Comparing the effects of vitamin E tocotrienol-rich fraction supplementation and α-tocopherol supplementation on gene expression in healthy older adults

OBJECTIVES This study aims to compare the differential gene expression resulting from tocotrienol-rich fraction and α-tocopherol supplementation in healthy older adults. METHODS A total of 71 eligible subjects aged 50 to 55 years from Gombak and Kuala Lumpur, Malaysia, were divided into three groups and supplemented with placebo (n=23), α-tocopherol (n=24) or tocotrienol-rich fraction (n=24). Blood samples were collected at baseline and at 3 and 6 months of supplementation for microarray analysis. RESULTS The number of genes altered by α-tocopherol was higher after 6 months (1,410) than after 3 months (273) of supplementation. α-Tocopherol altered the expression of more genes in males (952) than in females (731). Similarly, tocotrienol-rich fraction modulated the expression of more genes after 6 months (1,084) than after 3 months (596) and affected more genes in males (899) than in females (781). α-Tocopherol supplementation modulated pathways involving the response to stress and stimuli, the immune response, the response to hypoxia and bacteria, the metabolism of toxins and xenobiotics, mitosis, and synaptic transmission as well as activated the mitogen-activated protein kinase and complement pathways after 6 months. However, tocotrienol-rich fraction supplementation affected pathways such as the signal transduction, apoptosis, nuclear factor kappa B kinase, cascade extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2, immune response, response to drug, cell adhesion, multicellular organismal development and G protein signaling pathways. CONCLUSION Supplementation with either α-tocopherol or tocotrienol-rich fraction affected the immune and drug response and the cell adhesion and signal transduction pathways but modulated other pathways differently after 6 months of supplementation, with sex-specific responses.

Antioxidant effect of 4-nerolidylcatechol and -tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

The effects of alpha-tocopherol supplementation on fracture healing in a postmenopausal osteoporotic rat model

OBJECTIVE: Osteoporosis increases the risk of bone fractures and may impair fracture healing. The aim of this study was to investigate whether alpha-tocopherol can improve the late-phase fracture healing of osteoporotic bones in ovariectomized rats. METHOD: In total, 24 female Sprague-Dawley rats were divided into three groups. The first group was sham-operated, and the other two groups were ovariectomized. After two months, the right femora of the rats were fractured under anesthesia and internally repaired with K-wires. The sham-operated and ovariectomized control rat groups were administered olive oil (a vehicle), whereas 60 mg/kg of alpha-tocopherol was administered via oral gavage to the alpha-tocopherol group for six days per week over the course of 8 weeks. The rats were sacrificed, and the femora were dissected out. Computed tomography scans and X-rays were performed to assess fracture healing and callus staging, followed by the assessment of callus strengths through the biomechanical testing of the bones. RESULTS: Significantly higher callus volume and callus staging were observed in the ovariectomized control group compared with the sham-operated and alpha-tocopherol groups. The ovariectomized control group also had significantly lower fracture healing scores than the sham-operated group. There were no differences between the alpha-tocopherol and sham-operated groups with respect to the above parameters. The healed femora of the ovariectomized control group demonstrated significantly lower load and strain parameters than the healed femora of the sham-operated group. Alpha-tocopherol supplementation was not able to restore these biomechanical properties. CONCLUSION: Alpha-tocopherol supplementation appeared to promote bone fracture healing in osteoporotic rats but failed to restore the strength of the fractured bone.

Antioxidant supplementation decreases the cell death rate in the prostatic stromal tissue of long-term castrated rats

OBJECTIVE: The purpose of this study was to compare the effects of castration on cell death rate of the adult rat prostates and to evaluate the benefic action of alpha tocopherol supplementation to avoid apoptosis post-orchiectomy. MATERIAL AND METHODS: Thirty male Wistar rats weighing 250-300g were divided into three groups: group I - they were subjected to bilateral orchiectomy and sacrificed eight weeks after the procedure; group II - subjected to bilateral orchiectomy and alpha-tocopherol supplementation for four weeks preceding the procedure; and group III - subjected to bilateral orchiectomy and alpha-tocopherol supplementation for four weeks preceding the procedure and for eight weeks afterwards. At the end of the experiment, the prostatectomy was performed in all rats. The presence of oxidative stress was determined by assaying the blood level of 8-isoprostane and the occurrence of apoptosis was evaluated by identification of active caspase-3 through immunohistochemical analysis. RESULTS: The statistic analysis of active caspase-3 showed that in the long-term castrated group the detection was higher than in groups were the alpha-tocopherol was supplemented (p=0.007). Analysis of 8-isoprostane levels showed higher concentrations of reactive oxygen species in group I compared to other groups (p<0.05). Groups II and III presented active caspase-3 lower than in group I (p<0.05). CONCLUSION: Our exploratory analyses demonstrate a method to study the aging process and its influence on oxidative stress of prostatic tissue and cells death rate. Based on our results we can suggest that alpha tocopherol supplementation can decrease the apoptotic process as well as the oxidative stress levels induced by androgen deprivation of the prostate gland.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

-Tocopherol ameliorates cypermethrin-induced toxicity and oxidative stress in the nematode Caenorhabdtis elegans.

Oxidative stress and other effects induced by cypermethrin (CYP, 15 mM) and their amelioration by -tocopherol (400 M) was studied in the nematode Caenorhabditis elegans. The worms exposed for 4 h to CYP showed increased levels of reactive oxygen species (46%), H2O2 (37%) and protein carbonyls (29%), accompanied by decreased lifespan and brood size. However, exposure to both CYP and a-tocopherol resulted in diminution of above alterations with the worms exhibiting relatively lower levels of ROS (30%), H2O2 (15%), protein carbonyls (14%), altered antioxidant enzyme activities and normal lifespan and brood size. The results suggest that CYP induces oxidative stress in C. elegans and the strategy of intervention with -tocopherol could be exploited to offset this induced oxidative stress.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.

alpha-Tocopheryl succinate potentiates the paclitaxel-induced apoptosis through enforced caspase 8 activation in human H460 lung cancer cells

Paclitaxel is one of the chemotheraputic drugs widely used for the treatment of nonsmall cell lung cancer (NSCLC) patients. Here, we tested the ability of alpha-tocopheryl succinate (TOS), another promising anticancer agent, to enhance the paclitaxel response in NSCLC cells. We found that sub-apoptotic doses of TOS greatly enhanced paclitaxel-induced growth suppression and apoptosis in the human H460 NSCLC cell lines. Our data revealed that this was accounted for primarily by an augmented cleavage of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor) or z-IETD-FMK (a caspase-8 inhibitor) blocked TOS/paclitaxel cotreatment-induced PARP cleavage and apoptosis, suggesting that TOS potentiates the paclitaxel-induced apoptosis through enforced caspase 8 activation in H460 cells. Furthermore, the growth suppression effect of TOS/paclitaxel combination on human H460, A549 and H358 NSCLC cell lines were synergistic. Our observations indicate that combination of paclitaxel and TOS may offer a novel therapeutic strategy for improving paclitaxel drug efficacy in NSCLC patient therapy as well as for potentially lowering the toxic side effects of paclitaxel through reduced drug dosage.

Inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate: effect of pre-administration of alpha-tocopherol.

Rats pre-administered with alpha-tocopherol (10 mgs/day) for 7 days afforded a significant protection at the tissue level against the lowering of superoxide dismutase and glutathione peroxidase, especially the selenium-dependent glutathione peroxidase. The protective action of alpha-tocopherol in the diethyldithiocarbamate treated rats may be attributed to its antioxidant/free radical scavenging action. It is concluded that selenium-dependent glutathione peroxidase and alpha-tocopherol act in a complementary fashion to block free radical formation.

The effect of alpha-tocopherol on the oxidative stress and antioxidants in idiopathic IgA nephropathy.

OBJECTIVE: Nearly 25% of IgA nephropathy patients progress to end-stage renal disease over a 20-25 year follow-up period. IgA containing immune complex stimulates oxygen free radical production by mesangial cells in vitro, which may mediate glomerular injury in this disorder. Therefore, we studied whether dietary supplementation with the antioxidant agent, vitamin E, attenuates renal damage in patients with IgA nephropathy. MATERIAL AND METHOD: Twenty-eight patients with idiopathic IgA nephropathy were supplemented with vitamin E 400 mg/day for 6 months. Antioxidant enzymes, glutathione, plasma malondialdehyde (MDA), and renal function were studied after 3 and 6 months therapy. RESULT: The result of the study showed high plasma MDA and significant reduction after therapy (1.15 +/- 0.45 VS 0.86 +/- 0.30 microM, p < 0.0001). The RBC vitamin E was also elevated statistically significantly (5.07 +/- 2.42 VS 15.70 +/- 3.37 microM, p < 0.001). Glutathione peroxidase activities were decreased (38.52 +/- 15.53 VS 23.97 +/- 7.63 U/gHb, p < 0.001). Glutathione was also decreased (44.80 +/- 9.70 VS 32.45 +/- 6.74 mg/dl, p < 0.05) but there were no changes in red cell catalase and superoxide dismutase activities. Creatinine clearance, proteinuria, urine N-acetyl glucosaminidase and beta2-microglobulin also showed no improvement. CONCLUSION: Our data demonstrated the particular group of IgA nephropathy patients with low vitamin E level and high oxidative stress had significant reduction of oxidative stress after vitamin E therapy.

Effects of alpha-Tocopherol on Cadmium-Induced Toxicity in Rat Testis and Spermatogenesis

Cadmium is known to exert toxic effects on multiple organs, including the testes. To determine if alpha-tocopherol, an antioxidant, could protect testicular tissues and spermatogenesis from the toxic effects of cadmium, six-week old male Sprague-Dawley rats were randomized to receive cadmium at doses of 0 (control), 1, 2, 4 or 8 mg/kg by the intraperitoneal route (Group A) or alpha-tocopherol for 5 days before being challenged with cadmium (Group B) in an identical dose-dependent manner. When both groups received cadmium at 1 mg/kg, there were no changes in testicular histology relative to controls. When Group A received cadmium at 2 mg/kg, undifferentiated spermatids and dead Sertoli cells increased in the seminiferous tubules while interstitial cells decreased and inflammatory cells increased in the interstitial tissues. On flow cytometric analysis, the numbers of elongated spermatids (M1) and round spermatids (M2) decreased while 2c stage cells (M3, diploid) increased. In contrast, when Group B received cadmium at 2 mg/kg, the histological insults were reduced and the distribution of the germ cell population remained comparable to controls. However, alpha-tocopherol had no protective effects with higher cadmium doses of 4 and 8 mg/kg. These findings indicate that alpha-tocopherol treatment can protect testicular tissue and preserve spermatogenesis from the detrimental effects of cadmium but its effectiveness is dependent on the dose of cadmium exposed.

Modulatory effects of different doses of alpha-tocopherol on benzo(a)pyrene-DNA adduct formation in the pulmonary tissue of cigarette smoke inhaling mice.

Cigarette smoke (CS) has been established as one of the major risk factors for many pathologies including lung cancer in humans and experimental animals. In view of the discrepancy about the role of alpha-tocopherol (AT) in carcinogenesis, the present study was designed to investigate the effects of different doses of AT on benzo(a)pyrene-DNA [B(a)P-DNA] adduct formation in lungs of CS inhaling mice. Extent of carcinogen-DNA adduct formation has been considered as an index for carcinogenesis. Feeding of 35 IU AT/kg body weight increased B(a)P-DNA adducts formation significantly whereas feeding of 5 IU AT/kg body weight did not altered much the B(a)P-DNA adduct levels when both were compared to the control counterparts. With CS inhalation, the B(a)P-DNA adducts formation increased in all the groups when compared to their respective sham counterparts. Interestingly, in CS exposed groups, there was least increase in B(a)P-DNA adducts formation in 5 IU AT/kg fed animals followed by the control and 35 IU AT/kg body weight fed groups respectively. The results suggest that higher doses of AT accentuate DNA adduct formation in CS inhaling mice.

Effect of ascorbic acid, alpha-tocopherol, lecithin and L-ornithine-L-aspartate on ethanol induced hypoproteinemia and hyperlipidemia in rats.

We studied effect of exogenous ascorbic acid, alpha-tocopherol, lecithin and L-ornithine-L-aspartate on serum lipids and proteins in experimental hepatotoxic Wistar rats. Eleven groups (n = 6) of animals were used. Hepatotoxicity was induced by administering ethanol (1.6 g/kg/day) for 28 days. Both preventive and curative options were studied. Percentage increase in body weight was significantly lower in ethanol treated rats. Ethanol significantly (P<0.05) increased cholesterol, triglycerides and LDL, and decreased protein, albumin and A:G ratio in serum. Ascorbic acid, alpha-tocopherol, lecithin and L-ornithine-L-aspartate exhibited an ability to counteract the alcohol-induced changes in the body weight and biochemical parameters in preventive and therapeutic models in varying degree. Antioxidants showed better effect.

Histological and biochemical serum effects of alpha-tocopherol on ischemia/reperfusion-related injuries induced in the pelvic limb of rats

OBJETIVO: Avaliar ação protetora do alfa-tocoferol na lesão de isquemia e reperfusão em membro pélvico de ratos. MÉTODOS: Trinta ratos machos adultos da linhagem wistar foram distribuídos aleatoriamente, em três grupos experimentais, com 10 animais cada: Grupo I – Grupo controle sem isquemia ou reperfusão. Grupos II e III – quatro horas de isquemia e duas horas de reperfusão através clampeamento da aorta infra-renal. Os animais do grupo II foram tratados com solução salina e aqueles do grupo III, tratados com alfa-tocoferol 50 mg/kg por via endovenosa. Parâmetros estudados: Biópsias do músculo solear, dosagens da creatina fosfoquinase, da desidrogenasse láctica, do potássio, do cálcio e da hemogasometria arterial. RESULTADOS: Os resultados das biópsias dos músculos soleares estudados através da microscopia óptica, não foram significantes quanto a presença de edema entre os três grupos estudados. As variáveis inflamação e necrose não foram observadas e, portanto não analisáveis estatisticamente. Em relação às dosagens de cálcio e desidrogenase lática, pH, pO2, pCO2, não foram significantes em todos os grupos estudados. Observamos que os níveis de potássio (Grupo II > grupo I, F calculado = 5,84; F crítico = 3,33), creatina fosfoquinase (Grupo II > Grupo I e III, H calculado =13,92; Hcritico 5,99) , e bicarbonato (grupo I e III > grupo II, H calculado = 11,98; h critico 5.99 ) apresentaram resultados significantes entre os grupos. CONCLUSÃO: Tratamento com alfa-tocoferol do ponto de vista bioquímico sérico atenuou as lesões metabólicas na síndrome de isquemia e reperfusão neste modelo experimental.

Effect of D-alpha-tocopherol on tubular nephron acidification by rats with induced diabetes mellitus

The objective of the present study was to determine if treatment of diabetic rats with D-alpha-tocopherol could prevent the changes in glomerular and tubular function commonly observed in this disease. Sixty male Wistar rats divided into four groups were studied: control (C), control treated with D-alpha-tocopherol (C + T), diabetic (D), and diabetic treated with D-alpha-tocopherol (D + T). Treatment with D-alpha-tocopherol (40 mg/kg every other day, ip) was started three days after diabetes induction with streptozotocin (60 mg/kg, ip). Renal function studies and microperfusion measurements were performed 30 days after diabetes induction and the kidneys were removed for morphometric analyses. Data are reported as means ± SEM. Glomerular filtration rate increased in D rats but decreased in D + T rats (C: 6.43 ± 0.21; D: 7.74 ± 0.45; D + T: 3.86 ± 0.18 ml min-1 kg-1). Alterations of tubular acidification observed in bicarbonate absorption flux (JHCO3) and in acidification half-time (t/2) in group D were reversed in group D + T (JHCO3, C: 2.30 ± 0.10; D: 3.28 ± 0.22; D + T: 1.87 ± 0.08 nmol cm-2 s-1; t/2, C: 4.75 ± 0.20; D: 3.52 ± 0.15; D + T: 5.92 ± 0.19 s). Glomerular area was significantly increased in D, while D + T rats exhibited values similar to C, suggesting that the vitamin prevented the hypertrophic effect of hyperglycemia (C: 8334.21 ± 112.05; D: 10,217.55 ± 100.66; D + T: 8478.21 ± 119.81æm²). These results suggest that D-alpha-tocopherol is able to protect rats, at least in part, from the harmful effects of diabetes on renal function.

Oxyradicals under UV-b stress and their quenching by antioxidants.

Formation of oxyradicals under UV-B stress was investigated using cucumber cotyledons. UV-B radiation induced production of free radicals which were analyzed by ESR spectroscopy. Evidence was obtained for the formation of superoxide and hydroxyl radicals in the tissues by comparing PBN-adducts formed with radicals obtained by chemical autooxidation of KO2 and Fenton's reaction. Addition of superoxide dismutase (SOD) to the reaction mixture partially reduced the intensity of signals confirming the production of superoxide radical as well as hydroxyl radicals. These radicals were quenched in vitro by the natural antioxidants alpha-tocopherol, ascorbic acid and benzoquinone. Changes in the level of antioxidants were also monitored under UV-B stress. The endogenous level of ascorbic acid was enhanced and alpha-tocopherol level was reduced in the tissue after exposure to UV-B radiation. The present report happens to be the first direct evidence obtained for the formation of superoxide and hydroxyl radicals in plant tissues exposed to UV-B radiation.