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dc.contributor.authorLewin, Michelle Helenen
dc.date.accessioned2019-02-15T14:33:14Z
dc.date.available2019-02-15T14:33:14Z
dc.date.issued2004en
dc.identifier.urihttp://hdl.handle.net/1842/34959
dc.description.abstracten
dc.description.abstractReactive oxygen species (ROS) contribute to the pathogenesis of a number of common and important diseases which include atherosclerosis and skin cancer. Selenium (Se) supplementation can protect skin and the endothelium from oxidative damage possibly by increasing the synthesis of antioxidant selenoproteins such as the family of glutathione peroxidases (GPX) and thioredoxin reductase (TR). The relative importance of TR and GPX in protecting endothelial cells (EC) and skin cells from oxidative damage was studied using the EAhy926 and HaCaT cell lines as models of human endothelial cells and keratinocytes, respectively. [⁷⁵Se]-labelled human umbilical vein EC (HUVEC) had a similar selenoprotein profile to EAhy926 cells. In HUVEC, human coronary artery EC (HCAEC), bovine aortic EC (BAEC) and EAhy926 cells, the expression of TR, cytoplasmic GPX (cyGPX) and phospholipid hydroperoxide GPX (PHGPX) was increased by incubating cells with increasing sodium selenite concentrations for 48 hr. Basal and Se- induced levels of these selenoproteins were similar in EAhy926 to HUVEC. BAEC differed considerably from HUVEC and EAhy926 cells in their selenoprotein expression. Therefore, EAhy926 cells appear to be a better model than BAEC for studies of selenoprotein function in humans. In EAhy926 cells TR, cyGPX and PHGPX activities were induced 1.9-fold, 5.3 -fold, and 2.6- fold respectively by sodium selenite supplementation (40 nM for 48 hr). Se-deficient EAhy926 cells were susceptible to oxidative damage by tertiary butyl hydroperoxide (tBuOOH) and oxidised low density lipoprotein (oxLDL), as assessed using percentage retention of LDH. Cytotoxicity was attenuated (p < 0.001) by pre-incubation with 40 nM sodium selenite, a concentration which maximally induced TR and cyGPX. Treatment of Se-deficient EAhy926 cells with gold thioglucose (GTG) (1 μM) significantly inhibited TR activity (74.8 % activity retained) (p < 0.01) but not cyGPX or PHGPX. Treated cells were more susceptible to oxidative damage by t-BuOOH (p < 0.05) or oxLDL (p < 0.05), suggesting that TR may provide antioxidant protection. Cells treated with 10 pM GTG showed inhibition of both TR and the GPXs (14.02 % TR activity (p < 0.001), 40.2 % cyGPX activity (p < 0.001), and 77.5 % PHGPX activity (p < 0.01) retained). Such cells were more susceptible to t-BuOOH toxicity than cells treated with 1 μM GTG (p < 0.05). Hence, both TR and the GPXs may be involved in the prevention of oxidative damage to human EC. In HaCaT cells, expression of TR and cyGPX was optimally induced by incubation with sodium selenite concentrations of 10 nM and 100 nM (increased activities of 2.8-fold and3.8 -fold, respectively). Sodium selenite-treated HaCaT cells were significantly protected from oxidative damage mediated by UVB (p < 0.001) or menadione (p < 0.01). Using UVB as the oxidative agent, loss of protection occurred at sodium selenite concentrations greater than 100 nM. At 1000 nM no protective effect of selenite was observed. There was an accompanying loss of cyGPX activity (p < 0.05), but not of TR or PHGPX expression. No loss of protection was demonstrated at the higher sodium selenite concentrations using menadione as oxidative stressor. The concentration of Se used for protection against UVB thus appears crucial. Se-deficient HaCaT cells incubated with a GTG concentration (10 pM) that significantly inhibited TR activity (18.1 % activity retained) (p < 0.001) but not the GPXs were more susceptible to damage by menadione (p < 0.05), but showed no increase in susceptibility to UVB- mediated damage. Treatment with a GTG concentration (100 pM) which significantly inhibited both TR (3.18 % activity retained) (p < 0.001) and cyGPX activity (33.3 % activity retained) (p < 0.001) increased the susceptibility of HaCaT cells to UVB damage when compared to controls (p < 0.01). The data suggest that menadione, a model agent for UVB oxidative stress, may produce misleading results. TR appears to be important in protecting cells against damage mediated by menadione, but cyGPX to be more important in preventing damage caused by UVB. The two different oxidative stress agents may thus differ in their mechanism of toxicity. TR expression regulated by Se supply and the redox state of the cell may affect cell growth. Changes in TR and cyGPX activity were investigated in human foetal (16-20 weeks gestation) and neonatal (1 day-15 weeks postnatal) liver cytosols. TR activity and concentration, and cyGPX activity in human foetal liver were approximately 3-fold greater than in neonatal liver. These human findings contrast markedly with results in the rat where TR and cyGPX activities increase throughout the foetal, newborn and adult stages. These results cast doubt on the rat as a model for studying cyGPX and TR in human development. In conclusion, the data presented in this thesis suggest that both cyGPX and TR are important contributors to the antioxidant defence mechanisms of EAhy926 and HaCaT cells, and may therefore help to protect against atherogenesis and skin cancer formation respectively.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2019 Block 22en
dc.relation.isreferencedbyAlready catalogueden
dc.titleThioredoxin reductase and glutathione peroxidase in the prevention of oxidative damage to vascular endothelium and the skinen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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