Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

BACKGROUND AND OBJECTIVE: Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). MATERIALS AND METHODS: Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. RESULTS: Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. CONCLUSIONS: Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Targeting genes in insulin-associated signalling pathway, DNA damage, cell proliferation and cell differentiation pathways by tocotrienol-rich fraction in preventing cellular senescence of human diploid fibroblasts.

BACKGROUND AND OBJECTIVES: Tocotrienols have been known for their antioxidant properties besides their roles in cellular signalling, gene expression, immune response and apoptosis. This study aimed to determine the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs) by targeting the genes in senescence-associated signalling pathways. MATERIALS AND METHODS: Real time quantitative PCR (qRT-PCR) was utilized to evaluate the expression of genes involved in these pathways. RESULTS: Our findings showed that SOD1 and CCS-1 were significantly down-regulated in pre-senescent cells while CCS-1 and PRDX6 were up-regulated in senescent cells (p<0.05). Treatment with TRF significantly down-regulated SOD1 in pre-senescent and senescent HDFs, up-regulated SOD2 in senescent cells, CAT in young HDFs, GPX1 in young and pre-senescent HDFs, and CCS-1 in young, pre-senescent and senescent HDFs (p<0.05). TRF treatment also caused up-regulation of FOXO3A in all age groups of cells (p<0.05). The expression of TP53, PAK2 and CDKN2A was significantly increased in senescent HDFs and treatment with TRF significantly down-regulated TP53 in senescent cells (p<0.05). MAPK14 was significantly up-regulated (p<0.05) in senescent HDFs while no changes was observed on the expression of JUN. TRF treatment, however, down-regulated MAPK14 in young and senescent cells and up-regulated JUN in young and pre-senescent HDFs (p<0.05). CONCLUSIONS: TRF modulated the expression of genes involved in senescence-associated signalling pathways during replicative senescence of HDFs.

Comparable down-regulation of TYR, TYRP1 and TYRP2 genes and inhibition of melanogenesis by tyrostat, tocotrienol-rich fraction and tocopherol in human skin melanocytes improves skin pigmentation.

BACKGROUND AND OBJECTIVE: Antioxidant has been recognized to inhibit UV-induced melanogenesis. This study aimed to elucidate the molecular mechanism of tyrostat, tocopherol and tocotrienol-rich fraction in inhibiting melanogenesis in human skin melanocytes. MATERIALS AND METHODS: Primary culture of melanocytes was exposed to repeated doses of 0.6 J/cm2 UVA for 6 days and treated with tyrostat, tocotrienol-rich fraction or tocopherol alone or in combination. RESULTS: UVA irradiation increased melanin content and tyrosinase activity and up-regulated TYR, TYRP1 and TYRP2 genes. Treatment with tyrostat, tocotrienol-rich fraction or tocopherol decreased melanin content and down-regulated TYR, TYRP1 and TYRP2 genes with decreased tyrosinase activity. Combined treatment exerted better effects as compared to treatment with single compound in decreasing the melanin content and down-regulating TYR, TYRP1 and TYRP2 genes. These findings indicated that tyrostat, tocotrienol-rich fraction and tocopherol inhibit melanogenesis by modulating the expression of genes involved in the regulation of melanin synthesis and inhibiting tyrosinase activity. CONCLUSIONS: Tyrostat, tocopherol and tocotrienol-rich fraction possessed anti-melanogenic properties and might be useful in improving skin pigmentation caused by UVA exposure.

Efficacy of fasting and calorie restriction (FCR) on mood and depression among ageing men.

OBJECTIVE: An intervention study on the FCR (Fasting and Calorie Restriction) dietary regime was carried out to determine its efficacy in improving mood states and depression status among ageing men. SUBJECTS: A total of 32 healthy males (Mean±SD), aged 59.7±6.3 years, with a BMI of 26.7±2.2 kg/m2 were recruited to the study. METHOD: Participants were randomized to either the FCR group (and were instructed to follow a calorie restricted dietary regime with intermittent fasting) or to the control group (in which individuals were asked to maintain their current lifestyle), for a 3 month period. Mood was assessed using the Profile of Mood States and depression was assessed using Beck Depression Inventory-II and Geriatric Depression Scale-15 at baseline, week 6 and week 12 of the intervention. RESULTS: A total of 31 subjects completed the study (n=16, FCR and n=15, control). Significant decreases in tension, anger, confusion and total mood disturbance and improvements in vigor were observed in participants in the FCR group compared to the control group (p<0.05). No significant changes in mean depression scores were observed. Weight, BMI and percent body fat were reduced by 3.8%, 3.7% and 5.7% respectively in the FCR group. CONCLUSIONS: Our findings show that a FCR dietary regime is effective in improving mood states and nutritional status among ageing men.

Total testosterone and sex hormone-binding globulin are significantly associated with metabolic syndrome in middle-aged and elderly men.

Testosterone and sex hormone-binding globulin (SHBG) have been shown to be associated with metabolic syndrome (MS) in men. This study aimed at validating these relationships in a group of middle-aged and elderly men and assessing their strength of association to MS. A cross-sectional study of 332 Malaysian men aged 40 years and above was conducted. The blood of subject was collected under fasting condition for determination of testosterone, SHBG, glucose and lipid levels. Their medical history, smoking and alcohol consumption status, waist circumference (WC), body mass index (BMI) and blood pressure (BP) were recorded. All testosterone and SHBG levels were significantly reduced in MS subjects compared to non-MS subjects (p<0.05). Testosterone and SHBG were correlated significantly with most of the MS indicators without adjustments. In multiple regression analysis, the triglyceride level was the only MS indicator that was significantly, inversely and independently associated with all testosterone measurements and SHBG (p<0.05). Waist circumference was significantly and negatively associated with SHBG level (p<0.05) though not independent of BMI. Total testosterone and SHBG were significantly and inversely associated with the presence of MS. Testosterone and SHBG are potential intervention targets for the prevention of MS in men.

Tocotrienol rich fraction (TRF) supplementation protects against oxidative DNA damage and improves cognitive functions in Wistar rats.

AIM: Oxidative stress is caused by imbalance between the productions of reactive oxygen species (ROS) and antioxidant defense mechanisms. Palm oil antioxidants such as tocotrienol rich fraction (TRF) is known to have neuroprotective effects on neurones by acting against free radical induced neuronal cell death. This study was undertaken to elucidate the effect of TRF on oxidative DNA damage and cognitive functions in experimental rats. MATERIALS AND METHODS: A total of 20 male Wistar rats (aged 3 months) were divided into 2 groups: (i) control group fed with distilled water and (ii) experimental group fed with TRF (200 mg/ kg body weight) for 8 months. DNA damage was determined using Comet assay. Antioxidant enzymes like superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were assessed in the blood. The Morris Water Maze (MWM) test was used to evaluate the cognitive functions. RESULTS: DNA damage was significantly reduced in the experimental group supplemented with TRF compared to the control group (p <0.05). In the group supplemented with TRF, the percentage of DNA damage was 2.87 ± 0.48% compared to 5.96 ± 0.43% in the control group. SOD, GPx, and CAT enzyme activities increased in experimental group. Results from MWM showed improvement in cognitive functions as determined by latency to target platform, swim path and average speed between TRF and control groups. CONCLUSIONS: Continuous supplementation of TRF for 8 months reduced DNA damage and exhibited positive influence in spatial learning and memory.

Comparison of Chlorella vulgaris dressing and sodium alginate dressing: an experimental study in rats.

BACKGROUND AND AIMS: Chlorella vulgaris (CV) is a green microalgae enriched with nutrients, vitamins, minerals and chlorophyll. The aim of our study was to evaluate the potential wound healing effects of CV as a dressing while comparing it to sodium alginate dressing. MATERIALS AND METHODS: Fifteen Sprague-Dawley rats were divided into three groups consisting of control (normal-wound with no dressing), CV and sodium alginate dressings. Wound was created on the dorsal surface of rats using a 6-mm sterilized punch biopsy. The wounds were examined on days 3, 6, 9 and 12 for dryness, exudation, contraction, period of epithelialization, complete wound closure and scar formation. All rats were sacrificed on day 12 and the granulation tissue formed on the wound was excised for the histological examination using Hematoxylin and Eosin stain (H&E). RESULTS: The wound's size treated with CV dressing was significantly reduced (p<0.05) compared to the control and sodium alginate treated wounds. H&E staining showed that CV dressing minimized the formation of scar tissue during the healing process. Although there was no statistically significant difference in the complete wound closure and period of epithelialization among the groups, a significant difference was observed regarding the dryness, exudation and scar formation in the wound. CONCLUSION: CV extract can be used as an effective supplement for wound dressing.

Effect of Tai Chi exercise on DNA damage, antioxidant enzymes, and oxidative stress in middle-age adults.

BACKGROUND: The biochemical mechanisms involving oxidative stress to explain the relationship between exercise and healthy aging are still unclear. METHODS: Tai Chi participants and matched sedentary volunteers age 45 and above were enrolled. Glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) activities; levels of DNA damage using the comet assay; and malondialdehyde (MDA) and advanced glycation end products (AGE) were determined at 0, 6, and 12 months. RESULTS: Tai Chi subjects had decreased normal and increased mildly damaged DNA with elevated GPx activity after 6 months (n=25). Plasma MDA and AGE concentrations decreased significantly after 12 months (n=15) accompanied by increased SOD activity. This may be attributed to the hormesis effect, whereby mild induction of oxidative stress at the first 6 months of exercise resulted in stimulation of antioxidant defenses. These parameters were unchanged in the sedentary subjects in the first 6 months (n=27) except for elevated SOD activity. After 12 months, the sedentary subjects (n=17) had decreased normal DNA and increased severely damaged DNA with unaltered MDA and AGE levels while SOD and GPx activities were significantly elevated. CONCLUSION: Regular Tai Chi exercise stimulated endogenous antioxidant enzymes and reduced oxidative damage markers.

Apoptosis changes and SA-beta galactosidase expression in stress-induced premature senescence (SIPS) model of human skin fibroblasts.

Stress-induced premature senescence (SIPS) model is in vitro model of cellular aging. In this study, apoptosis was evaluated in SIPS model and in replicative senescent fibroblasts. We also compared the activity of senescence-associated beta-galactosidase (SA-beta gal) as a biomarker of cellular aging. Our results suggested that SIPS model and senescent fibroblasts might share similar mechanism of aging and apoptosis pathway.

GAPDH as housekeeping gene for human skin fibroblast senescent model.

Validation of housekeeping gene is important for accurate quantitation of RNA in real time RT-PCR technique. The purpose of this study was to determine the validity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for quantitative real time RT-PCR assessment in human skin fibroblast senescent model. The cells were divided into different treatment groups; young (passage 4), senescent (passage 30), treatment with H2O2 and treatment with A-tocotrienol prior to H2O2 treatment. Our results showed that the expression level of GAPDH was constant with different treatment groups. Therefore, we concluded that GAPDH was suitable to be used as housekeeping gene in human skin fibroblast senescent model.

Long term Tai Chi exercise reduced DNA damage and increased lymphocyte apoptosis and proliferation in older adults.

Effect of Tai Chi exercise on the level of DNA damage using the comet assay, lymphocyte viability and frequency of sister chromatid exchange (SCE) were determined in adults aged above 45. Tai Chi participants of 7 years (n=35), showed higher level of normal DNA and lower level of mild and severely damaged DNA as compared to the sedentary subjects (n=35). The former are suggested to have effective DNA repair mechanism as their frequency of SCE was markedly lower. Higher lymphocyte apoptosis and proliferation found in the Tai Chi participants also indicated that the exercise promotes renewal and regeneration of lymphocytes.

Comparative effects of palm vitamin E and alpha-tocopherol on healing and wound tissue antioxidant enzyme levels in diabetic rats.

The effect of supplementing 200 mg/kg body weight palm vitamin E (PVE) and 200 mg/kg body weight alpha-tocopherol (alpha-Toc) on the healing of wounds in streptozotocin-induced diabetic rats was evaluated. The antioxidant potencies of these two preparations of vitamin E were also evaluated by determining the antioxidant enzyme activities, namely, glutathione peroxidase (GPx) and superoxide dismutase (SOD), and malondialdehyde (MDA) levels in the healing of dermal wounds. Healing was evaluated by measuring wound contractions and protein contents in the healing wounds. Cellular redistribution and collagen deposition were assessed morphologically using cross-sections of paraffin-embedded day-10 wounds stained according to the Van Gieson method. GPx and SOD activities as well as MDA levels were determined in homogenates of day-10 dermal wounds. Results showed that PVE had a greater potency to enhance wound repair and induce the increase in free radical-scavenging enzyme activities than alpha-Toc. Both PVE and alpha-Toc, however, were potent antioxidants and significantly reduced the lipid peroxidation levels in the wounds as measured by the reduction in MDA levels.

Vitamin C and aloe vera supplementation protects from chemical hepatocarcinogenesis in the rat.

The effects of vitamin C and aloe vera gel extract supplementation on induced hepatocarcinogenesis in male Sprague-Dawley rats (120-150 g) by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) was investigated. The severity of the carcinogenesis process was determined by measuring gamma-glutamyl transpeptidase (GGT) and the placental form of glutathione S-transferase (GSTP) histochemically in situ and in plasma and liver fractions. In addition, plasma alkaline phosphatase (ALP) and liver microsomal uridine diphosphate glucuronyl transferase (UDPGT) activity were also determined. Administration of DEN/AAF caused an increase in the surface area and number of enzyme-positive foci (both GGT and GSTP) compared with control. Supplementation of vitamin C or aloe vera gel extract to the cancer-induced rats suppressed this increase significantly (P < 0.05; P < 0.001). Increases in liver UDPGT, GGT, and GSTP activities were also observed with cancer induction that were again suppressed with either vitamin C or aloe vera gel supplementation. Plasma GGT in the DEN/AAF rats were determined monthly for the duration of the experiment and found to be reduced as early as 1 mo with aloe vera gel supplementation and 2 mo with vitamin C supplementation. In conclusion, vitamin C and aloe vera gel extract supplementation were found to be able to reduce the severity of chemical hepatocarcinogenesis.

Different starting times of alpha-tocopherol and gamma-tocotrienol supplementation and tumor marker enzyme activities in the rat chemically induced with cancer.

1. alpha-Tocopherol (alpha-T) and gamma-tocotrienol (gamma-T) were supplemented continuously for 8 weeks in the diets of normal rats and rats chemically induced with cancer using diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF) and partial hepatectomy. Hepatocarcinogenesis was followed by determining the plasma gamma-glutamyl-transpeptidase (GGT) and alkaline phosphatase (ALP) activities as well as placental glutathione S-transferase (PGST) and GGT activities histochemically, at 4-week intervals. 2. Male Rattus norvegicus were supplemented alpha-T and gamma-T at two different doses of 30 and 300 mg/kg diet. The supplementation was started at three different times: simultaneously with DEN administration; 4 weeks; and 8 weeks after DEN administration. 3. Elevation of plasma GGT activities and formation of PGST and GGT positive foci were attenuated significantly (P < 0.05) when alpha-T and gamma-T were supplemented simultaneously with cancer induction. Supplementation begun 4 and 8 weeks after cancer induction did not affect plasma enzyme activities and formation of enzyme-positive foci. 4. alpha-T was more effective than gamma-T, and a lower dose of 30 mg/kg was found to be more effective in reducing the severity of hepatocarcinogenesis.

Nitrofurantoin-induced hepatic and pulmonary biochemical changes in mice fed different vitamin E doses.

The hepatic and pulmonary effects of nitrofurantoin (40 mg/kg, intraperitoneally) were determined at 4 and 24 hr following its administration in mice fed for 10 weeks with a vitamin E sufficient, deficient or enriched diet. Liver glutathione (GSH) was reduced by nitrofurantoin at 4 hr but was unchanged 20 hr later. Nitrofurantoin did not affect liver glutathione peroxidase, glutathione reductase or superoxide dismutase activities. Liver catalase activities were decreased by nitrofurantoin at 4 hr. Lung GSH levels were increased whilst glutathione peroxidase activity was decreased at 4 and 24 hr. Lung glutathione reductase activity was reduced in certain groups. Nitrofurantoin did not affect lung superoxide dismutase, but catalase was decreased at 24 hr. Liver malondialdehyde levels were increased by nitrofurantoin in the vitamin E deficient group whilst lung malondialdehyde levels remained unchanged. Both liver and lung malondialdehyde levels were unaffected by vitamin E supplementation when compared to the vitamin E-sufficient group. These results suggest that nitrofurantoin (40 mg/kg) was deleterious to the liver and lung. Nitrofurantoin-induced lipid peroxidation was seen in vitamin E deficiency but an increase in dietary vitamin E content did not provide additional protection compared to the recommended daily allowance. The antioxidant activities of alpha-tocopherol and gamma-enriched tocotrienol were similar.

Effect of ovariectomy and sex hormone replacement on glutathione and glutathione-related enzymes in rat hepatocarcinogenesis.

The effects of ovariectomy and hormone replacement in control and carcinogen treated female rats were investigated by measuring whole blood and liver glutathione (WGSH, HGSH), glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GRx) and histological evaluation. Hepatocarcinogenesis was induced by diethylnitrosamine and 2-acetylaminofluorene. In control rats not receiving carcinogen, ovariectomy significantly increased the GST and GRx activities. Replacement with either estrogen or progesterone reduced the GST activities to below intact female values whereas replacement of both hormones together brought the GST activities to that of intact females. GRx activities were brought to intact female values by replacement with estrogen or progesterone, either singly or in combination. Neither ovariectomy nor sex hormone/s replacement influenced the levels of WGSH, HGSH and GPx activities. Carcinogen administration to intact rats increased all the parameters measured. Ovariectomized rats treated with carcinogen showed lower GPx and GRx activities at 2 mths. However, replacement with either progesterone or combined estrogen and progesterone increased GPx and GRx activities to original values. On the other hand GST and GPx activities in ovariectomized rats which had carcinogen treatment were lower than intact rats after 5 mths. Replacement with hormones either singly or both brought GST and GPx activities up to intact rat levels receiving carcinogen. The levels of WGSH, HGSH and GRx activities (5 mths) in carcinogen treated rats were not influenced by ovariectomy and/or hormone/s replacement. The results from this study suggested that ovariectomy reduced the severity of hepatocarcinogenesis which was restored by sex hormone/s replacement.

Vitamin E, glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured hepatocytes of rats treated with carcinogens.

1. The effects of alpha-tocopherol and gamma-tocotrienol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (gamma-GT) activities in cultured hepatocytes prepared from rats treated with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated. 2. Both the alpha-tocopherol and gamma-tocotrienol treated hepatocytes showed significantly higher (P < 0.05) GST activities than untreated hepatocytes prepared from the carcinogen treated rats in the first 3 days of culture. Treatment with alpha-tocopherol and gamma-tocotrienol generally resulted in a tendency to increase the GST activities above that in the untreated hepatocytes. 3. Treatment with high doses (125-250 microM) of alpha-tocopherol and low doses (12.5-25 microM) of gamma-tocotrienol generally resulted in a significant reduction in gamma-GT activities at 1-3 days. gamma-GT activities are reduced as the dose of alpha-tocopherol and gamma-tocotrienol are increased.

Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol.

1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated. 2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1-2 days. 3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1-3 days. 4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 microM tocotrienol at 1-3 days in culture compared to the control.

Long-term administration of tocotrienols and tumor-marker enzyme activities during hepatocarcinogenesis in rats.

The effects of long-term administration of tocotrienol on hepatocarcinogenesis in rats induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated by determining the activities of gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), glutathione S-transferases (GSTs), and glutathione (GSH) levels in blood and liver. Twenty-eight male 7- to 8-wk-old Rattus norwegicus rats, weighing 120-160 g, were used in this study. The rats were divided into four treatment groups: a control group on a basal diet, a group fed a basal diet supplemented with tocotrienol (30 mg/kg food), a group treated with DEN/AAF, and a group treated with DEN/AAF and fed a diet supplemented with tocotrienol (30 mg/kg food). Blood was collected monthly, and GGT, ALP, and GSH levels were determined. The rats were killed after 9 mo, and the livers were examined morphologically. Grayish white nodules (2/liver) were found in all the DEN/AAF-treated rats (n = 10), but only one of the rats treated with DEN/AAF and supplemented with tocotrienol (n = 6) had liver nodules. A significant increase in the level of blood and liver GSH, ALP, and GGT activities was observed in the DEN/AAF-treated rats. Liver GSTs were similarly increased with DEN/AAF treatment. Tocotrienol supplementation attenuated the impact of the carcinogens in the rats.

Effect of tocotrienol on the activities of cytosolic glutathione-dependent enzymes in rats treated with 2-acetylaminofluorene.

The effect of tocotrienol on the activities of glutathione S-transferases (GSTs), glutathione reductase (GR) and glutathione peroxidase (GPx) in rats given 2-acetylaminofluorene (AAF) was investigated over a 20 week period. Liver and kidney GST and liver GR activities were significantly increased after AAF administration. Kidney GPx activities were significantly affected; activity assayed with cumene hydroperoxide (cu-OOH) was increased but activity assayed with H2O2 was reduced. Supplementation of the diet with tocotrienol in the AAF-treated rats reduced the increase in enzyme activities. Tocotrienol on its own had no effect on the enzyme activities.