Musalmah Mazlan

Comparative effects of α-tocopherol and γ-tocotrienol against hydrogen peroxide induced apoptosis on primary-cultured astrocytes

Oxidative stress is thought to be one of the factors that cause neurodegeneration and that this can be inhibited by antioxidants. Since astrocytes support the survival of central nervous system (CNS) neurons, we compared the effect of alpha-tocopherol and gamma-tocotrienol in minimizing the cytotoxic damage induced by H(2)O(2), a pro-oxidant. Primary astrocyte cultures were pretreated with either alpha-tocopherol or gamma-tocotrienol for 1 h before incubation with 100 microM H(2)O(2) for 24 h. Cell viability was then assessed using the MTS assay while apoptosis was determined using a commercial ELISA kit as well as by fluorescent staining of live and apoptotic cells. The uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes were also determined using HPLC. Results showed that gamma-tocotrienol is toxic at concentrations >200 microM but protects against H(2)O(2) induced cell loss and apoptosis in a dose dependent manner up to 100 microM. alpha-Tocopherol was not cytotoxic in the concentration range tested (up to 750 microM), reduced apoptosis to the same degree as that of gamma-tocotrienol but was less effective in maintaining the viable cell number. Since the uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes is similar, this may reflect the roles of these 2 vitamin E subfamilies in inhibiting apoptosis and stimulating proliferation in astrocytes.

Tocotrienol rich fraction supplementation improved lipid profile and oxidative status in healthy older adults: A randomized controlled study

BackgroundVitamin E supplements containing tocotrienols are now being recommended for optimum health but its effects are scarcely known. The objective was to determine the effects of Tocotrienol Rich Fraction (TRF) supplementation on lipid profile and oxidative status in healthy older individuals at a dose of 160 mg/day for 6 months.MethodsSixty-two subjects were recruited from two age groups: 35-49 years (n = 31) and above 50 years (n = 31), and randomly assigned to receive either TRF or placebo capsules for six months. Blood samples were obtained at 0, 3rd and 6th months.ResultsHDL-cholesterol in the TRF-supplemented group was elevated after 6 months (p < 0.01). Protein carbonyl contents were markedly decreased (p < 0.001), whereas AGE levels were lowered in the > 50 year-old group (p < 0.05). Plasma levels of total vitamin E particularly tocopherols were significantly increased in the TRF-supplemented group after 3 months (p < 0.01). Plasma total tocotrienols were only increased in the > 50 year-old group after receiving 6 months of TRF supplementation. Changes in enzyme activities were only observed in the > 50 year-old group. SOD activity was decreased after 3 (p < 0.05) and 6 (p < 0.05) months of TRF supplementation whereas CAT activity was decreased after 3 (p < 0.01) and 6 (p < 0.05) months in the placebo group. GPx activity was increased at 6 months for both treatment and placebo groups (p < 0.05).ConclusionThe observed improvement of plasma cholesterol, AGE and antioxidant vitamin levels as well as the reduced protein damage may indicate a restoration of redox balance after TRF supplementation, particularly in individuals over 50 years of age.

γ-Tocotrienol Prevents Oxidative Stress-Induced Telomere Shortening in Human Fibroblasts Derived from Different Aged Individuals

The effects of palm γ-tocotrienol (GGT) on oxidative stress-induced cellular ageing was investigated in normal human skin fibroblast cell lines derived from different age groups; young (21-year-old, YF), middle (40-year-old, MF) and old (68-year-old, OF). Fibroblast cells were treated with γ-tocotrienol for 24 hours before or after incubation with IC50 dose of H2O2 for 2 hours. Changes in cell viability, telomere length and telomerase activity were assessed using the MTS assay (Promega, USA), Southern blot analysis and telomere repeat amplification protocol respectively. Results showed that treatment with different concentrations of γ-tocotrienol increased fibroblasts viability with optimum dose of 80 µM for YF and 40 µM for both MF and OF. At higher concentrations, γ-tocotrienol treatment caused marked decrease in cell viability with IC50 value of 200 µM (YF), 300 µM (MF) and 100 µM (OF). Exposure to H2O2 decreased cell viability in dose dependent manner, shortened telomere length and reduced telomerase activity in all age groups. The IC50 of H2O2 was found to be; YF (700 µM), MF (400 µM) and OF (100 µM). Results showed that viability increased significantly (p < 0.05) when cells were treated with 80 µM and 40 µM γ-tocotrienol prior or after H2O2-induced oxidative stress in all age groups. In YF and OF, pretreatment with γ-tocotrienol prevented shortening of telomere length and reduction in telomerase activity. In MF, telomerase activity increased while no changes in telomere length was observed. However, post-treatment of γ-tocotrienol did not exert any significant effects on telomere length and telomerase activity. Thus, these data suggest that γ-tocotrienol protects against oxidative stress-induced cellular ageing by modulating the telomere length possibly via telomerase.

Effect of vitamin E (Tri E®) on antioxidant enzymes and DNA damage in rats following eight weeks exercise

BackgroundExercise is beneficial to health, but during exercise the body generates reactive oxygen species (ROS) which are known to result in oxidative stress. The present study analysed the effects of vitamin E (Tri E®) on antioxidant enzymes; superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (Cat) activity and DNA damage in rats undergoing eight weeks exercise.MethodsTwenty four Sprague-Dawley rats (weighing 320-370 gm) were divided into four groups; a control group of sedentary rats which were given a normal diet, second group of sedentary rats with oral supplementation of 30 mg/kg/d of Tri E®, third group comprised of exercised rats on a normal diet, and the fourth group of exercised rats with oral supplementation of 30 mg/kg/d of Tri E®. The exercising rats were trained on a treadmill for 30 minutes per day for 8 weeks. Blood samples were taken before and after 8 weeks of the study to determine SOD, GPx, Cat activities and DNA damage.ResultsSOD activity decreased significantly in all the groups compared to baseline, however both exercised groups showed significant reduction in SOD activity as compared to the sedentary groups. Sedentary control groups showed significantly higher GPx and Cat activity compared to baseline and exercised groups. The supplemented groups, both exercised and non exercised groups, showed significant decrease in Cat activity as compared to their control groups with normal diet. DNA damage was significantly higher in exercising rats as compared to sedentary control. However in exercising groups, the DNA damage in supplemented group is significantly lower as compared to the non-supplemented group.ConclusionsIn conclusion, antioxidant enzymes activity were generally reduced in rats supplemented with Tri E® probably due to its synergistic anti-oxidative defence, as evidenced by the decrease in DNA damage in Tri E® supplemented exercise group.

Evaluation of topical tocopherol cream on cutaneous wound healing in streptozotocin-induced diabetic rats

Diabetes is a common cause of delayed wound healing. The aim of the study was to determine the effect of topical administration of tocopherol cream on the wound healing process in diabetic rats. The study was conducted using 18 male Sprague Dawley rats which were divided into three groups: (I) diabetic rats receiving control cream (n = 6), (II) diabetic rats receiving 0.06% tocopherol cream (n = 6), and (III) diabetic rats receiving 0.29% tocopherol cream (n = 6). Four cutaneous wounds were created at the dorsal region of the rats. Wound healing was assessed by total protein content, rate of wound closure estimation, and histological studies on the tenth day after wounding. Tocopherol treatment enhanced the wound healing process by increasing rate of wound closure and total protein content significantly (P < 0.05) compared to the control group. Histological observation also showed better organized epithelium and more collagen fibers in the tocopherol treated groups. Application of tocopherol cream enhances wound healing process in diabetic condition which is known to cause delay in wound healing.

Gamma-tocotrienol acts as a BH3 mimetic to induce apoptosis in neuroblastoma SH-SY5Y cells.

Bcl-2 family proteins are crucial regulators of apoptosis. Both pro- and antiapoptotic members exist, and overexpression of the latter facilitates evasion of apoptosis in many cancer types. Bcl-2 homology domain 3 (BH3) mimetics are small molecule inhibitors of antiapoptotic Bcl-2 family members, and these inhibitors are promising anticancer agents. In this study, we report that gamma-tocotrienol (γT3), an isomer of vitamin E, can inhibit Bcl-2 to induce apoptosis. We demonstrate that γT3 induces cell death in human neuroblastoma SH-SY5Y cells by depolarising the mitochondrial membrane potential, enabling release of cytochrome c to the cytosol and increasing the activities of caspases-9 and -3. Treatment of cells with inhibitors of Bax or caspase-9 attenuated the cell death induced by γT3. Simulated docking analysis suggested that γT3 binds at the hydrophobic groove of Bcl-2, while a binding assay showed that γT3 competed with a fluorescent probe to bind at the hydrophobic groove. Our data suggest that γT3 mimics the action of BH3-only protein by binding to the hydrophobic groove of Bcl-2 and inducing apoptosis via the intrinsic pathway in a Bax- and caspase-9-dependent manner.

Vitamin E, γ-tocotrienol, Protects Against Buthionine Sulfoximine-Induced Cell Death by Scavenging Free Radicals in SH-SY5Y Neuroblastoma Cells.

ABSTRACT The induction of reactive oxygen species (ROS) to selectively kill cancer cells is an important feature of radiotherapy and various chemotherapies. Depletion of glutathione can induce apoptosis in cancer cells or sensitize them to anticancer treatments intended to modulate ROS levels. In contrast, antioxidants protect cancer cells from oxidative stress-induced cell death by scavenging ROS. The role of exogenous antioxidants in cancer cells under oxidative insults remains controversial and unclear. This study aimed to identify protective pathways modulated by γ-tocotrienol (γT3), an isomer of vitamin E, in human neuroblastoma SH-SY5Y cells under oxidative stress. Using buthionine sulfoximine (BSO) as an inhibitor of glutathione synthesis, we found that BSO treatment reduced the viability of SH-SY5Y cells. BSO induced cell death by increasing apoptosis, decreased the level of reduced glutathione (GSH), and increased ROS levels in SH-SY5Y cells. Addition of γT3 increased the viability of BSO-treated cells, suppressed apoptosis, and decreased the ROS level induced by BSO, while the GSH level was unaffected. These results suggest that decreasing GSH levels by BSO increased ROS levels, leading to apoptosis in SH-SY5Y cells. γT3 attenuated the BSO-induced cell death by scavenging free radicals.

DNA damage and protein oxidation associated with ageing correlate with cognitive dysfunction in a Malaysian population

Abstract Ageing is associated with increased oxidative stress accompanied by cognitive decline. The aim of this study was to evaluate oxidative stress biomarkers and their possible relationship with cognitive performances during ageing among the Malay population. Approximately 160 healthy Malay adults aged between 28 and 79 years were recruited around Selangor and Klang Valley. Cognitive function was assessed by Montreal Cognitive Assessment (MoCA), forward digit span (FDS), backward digit span (BDS), digit symbol, Rey Auditory Verbal Learning Test immediate recalled [RAVLT(I)] and delayed recalled [RAVLT(D)], and visual reproduction immediate recalled (VR-I) and delayed recalled (VR-II). DNA damage, plasma protein carbonyl and malondialdehyde (MDA) levels were also determined. Cognitive function test showed significant lower scores of MoCA, BDS, RAVLT(I), RAVLT(D), digit symbol, VR-I, and VR-II in the older age group (60 years old) compared with the 30-, 40-, and 50-year-old group. The extent of DNA damage was sequential with age: 60 > 50 > 40 > 30, whereas protein carbonyl was higher in 40-, 50-, and 60-year-old groups compared with the youngest group (30 years old). However, the MDA level was observed unchanged in all age groups. Approximately 21.88% of the participants had cognitive impairment. Multiple logistic regression analysis revealed that DNA damage and protein carbonyl levels are predictors for cognitive impairment in healthy Malays. In conclusion, cognitive decline occurred in healthy adult Malay population at an early age of 30 years old with corresponding higher DNA damage and protein oxidation.

Behavioral Assessment and Blood Oxidative Status of Aging Sprague Dawley Rats through a Longitudinal Analysis

BACKGROUND Cognitive frailty emerges as one of the threats to healthy aging. It is in continuum with advancing of age with uncertain indicator between pathological and physiological changes. Alterations in pathways associated with the aging process have been observed including oxidative stress, lipid metabolism, and inflammation. However, the exact mechanisms leading to cognitive decline are still unclear. OBJECTIVE This study was sought to assess the level of cognitive functions and linked with blood oxidative status during normal aging in rats. METHODS A longitudinal study using male Sprague Dawley rats was performed starting from the age of 14 months old to 27 months old. Cognitive functions tests such as open field, Morris water maze and object recognition were determined at the age of 14, 18, 23, and 27 months old and were compared with group 3 months old. Blood was collected from the orbital venous sinus and oxidative status was determined by measuring the level of DNA damage, lipid peroxidation, protein oxidation and antioxidant enzymes activity. RESULTS Aged rats showed declining exploratory behavior and increased in the level of anxiety as compared to the young rats. The level of DNA damage increased with increasing age. Interestingly, our study found that both levels of malondialdehyde and plasma carbonyl content decreased with age. In addition, the level of superoxide dismutase activity was significantly decreased with age whereas catalase activity was significantly increased from 18 months of age. However, no significant difference was found in glutathione peroxidase activity among all age groups. CONCLUSION The progressions of cognitive impairment in normal aging rats are linked to the increment in the level of DNA damage.

Effects of Aging and Tocotrienol-Rich Fraction Supplementation on Brain Arginine Metabolism in Rats

Accumulating evidence suggests that altered arginine metabolism is involved in the aging and neurodegenerative processes. This study sought to determine the effects of age and vitamin E supplementation in the form of tocotrienol-rich fraction (TRF) on brain arginine metabolism. Male Wistar rats at ages of 3 and 21 months were supplemented with TRF orally for 3 months prior to the dissection of tissue from five brain regions. The tissue concentrations of L-arginine and its nine downstream metabolites were quantified using high-performance liquid chromatography and liquid chromatography tandem mass spectrometry. We found age-related alterations in L-arginine metabolites in the chemical- and region-specific manners. Moreover, TRF supplementation reversed age-associated changes in arginine metabolites in the entorhinal cortex and cerebellum. Multiple regression analysis revealed a number of significant neurochemical-behavioral correlations, indicating the beneficial effects of TRF supplementation on memory and motor function.