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Title: Human vascular endothelial cells in culture : a model system for studying oxidative stress
Author: Millican, Stephanie A.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1993
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Cell culture systems were used to investigate the mechanisms of oxidant-induced cellular damage. The Girardi heart cell line was used to investigate possible mechanisms of ischaemic cell damage whilst human endothelial cells derived from the umbilical vein (HUVEC) were used to study the direct cellular effects of hydrogen peroxide (H2O2). A number of parameters including cytosolic enzyme leakage, ATP content and DNA and protein synthesis were used to assess cell damage. Girardi cells were found to be resistant to the effects of oxygen deprivation. In contrast, HUVEC were sensitive to H2O2 and concentrations of H2O2 250M resulted in irreversible cell damage. Irreversible damage produced by H2O2 was dependent upon the initial exposure time of the cells to H2O2, the cellular metabolism of H2O2 and the possible conversion of H2O2 to the hydroxyl radical via the iron catalysed Fenton reaction. Catalase activity was not detectable in HUVEC. In contrast, HUVEC contained relatively high concentrations of reduced glutathione (GSH) which decreased, with growth, during culture and were also reduced by the experimental treatment conditions. Cell damage appeared to occur independently of cellular GSH, however depletion of GSH with butathione sulfoximine, to below 10&'37 of control values did potentiate cytotoxicity in response to H2O2. Depletion of ATP, alone, did not correlate with irreversible cell damage. However, cells could recover from oxidant damage as long as the capability to synthesise ATP was maintained. The main mechanism by which ATP was depleted was via the activation of poly (ADP-ribose)polymerase, as demonstrated by the ability of 3-Aminobenzamide to prevent ATP depletion and cell lysis. The mechanism for this activation is most likely to be a consequence of hydroxyl radical-induced damage to DNA. However, activation of poly(ADP-ribose)polymerase was not the only mechanism which accounted for the depletion of ATP and a H2O2-dependent pathway appeared to exist.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available