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The metabolism of gamma-tocotrienol (gamma-TE) and gamma-tocopherol (gamma-T) was investigated in human A549 cells and in rats. Similar to gamma-T, A549 cells metabolized gamma-TE to sulfated 9'-, 11'-, and 13'-carboxychromanol and their unconjugated counterparts. After 72-h incubation with the cells, 90% of long-chain carboxychromanols in the culture media from gamma-TE, but <45% from gamma-T, were in the sulfated form. The formation of these metabolites was further investigated in rats gavaged by gamma-TE at 10 or 50 mg/kg, gamma-T at 10 mg/kg, or tocopherol-stripped corn oil in controls. Six hours after a single dosing, the supplemented rats had increased plasma concentrations of 13'-carboxychromanol and sulfated 9'-, 11'-, 13'-carboxychromanol, whereas none of these metabolites were detectable in the controls. Sulfated 11'-carboxychromanol was the most abundant long-chain metabolite in gamma-TE-supplemented rats. Sulfatase/glucuronidase hydrolysis revealed for the first time that >88% 2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), the terminal beta-oxidation metabolite, was in the conjugated form in the plasma. In all groups, conjugated gamma-CEHC accounted for >75% of total metabolites, whereas free CEHC was a minor metabolite. At 10 mg/kg, the plasma concentrations of total metabolites from gamma-TE-supplemented rats were higher (P < 0.05) than those from gamma-T-fed rats. These results demonstrate that in rats, conjugation such as sulfation occurs parallel to beta-oxidation in the liver and is quantitatively important to vitamin E metabolism. Conjugated long-chain carboxychromanols may be novel excreted metabolites during supplementation. Our data also provide in vivo evidence that gamma-TE is more extensively metabolized than gamma-T.

The aim of this experiment was to clarify the contribution of the alpha-tocopherol transfer activity of lipoprotein lipase (LPL) to vitamin E transport to tissues in vivo. We studied the effect of Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoproteins by LPL on vitamin E distribution in rats. Vitamin E-deficient rats fed a vitamin E-free diet for 4 wk were injected with Triton WR1339 and administered by oral gavage an emulsion containing 10 mg of alpha-tocopherol, 10 mg of gamma-tocopherol, or 29.5 mg of a tocotrienol mixture with 200 mg of sodium taurocholate, 200 mg of triolein, and 50 mg of albumin. alpha-Tocopherol was detected in the serum and other tissues of the vitamin E-deficient rats, but gamma-tocopherol, alpha- and gamma-tocotrienol were not detected. Triton WR1339 injection elevated (P<0.05) the serum alpha-tocopherol concentration and inhibited (P<0.05) the elevation of alpha-tocopherol concentration in the liver, adrenal gland, and spleen due to the oral administration of alpha-tocopherol. Neither alpha-tocopherol administration nor Triton WR1339 injection affected (P>or=0.05) the alpha-tocopherol concentration in the perirenal adipose tissue, epididymal fat, and soleus muscle despite a high expression of LPL in the adipose tissue and muscle. These data show that alpha-tocopherol transfer activity of LPL in adipose tissue and muscle is not important for alpha-tocopherol transport to the tissue after alpha-tocopherol intake or that the amount transferred is small relative to the tissue concentration. Furthermore, Triton WR1339 injection tended to elevate the serum gamma-tocopherol (P=0.071) and alpha-tocotrienol (P=0.053) concentrations and lowered them (P<0.05) in the liver and adrenal gland of rats administered gamma-tocopherol or alpha-tocotrienol. These data suggest that lipolysis of triacylglycerol-rich chylomicron by LPL is necessary for postprandial vitamin E transport to the liver and subsequent transport to the other tissues.

Vitamin E is a term that describes a group of compounds with similar yet unique chemical structures and biological activities. One interesting property possessed by certain vitamin E compounds-namely, delta-tocotrienol, RRR-alpha-tocopheryl succinate [vitamin E succinate (VES), a hydrolyzable ester-linked succinic acid analogue of RRR-alpha-tocopherol], and a novel vitamin E analogue referred to as alpha-TEA (alpha-tocopherol ether linked acetic acid analogue, which is a stable nonhydrolyzable analogue of RRR-alpha-tocopherol)-is their ability to induce cancer cells but not normal cells to undergo a form of cell death called apoptosis. In contrast, the parent compound, RRR-alpha-tocopherol, also referred to as natural or authentic vitamin E and known for its antioxidant properties, does not induce cancer-cell apoptosis. Efforts to understand how select vitamin E forms can induce cancer cells to undergo apoptosis have identified several nonantioxidant biological functions, including restoration of pro-death transforming growth factor-beta and Fas signaling pathways. Recent studies with alpha-TEA show it to be a potent inducer of apoptosis in a wide variety of epithelial cancer cell types, including breast, prostate, lung, colon, ovarian, cervical, and endometrial in cell culture, and to be effective in significantly reducing tumor burden and metastasis in a syngeneic mouse mammary tumor model, as well as xenografts of human breast cancer cells. Studies also show that alpha-TEA, in combination with the cyclooxygenase-2 inhibitor celecoxib and the chemotherapeutic drug 9-nitro-camptothecin decreases breast cancer animal model tumor burden and inhibits metastasis significantly better than do single-agent treatments.

1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] is important in the regulation of cell growth, differentiation, and apoptosis. Previous results from our laboratory demonstrate that 1,25(OH)(2)D(3) inhibits vitamin E succinate (VES) mediated apoptosis in untransformed C3H10T1/2 mouse fibroblast cells. The current work investigated cell survival signaling pathways that may be activated by 1,25(OH)(2)D(3), leading to protection from apoptosis. Results showed that nuclear factor kappaB (NFkappaB) transcriptional activity was significantly increased 1.8-fold over vehicle controls by 1,25(OH)(2)D(3) after 4 h of treatment. Protein kinase B/AKT, a downstream effector of phosphoinositide 3-kinase (PI3K), was activated 4-fold and 8-fold at 2 and 4 h, respectively, after treatment with 1,25(OH)(2)D(3). Pretreatment with two PI3K inhibitors, LY294002 and wortmannin, abolished the activation of NFkappaB by 1,25(OH)(2)D(3), suggesting that this pathway is essential for NFkappaB transcriptional activation. Additionally, the use of a p-21 activated kinase (PAK1) inhibitory construct (PAK(R299)) demonstrated that PAK1 was also required for NFkappaB transcriptional activation by 1,25(OH)(2)D(3). Inhibition of NFkappaB activity with transfection of the NFkappaB inhibitory construct (IkappaB(Ala32)) abolished the protective effect of 1,25(OH)(2)D(3) on VES-mediated apoptosis. In summary, NFkappaB transcriptional activation was essential to 1,25(OH)(2)D(3) protection from VES-mediated apoptosis and 1,25(OH)(2)D(3) regulated NFkappaB activity through PI3K and PAK pathways.

We previously showed that alpha- and gamma-tocotrienols accumulate in adipose tissue and skin but not in plasma or other tissues of rats fed a tocotrienol-rich fraction extracted from palm oil containing alpha-tocopherol and alpha- and gamma-tocotrienols. To clarify the nature of tocotrienol metabolism, we studied the distribution of alpha- or gamma-tocotrienol in rats fed alpha- or gamma-tocotrienol without alpha-tocopherol, and the effect of alpha-tocopherol on their distribution. Wistar rats (4-wk-old) were fed a diet with 50 mg alpha-tocotrienol/kg alone or with 50 mg alpha-tocopherol/kg in expt. 1, and a diet with 50 mg gamma-tocotrienol/kg alone or with 50 mg alpha-tocopherol/kg in expt. 2, for 8 wk. alpha-Tocotrienol was detected in various tissues and plasma of the rats fed alpha-tocotrienol alone, and the alpha-tocotrienol concentrations in those tissues and plasma decreased (P < 0.05) by the dietary alpha-tocopherol in the rats fed alpha-tocotrienol with alpha-tocopherol. However, gamma-tocotrienol preferentially accumulated in the adipose tissue and skin of the rats fed gamma-tocotrienol alone, and the dietary alpha-tocopherol failed either to decrease (P >/= 0.05) gamma-tocotrienol concentrations in the adipose tissue and skin or to increase (P >/= 0.05) in the urinary excretion of 2,7,8-trimethyl-2(2'-carboxymethyl)-6-hydroxycroman, a metabolite of gamma-tocotrienol, in the rats fed gamma-tocotrienol with alpha-tocopherol. These data suggest that alpha-tocopherol enhances the alpha-tocotrienol metabolism but not the gamma-tocotrienol metabolism in rats.

The metabolism of tocotrienol remains unclear. We studied the distribution of tocotrienol in rats fed the tocotrienol-rich fraction extracted from palm oil. We have previously shown that dietary sesame seeds markedly elevate the tocopherol concentration in rats. In this study, we also examined the effect of dietary sesame seeds on the tocotrienol concentration. In experiment 1, rats (4-wk-old) were fed the diet with alpha-tocopherol alone or with alpha- and gamma-tocotrienols. In experiment 2, the effect of dietary sesame seeds on tocopherol and tocotrienol concentrations in rats fed the diet with tocopherol and tocotrienol was studied. The rats were fed the experimental diet for 8 wk in both experiments. alpha- and gamma-Tocotrienols accumulated in the adipose tissue and skin, but not in plasma or other tissues, of the rats fed tocotrienols. Dietary sesame seeds elevated (P < 0.05) tocotrienol concentrations in the adipose tissue and skin, but did not affect their concentrations in other tissues or in plasma. The gamma-tocopherol concentration in all tissues and plasma of rats fed gamma-tocopherol was extremely low but was elevated (P < 0.05) in many tissues by feeding sesame seeds. These data suggest that the transport and tissue uptake of vitamin E isoforms are different. Dietary sesame seeds elevate the concentrations of both tocopherols and tocotrienols.

Vitamin E, the most important lipid-soluble antioxidant, was discovered at the University of California at Berkeley in 1922 in the laboratory of Herbert M. Evans (Science 1922, 55: 650). At least eight vitamin E isoforms with biological activity have been isolated from plant sources. Since its discovery, mainly antioxidant and recently also cell signaling aspects of tocopherols and tocotrienols have been studied. Tocopherols and tocotrienols are part of an interlinking set of antioxidant cycles, which has been termed the antioxidant network. Although the antioxidant activity of tocotrienols is higher than that of tocopherols, tocotrienols have a lower bioavailability after oral ingestion. Tocotrienols penetrate rapidly through skin and efficiently combat oxidative stress induced by UV or ozone. Tocotrienols have beneficial effects in cardiovascular diseases both by inhibiting LDL oxidation and by down-regulating 3-hydroxyl-3-methylglutaryl-coenzyme A (HMG CoA) reductase, a key enzyme of the mevalonate pathway. Important novel antiproliferative and neuroprotective effects of tocotrienols, which may be independent of their antioxidant activity, have also been described.

A tocotrienol-rich fraction (TRF(25)) and novel tocotrienols (d-P(21)-T3 and d-P(25)-T3) of rice bran significantly lowered serum and low density lipoprotein cholesterol levels in chickens. The present study evaluated the effects of novel tocotrienols on lipid metabolism in swine expressing hereditary hypercholesterolemia. Fifteen 4-mo-old genetically hypercholesterolemic swine were divided into five groups (n = 3). Four groups were fed a corn-soybean control diet, supplemented with 50 microg of either TRF(25), gamma-tocotrienol, d-P(21)-T3 or d-P(25)-T3 per g for 6 wk. Group 5 was fed the control diet for 6 wk and served as a control. After 6 wk, serum total cholesterol was reduced 32-38%, low density lipoprotein cholesterol was reduced 35-43%, apolipoprotein B was reduced 20-28%, platelet factor 4 was reduced 12-24%, thromboxane B(2) was reduced 11-18%, glucose was reduced 22-25% (P<0.01), triglycerides were reduced 15-19% and glucagon was reduced 11-17% (P<0.05) in the treatment groups relative to the control. Insulin was 100% greater (P<0.01) in the treatment groups than in the control group. Preliminary data (n = 1) indicated that hepatic activity of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase was lower in the treatment groups, and cholesterol 7alpha-hydroxylase activity was unaffected. Cholesterol and fatty acid levels in various tissues were lower in the treatment groups than in control. After being fed the tocotrienol-supplemented diets, two swine in each group were transferred to the control diet for 10 wk. The lower concentrations of serum lipids in these four treatment groups persisted for 10 wk. This persistent effect may have resulted from the high tocotrienol levels in blood of the treatment groups, suggesting that the conversion of tocotrienols to tocopherols may not be as rapid as was reported in chickens and humans.

We evaluated the effects of vitamin E and beta-carotene on apolipoprotein (apo)E +/- female mice, which develop atherosclerosis only when fed diets high in triglyceride and cholesterol. Mice were fed a nonpurified control diet (5.3 g/100 g triglyceride, 0.2 g/100 g cholesterol), an atherogenic diet alone (15.8 g/100 g triglyceride, 1.25 g/100 g cholesterol, 0.5 g/100 g Na cholate) or the atherogenic diet supplemented with either 0.5 g/100 g (+)-alpha-tocopherol (mixed isomers); 0.5 g/100 g palm tocopherols (palm-E; 33% alpha-tocopherol, 16.1% alpha-tocotrienol, 2.3% beta-tocotrienol, 32.2% gamma-tocotrienol, 16.1% delta-tocotrienol); 1.5 g/100 g palm-E; or 0.01 g/100 g palm-carotenoids (58% beta-carotene, 33% alpha-carotene, 9% other carotenoids). Compared with mice fed the control diet, plasma cholesterol was fourfold greater in mice fed the atherogenic diet. Mice fed the 1.5 g/100 g palm-E supplement had 60% lower plasma cholesterol than groups fed the other atherogenic diets. Mice fed the atherogenic diet had markedly higher VLDL, intermediate density lipoprotein (IDL) and LDL cholesterol and markedly lower HDL cholesterol than the controls. Lipoprotein patterns in mice supplemented with alpha-tocopherol or palm carotenoids were similar to those of the mice fed the atherogenic diet alone, but the pattern in mice supplemented with 1. 5 g/100 g palm-E was similar to that of mice fed the control diet. In mice fed the atherogenic diet, the hepatic cholesterol plus cholesterol ester concentration was 4.4-fold greater than in mice fed the control diet. Supplementing with 1.5 g/100 g palm-E lowered hepatic cholesterol plus cholesterol ester concentration 66% compared with the atherogenic diet alone. Mice fed the atherogenic diet had large atherosclerotic lesions at the level of the aortic valve. With supplements of 0.5 g/100 g palm-E or 1.5 g/100 g palm-E, the size of the lesions was 92 or 98% smaller, respectively. The 0.5 g/100 g alpha-tocopherol and palm carotenoid supplements had no effect. Supplements did not alter mRNA abundance for apolipoproteins A1, E, and C3. The beneficial effect of tocotrienols on atherogenesis, the plasma lipoprotein profile and accumulation of hepatic cholesterol esters cannot be attributed to their antioxidant properties.

The difference in bioavailabilities of the acetate and succinate esters of all-rac-alpha-tocopherol was investigated in a feeding experiment with broilers. The experiment was initiated with 96 12-d-old male Cobb broilers and lasted for 4 wk. The two sources of vitamin E were fed to eight groups of broilers at four different dietary levels (50, 100, 150 and 200 mg/kg feed, including the naturally occurring alpha-tocopherol). A total collection of droppings for determination of apparent tocopherol absorption were performed at two separate time periods (d 28-34 and d 35-41). There were no differences among the eight experimental groups with respect to animal performance or feed intake. At all dietary levels, the apparent absorption coefficient for all-rac-alpha-tocopherol succinate was significantly lower than that of the acetate ester. The mean (+/- SD) apparent absorption coefficient for all-rac-alpha-tocopherol succinate was 58.0 +/- 5.4 compared with 70. 8 +/- 5.6 for all-rac-alpha-tocopherol acetate. Furthermore, the apparent absorption coefficients for both esters was significantly lower in the first collection period (d 28-34) than in the second collection period (d 35-41). This difference in the apparent absorption coefficient between the succinate and the acetate ester was accompanied by significant differences in alpha-tocopherol concentrations in plasma, breast muscle, liver and adipose tissue of the broilers, which were lower in those fed the succinate ester. Based on a comparison of plasma and tissue responses, the succinate ester was utilized only 69-76% as efficiently as the acetate ester. In vitro studies showed a significantly higher capacity of pancreatic carboxyl ester hydrolase to hydrolyze alpha-tocopherol acetate compared to alpha-tocopherol succinate. This difference in intestinal hydrolysis of the two vitamin E sources may explain the observed differences in biopotency.

Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable and grain consumption on cancer risk.

The antioxidative role of Se-dependent cellular glutathione peroxidase (EC 1.11.1.9, GPX1) in vivo has not been established. Our objective was to determine the effects of GPX1 knockout or overexpression on the susceptibility of mice to paraquat toxicity and the contributions of GPX1, compared with other selenoproteins and vitamin E, to body defenses against such acute oxidative stress. Four experiments were conducted using 111 GPX1 knockout or overexpressing mice and the respective controls. Mice were fed diets supplemented with Se (as sodium selenite) at 0-0.4 mg/kg and/or all-rac-alpha-tocopheryl acetate at 0-75 mg/kg before intraperitoneal injections of 12.5, 50 or 125 mg paraquat/kg body weight. All mice that received 50 or 125 mg paraquat/kg died spontaneously, and the survival time of mice was (independent of dietary levels of Se per se or alpha-tocopheryl acetate) solely a function of tissue GPX1 activity (P < 0.001). Severe acute pulmonary interstitial necrosis was found only in the GPX1 overexpressing mice and the controls that had extended survival time. Thiobarbituric acid reacting substances in postmortem liver inversely correlated with the tissue GPX1 activity and dietary levels of Se and/or alpha-tocopheryl acetate. In contrast, all mice that received 12.5 mg paraquat/kg survived and were killed 2 wk after the injection for tissue collection. Compared with the saline injection, this low dose of paraquat resulted in greater (P < 0.001) liver and lung F2-isoprostanes in both the GPX1 knockout mice and the controls. However, there was no difference in plasma alanine transaminase (EC 2.6.1.2) activity or overt injuries in liver, lung and kidney in either group. Our data indicate that GPX1 is the major, if not the only, metabolic form of body Se that protects mice against the lethal oxidative stress caused by high levels of paraquat; it seems less important, however, in protecting mice against the moderate oxidative stress by the low level of paraquat.

The reaction of superoxide (a reactive oxygen species) and nitric oxide (one of the nitrogen oxides with numerous biological functions) results in the production of peroxynitrite. The characteristics of oxidation of alpha-tocopherol (vitamin E) in synaptosomes (nerve ending particles) and mitochondria by peroxynitrite were studied. The subcellular fractions were isolated from brain hemispheres of 4-month-old male Fischer 344 rats by standard centrifugation procedures involving Ficoll gradients. Peroxynitrite treatment oxidized alpha-tocopherol in <5 s. This reaction was selective because another membrane component, cholesterol, was not oxidized at the same time, as observed in our previous studies. Mitochondrial alpha-tocopherol was more susceptible to peroxynitrite-induced oxidation than synaptosomal tocopherol. Measurable and significant (P < 0.05) oxidation of tocopherol occurred when mitochondria or synaptosomes were incubated with peroxynitrite in concentrations as low as 5 or 10 micromol/L, respectively. The oxidation could be readily monitored by estimating the production of tocopherolquinone. Oxidation of tocopherol induced by ferrous iron and ascorbate was much slower and the yield of tocopherolquinone lower than by peroxynitrite. The fast and selective oxidation of alpha-tocopherol by peroxynitrite suggests that vitamin E may play an important role in preventing membrane oxidation induced by peroxynitrite. Literature reports indicate the existence of threshold concentrations of tocopherol below which functional alterations occur. Tocopherol oxidation by peroxynitrite could reduce tocopherol concentrations in tissues and subcellular structures to these threshold levels by different concentrations of peroxynitrite. Hence the sensitivity of tissues to peroxynitrite could vary over a wide range.

Sundry mevalonate-derived constituents (isoprenoids) of fruits, vegetables and cereal grains suppress the growth of tumors. This study estimated the concentrations of structurally diverse isoprenoids required to inhibit the increase in a population of murine B16(F10) melanoma cells during a 48-h incubation by 50% (IC50 value). The IC50 values for d-limonene and perillyl alcohol, the monoterpenes in Phase I trials, were 450 and 250 micromol/L, respectively; related cyclic monoterpenes (perillaldehyde, carvacrol and thymol), an acyclic monoterpene (geraniol) and the end ring analog of beta-carotene (beta-ionone) had IC50 values in the range of 120-150 micromol/L. The IC50 value estimated for farnesol, the side-chain analog of the tocotrienols (50 micromol/L) fell midway between that of alpha-tocotrienol (110 micromol/L) and those estimated for gamma- (20 micromol/L) and delta- (10 micromol/L) tocotrienol. A novel tocotrienol lacking methyl groups on the tocol ring proved to be extremely potent (IC50, 0.9 micromol/L). In the first of two diet studies, experimental diets were fed to weanling C57BL female mice for 10 d prior to and 28 d following the implantation of the aggressively growing and highly metastatic B16(F10) melanoma. The isomolar (116 micromol/kg diet) and the Vitamin E-equivalent (928 micromol/kg diet) substitution of d-gamma-tocotrienol for dl-alpha-tocopherol in the AIN-76A diet produced 36 and 50% retardations, respectively, in tumor growth (P < 0.05). In the second study, melanomas were established before mice were fed experimental diets formulated with 2 mmol/kg d-gamma-tocotrienol, beta-ionone individually and in combination. Each treatment increased (P < 0.03) the duration of host survival. Our finding that the effects of individual isoprenoids were additive suggests the possibility that one component of the anticarcinogenic action of plant-based diets is the tumor growth-suppressive action of the diverse isoprenoid constituents of fruits, vegetables and cereal grains.

Tocotrienols are a form of vitamin E, having an unsaturated isoprenoid side-chain rather than the saturated side-chain of tocopherols. The tocotrienol-rich fraction (TRF) from palm oil contains alpha-tocopherol and a mixture of alpha-, gamma- and delta-tocotrienols. Earlier studies have shown that tocotrienols display anticancer activity. We previously reported that TRF, alpha-, gamma- and delta-tocotrienols inhibited proliferation of estrogen receptor-negative MDA-MB-435 human breast cancer cells with 50% inhibitory concentrations (IC50) of 180, 90, 30 and 90 microg/mL, respectively, whereas alpha-tocopherol had no effect at concentrations up to 500 microg/mL. Further experiments with estrogen receptor-positive MCF-7 cells showed that tocotrienols also inhibited their proliferation, as measured by [3H] thymidine incorporation. The IC50s for TRF, alpha-tocopherol, alpha-, gamma- and delta-tocotrienols were 4, 125, 6, 2 and 2 microg/mL, respectively. Tamoxifen, a widely used synthetic antiestrogen inhibits the growth of MCF-7 cells with an IC50 of 0.04 microg/mL. We tested 1:1 combinations of TRF, alpha-tocopherol and the individual tocotrienols with tamoxifen in both cell lines. In the MDA-MB-435 cells, all of the combinations were found to be synergistic. In the MCF-7 cells, only 1:1 combinations of gamma- or delta-tocotrienol with tamoxifen showed a synergistic inhibitory effect on the proliferative rate and growth of the cells. The inhibition by tocotrienols was not overcome by addition of excess estradiol to the medium. These results suggest that tocotrienols are effective inhibitors of both estrogen receptor-negative and -positive cells and that combinations with tamoxifen should be considered as a possible improvement in breast cancer therapy.

The concentration-dependent impact of gamma-tocotrienol on serum cholesterol can be traced to the posttranscriptional down-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. gamma-Tocotrienol also suppresses tumor growth. Palmvitee, the tocopherol and tocotrienol-rich fraction of palm oil, is the sole commercial source of gamma-tocotrienol. Contrary to the universal findings of the efficacy of gamma-tocotrienol there are conflicting reports of the impact of Palmvitee on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, serum cholesterol concentrations and tumor development. These conflicting reports led us to examine the impact of alpha-tocopherol on the cholesterol-suppressive action of gamma-tocotrienol. Control and experimental diets were fed to groups of White Leghorn chickens (n = 10) for 26 d. The control diet was supplemented with 21 nmol alpha-tocopherol/g. All experimental diets provided 141 nmol of blended tocols/g diet. The alpha-tocopherol and gamma-tocotrienol concentrations of the experimental diets ranged from 21 to 141 and 0 to 120 nmol/g, respectively. We now report that including alpha-tocopherol in tocol blends containing adequate gamma-tocotrienol to suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase activity results in an attenuation of the tocotrienol action (P < 0.001). A summary of results from studies utilizing different Palmvitee preparations shows that effective preparations consist of 15-20% alpha-tocopherol and approximately 60% gamma- (and delta-) tocotrienol, whereas less effective preparations consist of > or = 30% alpha-tocopherol and 45% gamma- (and delta-) tocotrienol.

The effect of age, dietary fat type and all-rac-alpha-tocopheryl acetate (vitamin E) supplementation on ex vivo synthesis of lung eicosanoids was measured in C57BL/6NIA mice using a 2 (age) x 3 (fat) x 3 (vitamin E) factorial design. Young (3-mo-old) and old (24-mo-old) mice were fed a semipurified diet containing 5% (by wt) corn oil, coconut oil or fish oil supplemented with 30, 100 or 500 mg vitamin E/kg for 4 wk. Ex vivo synthesis of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (PGI2) were measured by RIA in lung homogenates. Old mice had significantly higher concentrations of TXB2 and PGI2 than did young mice, resulting in a significant increase in the TXB2:PGI2 ratio with aging. Young and old mice fed fish oil had significantly lower concentrations of PGI2 and TXB2 than those fed corn oil or coconut oil. The degree of reduction varied according to age and vitamin E status. Old mice fed fish oil and 30 mg vitamin E/kg diet had the lowest plasma vitamin E concentration and the highest TXB2:PGI2 ratio. The TXB2:PGI2 ratio was significantly reduced in old mice fed coconut oil or fish oil by vitamin E supplementation. Vitamin E supplementation (100 mg/kg) significantly increased PGI2 concentration in young mice fed coconut oil. Thus, significant changes in the capacity of lung to synthesize eicosanoids occur with age and are influenced by dietary fat type and vitamin E. J. Nutr.

I(indigenous)-compounds are age-related, carcinogen adduct-like, putative indigenous DNA modifications detectable by 32P-postlabeling assay in untreated animals. To investigate the origins of these DNA derivatives, we examined the effects of dietary vitamin E, a natural antioxidant, on I-compounds of rat liver and kidney DNA. Weanling female Sprague-Dawley rats were fed Draper's diets containing 0, 100, 1000, or 10,000 mg/kg alpha-tocopheryl acetate for 6 mo. The DNA from four individual rats of each group was analyzed by a nuclease P1-enhanced version of the 32P-postlabeling assay for DNA adducts. The amount of vitamin E in the liver was measured by high performance liquid chromatography. Rats fed vitamin E-deficient diet (0 mg/kg) showed identical profiles and similar levels of I-compounds as those fed the 100 mg/kg diet. Most I-spots were significantly intensified and one tissue-specific extra spot was found in both liver and kidney DNA of rats fed the 1000 or 10,000 mg/kg vitamin E diet. However, one of the five major I-spots detected in the kidney was weaker in the 1000 and 10,000 mg/kg groups than in the 0 and 100 mg/kg groups. These results show that formation of most I-compounds was not affected by vitamin E-deficient diet, and that long-term feeding of diet containing high levels of vitamin E may cause metabolic alterations leading to an increased formation of DNA-reactive (potentially mutagenic or carcinogenic) electrophiles.

Studies have been done to determine the optimal dosage of vitamin E. Vitamin E is generally considered to be relatively nontoxic at high dosage in spite of the fact that it is a fat-soluble vitamin. From our experiments using mice, when various dosages of all-rac-alpha-tocopherol were injected into the intraperitoneal cavity every day, 1) the body weight decreased when the dose was more than 100 IU/kg per day, and all the mice died within 3 days at 400 IU/kg per day; 2) immune responses investigated by lymphoproliferative assays with phytohemagglutinin, concanavalin A and lipopolysaccharide were enhanced significantly between 5 and 20 IU/kg per day, but were inhibited by 80 IU/kg per day. When the immunopotentiation effect of vitamin E was discernible, serum tocopherol levels were about twice the control values. From our results, the optimal dosage of vitamin E was between 5 and 20 IU/kg per day, and dosages over 80 IU/kg per day were toxic to mice. We then experimented similarly with vitamin K, which is fat soluble and possesses a quinone structure resembling vitamin E. When doses between 12.5 and 150 mg/kg per day of vitamin K were injected into the intraperitoneal cavity daily fore 14 days, increase of body weight was generally inhibited. This did not depend on the dose, and there was no definite relationship between mitogen responses and vitamin K.

By comparing the activities of tocopherol with other antioxidants and tocopherol analogs on mitogenesis of murine spleen cells, an attempt was made to understand the mechanism of tocopherol stimulation. N,N'-diphenyl-p-phenylene diamine (DPPD) was very active in this system, whereas butylated hydroxytoluene, another antioxidant, was much less active. Trolox C, an analog of alpha-tocopherol with a carboxyl group instead of the isoprene side chain was totally inactive. Tocopherol quinone, an oxidized form of tocopherol, was almost as active as tocopherol. Menadione (vitamin K-3), a quinone without a side chain, was also active. It was suggested therefore, that although in tocopherol-like compounds the side chain seems important, the activities of DPPD and menadione clearly do not require it. On the other hand, the stimulation by tocopherol quinone and menadione appears to rule out a requirement for an antioxidant function in mitogenic stimulation. A comparison of the action of tocopherol with indomethacin ruled out regulation of the peroxidation step leading to prostaglandin synthesis as the mechanism for the tocopherol effect.

This study was designed to evaluate the effects of dietary vitamin E and synthetic antioxidants on prostacyclin (PGI2) synthesis in isolated aorta segments and perfused hearts as well as thromboxane (TxA2) synthesis in thrombin-stimulated washed platelets. Weanling male New Zealand rabbits were fed a vitamin E-deficient basal diet or the basal diet supplemented with either all-rac-alpha-tocopherol acetate or propyl gallate or DPPD (N,N'-diphenyl-p-phenylenediamine). After 30 days on the diet, plasma tocopherol level, pyruvate kinase and liver microsomal NADPH oxidase were determined. DPPD but not propyl gallate prevented the development of myopathy. None of the synthetic antioxidants could substitute for vitamin E in decreasing enzymatic lipid peroxidation. PGI2 release by the aorta was lowered in vitamin E deficiency and was highest with DPPD supplementation. In the Langendorff perfused heart, however, PGI2 release was highest in the vitamin E-deficient group, possibly due to cardiomyopathy. TxA2 synthesis by washed platelets challenged with thrombin was independent of the antioxidant status of the animal. The data showed that dietary antioxidants selectively affect eicosanoid synthesis in different tissues.

The appearance of radioactivity after the oral administration of 3 microCi D-alpha-[5-methyl-3H]tocopherol and 10 microCi DL-alpha-[3',4'-14C]tocopheryl acetate in plasma, liver, kidney, spleen and heart of rainbow trout showed an exponential increase up to 32 hours, followed by a plateau or slight decline from 32 to 64 hours. Radioactivity in the skeletal muscle increased exponentially up to 8 hours followed by a slower liner increase up to 64 hours. Comparisons of plasma 3H and 14C radioactivity suggested that the uptake of D-alpha-tocopherol (EOH) was 6 to 18 times greater than DL-alpha-tocopheryl acetate (EAc) in the first 4 hours and 2 to 3 times greater between 8 and 64 hours. At the plateau, the amount of 3H and 14C radioactivity incorporated per unit wet weights of tissue decreased in the order liver greater than kidney greater than plasma greater than spleen greater than heart much greater than skeletal muscle. More than 87% of the 3H and 14C radioactivity after 16 hours was found to be free alpha-tocopherol in both plasma and liver. The radioactivity labeled vitamins were bound primarily to plasma low-density lipoprotein (density 1.015 to 1.085). These studies support the hypothesis that the uptake, transport and distribution fo EAc after hydrolysis in the gastrointestinal tract of trout follows a pattern similar to that of EOH.

A factorial experiment was conducted using two degrees of oxidation of the 7.5% supplemental fish oil (peroxide values of 5 and 120 meq/kg oil), two levels of supplemental DL-apha-tocopheryl acetate (0 and 33 mg/kg diet) and two levels of ethoxyquin (0 and 125 mg/kg diet) supplementation. Dietary thiobarbituric acid number, weight percentage of polyunsaturated fatty acids and omega-three fatty acids in the total fatty acids were significantly (P less than 0.05) different between diets with fresh and highly oxidized oil. Dietary RRR-alpha-tocopherol was significantly (P less than 0.05) reduced by the addition of highly oxidized oil after 24 weeks storage of the feed while supplemental DL-alpha-tocopheryl acetate level was not changed. Fish fed the various diets showed no differences in growth, feed:gain ratio, carcass composition or plasma glutathione peroxidase activity. The mortality, percent red cells hemolyzed by hydrogen peroxide, plasma and liver RRR-alpha-tocopherol concentrations were significantly (P less than 0.05) affected by the addition of highly oxidized oil or DL-alpha-tocopheryl acetate but not by ethoxyquin except that mortality was reduced by supplementary ethoxyquin. The results of this study suggested that no vitamin E or ethoxyquin supplementation was needed to prevent a deficiency of vitamin E in rainbow trout fed a practical diet containing 7.5% of a good quality herring oil for 24 weeks.

The dystrophic RCS rat is one of the most important animal models available for investigating retinal degeneration. In addition to the characteristic progressive loss of neural retina the strain is hampered by a high rate of mortality during the first week of life. Death rate during this period is greatly influenced by diet. A 69% reduction in mortality was achieved by supplementing a purified diet with double the amount of AIN-76 vitamin mix. The objective of this study was to identify vitamin(s) in the AIN-76 mix responsible for the enhanced survival. The experiment determined the effect on survival of independently doubling the concentration of each vitamin present in the AIN-76 vitamin mix. This was done by single addition of individual vitamins to a complete purified diet. Survival was determined in litters whose mothers and grandmothers had been provided the supplemented diets as their sole source of food. Supplementation with riboflavin increased mortality by 19%, whereas RRR-alpha-tocopheryl acetate supplementation reduced the mortality by 73%. The effect of RRR-alpha-tocopheryl acetate was equivalent to that achieved by supplementation with complete vitamin mix. First-week survival of pups (born alive) rose from 72.3% +/- 11.0 to 92.5% +/- 3.8 when the level of vitamin E was increased from 50 to 100 IU/kg diet.

The decrease in plasma pyruvate kinase activity after administration of various tocopherols to vitamin E-deficient rats provided the basis for a 3-point parallel-slope assay for vitamin E activity. Based on 6 replicate assays, RRR-alpha-tocopheryl acetate (RRR-alpha-TA) was 141% as active as all-rac-alpha-tocopheryl acetate (all-rac-alpha-TA). This confirms many previous reports with other bioassays and is in agreement with the unit-weight relationships assigned by The National Formulary. 2-ambo-alpha-Tocopheryl acetate was 97% as active as all-rac-alpha-TA indicating that only the configuration of the 2-position on the chromanol ring is important in determining the biological activity of alpha-tocopherol.