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Molecular Mechanisms of Vitamin E Transport in Hepatocytes
levels (p<0.01) in diabetic wounds treated with Se compared to vehicle. Conclusions: Enhanced wound healing occurs in the presence of the novel selenium compound. This enhancement was more marked in diabetic compared to wildtype mice. The decrease in monocyte chemotactic activity, IL-6 expression and improvements in tissue elasticity and tensile strength, suggest that this selenium compound may aid wound healing.
the oxidative stress. DMT1 may not work in the iron uptake.
doi: 10.1016/j.freeradbiomed.2016.10.188 148 Fluorescence Imaging and Flow Cytometric Analysis of Hydrogen Peroxide in Living Cells Using a Novel Fluorescent Probe
doi: 10.1016/j.freeradbiomed.2016.10.186 146 The Exposure of 5-Aminolevulinic Acid Induced Apoptotic Cell Death via Oxidative Stress in Normal Gastric Epithelial Cells Hiromu Ito1 and Hirofumi Matsui1,2 University of Tsukuba, Japan, 2Kyoto Prefectural University of Medicine, Japan 1
Heme is an essential substance for oxygen metabolism. 5aminolevulinic acid (ALA) is a precursor of heme and accelerates plants, hair and sperm growth. However, ALA is reported to be an oxidative stressor. We previously reported that reactive oxygen species (ROS) enhanced cancer cellular bioactivity such as metastasis and invasion. On the other hand, over-generation of ROS induces apoptotic cell death in normal cells. Thus, ROS caused by ALA may promote cancerous malignancy and give damage to normal cells. In this study, we investigated the different effect of ALA between normal cells and cancer cells using a rat normal gastric epithelial cell line, RGM and its chemically mutated cell line, RGK. 50 μM ALA for 48h induced normal cellular apoptosis, not in cancer cells. In addition, the normal cell death was suppressed by treatment with N-acetylcysteine. Therefore, we concluded that ALA induced ROS and subsequent normal cell specific death via oxidative stress. From this result, administration of ALA for cancer patients may bring getting worse.
Zhen Luo1, Jixiang Liu1, Qin Zhao1, Yunting Xi1, Ruogu Peng1, Jennifer Liao1, and Jack Diwu1 1 AAT Bioquest, Sunnyvale, USA As a type of reactive oxygen species (ROS), hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related states. It is involved in many biological events that are linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down’s syndrome. The measurement of this reactive species is helpful for determining how oxidative stress modulates various intracellular pathways. However, fluorescent probes which can detect H2O2 produced in mitochondrial remain rare. OxiVision Blue was developed to detect mitochondrial H2O2 in living cells. Cervial cancer HeLa cells stained with the novel fluorescent probe show negligible fluorescence in absence of peroxide stimulants. In contrast, the treatment of OxiVision Blue -loaded cells with H2O2 displayed strong blue fluorescence in mitochondria. The probe has also been successfully applied in flow cytometer for the quantitative detection of H2O2 in individual cells. Taken together, these results demonstrate that OxiVision Blue is mitochondria-targeted H2O2 probe, and the exogenous and endogenous changes of H2O2 levels in living cells can be monitored in real time and quantitatively measured by fluorescence imaging and flow cytometer.
doi: 10.1016/j.freeradbiomed.2016.10.189
doi: 10.1016/j.freeradbiomed.2016.10.187
149
147
Molecular Mechanisms of Vitamin E Transport in Hepatocytes
Gain of Tolerance to Iron-Induced Oxidative Injury in Neuronal PC12 Cells Teruyuki Kawabata1, Daiki Utsunomiya2, Natsuki Kawaguchi2, Shouma Nakatani2, Touma Murata2, Takeru Yasuhara2, and Yoshie Asahara3 1 Grad. School of Sci., Okayama Univ. of Sci., Japan, 2Okayama Univ. of Sci., Japan, 3Med. Sci. Edu. Center, Kake Edu. Inst., Japan To elucidate a mechanism of iron-induced oxidative stress on nervous system, we studied cell injuries of undifferentiated PC12 cell (PC12U) and neural one (PC12N), and reported in SFRRI conference 2014 that PC12U were more vulnerable to iron-induced oxidative stress than PC12N. And we suggested that the labile iron pool (LIP) was larger in PC12U than in PC12N. In this study we measured the amount of labile iron pool (LIP) in the cells using a newly synthesized fluorescent, FeRhoNox-1 (Goryo Chemical). Iron nitrilotriacetate was added to the cells in a serum-free medium. We also checked mRNA contents of TFR1 and DMT1 in PC12 cells using a real-time RT-PCR. We conclude that PC12U cells take a larger amount of iron than PC12N cells and are more vulnerable to
D. Manor1 and S. Chung1 1 Case Western Reserve University, Cleveland, USA Alpha-tocopherol (vitamin E) is an essential plant lipid that scavenges free radicals in biological membranes, thereby preventing oxidative stress. In the liver, the alpha-tocopherol transfer protein (TTP) facilitates the secretion of ingested αtocopherol to the circulation for uptake by extra hepatic tissues. We aim to understand the molecular mechanisms that regulate TTP activity in hepatocytes, and report here on our findings regarding intracellular trafficking of the protein and its ligands. In the absence of α-tocopherol, TTP localizes to lysosomal vesicles. After treatment with α-tocopherol, the protein redistributes to a diffuse cytosolic pattern, and this re-distribution is altered in TTP mutants that cause ataxia with vitamin E deficiency (AVED) in humans. Kinetic microscopy experiments show that upon binding tocopherol, TTP is transported to cell periphery by vesicles derived from recycling endosomes. The directionality of this vectorial transport of TTP to the plasma membrane id directed by inverse concentration gradients of -tocopherol and PI(4,5)P2. These data
SfRBM / SFRRI 2016
S73
allow us to develop a mechanistic model for TTP action, its modulation by physiological triggers, and the impact of diseasecausing mutations in the protein.
doi: 10.1016/j.freeradbiomed.2016.10.191
doi: 10.1016/j.freeradbiomed.2016.10.190 150 Molecular Target(S) Responsible for the Alveolar Tight Junction Proteins Disruption Under Hypoxia: Prophylactic Role of Curcumin in Refurbishing the Alveolar Barrier Integrity Under Hypoxia? Titto Mathew1, Saumya Bhagath2, Gausal Khan2, and Sarada SK Sagi1 1 Defence Institute of Physiology and Allied Sciences, Delhi, India, 2 Defence Research and Development Organization, India Pulmonary edema (PE) is attributed to ruptured alveolar-epithelial barrier integrity together with impaired alveolar fluid clearance (AFC). Disruption of tight junction (TJ) integrity can occur from loss of TJ protein expression and/or disorganization. TJ determine the barrier properties of cell-cell contact existing between two neighbouring cells and regulate paracellular permeability. The TJ disruption has been related to multiple signal transduction pathways; understanding such mechanisms in hypoxia induced disruption of epithelial and endothelial barrier functions is likely to provide insight into the pathogenesis of various inflammatory diseases, and may form a basis for the design of treatment strategies for different diseases involving barrier dysfunction. The present study was undertaken to determine whether differential expression of tight junction protein(s) is a mechanism for regulation of hypoxia-induced pulmonary epithelial permeability both in-vitro and in-vivo. For in-vitro studies, Human lung adenocarcinoma cell line (A549) was exposed to 3% O2 for different time periods viz. 1 h, 3h, 6h, 12h, 24h and 48 h. For in-vivo studies male Sprague Dawley rats were used as an animal model and exposed them to 25000 ft at 25°C for 6 hrs. Simultaneously, to rule out the fact that inflammation causes impaired tight junction proteins integrity leading to pulmonary edema, A549 cells were pre-treated with 10 µM curcumin and the rats were pre-treated with curcumin (50 mg/kg body weight) 1 h prior to 6-h hypoxia. Hypoxia exposure resulted into increased RBC, WBC, Hb, monocytes, lymphocytes and HCT. Blood gas analysis revealed that PaO2 mmHg, PaCO2 mmHg and SaO2 (%) were significantly (P≤0.001) altered compared to control. Hypoxia induced a progressive time dependent decrease in TJ proteins ZO-1, Claudin-4, and JAM-C proteins expression over control. Further, hypoxic exposure also increased the NFkB, TNF-α, Claudin -5 and Occludin protein expression levels with time as compared to control. However prior treatment with curcumin both in-vitro and in-vivo showed significant changes in tight junction proteins integrity and attenuated NFkB activity with reduced expression of its regulatory genes in lung tissue, serum and BALF. Further we confirmed by pre-treatment with NFkB inhibitor MG132 or siRNA mediated knock down of p65 significantly abrogated (p≤0.001) hypoxia induced NFkB expression followed by significant reduction in (p ≤0.05 ) in dextran FITC efflux in to the lungs. Similarly, HIF-1α reporter gene studies further, confirmed that curcumin stabilized the lung HIF-1α levels under hypoxia. Immunohistochemistry and immunofluorescence observations supports the notion that curcumin restored the brain ZO-1, JAMC, claudin 4 and claudin 5 levels (p ≤ 0.001) under hypoxia. These results indicate that curcumin is a potent drug in amelioration of pulmonary edema as it effectively attenuated inflammation with enhanced alveolar fluid clearance via maintaining the tight junction proteins integrity under hypoxia.
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SfRBM / SFRRI 2016
the oxidative stress. DMT1 may not work in the iron uptake.
doi: 10.1016/j.freeradbiomed.2016.10.188 148 Fluorescence Imaging and Flow Cytometric Analysis of Hydrogen Peroxide in Living Cells Using a Novel Fluorescent Probe
doi: 10.1016/j.freeradbiomed.2016.10.186 146 The Exposure of 5-Aminolevulinic Acid Induced Apoptotic Cell Death via Oxidative Stress in Normal Gastric Epithelial Cells Hiromu Ito1 and Hirofumi Matsui1,2 University of Tsukuba, Japan, 2Kyoto Prefectural University of Medicine, Japan 1
Heme is an essential substance for oxygen metabolism. 5aminolevulinic acid (ALA) is a precursor of heme and accelerates plants, hair and sperm growth. However, ALA is reported to be an oxidative stressor. We previously reported that reactive oxygen species (ROS) enhanced cancer cellular bioactivity such as metastasis and invasion. On the other hand, over-generation of ROS induces apoptotic cell death in normal cells. Thus, ROS caused by ALA may promote cancerous malignancy and give damage to normal cells. In this study, we investigated the different effect of ALA between normal cells and cancer cells using a rat normal gastric epithelial cell line, RGM and its chemically mutated cell line, RGK. 50 μM ALA for 48h induced normal cellular apoptosis, not in cancer cells. In addition, the normal cell death was suppressed by treatment with N-acetylcysteine. Therefore, we concluded that ALA induced ROS and subsequent normal cell specific death via oxidative stress. From this result, administration of ALA for cancer patients may bring getting worse.
Zhen Luo1, Jixiang Liu1, Qin Zhao1, Yunting Xi1, Ruogu Peng1, Jennifer Liao1, and Jack Diwu1 1 AAT Bioquest, Sunnyvale, USA As a type of reactive oxygen species (ROS), hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related states. It is involved in many biological events that are linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down’s syndrome. The measurement of this reactive species is helpful for determining how oxidative stress modulates various intracellular pathways. However, fluorescent probes which can detect H2O2 produced in mitochondrial remain rare. OxiVision Blue was developed to detect mitochondrial H2O2 in living cells. Cervial cancer HeLa cells stained with the novel fluorescent probe show negligible fluorescence in absence of peroxide stimulants. In contrast, the treatment of OxiVision Blue -loaded cells with H2O2 displayed strong blue fluorescence in mitochondria. The probe has also been successfully applied in flow cytometer for the quantitative detection of H2O2 in individual cells. Taken together, these results demonstrate that OxiVision Blue is mitochondria-targeted H2O2 probe, and the exogenous and endogenous changes of H2O2 levels in living cells can be monitored in real time and quantitatively measured by fluorescence imaging and flow cytometer.
doi: 10.1016/j.freeradbiomed.2016.10.189
doi: 10.1016/j.freeradbiomed.2016.10.187
149
147
Molecular Mechanisms of Vitamin E Transport in Hepatocytes
Gain of Tolerance to Iron-Induced Oxidative Injury in Neuronal PC12 Cells Teruyuki Kawabata1, Daiki Utsunomiya2, Natsuki Kawaguchi2, Shouma Nakatani2, Touma Murata2, Takeru Yasuhara2, and Yoshie Asahara3 1 Grad. School of Sci., Okayama Univ. of Sci., Japan, 2Okayama Univ. of Sci., Japan, 3Med. Sci. Edu. Center, Kake Edu. Inst., Japan To elucidate a mechanism of iron-induced oxidative stress on nervous system, we studied cell injuries of undifferentiated PC12 cell (PC12U) and neural one (PC12N), and reported in SFRRI conference 2014 that PC12U were more vulnerable to iron-induced oxidative stress than PC12N. And we suggested that the labile iron pool (LIP) was larger in PC12U than in PC12N. In this study we measured the amount of labile iron pool (LIP) in the cells using a newly synthesized fluorescent, FeRhoNox-1 (Goryo Chemical). Iron nitrilotriacetate was added to the cells in a serum-free medium. We also checked mRNA contents of TFR1 and DMT1 in PC12 cells using a real-time RT-PCR. We conclude that PC12U cells take a larger amount of iron than PC12N cells and are more vulnerable to
D. Manor1 and S. Chung1 1 Case Western Reserve University, Cleveland, USA Alpha-tocopherol (vitamin E) is an essential plant lipid that scavenges free radicals in biological membranes, thereby preventing oxidative stress. In the liver, the alpha-tocopherol transfer protein (TTP) facilitates the secretion of ingested αtocopherol to the circulation for uptake by extra hepatic tissues. We aim to understand the molecular mechanisms that regulate TTP activity in hepatocytes, and report here on our findings regarding intracellular trafficking of the protein and its ligands. In the absence of α-tocopherol, TTP localizes to lysosomal vesicles. After treatment with α-tocopherol, the protein redistributes to a diffuse cytosolic pattern, and this re-distribution is altered in TTP mutants that cause ataxia with vitamin E deficiency (AVED) in humans. Kinetic microscopy experiments show that upon binding tocopherol, TTP is transported to cell periphery by vesicles derived from recycling endosomes. The directionality of this vectorial transport of TTP to the plasma membrane id directed by inverse concentration gradients of -tocopherol and PI(4,5)P2. These data
SfRBM / SFRRI 2016
S73
allow us to develop a mechanistic model for TTP action, its modulation by physiological triggers, and the impact of diseasecausing mutations in the protein.
doi: 10.1016/j.freeradbiomed.2016.10.191
doi: 10.1016/j.freeradbiomed.2016.10.190 150 Molecular Target(S) Responsible for the Alveolar Tight Junction Proteins Disruption Under Hypoxia: Prophylactic Role of Curcumin in Refurbishing the Alveolar Barrier Integrity Under Hypoxia? Titto Mathew1, Saumya Bhagath2, Gausal Khan2, and Sarada SK Sagi1 1 Defence Institute of Physiology and Allied Sciences, Delhi, India, 2 Defence Research and Development Organization, India Pulmonary edema (PE) is attributed to ruptured alveolar-epithelial barrier integrity together with impaired alveolar fluid clearance (AFC). Disruption of tight junction (TJ) integrity can occur from loss of TJ protein expression and/or disorganization. TJ determine the barrier properties of cell-cell contact existing between two neighbouring cells and regulate paracellular permeability. The TJ disruption has been related to multiple signal transduction pathways; understanding such mechanisms in hypoxia induced disruption of epithelial and endothelial barrier functions is likely to provide insight into the pathogenesis of various inflammatory diseases, and may form a basis for the design of treatment strategies for different diseases involving barrier dysfunction. The present study was undertaken to determine whether differential expression of tight junction protein(s) is a mechanism for regulation of hypoxia-induced pulmonary epithelial permeability both in-vitro and in-vivo. For in-vitro studies, Human lung adenocarcinoma cell line (A549) was exposed to 3% O2 for different time periods viz. 1 h, 3h, 6h, 12h, 24h and 48 h. For in-vivo studies male Sprague Dawley rats were used as an animal model and exposed them to 25000 ft at 25°C for 6 hrs. Simultaneously, to rule out the fact that inflammation causes impaired tight junction proteins integrity leading to pulmonary edema, A549 cells were pre-treated with 10 µM curcumin and the rats were pre-treated with curcumin (50 mg/kg body weight) 1 h prior to 6-h hypoxia. Hypoxia exposure resulted into increased RBC, WBC, Hb, monocytes, lymphocytes and HCT. Blood gas analysis revealed that PaO2 mmHg, PaCO2 mmHg and SaO2 (%) were significantly (P≤0.001) altered compared to control. Hypoxia induced a progressive time dependent decrease in TJ proteins ZO-1, Claudin-4, and JAM-C proteins expression over control. Further, hypoxic exposure also increased the NFkB, TNF-α, Claudin -5 and Occludin protein expression levels with time as compared to control. However prior treatment with curcumin both in-vitro and in-vivo showed significant changes in tight junction proteins integrity and attenuated NFkB activity with reduced expression of its regulatory genes in lung tissue, serum and BALF. Further we confirmed by pre-treatment with NFkB inhibitor MG132 or siRNA mediated knock down of p65 significantly abrogated (p≤0.001) hypoxia induced NFkB expression followed by significant reduction in (p ≤0.05 ) in dextran FITC efflux in to the lungs. Similarly, HIF-1α reporter gene studies further, confirmed that curcumin stabilized the lung HIF-1α levels under hypoxia. Immunohistochemistry and immunofluorescence observations supports the notion that curcumin restored the brain ZO-1, JAMC, claudin 4 and claudin 5 levels (p ≤ 0.001) under hypoxia. These results indicate that curcumin is a potent drug in amelioration of pulmonary edema as it effectively attenuated inflammation with enhanced alveolar fluid clearance via maintaining the tight junction proteins integrity under hypoxia.
S74
SfRBM / SFRRI 2016