A large number of studies have shown that ROS can act as intracellular signaling molecules in many normal physiological processes; however, an increase in such ROS can lead to an imbalance causing oxidative stress . Several factors such as the diet composition, increased energy intake, intense physical exercise, and hypercatabolic conditions are associated with increased cellular levels of ROS and /or decrease in antioxidant defense systems triggering a state of oxidative stress in different tissues/organs [17, 18]. The oxidative process such as lipid peroxidation of biomembranes, produces several compounds that are used as molecular markers, among them MDA is one of the most widely used indicators of the cellular redox state . In this study, the concentration of the lipid peroxidation marker MDA in the liver was higher in the group fed with the commercial diet. According to Sohet et al. , commercial diets contained lower levels of antioxidant vitamins such as vitamin E, resulting in higher values of MDA in hepatic tissue of animals. In another report using mice treated with 0.004, 0.008 and 0.032% vitamin E, a progressive decrease in MDA levels was detected . In a study where male Wistar rats were submitted to exhaustive stress and treated with gavage administration of vitamin E, decreased production of MDA in the kidney tissue was observed when compared with the control group .
As well as MDA, the concentration of H2O2 has also been used as an indicator of oxidative stress. High concentrations of H2O2 are closely related with lipid peroxidation, where the H2O2 in the cell can be converted by the Fenton reaction to the hydroxyl radical, a highly reactive compound involved in the initiation of lipid peroxidation . H2O2 can be formed from the degradation of superoxide produced during aerobic respiration, and by the exposure of cells to physical, chemical and biological agents .
Venditti et al.  showed that vitamin E decreased H2O2 release during basal respiration. This effect led to reduced ROS flow from the mitochondria to the cytosol, limiting oxidative damage to the liver. In this current study, the vitamin E content of the AIN 93 diet was 2.5-fold higher (0.015%) than that of the commercial diet (0.006%), which might suggest participation of Vitamin E, along with other nutrients in the diet, in the responses observed. For instance, in the livers of the animals fed with AIN 93 there was less accumulation of H2O2, when compared to the animals on the commercial diet, indicating that vitamin E may be interfering in ROS levels during normal cell metabolism.
Vitamin E is an important fat-soluble antioxidant in the body and operates with some of the antioxidant enzymes tested in this study, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), to protect cells from attack by ROS . SOD provides the first line of defense against oxygen derived free radicals . Under stress conditions, high SOD activity reflects a compensatory mechanism to reduce the superoxide radical. Male rats Wistar fed with the control diet supplemented with 0.01% of vitamin E showed a reduction in SOD activity . In the results presented here, there was lower SOD activity in the livers of the AIN 93 fed group compared to the commercial diet group, suggesting that vitamin E might play an important role in lipid peroxidation and, indirectly, in regulating SOD activity by maintaining a reduced level of superoxide in the cell system. The SOD isoforms II and V were hardly detectable following PAGE of liver extracts of mice fed on the AIN 93 diet, which could account for the reduction in total SOD activity observed in Table 2. This is an important result, since the increased concentration of vitamin E and possibly other compounds of the AIN 93 diet clearly affected specific SOD isoforms, one located in the mitochondria (SOD II) and more strongly one located in the cytosol (SOD V), since the latter accounts for a higher SOD activity when compared to SOD II. Within the cell, vitamin E partitions into the hydrophobic core of the various cell membranes, including the inner and outer membrane of the mitochondria , although the relative concentration of vitamin E differs from one membrane to another . Furthermore, α-tocopherol supplementation in human subjects and animal models has been shown to decrease lipid peroxidation and superoxide production by impairing the assembly of NADPH oxidase, as well as by decreasing the expression of scavenger receptors (SR-A and CD36) , to which our results appear to match such a possibility. The reduction observed in SOD activity in the livers of animals subjected to the increased vitamin E diet also suggests that the production of the superoxide radical is likely to be diminished more likely in the cytosol and the mitochondria, which agrees and can be clearly correlated to the specific depletion of SOD II and SOD V. Moreover, vitamin E has also been shown to prevent the induction of metallothionein synthesis as well as lipid peroxidation in the liver of mice administered the mitochondrial inhibitor 2,4-dinitrophenol , which agrees with the findings observed here of the depletion of the specific SOD isoforms and reduction in lipid peroxidation. Moreover, Fe-SOD isoforms, which can be found in living cells, but not necessarily in all living organisms, were not detected following PAGE in this work.
The enzymes CAT and GSH-Px are part of the next step of the antioxidant defense mechanism, converting H2O2 to water . Alper et al.  reported a decrease in CAT activity in rats fed with a diet deficient in vitamin E. The authors suggested that the decrease in CAT activity might be due to the suppression of heme biosynthesis. Heme is a prosthetic group that consists of an iron atom contained in the center of a heterocyclic organic ring termed porphyrin, which is present in the molecule of CAT, and is synthesized in the liver and erythroid tissues . Studies with animals deficient in vitamin E showed a decrease in hepatic activity of heme proteins such as CAT and microsomal cytochrome P450 and b5. In this study, there was a significant increase in CAT activity in the livers of mice grown on the AIN 93 diet, which contained 2.5 fold higher more vitamin E than the commercial diet, supporting a role for this vitamin in improving the rate of removal of H2O2 in metabolically normal animals. Thus, the higher CAT activity observed in animals fed with the AIN 93 diet could explain the lower concentration of H2O2 observed (Figure 1B). Similar results were described by Ryan et al.  in a study on the muscles of rats fed with supplemented vitamin E or normal non-supplemented rat chow. GSH-Px can act directly on H2O2, however, this enzyme also acts in the inactivation of organic hydroperoxides . There was little difference in the activity of GSH-Px in the livers of the mice fed with the two different diets. Similar results were reported in a study with rats fed with diets supplemented with vitamin E and a control diet . GR is an enzyme that is used for the regeneration of reduced glutathione from oxidized glutathione, especially when the cell is exposed to free radicals. Shireen et al.  working with rats fed with an AIN 76 diet and AIN 76 supplemented with vitamin E and C, demonstrated that there was a significant increase in the activity of both GSH-Px and GR, in the animals fed with the vitamin supplemented diet. In this study, the activity of GR in the livers of animals fed with the AIN 93 diet was significantly lower than in animals fed with the commercial diet, suggesting that the AIN 93 diet group has a lower demand for reduced glutathione in the cellular defense mechanisms, or that GR may not have a major role as a defense enzyme under the conditions tested. A wide range of antioxidant enzymes exist in order to keep the redox state of the cell, so they are important not only under normal metabolic conditions, but also when dealing with stressful conditions. Although interesting responses in enzyme activities were detected in this research, the role of other peroxidases or antioxidant enzymes cannot be ruled out and should be considered in future research.
Before any major conclusions can be drawn, it is important to state that this study did not investigate varying concentrations of vitamin E or of any other individual components of the diets, which limits the impact of the results and how they are correlated with the major changes observed. Even with such a limitation, the data obtained are not invalidated. However, future studies should be performed in a way that varying concentrations of vitamin E and other components of both diets are tested so that the effects on the antioxidant responses are more clearly understood and the putative key role of vitamin E is confirmed. Based on the composition of both diets, it cannot be ruled out that other components may have had an indirect effect on mouse metabolism, which might have resulted in a response by the antioxidant system. Nevertheless, we must state that the fact that a high vitamin E concentration was used and that among the components of the diet vitamin E is clearly the only direct and the major non-enzymatic antioxidant included, we may assume that Vitamin E appears to have an important participation in the responses observed.