Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells |
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Authors: | Nunes Viviane A Gozzo Andrezza J Cruz-Silva Ilana Juliano Maria A Viel Tania A Godinho Rosely O Meirelles Flavio V Sampaio Misako U Sampaio Claudio A M Araujo Mariana S |
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Affiliation: | Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Tres de Maio, 100, 6. andar, P.O. Box O4044-020, Sao Paulo, SP, Brazil. vanunes@ib.usp.br |
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Abstract: | Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells. |
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Keywords: | Abz Aminobenzoic acid BSA bovine serum albumin CHAPS 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate CHX cycloheximide DAPI 4′-6-diamidino-2-phenylindole DMEM Dulbecco's modified Eagle's medium DTT dithiothreitol ECL Enhancer of Chemiluminescence EDDnp EDTA ethylenediaminetetraacetic acid GSH reduced glutathione HEPES [4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid] hydrogen peroxide MDN (Menadione) PI propidium iodide PMSF phenylmethylsulfonyl fluoride ROS reactive oxygen species SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis STP staurosporine TNF-α tumor necrosis factor α Vit E vitamin E Z-VAD-fmk benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone Apoptosis Necrosis Oxidative stress Vitamin E Muscle cells Fibroblasts Chicken Bax and Bcl-2 |
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