily to endocrine-disrupting chemicals (EDCs). EDCs induce multiple adverse effects, not only on endocrine organs, but also on non-endocrine organs, with the P2X7 cell death receptor being potentially the common key element. Our objective was first to investigate mechanisms of EDCs toxicity in both endocrine and non-endocrine cells through P2X7 receptor activation, and second, to compare the level of activation in lung, skin and placental cells. In addition, apoptosis in placental cells was studied because the placenta is the most exposed organ to EDCs and has essential endocrine functions. A total of nine EDCs were evaluated on three human cell models. We observed that the P2X7 receptor was not activated by EDCs in lung non-endocrine cells but was activated in skin and placenta cells, with the highest activation in placenta cells. P2X7 receptor activation and apoptosis are pathways shared by all tested EDCs in endocrine placental cells. P2X7 receptor activation along with apoptosis induction could be key elements in understanding endocrine placental and skin disorders induced by EDCs.
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Disruptores Endócrinos , Disruptores Endócrinos/metabolismo , Disruptores Endócrinos/toxicidade , Sistema Endócrino , Feminino , Humanos , Placenta/metabolismo , Gravidez , Gestantes , Receptores Purinérgicos P2X7/metabolismoRESUMO
BACKGROUND: Bisphenol A (BPA), a reprotoxic and endocrine-disrupting chemical, has been substituted by alternative bisphenols such as bisphenol F (BPF) and bisphenol S (BPS) in the plastic industry. Despite their detection in placenta and amniotic fluids, the effects of bisphenols on human placental cells have not been characterized. Our objective was to explore in vitro and to compare the toxicity of BPA to its substitutes BPF and BPS to highlight their potential risks for placenta and then pregnancy. METHODS: Human placenta cells (JEG-Tox cells) were incubated with BPA, BPF, and BPS for 72 h. Cell viability, cell death, and degenerative P2X7 receptor and caspases activation, and chromatin condensation were assessed using microplate cytometry and fluorescence microscopy. RESULTS: Incubation with BPA, BPF, or BPS was associated with P2X7 receptor activation and chromatin condensation. BPA and BPF induced more caspase-1, caspase-9, and caspase-3 activation than BPS. Only BPF enhanced caspase-8 activity. CONCLUSIONS: BPA, BPF, and BPS are all toxic to human placental cells, with the P2X7 receptor being a common key element. BPA substitution by BPF and BPS does not appear to be a safe alternative for human health, particularly for pregnant women and their fetuses.
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Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.
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Benzo(a)pireno/antagonistas & inibidores , Benzo(a)pireno/toxicidade , Phaeophyceae , Receptores Purinérgicos P2X7/metabolismo , Taninos/farmacologia , Células A549 , Quimioprevenção/métodos , Relação Dose-Resposta a Droga , Humanos , Taninos/isolamento & purificaçãoRESUMO
Placental alterations are responsible for adverse pregnancy outcomes like preeclampsia and intrauterine growth restriction. And yet, placenta toxicology has not become a fully-fledged toxicology field. Because placenta is very often seen only as a barrier between the mother and the fetus, there is a lack and therefore a need for an experimental human model with technical recommendations to study placenta toxicology. In vitro approaches are recommended in experimental toxicology as they focus on a specific biological process and yield high-throughput screening methods. In the present study, we first established incubation conditions to preserve signatures of the human JEG-3 cell line identity while enabling toxicity detection. JEG-3 cells prepared in our incubation conditions were renamed JEG-Tox cells. As placental alterations are mainly triggered by uncontrolled apoptosis, we second used known apoptotic agents pregnant women are exposed to, to check that JEG-Tox cells can trigger apoptosis. Ethanol, bisphenol F, quinalphos, 4,4'-DDT, benzalkonium chloride, phenoxyethanol, propylparaben, and perfluorooctanic acid all induced chromatin condensation in JEG-Tox cells. Our incubation conditions allow JEG-Tox cells to keep placental cell identity and to respond to toxic chemicals. JEG-Tox cells are a pertinent model for placenta toxicology and could be used to better understand pregnancy alterations.
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Detergent chemicals, widely used in household products, in pharmaceutical, medical, cosmetic and industrial fields, have been linked to side effects and involved in several eye diseases. On the ocular surface, detergents can interfere with the corneal epithelium, the most superficial layer of the cornea, representing a line of defence against external aggression. Despite its major role in numerous biological functions, there is still little data regarding disruption of lipid homeostasis induced by ocular irritants. To this purpose, a lipidomic analysis using UPLC-HRMS/MS-ESI ± was performed on human corneal epithelial (HCE) cells incubated with three widely known ocular irritants: benzalkonium chloride (BAK), sodium lauryl sulfate (SLS) and Triton X-100 (TXT). We found that these ocular irritants lead to a profound modification of the HCE cell lipidome. Indeed, the cell content of ceramide species increased widely while plasmalogens containing polyunsaturated fatty acid species, especially docosahexaenoic acids, decreased. Furthermore, these irritants upregulated the activity of phospholipase A2. The present study demonstrates that BAK, SLS and TXT induced disruption of the cell lipid homeostasis, highlighting that lipids mediate inflammatory and cell death processes induced by detergents in the cornea. Lipidomics may thus be regarded as a valuable tool to investigate new markers of corneal damage.
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Detergentes/toxicidade , Epitélio Corneano/química , Epitélio Corneano/patologia , Oftalmopatias/induzido quimicamente , Irritantes/toxicidade , Lipidômica , Fosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Compostos de Benzalcônio/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Oftalmopatias/metabolismo , Humanos , Inflamação/induzido quimicamente , Metabolismo dos Lipídeos/efeitos dos fármacos , Octoxinol/toxicidade , Plasmalogênios/metabolismo , Dodecilsulfato de Sódio/toxicidadeRESUMO
Mydriasis is required prior to many eye examinations and ophthalmic surgeries. Nowadays, phenylephrine hydrochloride (PHE) and tropicamide (TPC) are extensively used to induce mydriasis. Several pharmaceutic dosage forms of these two active ingredients have been described. However, no optimal therapeutic strategy has reached the market. The present work focuses on the formulation and evaluation of a mucoadhesive ion-activated in situ gelling delivery system based on gellan gum and hydroxyethylcellulose (HEC) for the delivery of phenylephrine and tropicamide. First, in vitro drug release was studied to assess appropriate sustained drug delivery on the ocular surface region. Drug release mechanisms were explored and explained using mathematical modeling. Then, in situ gelling delivery systems were visualized using scanning electron microscopy illustrating the drug release phenomena involved. Afterward, cytotoxicity of the developed formulations was studied and compared with those of commercially available eye drops. Human epithelial corneal cells were used. Finally, mydriasis intensity and kinetic was investigated in vivo. Mydriasis pharmacodynamics was studied by non-invasive optical imaging on vigilant rabbits, allowing eye blinking and nasolacrimal drainage to occur physiologically. In situ gelling delivery systems mydriasis profiles exhibited a significant increase of intensity and duration compared with those of conventional eye drops. Efficient mydriasis was achieved following the administration of a single drop of in situ gel reducing the required amount of administered active ingredients by four- to eight-fold compared with classic eye drop regimen.
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This paper reports the isolation and structural characterization of four new ent-kaurane derivatives from the Lamiaceae plant Sideritis hyssopifolia. Planar structures and relative configurations were determined using both mass spectrometry and nuclear magnetic resonance (1D and 2D). Absolute configurations were determined by comparing experimental and theoretical electronic circular dichroism spectra. The cytotoxic and microbial activities of all new compounds were tested. Compounds that were non-cytotoxic were further evaluated for anti-inflammatory activity.
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Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Diterpenos do Tipo Caurano/farmacologia , Extratos Vegetais/farmacologia , Sideritis/química , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/isolamento & purificação , Humanos , Modelos Moleculares , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Análise EspectralRESUMO
Degenerative diseases are characterized by both cell death and inflammation, which involve different pathways such as apoptosis and pyroptosis. Oxysterols, oxidized derivatives of cholesterol, are known to act as key actors in degenerative disorders such as skin photoaging. We hypothesize that oxysterols could be implicated in either apoptosis or pyroptosis, or both. The aim of our study was first to quantify oxysterol levels in keratinocytes as a function of aging and UV irradiation. Second, we evaluated the effects of 25-OH oxysterol on apoptosis and pyroptosis hallmarks in keratinocytes. Our results showed that 25-OH exhibited an increasing after UV irradiation, highlighting the pivotal role of this oxysterol in skin degeneration. In our model, 25-OH induced not only caspases-dependent apoptosis associated to granzyme B release but also P2X7 receptor-dependent pyroptosis in skin cells. 25-OH seems to be at the origin of the main toxic pathways responsible for degenerative disorders; therefore, it could be the target of antidegenerative treatments, opening new potential therapeutic strategies.
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Apoptose/efeitos dos fármacos , Caspases/metabolismo , Hidroxicolesteróis/farmacologia , Modelos Biológicos , Piroptose/efeitos dos fármacos , Receptores Purinérgicos P2X7/metabolismo , Pele/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Pré-Escolar , Relação Dose-Resposta a Droga , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pessoa de Meia-Idade , Pele/metabolismo , Pele/patologia , Relação Estrutura-Atividade , Raios UltravioletaRESUMO
The YO-PRO-1 assay provides a quantitative estimation of P2X7 receptor activation. P2X7 receptor is associated to pathological conditions including infectious, inflammatory, neurological, musculoskeletal disorders, pain and cancer. Most primary cells and cell lines from diverse origin may be used thanks to the ubiquitous distribution of P2X7 receptor. To study the activation of P2X7 receptor by chemicals or biological agents, we established a microplate-based cytometry protocol to accurately and rapidly quantify the activation of P2X7 receptor that leads to the formation of large pores in cell membranes. The YO-PRO-1 assay is based on the ability of cells to incorporate and bind YO-PRO-1 dye to DNA after activation of P2X7 receptor through pore formation. Cells are seeded in 96-well plates and incubated with the compound being tested for the appropriate time. The microplate is then incubated for 10 min with YO-PRO-1 staining solution. After the 10 min staining time, fluorescence signal is read using a microplate reader in 1 min. This procedure is easier and requires less handling steps than flow cytometry. 96-well plate based YO-PRO-1 assay is a reproducible and fast method to study both P2X7 receptor activation by toxic agents at subnecrotic concentrations and P2X7 receptor inhibition by antagonists.
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The P2X7 receptor is expressed in both anterior and posterior segments of the eyeball. In the ocular surface, the P2X7 receptor is activated in case of external aggressions: preservatives and surfactants induce the activation of P2X7 receptors, leading to either apoptosis, inflammation, or cell proliferation. In the retina, the key endogenous actors of age-related macular degeneration, diabetic retinopathy, and glaucoma act through P2X7 receptors' activation and/or upregulation of P2X7 receptors' expression. Different therapeutic strategies aimed at the P2X7 receptor exist. P2X7 receptor antagonists, such as divalent cations and Brilliant Blue G (BBG) could be used to target either the ocular surface or the retina, as long as polyunsaturated fatty acids may exert their effects through the disruption of plasma membrane lipid rafts or saffron that reduces the response evoked by P2X7 receptor stimulation. Treatments against P2X7 receptor activation are proposed by using either eye drops or food supplements.
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Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.
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Hidroxicolesteróis/química , Queratinócitos/citologia , Lipídeos/química , Envelhecimento da Pele , Pele/citologia , Autofagia , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Ceramidas/química , Meios de Cultura , Citoesqueleto/metabolismo , Citometria de Fluxo , Humanos , Queratinócitos/efeitos da radiação , Luz , Microscopia Confocal , Necrose , Oxisteróis/química , Precursores de Proteínas/química , Raios UltravioletaRESUMO
Age-related macular degeneration (AMD) is the most common cause of severe vision loss worldwide. Amyloid ß involvement in degenerative diseases such as AMD is well known and its toxicity has been related to P2X7 receptor-pannexin-1. Recently, oxysterols (oxidized derivatives of cholesterol) have been implicated in AMD pathogenesis. The aim of our study was to highlight amyloid ß/oxysterols relationship and to describe P2X7 receptor-pannexin-1 role in oxysterols toxicity. Using retinal epithelial cells, we first quantified sterols levels after amyloid ß incubation and second we investigated the cytotoxic effects induced by oxysterols. For the first time, our results showed that amyloid ß induced oxysterols formation in human retinal pigmented epithelial cells. We showed that oxysterol toxicity is mediated by P2X7 receptor activation. This activation was dependent on pannexin-1 with 25-hydroxycholesterol whereas P2X7 receptor signaling pathway was pannexin-1-independent for 7-ketocholesterol. Taken together our data suggest a pivotal role of P2X7 receptor-pannexin-1 in oxysterols toxicity in retinal cells which could be an important target to develop new treatments for AMD.
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Peptídeos beta-Amiloides/química , Conexinas/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Proteínas do Tecido Nervoso/metabolismo , Oxisteróis/toxicidade , Receptores Purinérgicos P2X7/metabolismo , Retina/patologia , Linhagem Celular , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Humanos , Necrose , Estresse Oxidativo/efeitos dos fármacos , Retina/metabolismoRESUMO
A salbutamol sulfate (SS)-Poloxamer bioadhesive hydrogel specially developed for buccal administration was investigated by studying interactions with TR146 human buccal epithelium cells (i.e. cellular toxicity (i) and trans-epithelial SS diffusion (ii)). The assessment of cell viability (MTT, Alamar Blue), membrane integrity (Neutral Red), and apoptosis assay (Hoechst 33342), were performed and associated to Digital Holographic Microscopy analysis. After the treatment of 2h, SS solution induced drastic cellular alterations that were prevented by hydrogels in relation with the concentrations of poloxamer and xanthan gum. The formulation containing P407 19%/P188 1%/Satiaxane 0.1% showed the best tolerance after single and multiple administrations and significantly reduced the trans-epithelial permeability from 5.00±0.29 (×10(3)) (SS solution) to 1.83±0.22 cm/h. Digital Holographic Microscopy images in good agreement with the viability data confirmed the great interest of this direct technique. In conclusion, the proposed hydrogels represent a safe and efficient buccal drug delivery platform.