Cytochrome P4502E1 or menadione-induced oxidative stress, cytotoxicity, and apoptosis to Hep G2 cells.
Item
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Title
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Cytochrome P4502E1 or menadione-induced oxidative stress, cytotoxicity, and apoptosis to Hep G2 cells.
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Identifier
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AAI9807913
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identifier
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9807913
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Creator
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Chen, Qi.
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Contributor
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Adviser: Arthur I. Cederbaum
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Cell | Biology, Molecular
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Abstract
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Menadione generates reactive oxygen intermediates and causes oxidative injury. Preincubation of Hep G2 cells with low, non-toxic concentrations of menadione increased the viability of the cells against toxic doses of menadione or H{dollar}\rm\sb2O\sb2.{dollar} Menadione activated NF-{dollar}\kappa{dollar}B, and this activation was prevented by antioxidants or salicylate. Transfection with an expression vector containing cDNA encoding mouse {dollar}\rm I\kappa B\beta{dollar} resulted in increased toxicity by menadione. An increased level of glutathione (GSH) was observed after menadione pretreatment; this increase was blocked by salicylate, thereby linking the GSH increase to activation of NF-{dollar}\kappa{dollar}B by menadione. This study suggests that menadione pretreatment protects Hep G2 cells from oxidative injury through an NF-{dollar}\kappa{dollar}B related mechanism, which may involve, in part, increased production of GSH.;Arachidonic acid produced a concentration- and time-dependent toxicity to previously established E9 cells, which express cytochrome P4502E1 (CYP2E1), but little or no toxicity was found with control MV5 cells, which were infected with retrovirus lacking CYP2E1 cDNA. In contrast to arachidonic acid, oleic acid was not toxic to both subclones. The cytotoxicity of arachidonic acid appeared to involve lipid peroxidation, the toxicity was enhanced after depletion of cellular glutathione, and toxicity was prevented by the antioxidants. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), which prevented toxicity of arachidonic acid, also prevented the apoptosis, suggesting a role for lipid peroxidation to apoptosis. Transfection with a plasmid containing bcl-2 cDNA resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. This study reproduced several of the key features associated with ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment.;New Hep G2 subclones were established by transfecting Hep G2 cells with various vectors and limited dilution screening. Experiments demonstrated a growth inhibition effect and a cytotoxic effect of CYP2E1 in E47 and E43 cells, which express CYP2E1 at levels 4-10 times higher compared to the previously established E9 cells. These effects occur in the absence of externally added toxin or agent and therefore are directly due to high levels of expression of CYP2E1 itself. The slow growth may be a result of mitochondrial damage, the need to maintain cellular GSH level, and lower level of intracellular ATP content. The cytotoxicity is apoptotic in nature, and is initiated by the depletion of GSH by CYP2E1-related oxidative stress and elevated lipid peroxidation. The direct toxicity of overexpressed CYP2E1 is probably a reflection of the ability of this isoform to produce reactive oxygen species (ROI) even in the absence of substrate. It would be anticipated that substrates which are oxidized by CYP2E1 to reactive metabolites, e.g. ethanol, acetaminophen, and CCl{dollar}\sb4,{dollar} or PUFA which produces elevated lipid peroxidation in the presence of CYP2E1, would show strong toxicity in the E47 and E43 cells. Preliminary studies showed that these agents are indeed strongly toxic in the E47 cells even without the necessity of depleting cellular GSH.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
