Reactive 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