Normal human cells exhibit a limited capacity for proliferation in culture and this finite replicative lifespan has frequently been used as a model of human aging in mitotic tissues and organs.¹ This phenomenon is considered to be associated with reduction in telomere length as an indicator of the number of cell divisions undergone. The human telomere is a simple repeating sequence of six bases, TTAGGG, located at the ends of chromosomes.² It is thought that telomeres have multiple roles, including protection against degeneration, reconstruction, fusion and loss.³,⁴ The telomeric repeat sequences are added on by the enzyme telomerase which, when present, compensates for the loss of DNA from the end of chromosomes due to incomplete replication. In normal human somatic cells, because of inherent limitations in the mechanics of DNA replication, telomeres shorten at each cell division. In the absence of telomerase, when telomere shortening reaches a critical limit, cells are susceptible to chromosomal aberrations such as end-to-end fusion and aneuploidy. In such a situation, the cells cease to divide and reach replicative senescence.⁵,⁶ Telomere length may restrict the replicative potential of hemopoietic cells contributing to the decline in immune function with age.⁷ The erosion of chromosome ends, or telomeres, was proposed as a major mechanism that contributes to cellular aging.⁸

While convincing evidence has been obtained that telomere shortening occurs during aging both in vitro and in vivo, additional factors are likely to contribute to aging processes. Thus, it has been hypothesized that oxidative stress is a major determinant of human aging in vivo and that the resistance of animals to oxidative stress may decrease with advancing age.⁹,¹⁰ There is increasing evidence that this aspect of the aging process can be reproduced in vitro. For example, it has been shown that increasing the concentration of reactive oxygen species (ROS), either by increasing the oxygen partial pressure, or by treating cells with H₂O₂, can induce premature senescence in young diploid human fibroblasts.¹¹ H₂O₂ is one of the oxidant species and it can be genotoxic. Treatment of human fibroblast with H₂O₂ which induced oxidative damage results in a growth arrest state that is similar to senescence cell.¹²

If free radicals and oxidative stress are at least partly responsible for lifespan and aging, it follows that antioxidant should prolong life and retard aging. The biological activity of vitamin E has generally been associated with it well-defined antioxidant property. Vitamin E is the major chain-breaking antioxidant preventing the propagation of oxidative stress, especially in biological membranes.¹³ Vitamin E also has revealed many important molecular properties, such as the scavenging of ROS to prevent oxidative damage that is associated with many diseases, or the modulation of signal transduction and gene expression in antioxidant and non-antioxidant manners.¹⁴ The other biological functions of vitamin E that are unrelated to its antioxidant properties include its roles in cellular signalling, gene expression, immune response, and apoptosis are now considered to be of importance.¹⁵,¹⁶ In the vitamin E group, α-tocopherol is considered to be the most active form. However, recently, tocotrienol have gained increasing scientific interest due to their eminent antioxidant effects and a nonantioxidant activity profile that differs somewhat from that of tocopherol.¹³ Tocotrienols are identical in structure to tocopherols except for the degree of saturation in their side chain. Tocopherol has a saturated phytyl tail, while tocotrienols posses an unsaturated isoprenoid side chain. The prenyl side chain of tocotrienol has been postulated to be responsible for the differential membrane distribution and metabolism of tocotrienols when compared with tocopherols. α-Tocotrienol was observed in vitro to possess a remarkably higher antioxidant activity against lipid peroxidation than α-tocopherol. This was due to a more uniform distribution in the membrane lipid bilayer providing a more efficient interaction of the chromanol ring with lipid radicals.¹⁷

In view of this, it is of interest to investigate the effects of tocotrienol in preventing oxidative stress-induced cellular aging by determining telomere lengths and telomerase activities in normal skin fibroblast derived from young, middle and old age humans. Therefore the objective of this study was to determine the protective effects of palm γ-tocotrienol treatment before or after H₂O₂-induced oxidative stress on cell viability, telomere length and telomerase activity in normal human skin fibroblast cell lines derived from different age groups viz; young (21-yearold), middle (40-year-old) and old (68-year-old).

Accumulation of oxidative damage plays an important role in aging and associated diseases.¹⁸ The pioneering discovery of Hayflick and Moorhead identified the cultured human diploid fibroblast (HDF) as a potential model for aging.¹ There is evidence that in vitro cellular senescence may directly correlate with aging in vivo. In this study, we explored the effect of H₂O₂ on fibroblast cell lines derived from young, middle and old age humans and determined the protective effect of γ-tocotrienol against H₂O₂-induced oxidative stress by determining cell viability as measured by the MTS assay, and examining the morphology of cells by staining with specific fluorescence dyes that differentiate live from apoptotic cells, determination of telomere length as well as telomerase activity using Southern blot analysis and telomere repeat amplification protocol respectively.

The data showed that H₂O₂ at concentrations 700 µM, 400 µM and 100 µM were cytotoxic to fibroblast cells derived from young, middle and old age humans respectively as it increased the number of cell loss as well as the number of apoptotic cells as shown by the fluorescence dyes staining. Almost all the cells were not viable at H₂O₂ concentration of 1000 µM, 900 µM and 800 µM for fibroblast cells derived from young, middle and old age humans respectively. This is in agreement with earlier observations.¹⁹ H₂O₂ has been reported to directly stimulate endonuclease activity in renal tubular epithelial cells, leading to DNA fragmentation and cell death via apoptosis.²⁰ The present data showed that fibroblast cells derived from old age human was more susceptible to the action of H₂O₂. This is in agreement with earlier observations.²¹ Old fibroblasts were reported to be more sensitive to oxidative stress due to decreased antioxidant activity, less capability to repair, increased reactive oxygen species (ROS) production, and increased sensitivity of DNA towards the action of ROS.²²

The data showed that at low concentration, γ-tocotrienol increased the number of viable cell. At γ-tocotrienol concentration of 80 µM the number of cell viable is maximum for young fibroblasts and 40 µM for middle and old fibroblasts. However, high concentration of γ-tocotrienol is cytotoxic. There was more than 50% reduction in viable cell number at concentrations greater than 230 µM, 280 µM and 110 µM γ-tocotrienol for young, middle and old fibroblasts respectively. This probably reflects the pro-oxidant activity of γ-tocotrienol.

The present data also showed that γ-tocotrienol provides protection against H₂O₂-induced cell loss in fibroblast cells derived from all age groups. The protection was more effective in middle and old fibroblasts. Previous study reported that vitamin E reduced H₂O₂-induced HO^· generation and subsequent DNA base pair modification in human oral epithelial cells.²³ Vitamin E was found to decrease H₂O₂-induced DNA strand breaks in human skin cell line VH10.²⁴ Konopacka et al. (1998) reported that the addition of vitamin E immediately after radiation treatment to bone marrow polychromatic erythrocytes, reduced radiation-induced micronucleus formation.²⁵ It has been suggested that the inhibitory effect of vitamin E micronucleus formation may be due to modulation of the DNA repair system. Indeed, measurement of DNA repair ability tested in lymphocytes indicated that vitamin E increased the removal rate of damaged DNA compared to cells that are not treated with vitamin E.

Telomere shortening is one of a few mechanisms mediating the development of the senescent phenotype. Telomere effects have been reported to be the predominat trigger of premature senescence in Werner syndrome, a disease that caused many clinical features associated with aging in human. Microarray analyses showed that mRNA expression patterns induced in senescent Werner syndrome cells appeared similar to those in normal strains.²⁶

Data in the present study seems to suggest that both telomere shortening and decrease telomerase activity to be contributing factors in cellular aging. In the present study, treatment of fibroblast cells with H₂O₂ resulted in shortening of telomere length and reduction of telomerase activity. However, γ-tocotrienol treatment protected against H₂O₂-induced telomere shortening. γ-tocotrienol was also shown to protect against H₂O₂-induced telomerase activity loss in fibroblast cells derived from all age groups. Therefore the anti aging effect of tocotrienols may not only be due to their potent antioxidant properties but may also involve its role in maintaining telomere length possible mediated by telomerase. Consistent with the telomere theory of aging, telomerase positive human diploid fibroblasts were resistant to replicative senescence.⁵ Our findings showed similar results whereby increased telomerase activity induced by γ-tocotrienol cause a significantly increased of telomere length in skin fibroblast derived from young and old age humans.

Our data that showed shortening of telomere length with H₂O₂ treatment is consistent with previous findings which reported that telomeres are particularly sensitive to oxidative stress.²⁷ A study by Rubio et al. (2004) showed that telomerase influences the stress response indirectly by altering telomere length, mainly by elongating shorter telomeres.²⁸ Thus, telomere length does not only determine replicative senescence, but also influences the senescence response to genotoxic agents.

Telomere shortening has been proven to be a cell division counter in proliferating fibroblast, or ‘replicometer’. Telomere shortening down to a threshold length seems to be the best known predictor of senescence.²⁹ A study by Von Zglinicki (2002) showed that telomere shortening is largely dependent on the interplay of oxidative stress and antioxidant defense rather than to counter clock of numbers of cell divisions.²⁷ H₂O₂ has been the most commonly used inducer of stress-induced premature senescence (SIPS), which shares features of replicative senescence including similar morphology, increase senescence-associated γ-galactosidase activity and cell cycle regulation.¹²

It has been reported that treating cells exogenously with a certain hydrogen peroxide (H₂O₂) concentration can trigger entry into a senescent-like state.³⁰ Sublethal dose (IC₅₀) of H₂O₂ used in this study induced shortening of telomere length and reduction in telomerase activity of skin fibroblast cells derived from young and old age humans.

Our data that showed γ-tocotrienol protected against H₂O₂-induced telomere shortening and protected skin fibroblast against H₂O₂-induced telomerase activity loss suggesting its effect in protecting and recovering the skin fibroblast from oxidative stress. Tocotrienols are capable of scavenging and quenching reactive oxygen species. This antioxidant activity however, remains mainly with its ‘chain-breaking’ property which neutralizes peroxyl and alkoxyl radicals generated during lipid peroxidation.¹³ As vitamin E is also involved in cell signaling, we suggested that elongation of telomere length and activation of telomerase activity by γ-tocotrienol may be mediated by cell signaling pathway. Previous study showed that vitamin E supplementation inhibits chromosomal alterations that are measured by chromosome gaps and breaks in c-myc proto-oncogene and transforming growth factor co-overexpressing transgenic mice.³¹ Additional studies have shown that c-myc may also be a ‘key switch’ for the induction of telomerase activity.³² The c-fos expression is induced when cells are exposed to H₂O₂ and UV, and both H₂O₂ and UV induce DNA damage.³³ The c-fos encodes a nuclear protein that forms heterodimer with jun and binds a common DNA site (AP-1) to regulate various transcriptional activity. Vitamin E treatment also has been shown to induce c-jun expression as well as AP-1 binding activity in human breast cancer cells.³⁴,³⁵ These studies suggest that the protective role of vitamin E against DNA damage induced by H₂O₂ and UV is in part, mediated by upregulation of c-fos expression and AP-1 binding activity. Hadshiew et al. (2000) in their study showed that there are homologies between certain protein kinases and telomeric protein suggest a link between chromosome stability and cell cycle regulation.³⁶ In fact, a critical reduction in telomere length appears to interfere with transcription of subtelomeric genes and leads to cell cycle arrest or apoptosis and finally the aging phenotype.

The results obtained showed that γ-tocotrienol at low concentration was able to protect against H₂O₂-induced cell loss in fibroblast cell lines derived from different age groups. γ-tocotrienol was also able to protect against H₂O₂-induced telomere shortening and telomerase activity loss. Therefore we suggest that γ-tocotrienol protects against oxidative stress-induced cellular aging by modulating the telomere length possibly via telomerase. However more studies need to be done in order to understand the mechanisms.

Previously published online: www.landesbioscience.com/journals/oximed/article/9940

We thank Professor Dr. Hayati Abdul Rahman (Department of Pathology) and Associate Professor Dr. Azian Abdul Latif (Department of Anatomy) for their help in interpreting the morphological study. This research was supported by the Ministry of Science, Technology and Innovation under the Intensified Research in Prioritised Area (IRPA) grant 06-02-02-0021/PR0008/09-7.