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LDH Release (ldh + release)
Selected AbstractsLabeling of Adipose-Derived Stem Cells by Oleic-Acid-Modified Magnetic NanoparticlesADVANCED FUNCTIONAL MATERIALS, Issue 8 2009Lian Cen Abstract The in vivo tracking of adipose derived stem cells (ASCs) is of essential concern when they are used as seed cells in tissue engineering. This study explores the feasibility of using magnetic nanoparticles (MNs), a type of contrast agents in magnetic resonance imaging (MRI), to label ASCs such that the labeled ASCs could be tracked in vivo by MRI non-invasively and repeatedly. To do this, MNs of <10,nm surface-coated with oleic acid are synthesized via a high-temperature solution-phase reaction. Cytotoxicity of the as-synthesized MNs at concentrations up to 0.1,mg,mL,1 on 104,cells,mL,1 ASCs is evaluated by LDH release. Since only minor cytotoxicity is detected, the effects of the labeling technique on cellular behaviors and uptake by labeled cells are investigated. Cell proliferation and differentiation with and without MNs are compared. The results show that proliferation of ASCs (104,cells,mL,1) labeled by MNs (0.05,mg,mL,1) is significantly enhanced and dependent on the labeling time. The MNs are located in the vesicles within cytoplasm of ASCs. The cellular uptake reaches as high as ,180,pg/cell. Nevertheless, the labeled ASCs still maintained adipogenic and osteogenic differentiation. Hence, the feasibility of labeling ASCs by oleic acid coated MNs is ascertained and it was better to label the cells during their quiescent stage. The labeled ASCs can also be in vivo detected by MRI in a subcutaneous model in vivo. Further MRI tracking of the labeled ASCs in long-term follow-up would thus follow this current study. [source] Urinary macromolecules and renal tubular cell protection from oxalate injury: Comparison of normal subjects and recurrent stone formersINTERNATIONAL JOURNAL OF UROLOGY, Issue 3 2006MASAO TSUJIHATA Aim:, To determine whether urinary macromolecules (UMM), which are the high molecular weight substances in urine, can provide protection against the oxalate-associated injury to the renal tubular cells. Methods:, UMM were extracted from 24-h urine of 12 healthy adult male volunteers and 13 recurrent-stone-former male patients. Urine parameters in relation to urolithiasis were measured, including the level of glycosaminoglycans (GAG) in the UMM. Madin-Darby canine kidney (MDCK) cells were used to evaluate the protective activity of UMM from oxalate-induced cytotoxicity by LDH release measurement and methyl-thiazolyl tertrazolium (MTT) assay. Results:, Considering urinary parameters, citrate was significantly higher in urine from normal subjects than stone-former subjects; the other parameters show no differences between the groups. Total UMM and the level of GAG in the UMM were also significantly higher in the normal subject group. Compared with normal subject and stone-former subject UMM, after cells were treated with the UMM and then exposed to oxalate solution, LDH release was significantly higher in stone-former group. In the MTT assay, we found that more viable cells were observed after treatment with UMM compared to control in both groups. Moreover, UMM from the normal subjects showed higher protective activity against oxalate-related cytotoxicity than UMM from the stone-former subjects. Conclusion:, UMM protected renal epithelial cells from oxalate-related injury. This protective activity was found to be higher in normal subject UMM than stone-former UMM. Among other factors, a higher concentration of GAG and citrate in normal subject UMM might affect some parts in this finding. [source] Butterfat fatty acids differentially regulate growth and differentiation in Jurkat T-cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Paolo Bergamo Abstract Synthetic Conjugated Linoleic Acid mixture (CLA; c9,t11; t10,c12-18:2) has been previously shown to inhibit growth, and enhance apoptosis and IL-2 mRNA synthesis in human lymphoblastic Jurkat T-cells. In this study, two different butterfat types were evaluated and compared for their effects on Jurkat cell viability, oxidative stress, pro-apoptotic activity, and cytokine synthesis: the conventionally produced butterfat (CBF), and organic butterfat (OBF) containing significantly higher amounts of c9,t11 (Rumenic Acid, RA), trans-vaccenic acid (VA; t11-18:1), ,-linolenic acid (ALA), and lower levels of linoleic acid (LA). Results from cell treatment with both butterfat mixtures showed comparable oxidative stress (superoxide production, intracellular GSH depletion,and lipid peroxides yield), NADPH oxidase activation, cytotoxicity (LDH release), and IL-2 transcript level, whereas the effects of enhanced growth-inhibitory and pro-apoptotic activities were associated with OBF treatment. To then investigate each butterfat-induced effect caused by RA, VA, LA, and ALA, cells were exposed to synthetic FA concentrations similar to those from the different butterfats. Higher oxidative stress (superoxide production, intracellular GSH depletion) was induced by ,-linolenic (ALA) and linoleic (LA) incubation (P,<,0.01) and superoxide production was suppressed by specific PKC, inhibitor (Gö 6976) and linked to increased toxicity and IL-2 synthesis inhibition. By contrast, cell treatment with RA increased apoptosis and IL-2 synthesis. These results suggest that a supply of ALA and LA is responsible for BF-induced oxidative stress via PKC,-NADPH oxidase pathway, and that enhanced antiproliferative effects in OBF treated cells is essentially determined by RA-induced pro-apoptotic activity. © 2005 Wiley-Liss, Inc. [source] Antioxidative and Anti-Inflammatory Protection of Oleanolic Acid and Ursolic Acid in PC12 CellsJOURNAL OF FOOD SCIENCE, Issue 7 2008Shih-Jei Tsai ABSTRACT:, PC12 cells were used to examine the in vitro antioxidative and anti-inflammatory effects of oleanolic acid (OA) and ursolic acid (UA). PC12 cells were pretreated with OA or UA at 20 and 40 ,M and followed by exposure of hydrogen peroxide (H2O2) or 1-methyl-4-phenylpyridinium ion (MPP+) to induce cell injury. Results showed that H2O2 - or MPP+ -treatment significantly decreased cell viability and increased lactate dehydrogenase (LDH) release (P < 0.05). The pretreatment from OA or UA significantly and concentration-dependently reduced subsequent H2O2 - or MPP+ -induced cell death and LDH release (P < 0.05). Either H2O2 - or MPP+ -treatment significantly increased malonyldialdehyde (MDA) formation, decreased glutathione (GSH) content, and diminished glutathione peroxidase (GPX), catalase, and superoxide dismutase (SOD) activities (P < 0.05). The pretreatment from OA or UA significantly retained GSH, and reversed H2O2 - and MPP+ -induced impairment in catalase and SOD activities (P < 0.05), and decreased MDA formation (P < 0.05). Either H2O2 - or MPP+ -treatment significantly elevated interleukin-6 (IL-6) and tumor necrosis factor (TNF)-, levels (P < 0.05). The pretreatments from OA or UA significantly attenuated subsequent H2O2 - or MPP+ -induced release of IL-6 and TNF-, (P < 0.05). Based on the observed antioxidative and anti-inflammatory activities from OA and UA, these 2 compounds were potent agents against neurodegenerative disorder. [source] Neuroprotective actions of noradrenaline: effects on glutathione synthesis and activation of peroxisome proliferator activated receptor deltaJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Jose L. M. Madrigal Abstract The endogenous neurotransmitter noradrenaline (NA) can protect neurons from the toxic consequences of various inflammatory stimuli, however the exact mechanisms of neuroprotection are not well known. In the current study, we examined neuroprotective effects of NA in primary cultures of rat cortical neurons. Exposure to oligomeric amyloid beta (A,) 1-42 peptide induced neuronal damage revealed by increased staining with fluorojade, and toxicity assessed by LDH release. A,-dependent neuronal death did not involve neuronal expression of the inducible nitric oxide synthase 2 (NOS2), since A, did not induce nitrite production from neurons, LDH release was not reduced by co-incubation with NOS2 inhibitors, and neurotoxicity was similar in wildtype and NOS2 deficient neurons. Co-incubation with NA partially reduced A,-induced neuronal LDH release, and completely abrogated the increase in fluorojade staining. Treatment of neurons with NA increased expression of ,-glutamylcysteine ligase, reduced levels of GSH peroxidase, and increased neuronal GSH levels. The neuroprotective effects of NA were partially blocked by co-treatment with an antagonist of peroxisome proliferator activated receptors (PPARs), and replicated by incubation with a selective PPARdelta (PPAR,) agonist. NA also increased expression and activation of PPAR,. Together these data demonstrate that NA can protect neurons from A,-induced damage, and suggest that its actions may involve activation of PPAR, and increases in GSH production. [source] Macrophage migration inhibitor factor (MIF) can induce oxidative injury and apoptotic cell death of spinal cord neuronsJOURNAL OF NEUROCHEMISTRY, Issue 2003M. Toborek MIF is a cytokine produced by a variety of cells and tissues including the CNS. It can exert a variety of biological functions, such as induction of inflammatory responses and counterregulation of glucocorticoid effects. However, the role of MIF in the pathogenesis of spinal cord trauma is not fully understood. Therefore, the aim of the present study was to evaluate the cellular effects of MIF in cultured spinal cord neurons. A 3-h exposure to MIF significantly enhanced intracellular calcium levels; an effect which was prevented by TMB-8, an antagonist of the IP3 receptor. In addition, MIF treatment increased oxidative stress, decreased viability of spinal cord neurons, increased LDH release and stimulated apoptosis. These results suggest that MIF may play an important role in secondary spinal cord injury. Acknowledgements:, Supported by grants from KSCHIRT and Philip Morris External Research Program. [source] Pregnenolone Sulfate, a Naturally Occurring Excitotoxin Involved in Delayed Retinal Cell DeathJOURNAL OF NEUROCHEMISTRY, Issue 6 2000C. Cascio Abstract: The present study was designed to investigate the neurosteroid pregnenolone sulfate (PS), known for its ability to modulate NMDA receptors and interfere with acute excitotoxicity, in delayed retinal cell death. Three hours after exposure of the isolated and intact retina to a 30-min PS pulse, DNA fragmentation as assessed by genomic DNA gel electrophoresis and a modified in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method appeared concurrently with an increase in superoxide dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBARS) levels. At 7 h, the increased amount of DNA laddering was accompanied by a higher number of TUNEL-positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lactate dehydrogenase (LDH) release, and damage mainly in the inner nuclear layer. PS-induced delayed cell death was markedly reduced by the NMDA receptor antagonists 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid and 3,-hydroxy-5,-pregnan-20-one sulfate but completely blocked after concomitant addition of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Steroids with antioxidant properties (progesterone, dehydroepiandrosterone and its sulfate ester, and 17,-estradiol) differently prevented PS-induced delayed cell death. Cycloheximide treatment protected against DNA fragmentation and LDH release but failed to prevent the rise in SOD activity and TBARS level. We conclude that a brief PS pulse causes delayed cell death in a slowly evolving apoptotic fashion characterized by a cycloheximide-sensitive death program downstream of reactive oxygen species generation and lipid peroxidation, turning into secondary necrosis in a retinal cell subset. [source] Deletion of tau attenuates heat shock-induced injury in cultured cortical neuronsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2010Yanying Miao Abstract The microtubule-associated protein tau has been implicated in ,-amyloid- and glutamate-induced neurotoxicity. However, the potential role of tau in response to other insults to neurons remains unclear. In this study, we examined whether deletion of tau would change cell injury induced by heat shock in primary cultures of cortical neurons. After 30 min of a 45°C heat shock, lactate dehydrogenase (LDH) release increased, reaching a peak at 6 hr in wild-type (WT) neurons. A significantly lower LDH release, with a peak delayed by 24 hr, was detected in tau knockout (TKO) neurons. After heat shock treatment, MAP-2 and tubulin staining of the processes of WT neurons revealed more dramatic abnormalities than in TKO neurons. Both WT and TKO neurons exhibited a similar elevation of HSP70 level but different time courses of Akt phosphorylation. In contrast to an early, brief response in WT neurons, TKO neurons displayed a late, but long-lasting increase in phosphorylation of Akt and its downstream target, glycogen synthase kinase 3,. Additionally, inhibition of Akt activity aggravated the cell morbidity caused by heat shock exposure in both WT and TKO neurons, indicating a protective role of Akt against cell injury. In conclusion, our results demonstrate that deletion of tau attenuated heat shock-induced neuronal injury. Enhanced Akt response in the absence of endogenous tau is suggested to represent a compensatory mechanism for regulating cell reactions to stress stimuli. © 2009 Wiley-Liss, Inc. [source] Protective effects of sesamin and sesamolin on hypoxic neuronal and PC12 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2003Rolis Chien-Wei Hou Abstract Reactive oxygen species (ROS) are important mediators of a variety of pathological processes, including inflammation and ischemic injury. The neuroprotective effects of sesame antioxidants, sesamin and sesamolin, against hypoxia or H2O2 -induced cell injury were evaluated by cell viability or lactate dehydrogenase (LDH) activity. Sesamin and sesamolin reduced LDH release of PC12 cells under hypoxia or H2O2 -stress in a dose-dependent manner. Dichlorofluorescein (DCF)-sensitive ROS production was induced in PC12 cells by hypoxia or H2O2 -stress but was diminished in the presence of sesamin and sesamolin. We evaluated further the role of mitogen-activated protein kinases (MAPKs) and caspase-3 in hypoxia-induced PC12 cell death. Extracellular signal-regulated protein kinase (ERK) 1, c-jun N-terminal kinase (JNK), and p38 MAPKs of signaling pathways were activated during hypoxia. We found that the inhibition of MAPKs and caspase-3 by sesamin and sesamolin correlated well with the reduction in LDH release under hypoxia. Furthermore, the hypoxia-induced apoptotic-like cell death in cultured cortical cells as detected by a fluorescent DNA binding dye was reduced significantly by sesamin and sesamolin. Taken together, these results suggest that the protective effect of sesamin and sesamolin on hypoxic neuronal and PC12 cells might be related to suppression of ROS generation and MAPK activation. © 2003 Wiley-Liss, Inc. [source] Evaluation of buccal methyl-,-cyclodextrin toxicity on human oral epithelial cell culture modelJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2005Laïla Boulmedarat Abstract Cyclodextrins, especially methylated ,-cyclodextrins offer several advantages for drug delivery which include improved drug solubilization, protection against physicochemical and enzymatic degradation, as well as a potential for absorption improvement. However, little or no data are available for their use as drug penetration enhancer via the buccal route. This study focuses on the toxicity of randomly methylated ,-cyclodextrin (RAMEB) on buccal mucosa using a reconstituted human oral epithelium model composed of TR 146 cells. Toxicity of RAMEB on TR 146 cells was evaluated by measuring cell viability (MTT assay) and membrane damages followed by LDH release after single and repeated exposures to RAMEB solutions. Inflammatory effects of RAMEB are also considered by measuring expression of interleukin-1, and are supported by histological examination. The present results indicate that 10% RAMEB results in cytotoxic and inflammatory effects depending on time exposure, whereas 2% and 5% RAMEB do not induce tissue damages even after 5 days of repeated exposures. Therefore, the highly water-soluble RAMEB is thought to be a safe candidate as an excipient for buccal mucosal drug delivery. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1300,1309, 2005 [source] In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studiesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2001Remigius Uchenna Agu The aim of this study was to establish a collagen matrix-based nasal primary culture system for drug delivery studies. Nasal epithelial cells were cultured on derivatised (Cellagen membrane CD-24), polymerised (Vitrogen gel) and fibrillar (Vitrogen film) collagen substrata. Cell morphology was assessed by microscopy. The cells were further characterised by measurement of ciliary beat frequency (CBF), transepithelial resistance (TER), permeation of sodium fluorescein, mitochondrial dehydrogenase (MDH) activity and lactate dehydrogenase (LDH) release upon cell exposure to sodium tauro-24, 25 dihydrofusidate (STDHF). Among the three collagen substrata investigated, the best epithelial differentiated phenotype (monolayer with columnar/cuboidal morphology) occurred in cells grown on Cellagen membrane CD-24 between day 4 and day 11. Cell culture reproducibility was better with Cellagen membrane CD-24 (90%) in comparison with Vitrogen gel (70%) and Vitrogen film (< 10%). TER was higher in cells grown on Vitrogen gel than on Cellagen membrane CD-24 and Vitrogen film. The apparent permeability coefficient (Papp × 10,7 cm s,1) of sodium fluorescein in these conditions was 0.45 ± 0.08 (Vitrogen gel) and 1.91 ± 0.00 (Cellagen membrane CD-24). Except for LDH release, CBF and cell viability were comparable for all the substrata. Based on MDH activity, LDH release, CBF, TER and permeation studies, Cellagen membrane CD-24- and Vitrogen gel-based cells were concluded to be functionally suitable for in-vitro nasal drug studies. Vitrogen film-based cultures may be limited to metabolism and cilio-toxicity studies. [source] Ethanol Effects on Nitric Oxide Production in Cerebral Pial CulturesALCOHOLISM, Issue 4 2001Chin-Lung Shih Background: Although alcohol abusers are known to have higher incidences of hemorrhagic cerebrovascular diseases, it is not known whether these changes are associated with ethanol (EtOH) action on nitric oxide (NO) production in the cerebrovascular cells. The purpose of this study was to examine the effects of EtOH treatment on basal and cytokine-induced NO production in cortical pial cultures. Methods: Cell cultures for this study included murine primary pial vascular cells, primary glial cells and cortical neurons. These cells were exposed to cytokines or EtOH for 24 to 48 hr. The culture media were used for measurement of nitrite, as an indication for NO release, and lactate dehydrogenase (LDH), as an index of cell membrane integrity. In addition, immunocytochemical determinations were carried out to identify cell types and to assess inducible nitric oxide synthase (iNOS). Results: Exposure of primary pial vascular cultures to cytokines that consisted of interleukin-1, (IL-1,; 250 pg/mL) and interferon-, (IFN,; 2 ng/mL) or to EtOH (50 to 100 mM) for 24 to 48 hr significantly elevated NO production. NO production could be attenuated by N -nitro-L-arginine (N-arg), a nonspecific NOS inhibitor, or aminoguanidine (AG), an iNOS inhibitor. Increased iNOS immunoreactivity was observed in cytokines- or EtOH-treated pial cells. When pial cells were cocultured with cortical neurons, prolonged EtOH exposure led to a large increase in NO production as well as LDH release. However, this increase was not observed in pial culture alone or in mixed cortical culture. Nevertheless, inhibition of NO production with N-arg or AG did not alter the EtOH-induced LDH release in the pial cells cocultured with cortical neurons. Conclusion: These results show that EtOH exposure led to increased production of NO in primary pial cell culture. In mixed culture that contained cortical neurons and pial cells, EtOH induced increase in NO as well as LDH release, which is an indication of loss of cell membrane integrity. However, EtOH-mediated LDH release in mixed cortical pial cultures was not a consequence of the increase in NO production by these cells. Studies that use mixed cortical-pial cultures may provide a unique in vitro system for examining the interactions among glial cells, neurons, and cerebrovascular cells. [source] S -Allyl cysteine, S -ethyl cysteine and S -propyl cysteine alleviate oxidative stress-induced damage within PC12 cellsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2008Chiu-mei Chen Abstract BACKGROUND: The PC12 cell line is a suitable model for the investigation of neurodegenerative diseases. In this study, PC12 cells were used to examine in vitro antioxidative and antiapoptotic protection by S -allyl cysteine (SAC), S -ethyl cysteine (SEC) and S -propyl cysteine (SPC). PC12 cells were treated with these agents at 5 and 10 µmol L,1 before exposure to hydrogen peroxide (H2O2). RESULTS: H2O2 treatment significantly decreased mitochondrial membrane potential (MMP) and cell viability and increased lactate dehydrogenase (LDH) release and DNA fragmentation (P < 0.05). The pre-treatments with SAC, SEC and SPC significantly and concentration-dependently elevated cell viability and MMP and lowered LDH release and DNA fragmentation (P < 0.05). H2O2 treatment also significantly increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and oxidised glutathione (GSSG) and decreased glutathione (GSH) content (P < 0.05). The pre-treatments with SAC, SEC and SPC significantly decreased subsequent H2O2 -induced formation of MDA, ROS and GSSG (P < 0.05) and also alleviated H2O2 -induced GSH depletion (P < 0.05). Finally, H2O2 treatment significantly decreased Na+ -K+ -ATPase activity and elevated caspase-3 activity (P < 0.05). The pre-treatments with SAC, SEC and SPC significantly attenuated H2O2 -induced Na+ -K+ -ATPase activity reduction and caspase-3 activity elevation (P < 0.05). CONCLUSION: The results obtained support that the three cysteine-containing compounds studied are potent neuroprotective agents. Copyright © 2008 Society of Chemical Industry [source] Effect of intracellular lipid droplets on cytosolic Ca2+ and cell death during ischaemia,reperfusion injury in cardiomyocytesTHE JOURNAL OF PHYSIOLOGY, Issue 6 2009Ignasi Barba Lipid droplets (LD) consist of accumulations of triacylglycerols and have been proposed to be markers of ischaemic but viable tissue. Previous studies have described the presence of LD in myocardium surviving an acute coronary occlusion. We investigated whether LD may be protective against cell death secondary to ischaemia,reperfusion injury. The addition of oleate,bovine serum albumin complex to freshly isolated adult rat cardiomyocytes or to HL-1 cells resulted in the accumulation of intracellular LD detectable by fluorescence microscopy, flow cytometry and 1H-nuclear magnetic resonance spectroscopy. Simulated ischaemia,reperfusion of HL-1 cells (respiratory inhibition at pH 6.4 followed by 30 min of reperfusion) resulted in significant cell death (29.7 ± 2.6% of total lactate dehydrogenase release). However, cell death was significantly attenuated in cells containing LD (40% reduction in LDH release compared with control cells, P= 0.02). The magnitude of LD accumulation was inversely correlated (r2= 0.68, P= 0.0003) with cell death. The protection associated with intracellular LD was not a direct effect of the fatty acids used to induce their formation, because oleate added 30 min before ischaemia, during ischaemia or during reperfusion did not form LD and did not protect against cell death. Increasing the concentration of free oleate during reperfusion progressively decreased the protection afforded by LD. HL-1 cells labelled with fluo-4, a Ca2+ -sensitive fluorochrome, fluorescence within LD areas increased more throughout simulated ischaemia and reperfusion than in the cytosolic LD-free areas of the same cells. As a consequence, cells with LD showed less cytosolic Ca2+ overload than control cells. These results suggest that LD exert a protective effect during ischaemia,reperfusion by sequestering free fatty acids and Ca2+. [source] Toxicological effects of iron on intestinal cellsCELL BIOCHEMISTRY AND FUNCTION, Issue 3 2004Bettina Zödl Abstract The aim of the present study was to investigate whether iron, which is involved in the formation of free radicals in vitro, can initiate cellular injury in human intestinal cells. The effects of various concentrations of iron were studied in preconfluent, colonic-cancerogenous cells, and also in postconfluent, differentiating cells. Cellular damage was assessed using cell proliferation (serial cell counting), tetrazolium dye (MTT) uptake, lactate dehydogenase (LDH) release and apoptosis studies based on caspase-3 activities. Also the activities of the major antioxidative enzymes, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were measured after the cells had been exposed to iron. Our results indicated that preconfluent cells were more susceptible to iron toxicity, as assessed by a significant reduction in cell proliferation and MTT uptake in a concentration-dependent manner compared to the control. However, no evidence for MTT uptake was observed in postconfluent cells. Caspase-3 activity, an indicator of cell apoptosis, considerably increased in preconfluent cells at high iron levels compared to the control (p <,0.05), whereas postconfluent cells were not significantly affected. LDH release was similar for both groups and was significantly higher than the control at 900,,M iron and above. SOD activities were not affected by iron in either group, whereas GPx was considerably higher in iron-treated cells in both groups compared with the control (because of relatively high standard deviations this effect was not significant). In conclusion we suggest that iron exerts its toxic effects intracellularly especially in preconfluent Caco-2 cells, whereas only high iron doses were able to alter the viability of differentiating, enterocyte-like cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] Effect of nitric oxide on iron-mediated cytotoxicity in primary cultured renal proximal tubulesCELL BIOCHEMISTRY AND FUNCTION, Issue 4 2001Zhao-long Wu Abstract Nitric oxide (NO) has been proved to be a mediator of hypoxic injury in renal proximal tubules (PT), but its effect on iron-induced cytotoxicity has remained little known. In this study, we observed the relationship between NO production and lactate dehydrogenase (LDH) release in primary proximal tubular epithelia co-incubated with different doses of NTA-Fe and lipopolysaccharide (LPS) alone or in combination. NO production was monitored by NO2 concentration in supernatants based on the Griess reaction; while the semi-quantitative RT-PCR was applied to detect the inducible nitric oxide synthase (iNOS) mRNA level induced by NTA-Fe and LPS together. In addition, experimental groups were subjected to reactive oxygen species (ROS) scavengers to determine the impact of the interaction between NO and ROS on iron-mediated cytotoxicity. After a 12-h co-incubation, we found that NTA-Fe increased both LDH release and 2, production in a dose-dependent manner (P,<,0.001). The level of iNOS mRNA induced by LPS was enhanced by 500 ,m NTA-Fe (P,<,0.01), lower or higher concentrations had no effect. However, the supernatant 2, level in the same group did not change significantly (P,>,0.05) although tubular injury was aggravated (P,<,0.001). The addition of l -arginine increased LDH release from 25.05,±,8.36% in the iron group to 38.67,±,7.67% in iron plus LPS group (P,<,0.05); concomitantly, l -NAME mitigated iron toxicity in LPS-treated PT (P,<,0.05). Hydroxyl scavengers provided complete protection against iron-mediated cytotoxicity (P,<,0.001), but the decrease of 2, production was only significant in the LPS-treated group. In contrast, SOD was partially effective in the LPS group (P,<,0.05) whereas the 2, level in the supernatant was inversely raised (P,<,0.05). GSH had no effect on either iron toxicity or 2, production. Thus, we conclude that NO can exacerbate the cytotoxicity caused by NTA-Fe in cultured proximal tubular epithelia, but NO is not the only factor. NTA-Fe could enhance the upregulation of iNOS transcription induced by LPS in a specific concentration range, and its regulation of NO production might also involve a post-transcription mechanism. The hydroxyl group is the major mediator in our model and the pro-oxidant role of NO is probably due to its ability to promote the Fenton reaction and form both ONOO, and ,OH via its interaction with ROS. Copyright © 2001 John Wiley & Sons, Ltd. [source] Neuroprotective effects of pramipexole against tunicamycin-induced cell death in PC12 cellsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2009Hitoshi Nakayama Summary 1Pramipexole (PPX), a dopamine D2 and D3 receptor agonist, exerts neuroprotective effects via both dopamine receptor-mediated and non-dopaminergic mechanisms. In the present study, we demonstrate that PPX reduces the toxicity of tunicamycin, a typical endoplasmic reticulum (ER) stressor, in PC12h cells, a subline of PC12 cells. 2The PC12h cells were treated with 300 ,mol,/,L PPX in the presence of 0.5 ,mol,/,L tunicamycin for 24 h. The neuroprotective effects of PPX against tunicamycin-induced cell death were evaluated using 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, Hoechst 33258 staining and western blot analysis. 3Tunicamycin (0.2, 0.3 and 0.5 ,g,/,mL) dose-dependently decreased MTT activity and increased LDH release from PC12h cells. Treatment with 300 ,mol,/,L PPX rescued the tunicamycin-induced decrease in cell viability. 4Spiperone (10 ,mol,/,L), a dopamine D2 and D4 receptor antagonist, had no effect on PPX neuroprotection against tunicamycin in these cells. Marker proteins of ER stress and apoptosis are known to be upregulated by tunicamycin, but we detected no significant effects of PPX on these factors. 5In conclusion, we speculate that a combination of several mechanisms may be involved in PPX-induced neuroprotection. [source] | ||||||||||||||||