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1	The effect of nonylphenol and bisphenol A on calcium signaling and viability in cultured cells Kuo, Chun-Chi 23 June 2010 (has links) Environmental chemicals may affect human health by disrupting endocrine function. Many of the endocrine disrupting chemicals (EDCs) are estrogens or estrogen-like molecules that have been classified as environmental estrogens or xenoestrogens (XEs). XEs include endosulfan, chlordance, nonylphenol, bisphenol A, octylphenol, and coumestrol, etc. Although these compounds have wide structural diversity, but all have in common the and/or other hydrophobic components. Many studies have shown that XEs affect cell viability. For instance, Nonylphenol is used in surfactants or plasticizers and bisphenol A (4, 4¡¦-isopropylidene-2-diphenol) is used as protective coatings on food containers and for composites and sealants in dentistry. Most previous studies have focused on the toxicity of XEs on development process and reproductive system, especially in aquatic ecosystems. Thus, the effects of these two environmental chemicals on the toxicological effect are still controversial. The aim of this study is to investigate the molecular mechanisms of nonylphenol and bisphenol A in induction of cell death in human gastric cancer (SCM-1) cells and Madin Darby canine renal tubular (MDCK) cells. First, WST-1 reduction assays and propidium iodide-staining assay were used to determine cell viability and apoptosis in the present of nonylphenol and bisphenol A. Furthermore, we will use immunoblotting to measure the activity of apoptotic markers caspase-3, mitogen-activated protein kinases (MAPKs) to survey how nonylphenol affects apoptotic pathways. Besides, I will explore bisphenol A whether induces cell death and the mechanisms underlying the [Ca2+]i rise in MDCK cells. The results may be helpful for understanding the pharmacological and toxicological effects of these two environmental chemicals in cells from important organs. Results showed that nonylphenol caused apoptosis via the activation of caspase-3 in cultured human gastric cancer (SCM-1) cells. Although nonylphenol could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Nonylphenol was also found to induce [Ca2+]i increases and pretreatment with BAPTA/AM, a Ca2+ chelator, prevented nonylphenol-induced [Ca2+]i increases, and protect cells from death. These results suggest that nonylphenol induced apoptosis via a Ca2+- and p38 MAPK-dependent pathway. On the other hand, the effect of the environmental contaminant bisphenol A on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Bisphenol A at concentrations between 50-300 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Bisphenol A induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholiapase A2 inhibitor aristolochic acid, store-operated Ca2+ channel blockers nifedipine and SK&F96365; and protein kinase C inhibitor GF109203X. In Ca2+-free medium, pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) and the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited bisphenol A-induced Ca2+ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca2+]i rise. Bisphenol A caused concentration-dependent decrease in cell viability via apoptosis in a Ca2+-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca2+]i rises by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and mitochondria and Ca2+ influx via phospholipase A2-protein kinase C-sensitive store-operated Ca2+ channels. Key words: calcium, apoptosis, human gastric cancer cells (SCM-1), Madin Darby canine kidney (MDCK), nonylphenol, bisphenol A. human gastric cancer cells (SCM-1) Madin Darby canine kidney (MDCK) nonylphenol bisphenol A apoptosis calcium
2	Inhibition of Phorbol Ester-Stimulated Arachidonic Acid Release by Alkylglycerols Robinson, Mitchell, Burdine, Robin, Warne, Thomas R. 09 February 1995 (has links) Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C. alkylglycerol arachidonic acid madin-darby canine kidney monoglyceride phorbol myristate acetate protein kinase C
3	Henry Madin. Traitement des lacunes dans l'orchestre des motets à grand chœur: Spécificités et problématiques d’une pratique entre science et art Balthazart, Fabian 03 October 2015 (has links) S'il est aujourd'hui moins connu que ses contemporains André Campra (1660-1744) ou Jean-Philippe Rameau (1683-1764), l'abbé Madin (1698-1748), selon Évrard Titon du Tillet « un des meilleurs compositeurs de ce siècle pour les motets », est une des figures les plus représentatives de la vie musicale sous Louis XV. Il fut, post mortem, le compositeur le plus joué à la Chapelle royale de Versailles. Comme une partie du répertoire de cette institution, ses motets à grand chœur nous sont parvenus sous forme de partitions réduites. Cet usage, propre à la musique française de l'Ancien Régime, consistait à copier les chœurs, les solistes, les dessus de violon et la basse continue en omettant les parties intérieures de l'orchestre. En nombre variable, de deux à trois, ces parties, qui existaient sur le matériel d'orchestre, ont très souvent été perdues. Pour restituer cette musique aujourd'hui, une des options possibles consiste à recomposer ces parties selon les critères esthétiques de l'époque et le style propre à chaque compositeur.La présente thèse, tout d'abord, entame une réflexion sur l'acte d'ajout de parties manquantes dans les œuvres lacunaires du passé. Elle propose ensuite, une restitution de l'intégrité orchestrale de trois motets à grand chœur de Henry Madin (Beatus vir, De profundis, Te Deum) qui servirent de terrain d'expérimentation. Ils sont présentés dans leur intégralité, en partitions et, pour certains extraits, en enregistrements. Enfin, la thèse tend à démontrer qu'une telle démarche éclaire les œuvres d'une nouvelle lumière et leur redonne un éclat nouveau que la patine avait terni.Bien que l'ajout de parties ne puisse rendre son état originel aux motets abordés, le postulat de départ est de se positionner dans un contexte historico-stylistique le plus proche possible des sources étudiées. Cette attitude passe par l'étude des formes musicales, du contrepoint et de l'harmonie au XVIIIe siècle, des sources musicales, des effectifs et des traditions de la Chapelle royale de Versailles. Subséquemment, ces recherches, indispensables à la mise en œuvre de l'expérimentation, apportent un éclairage original sur les pratiques musicales et les œuvres de cette institution dans la première moitié dudit siècle.Aussi poussées que puissent être les recherches et analyses, l'expérimentation démontre que les prises de décisions relèvent du seul libre arbitre du musicien intervenant dans les œuvres, acte éminemment artistique. La thèse positionne ainsi la démarche entre science et art. / Doctorat en Art et Sciences de l'Art / info:eu-repo/semantics/nonPublished Arts
4	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology
5	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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