In this study, the impact of the antioxidants, ebselen and NAC, on established monoclonal CD4+ T cell cultures and primary cultures of human PBMCs and CD4+ T cells has been investigated. The impact of age (in vitro lifespan) and/or age of cell donor on markers of T cell integrity and function (DNA damage and cell proliferation) was examined.
Supplementation of CD4+ TCCs, derived from a healthy 26 year old donor (400–23) and a healthy 45 year old donor (385–7) with 30 μM ebselen or 7.5 mM NAC (Table 1) from a young in vitro (34.5 – 31.0 Initial PD) age significantly extended their lifespan. TCCs derived from a healthy 26 year old donor and a healthy 45 year old donor supplemented with 30 μM ebselen had cumulative PDs of 56.2 and 50.8 respectively compared to PDs 44.9 and 44.4 in non-supplemented clones. 7.5 mM NAC supplementation enabled the TCCs from a healthy 26 year old donor and a healthy 45 year old donor to extend their lifespan evidenced by their cumulative PDs (56.1 and 55.8 respectively) compared to the non-supplemented clones (cumulative PDs 49.1 and 47.2).
The impact of supplementation on intracellular redox status (GSH: GSSG ratio) and total glutathione levels was determined. Intracellular redox status is an important mechanism having an invaluable role as a mediator in apoptosis in many cell systems . The GSH: GSSG redox couple maintains the redox environment of the cell and GSH is abundant in the cell  serving as an indicator of the cellular redox environment. Redox status of the cell is hindered on oxidation of even a small amount of GSH. This oxidation results in the formation of GSSG and mixed disulfides between protein sulfhydryl groups in biological systems. It will also decrease the levels of GSH, resulting in an increase in levels of GSSG and so lowering the GSH: GSSG ratio, which has been suggested to be responsible for several human diseases . Supplementation with 30 μM ebselen or 7.5 mM NAC (Figures 3 and 4, A, B) resulted in a significantly higher GSH: GSSG ratio in the CD4+ TCCs examined. These results suggest that in the presence of this level of ebselen, intracellular radical levels were lowered as inferred from the higher GSH: GSSG ratio. NAC is a derivative of GSH formation, explaining the higher GSH: GSSG ratio on supplementation. However, neither of the antioxidants significantly altered the total glutathione levels in either of the CD4+ TCCs.
The results of the analysis of DNA damage levels in TCCs at various point in their lifespan revealed, as has been previously reported , an in vitro age-related increase in DNA damage levels in non-supplemented TCCs (Figures 7 and 8). The enhanced antioxidant status of the supplemented cells, higher GSH: GSSG ratio on supplementation with 30 μM ebselen or 7.5 mM NAC, was in parallel to the significant decreases in oxidative DNA damage levels after 14 weeks of supplementation in CD4+ TCCs from donors of both age groups, compared to the non-supplemented clones. These results do suggest that the DNA damaging free radical/oxidant burden in the supplemented cells was less than for non-supplemented cells.
Previous findings reveal that intracellular reduced GSH levels become depleted before the onset of apoptosis . The maintenance of GSH levels could explain the observed delayed time to apoptosis/cell death in the supplemented cells. This is consistent with the findings that CD4+ TCCs in vitro self-delete via apoptosis at the end of their in vitro lifespan.
In contrast to clones supplemented from an early point in their in vitro lifespan, there was no significant change to the GSH: GSSG ratio or total glutathione levels, proliferative capacity, lifespan or oxidative DNA damage levels, in TCCs derived from a 26 year old donor or a 45 year old donor on supplementation from the midpoint of their in vitro age (58.7 & 63.4 Initial PD) with any of the concentrations of ebselen or NAC, compared to the non-supplemented clones. These results were in line with the previous findings from our group that revealed no significant changes in longevity in CD4+ TCCs on long term culture, from the midpoint of their in vitro lifespan, with 20 mM carnosine . The suggested reason for the failure of carnosine to reveal its antioxidant potential may have been due to the high background of biomolecule damage that already existed in these T cells, accumulated during earlier stages of their lifespan under conditions of 20% O2, that may have decreased the antioxidant or free radical scavenging potential of carnosine. Previous findings from the group of Barnett have demonstrated an age-related increase in levels of oxidised purine and pyrimidine bases within T cells  which is in line with the findings reported here. Greater levels of radicals in older TCCs may affect the function of their defence systems. Published papers have reported a decrease in other markers of T cell integrity with age. The DNA repair capacity in T cells in vitro has been shown to decline with age, revealed in previous studies by this group [6, 7]. Furthermore, Heat Shock Protein activity has been demonstrated to decline in response to age making the body more susceptible to stress-induced damage [17, 18].
In PBMCs and CD4+ T cells ex vivo derived from donors from either age groups, supplementation with 30 μM ebselen or 7.5 mM NAC resulted in a significantly higher GSH: GSSG ratio (Figures 5 and 6), total glutathione levels and proliferative capacity (Figures 1 and 2) at different time points during their life in culture, compared to non-supplemented cells ex vivo. The levels of oxidative DNA damage were significantly decreased after two weeks of culture in human PBMCs and CD4+ T cells ex vivo (Figures 9 and 10), when compared to non-supplemented cells ex vivo.
The significant increase in total glutathione levels on supplementation with 7.5 mM NAC demonstrates the ability of NAC to contribute to enhanced levels of glutathione synthesis in these cells ex vivo, thereby enhancing the intracellular antioxidant status. A significant increase in total glutathione levels was also observed on supplementation with 30 μM ebselen. Ebselen is glutathione peroxide (GPx) mimetic. GPx has the ability to scavenge organic and inorganic peroxides and hydroxyl radicals using GSH as substrate, which in turn becomes depleted. From the results reported in this paper, ebselen may have scavenged the existing intracellular radicals, in so doing the need for GSH to scavenge them would be lessened, leading to higher GSH levels in the cells supplemented with ebselen.
The results reported in this paper provide evidence for the antioxidant potential of 30 μM ebselen or 7.5 mM NAC, against endogenously generated oxidants and those associated with the 95% air, 5% CO2 culture conditions in human PBMCs and CD4+ T cells ex vivo. The higher GSH: GSSG ratio represents an increased antioxidant state on supplementation with ebselen or NAC. This increase in antioxidant status helps explain the decrease in levels of oxidative DNA damage and the increase in proliferative capacity of the supplemented cells. 5 mM NAC supplementation also revealed a significant antioxidant potential, similar to supplementation with 7.5 mM NAC, in human PBMCs and CD4+ T cells ex vivo.
A comparative analysis did not reveal any differences in the effects of 30 μM ebselen or 7.5 mM NAC in human CD4+ T cells ex vivo or CD4+ TCCs in vitro in the markers of T cell integrity and function examined. Interestingly, 5 mM NAC supplementation did not reveal any significant antioxidant potential in CD4+ TCCs in vitro, unlike the potential demonstrated in the ex vivo polyclonal PBMC population and CD4+ T cells ex vivo. Human TCCs are maintained in culture throughout their lifespan over several months. This is not the case in cells ex vivo. It may be that the longer span of culture in vitro altered certain aspects of the biochemical/molecular status of the cells. The in vitro TCCs may have steadily accumulated biomolecule damage during their lifespan under conditions of 20% O2 that may have decreased the antioxidant or free radical scavenging potential of NAC at this specific concentration on long-term supplementation. Under these extensive culture conditions, antioxidant or free radical scavenging effects of NAC at 5 mM concentrations may be insufficient to reveal any significant antioxidant potential in TCCs in vitro.
The impact of long term supplementation of ebselen or NAC has not yet been reported for other cell lines. The previous studies investigating the impact of ebselen in HepG2, HL-60  and glioblastoma cell lines  or the impact of NAC in HepG2 and HeLa  cell lines to name a few, revealed their potential impact only up to 24 hours after the antioxidant supplementation.
Furthermore, in this investigation we found that higher concentrations of ebselen (60-100 μM) or NAC (10 mM) resulted in complete inhibition of the growth of TCCs. It may be that higher concentrations of ebselen or NAC scavenged intracellular oxygen free radicals to such an extent that those necessary for the proliferative pathways were severely/completely limited. Other studies have demonstrated a significant increase in intracellular GSH depletion with an increase in ebselen concentration in HepG2 cells . Ebselen has the ability to bind with GSH to form ebselen selenyl sulphide which reacts with excess of GSH to form a ebselen selenol intermediate that in turn reacts with peroxides . The ebselen selenol intermediate can further react with ebselen selenyl sulphide to form ebselen diselenide. This in turn can further react with peroxides resulting in intracellular GSH depletion that leads to induction of apoptosis  or cells succumbing to stress . This mechanism may also contribute to the complete inhibition of growth of TCCs on supplementation with higher concentrations of ebselen.
The dose- and time-dependent differences on the impact of radical scavenging capacity or pro-oxidant effect of antioxidants has been previously demonstrated . Although ROS are generally thought of as harmful molecules, they play an important role in T cell signalling events such as protein tyrosine phosphorylation and activation of JNK . One of the prominent families of protein kinases, the MAP kinases, includes ERK, JNK (SAPK) and P38 kinase involved in proliferation, differentiation and apoptosis in cells. The impact of ebselen or NAC on these pathways in CD4+ TCCs of different age group maintained at 20% O2 tension is currently under investigation.