Exercise is beneficial for the maintenance of a good health but it generates reactive oxygen species (ROS) that may result in oxidative stress [¹]. ROS are continuously produced in the normal process of cellular metabolism, but in healthy individuals these are destroyed immediately well within the body's antioxidant defense system. Physical activity increases the generation of ROS in several ways. Two to five percent of oxygen used in the mitochondrial oxidative phosphorylation forms ROS. As the oxidative phosphorylation increases in response to exercise due to increased oxygen consumption, there will be a concomitant increase in free radicals.

Potential sources of ROS during exercise include leakage of electrons from the mitochondrial electron transport chain [²], enhanced purine oxidation, damage to iron-containing proteins, and disruption of Ca²⁺ homeostasis [³]. Other sources of free radicals that increase with exercise include prostanoid metabolism, xanthine oxidase, NAD(P)H oxidase, and several secondary sources, such as the release of radicals by macrophages recruited to repair damaged tissue [⁴]. Hence, exercise can produce an imbalance between ROS and antioxidants, which is referred to as oxidative stress.

Oxidative stress defined as the imbalance in the oxidants and antioxidants, in favour of the oxidants potentially leading to cellular damage [⁵]. Oxidative damage results when the generation of ROS produced exceeds the cellular capacity to destroy them to protect or repair it. ROS lead to alterations in membrane protein structure and also brings changes in enzymatic activity [⁶]. These events may promote damage to cells by causing alterations in mitochondrial and sarcoplasmic reticular membranes and breakdown of lysosomal membranes. An increase in ROS production may occur during and after exercising by an increase of oxygen consumption, increase of catecholamine levels, lactic acid production, elevated rate of hemoglobin auto oxidation and hyperthermia [^7-10]. If the free radical generation is greater than the cell's ability to neutralise them, the radicals will attack cellular components, especially membranous lipids. They initiates a chain reaction involving oxidation of membranous lipids called lipid peroxidation, which leads to generation of more toxic radicals which may harm other cellular components [¹¹].

Antioxidant defence system comprises of enzymes such as catalase, superoxide dismutase, glutathione peroxidase and non-enzymatic antioxidants including vitamin A, vitamin C, vitamin E, ubiquinone and flavonoids. Antioxidants are molecules which interact with ROS and scavenge the free radicals before cellular vital molecules are damaged preventing cellular damage and disease. Vitamin E, a potent naturally occurring lipid-soluble antioxidant possesses the ability to directly quench free radicals and function as a membrane stabilizer. It protects critical cellular structures against the damage from oxygen free radicals and reactive products of lipid peroxidation. The protective effect of vitamin E supplementation against exercise-induced oxidative stress has been reported in humans and rats [^12-14].

Vitamin E (Tri E^®) used in this study was tocotrienol-rich fraction (TRF) palm oil which contains 70% δ-tocotrienol and 30% α-tocopherol. The TRF from palm oil proved to be a more economical and efficient substitute for alpha-tocopherol, significantly inhibited oxidative damage in-vitro to both lipids and proteins in rat brain mitochondria. Studies have shown the ability of vitamin E supplementation to reduce oxidative stress or muscle damage caused by exercise [¹¹]. TRF deficiency can increase free-radical induced tissue injury to levels comparable to those found after exercise, so an adequate status of TRF is important for maintaining membrane integrity during exercise [¹⁵,¹⁶].

In this research, training protocols with rats running on the specially designed treadmill 30 minutes per day for eight weeks were designed to simulate exercise conditions common to rat. The aim of the study was to demonstrate the oxidative stress and DNA damage due to the treadmill exercise protocols on rats and the effect of supplementation with Tri- E^®.

The parameter values were all expressed as the mean ± standard deviation. Significant differences among the groups were determined by one-way ANOVA using SPSS 12.0 software package programme. The results were considered significant if the value is p < 0.05.

Dietary antioxidants play a crucial role in preventing the toxic effects of endogenous reactive oxygen species and studies support a protective role of vitamin E against oxidative stress induced by exercise [²²]. In rats, deficiency in vitamin E can increase the susceptibility of these animals to oxidative stress induced toxicity and human studies have shown that vitamin E supplementation reduces the oxidative stress and lipid peroxidation induced by exercise [²³].

Most studies suggest that stressors like exercise increases the free radical generation in the body which in turn stimulates the increased production of antioxidant enzymes GPx and SOD [²⁴]. Antioxidant enzymes SOD, CAT and GPx prevent the cellular toxic effects of free radicals, generated during oxidative stress by scavenging ROS immediately at the site of their production. A balance between antioxidants and oxidant production ensures a protective cellular environment.

SOD functions as one of the primary enzymatic antioxidant defense against highly reactive superoxide radicals. It catalyses the dismutation of superoxide into oxygen and H₂O₂. SOD converts superoxide to H₂O₂, which is in turn catabolised to water mainly by CAT, preventing the formation of the highly reactive hydroxyl radical (OH^.- ) [²⁵].

Previous studies reported that increased levels of SOD activity in blood and muscle at rest are common in trained individuals, and SOD activity is increased in response to exercise interventions in a trained population [¹¹].

Endurance training has been shown to increase SOD activity in skeletal muscle [²⁶,²⁷]. However, not all studies are consistent with these conclusions. This is consistent with our results which showed that the level of SOD is decreased in exercise group as compared to sedentary group. The reduction in SOD activity is unclear, but it could be due to the age related changes and also probably due to the activity of rats have been reduced during the 8 wks of the confinement in the cage. In reduce physical activity of rats the oxygen radical produced will be less and thus reducing the SOD activity. Supplementation with Tri E^® showed a further decrease in SOD activity as compared to non-supplemented group. Tri E^® may have induced adaptive changes in the antioxidative defenses in order to compensate for greater free radical generation during exercise [²²,²⁸]. The ROS may have been removed by the antioxidant action which then reduced the need for the SOD to be induced.

In GPx catalysed reaction, glutathione is oxidised inactivating H₂O₂ to H₂O. GPx belongs to peroxidase class of enzymes found in the erythrocytes of mammals that prevents lipid peroxidation of the cellular membrane. GPx reduces lipid hydroperoxides generated during the lipid peroxidation to their corresponding alcohols and reduces free hydrogen peroxide to water [²⁹].

It has been reported that there is a significant correlation of GPx activity and weekly training in runners [³⁰]. Ortenblad et al. observed no difference in erythrocyte GPx or glutathione reductase activity between trained and untrained subjects, however muscle activities of these enzymes were higher in the trained subjects [³¹]. In correspondence to our study, we found a significant decrease in the level of GPx activity in exercise group compared to sedentary group, with or without supplementation. GPx activity was also reported to decrease as a result of adaptive changes in trained rats compared to untrained rats [³²]. Vitamin E has glutathione(GSH) sparing effect. By supplementation with Tri E^® GSH is spared. GPx uses GSH and when there is sparing of GSH, action of GPx is not affected by exercise, so we would expect no change in the exercising group with and without vitamin e supplementation.

Reactive oxygen species itself can act as a signal during exercise which upregulates the expression of GPx to prevent the oxidative stress. Catalase is widely distributed in the cell, with the majority of the activity occurring in the mitochondria and peroxisomes. Catalase activity in response to a single bout of exercise is variable. Catalase activity undergoes adaptive changes during the exercise [³³,³⁴].

Reports have also depicted that there is no difference in catalase activity levels following marathon running [¹²]. Our results also showed that the level of catalase activity decreased between the exercise group compared to sedentary group. The results also showed that Cat activity decreased in supplemented Tri E^® compared to non-supplemented and the reason being Tri E^® may have taken over oxidative defense and thus reduced the induction of Cat [²¹]. The mechanism is unclear, however it has been found that tocotrienol in Tri E^® has effect on gene expression of antioxidant response elements.

Comet assay showed that exercise groups had significantly increased DNA damage as compared to the sedentary group. Oxidative tissue damage in vitamin E deficient animals is exacerbated by endurance training and, conversely, it is reduced by high-dose vitamin E supplementation; also, preliminary studies in humans have demonstrated antioxidant protection by high-dose vitamin E supplementation [³⁵].

This is consistent with the study done by Tsai et al, 2001, on human study who reported an increase in DNA damage 24 hour post exercise that persisted through day 7 in response to a 42 km run (average run time 3 hours) [³⁶]. Supplementation with Tri E^® had decreased the DNA damage significantly. This is in accordance with the human studies done earlier which found that supplementation with vitamin E for 8 weeks was effective in reducing DNA damage after an incremental exercise test to exhaustion in healthy non smokers aged between 29-34 years [³⁷]. Supplementation with vitamin E before the exercise seemed to have the good effect, leading the investigators to conclude that vitamin E prevents exercise-induced DNA damage [¹³]. Vitamin E prevents the leakage of the cellular enzymes and content due to ROS [³⁵].

In conclusions, this study showed that eight week exercise training had adaptive effects on antioxidant enzymes activity and supplementation with Tri E^® showed a synergistic effect by further reducing their activity as proven by the reduction in DNA damage in supplemented exercising rats as compared to the non-supplemented group.

The authors declare that they have no competing interests.

MHA and RRJ did the experimental work, while IAI and PSB drafted the manuscript. MMZ and YAMY contributed to the study design. NAH and WZWN took part in planning the study design, supervising the study design, contributed to the analysis and writing of manuscript, edited the paper, revising it critically for important intellectual content and provided the final version. All authors read and approved the final manuscript.

We wish to thank the staff members of Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia for providing technical and financial support.