Stable expression of human cytochrome P4502E1 in HepG2 cells: Characterization of catalytic activities, cytotoxicity with acetaminophen and degradation by carbon tetrachloride.
Item
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Title
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Stable expression of human cytochrome P4502E1 in HepG2 cells: Characterization of catalytic activities, cytotoxicity with acetaminophen and degradation by carbon tetrachloride.
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Identifier
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AAI9510652
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identifier
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9510652
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Creator
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Dai, Yan.
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Contributor
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Adviser: Arthur I. Cederbaum
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Date
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1994
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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
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Abstract
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To study the function of the human isoform of P450-2E1, the following series of experiments were carried out: (A) A stable cell line that constitutively expresses the human P450-2E1 in HepG2 cells was established by a recombinant retroviral expression method. Western blot analysis indicated that the transduced clones produced a protein band with molecular weight of 54,000. Northern blot and Southern blot analyses confirmed the expression and that the CYP2E1 was transcribed from a single copy of CYP2E1 cDNA integrated into the chromosome. The expressed human 2E1 was functionally active and could metabolize p-nitrophenol, N-dimethylnitrosamine, aniline and ethanol in the microsomal fraction of the transduced cells. The MVh2E1-9 clone expressed the P450 at a level of about 15 pmol/mg microsomal protein. No such activities were found with the MV-5 clone in which HepG2 cells were infected with retrovirus lacking the CYP2E1 cDNA. (B) The microsomal fraction of this clone was able to produce superoxide radical as measured by ESR spectroscopy and generate increased levels of hydrogen peroxide as compared to the control clone, MV-5 cells. Microsomes from MVh2E1-9 cells could initiate lipid peroxidation with a low iron concentration while microsomes from MV-5 cells essentially showed no lipid peroxidation at such low concentrations of iron catalyst. This suggests that human P450-2E1 is especially reactive in production of reactive oxygen species and in catalysis of lipid peroxidation at physiologically relevant low iron concentration. (C) When the cells were treated with acetaminophen, a typical hepatotoxin, MVh2E1-9 cells showed a dose-dependent toxicity as measured by an LDH leakage method while no toxicity was observed with MV-5 cells. Cytotoxicity by acetaminophen required removal of cellular GSH with BSO. This toxicity could be blocked by 2E1 ligands such as 4-methylpyrazole and ethanol, by antioxidants such as N-acetylcysteine and by a spin trapping agent, PBN, but not by inhibitors of lipid peroxidation, suggesting the involvement of reactive species such as the benzoquinone imine which are produced during the P450-dependent metabolism of acetaminophen. Using {dollar}\sp{14}{dollar}C-labeled acetaminophen, protein adducts were produced in the MVh2E1-9 cells. Moreover, this adduct formation was inhibited by the same agents which prevented the cytotoxicity induced by acetaminophen, suggesting a close correlation between adduct formation and cytotoxicity. Acetaminophen at a lower concentration inhibited cell proliferation of both MVh2E1-9 and MV-5; this anti-proliferative effect of acetaminophen was similar in the two cell lines and was independent of CYP2E1. Thus acetaminophen has a dual action in the MVh2E1-9 cells, a CYP2E1-independent effect which prevents cell proliferation and a CYP2E1-dependent action which results in cellular cytotoxicity. (D) When the cells were treated in situ with CCl{dollar}\sb4{dollar}, the CYP2E1 expressed in MVh2E1-9 cells was inactivated and then degraded. This CCl{dollar}\sb4{dollar}-dependent inactivation and degradation could be prevented by 2E1 ligands but not by antioxidants or inhibitors of lipid peroxidation. These actions of CCl{dollar}\sb4{dollar} were enhanced in the presence of cycloheximide to prevent new enzyme synthesis. CCl{dollar}\sb4{dollar} was able to form adducts with cellular macromolecules suggesting that the human P450-2E1 expressed in MVh2E1-9 cells metabolizes CCl{dollar}\sb4{dollar}, generating reactive metabolites which directly inactivate and labilize the P450 for degradation by proteases, including lysosomal protease, present in the HepG2 cells. (Abstract shortened by UMI.).
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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.
