Tissue distribution of α- and γ-tocotrienol and γ-tocopherol in rats and interference with their accumulation by α-tocopherol.

The aim of this study was to evaluate tissue distribution of vitamin E isoforms such as α- and γ-tocotrienol and γ-tocopherol and interference with their tissue accumulation by α-tocopherol. Rats were fed a diet containing a tocotrienol mixture or γ-tocopherol with or without α-tocopherol, or were administered by gavage an emulsion containing tocotrienol mixture or γ-tocopherol with or without α-tocopherol. There were high levels of α-tocotrienol in the adipose tissue and adrenal gland, γ-tocotrienol in the adipose tissue, and γ-tocopherol in the adrenal gland of rats fed tocotrienol mixture or γ-tocopherol for 7 weeks. Dietary α-tocopherol decreased the α-tocotrienol and γ-tocopherol but not γ-tocotrienol concentrations in tissues. In the oral administration study, both tocopherol and tocotrienol quickly accumulated in the adrenal gland; however, their accumulation in adipose tissue was slow. In contrast to the dietary intake, α-tocopherol, which has the highest affinity for α-tocopherol transfer protein (αTTP), inhibited uptake of γ-tocotrienol to tissues including adipose tissue after oral administration, suggesting that the affinities of tocopherol and tocotrienol for αTTP in the liver were the critical determinants of their uptake to peripheral tissues. Vitamin E deficiency for 4 weeks depleted tocopherol and tocotrienol stores in the liver but not in adipose tissue. These results indicate that dietary vitamin E slowly accumulates in adipose tissue but the levels are kept without degradation. The property of adipose tissue as vitamin E store causes adipose tissue-specific accumulation of dietary tocotrienol.

Tissue distribution of vitamin E metabolites in rats after oral administration of tocopherol or tocotrienol.

We previously found that 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (γCEHC), a metabolite of the vitamin E isoforms γ-tocopherol or γ-tocotrienol, accumulated in the rat small intestine. The aim of this study was to evaluate tissue distribution of vitamin E metabolites. A single dose of α-tocopherol, γ-tocopherol or a tocotrienol mixture containing α- and γ-tocotrienol was orally administered to rats. Total amounts of conjugated and unconjugated metabolites in the tissues were measured by HPLC with an electrochemical detector, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) was used as an internal standard. Twenty-four hours later, the vitamin E isoforms were detected in most tissues and in the serum. However, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (αCEHC), a metabolite of α-tocopherol or α-tocotrienol, and γCEHC accumulated in the serum and in some tissues including the liver, small intestine and kidney. Administration of α-tocopherol increased the γCEHC concentration in the small intestine, suggesting that α-tocopherol enhances γ-tocopherol catabolism. In contrast, ketoconazole, an inhibitor of cytochrome P450 (CYP)-dependent vitamin E catabolism, markedly decreased the γCEHC concentration. These data indicate that vitamin E metabolite accumulates not only in the liver but also in the small intestine and kidney. We conclude that some dietary vitamin E is catabolized to carboxyethyl-hydroxychroman in the small intestine and is secreted into the circulatory system.

Complexation of tocotrienol with gamma-cyclodextrin enhances intestinal absorption of tocotrienol in rats.

To determine the bioavailability of tocotrienol complex with gamma-cyclodextrin, the effects of tocotrienol/gamma-cyclodextrin complex on tocotrienol concentration in rat plasma and tissues were studied. Rats were administered by oral gavage an emulsion containing tocotrienol, tocotrienol with gamma-cyclodextrin, or tocotrienol/gamma-cyclodextrin complex. At 3 h after administration, the plasma gamma-tocotrienol concentration of the rats administered tocotrienol/gamma-cyclodextrin complex was higher than that of the rats administered tocotrienol and gamma-cyclodextrin. In order to determine the effect of complexation on tocotrienol absorption, rats were injected with Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoprotein by lipoprotein lipase, and then administered by oral gavage an emulsion containing tocotrienol, tocotrienol with gamma-cyclodextrin, or tocotrienol/gamma-cyclodextrin complex. The plasma gamma-tocotrienol concentration of the Triton-treated rats administered tocotrienol/gamma-cyclodextrin complex was higher than that of the other Triton-treated rats. These results suggest that complexation of tocotrienol with gamma-cyclodextrin elevates plasma and tissue tocotrienol concentrations by enhancing intestinal absorption.

Molecular characterization and gene disruption of a novel zinc-finger protein, HIT-4, expressed in rodent brain.

To identify a novel regulatory factor involved in brain development or synaptic plasticity, we applied the differential display PCR method to mRNA samples from NMDA-stimulated and un-stimulated neocortical cultures. Among 64 cDNA clones isolated, eight clones were novel genes and one of them encodes a novel zinc-finger protein, HIT-4, which is 317 amino acid residues (36-38 kDa) in length and contains seven C2H2 zinc-finger motifs. Rat HIT-4 cDNA exhibits strong homology to human ZNF597 (57% amino acid identity and 72% homology) and identity to rat ZNF597 at the carboxyl region. Furthermore, genomic alignment of HIT-4 cDNA indicates that the alternative use of distinct promoters and exons produces HIT-4 and ZNF597 mRNAs. Northern blotting revealed that HIT-4 mRNA (approximately 6 kb) is expressed in various tissues such as the lung, heart, and liver, but enriched in the brain, while ZNF597 mRNA (approximately 1.5 kb) is found only in the testis. To evaluate biological roles of HIT-4/ZNF597, targeted mutagenesis of this gene was performed in mice. Homozygous (-/-) mutation was embryonic lethal, ceasing embryonic organization before cardiogenesis at embryonic day 7.5. Heterozygous (+/-) mice were able to survive but showing cell degeneration and vacuolization of the striatum, cingulate cortex, and their surrounding white matter. These results reveal novel biological and pathological roles of HIT-4 in brain development and/or maintenance.

Dietary sesame seed and its lignan increase both ascorbic acid concentration in some tissues and urinary excretion by stimulating biosynthesis in rats.

We previously showed that the intake of sesamin, a major lignan in sesame seed, decreased lipid peroxidation and elevated tocopherol concentration in rat tissues. In this study, we examined the effect of dietary sesame seed and sesamin on the ascorbic acid concentration in rat tissues. Rats (4-wk-old) were fed either a vitamin E-free diet, or a diet containing 50 mg gamma-tocopherol/kg, one containing 2 g sesamin/kg, one containing 50 mg gamma-tocopherol/kg and 2 g sesamin/kg, or one containing 200 g sesame seed/kg for 28 d. The dietary sesamin and sesame seed elevated ascorbic acid concentrations in the liver and kidney, and increased urinary excretion in those Wistar rats. The dietary sesamin also elevated the hepatic mRNA levels of cytochrome P450 (CYP) 2B, and UDP-glucuronosyltransferase (UGT) 1A and 2B. In contrast, neither the sesamin nor the sesame seed affected the liver concentration of ascorbic acid in ODS rats with a hereditary defect in ascorbic acid synthesis, though the dietary sesame seed elevated the UGT1A and 2B mRNA levels in the liver. In addition, the sesame seed elevated the gamma-tocopherol concentration in the various ODS rat tissues and the ascorbic acid concentrations in the kidney, heart and lung, while reducing the thiobarbituric acid reactive substance concentration in the heart and kidney. These results suggest that dietary sesame seed and its lignan stimulate ascorbic acid synthesis as a result of the induction of UGT1A and the 2B-mediated metabolism of sesame lignan in rats. The data of ODS rat studies also suggest that dietary sesame seed enhances antioxidative activity in the tissues by elevating the levels of two antioxidative vitamins, vitamin C and E.

Dietary sesame seed decreases urinary excretion of alpha- and gamma-tocopherol metabolites in rats.

We previously showed that dietary sesame seed and its lignan inhibited gamma-tocopherol metabolism to 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a gamma-tocopherol metabolite, and markedly elevated tissue gamma-tocopherol concentration in rats. The aim of this study was to clarify the effect of dietary sesame seed on alpha-tocopherol metabolism. Vitamin E-deficient rats fed a vitamin E-free diet for 4 wk were fed a diet containing alpha-tocopherol, alpha- and gamma-tocopherol, or alpha-tocopherol with sesame seed for 7 d. Urinary excretion of 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a alpha-tocopherol metabolite, in rats fed alpha-tocopherol with sesame seed was inhibited (p<0.05) as compared with that in rats fed alpha-tocopherol alone, or alpha- and gamma-tocopherol. The gamma-CEHC excretion was also less (p<0.05) in rats fed alpha-tocopherol with sesame seed than that in rats fed alpha- and gamma-tocopherol. The inhibition of alpha- and gamma-CEHC excretion by sesame seed was accompanied by elevation (p<0.05) of the alpha- and gamma-tocopherol concentration in the liver. These results suggest that dietary sesame seed inhibits not only gamma-tocopherol metabolism to gamma-CEHC but also alpha-tocopherol metabolism to alpha-CEHC in rats.

Cytochrome P450-dependent metabolism of vitamin E isoforms is a critical determinant of their tissue concentrations in rats.

The aim of this study was to clarify the contribution of cytochrome P450 (CYP)-dependent metabolism of vitamin E isoforms to their tissue concentrations. We studied the effect of ketoconazole, a potent inhibitor of CYP-dependent vitamin E metabolism in cultured cells, on vitamin E concentration in rats. Vitamin E-deficient rats fed a vitamin E-free diet for 4 weeks were administered by oral gavage a vitamin E-free emulsion, an emulsion containing alpha-tocopherol, gamma-tocopherol or a tocotrienol mixture with or without ketoconazole. Alpha-tocopherol was detected in the serum and various tissues of the vitamin E-deficient rats, but gamma-tocopherol, alpha- and gamma-tocotrienol were not detected. Ketoconazole decreased urinary excretion of 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman after alpha-tocopherol or a tocotrienol mixture administration, and that of 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC) after gamma-tocopherol or a tocotrienol mixture administration. The gamma-tocopherol, alpha- and gamma-tocotrienol concentrations in the serum and various tissues at 24 h after their administration were elevated by ketoconazole, while the alpha-tocopherol concentration was not affected. The gamma-tocopherol or gamma-tocotrienol concentration in the jejunum at 3 h after each administration was also elevated by ketoconazole. In addition, significant amount of gamma-CEHC was in the jejunum at 3 h after gamma-tocopherol or gamma-tocotrienol administration, and ketoconazole inhibited gamma-tocopherol metabolism to gamma-CEHC in the jejunum. These results showed that CYP-dependent metabolism of gamma-tocopherol and tocotrienol is a critical determinant of their concentrations in the serum and tissues. The data also suggest that some amount of dietary vitamin E isoform is metabolized by a CYP-mediated pathway in the intestine during absorption.

Triton WR1339, an inhibitor of lipoprotein lipase, decreases vitamin E concentration in some tissues of rats by inhibiting its transport to liver.

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.

Delayed maturation of neuronal architecture and synaptogenesis in cerebral cortex of Mecp2-deficient mice.

We detected morphologic abnormalities in the cerebral cortex of Mecp2-hemizygous (Mecp2(-/y)) mice. The cortical thickness of both somatosensory and motor cortices in mutants did not increase after 4 weeks of age, as compared with that in wild-type male mice. The density of neurons in those areas was significantly higher in layers II/III and V of Mecp2(-/y) mice than in wild-type mice, particularly in layers II/ III after 4 weeks of age. In layer II/III of the somatosensory cortex of Mecp2(-/y) mice, the diameter of the apical dendrite was thin and the number of dendritic spines was small. Electron microscopy revealed that two-week-old mutants already had numerous premature postsynaptic densities. These results indicate that Mecp2(-/y) mice suffered delayed neuronal maturation of the cerebral cortex and that the initial neuronal changes were caused by premature synaptogenesis. Rett syndrome patients with a heterozygous mutation of Mecp2 display developmental disorders including cortical malfunctions such as mental retardation, autism, and epilepsy. Our results provide evidence of the similarity with Rett syndrome brains in some respects and suggest that MeCP2/Mecp2 plays some role in synaptogenesis.

Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxypropyl-alpha-tocotrienol, a redox-silent derivative of alpha-tocotrienol.

Tocotrienols are one of the most potent anticancer agents of all natural compounds and the anticancer property may be related to the inactivation of Ras family molecules. The anticancer potential of tocotrienols, however, is weakened due to its short elimination half life in vivo. To overcome the disadvantage and reinforce the anticancer activity in tocotrienols, we synthesized a redox-silent analogue of alpha-tocotrienol (T3), 6-O-carboxypropyl-alpha-tocotrienol (T3E). We estimated the possibility of T3E as a new anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras gene. T3E showed cytotoxicity against A549 cells, a human lung adenocarcinoma cell line with a ras gene mutation, in a dose-dependent manner (0-40 microM), whereas T3 and a redox-silent analogue of alpha-tocopherol (T), 6-O-carboxypropyl-alpha-tocopherol (TE), showed much less cytotoxicity in cells within 40 microM. T3E cytotoxicity was based on the accumulation of cells in the G1-phase of the cell-cycle and the subsequent induction of apoptosis. Similar to this event, 24-hr treatment of A549 cells with 40 microM T3E caused the inhibition of Ras farnesylation, and a marked decrease in the levels of cyclin D required for G1/S progression in the cell-cycle and Bcl-xL, a key anti-apoptotic molecule. Moreover, the T3E-dependent inhibition of RhoA geranyl-geranylation is an inducing factor for the occurrence of apoptosis in A549 cells. Our results suggest that T3E suppresses Ras and RhoA prenylation, leading to negative growth control against A549 cells. In conclusion, a redox-silent analogue of T3, T3E may be a new candidate as an anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras genes.

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

Genistein, a soy isoflavone, enhances necrotic-like cell death in a breast cancer cell treated with a chemotherapeutic agent.

Genistein is a major component of soybean isoflavone and has preventive effect against breast cancer. In breast cancer, the over-expression of HER-2 contributes to malignant transformation of the cancer cells. The present study was undertaken to estimate if genistein could act as a useful anti-cancer agent against a breast cancer cell overexpressing HER-2 in combination with a conventional chemotherapy agent, adriamycin (ADR). Genistein enhanced cytotoxic effect of ADR at low doses less than IC50 against the human breast cancer cell. The enhancing effect was mainly dependent on the elevation of necrotic-like cell death but not apoptotic cell death. In conjugation with this event, remarkable inactivation of HER-2 and Akt in the breast cancer cell was caused by the combination of genistein and ADR. These results suggest that genistein enhances necrotic-like cell death of the breast cancer cells through the inactivation of HER-2 receptor and Akt in combination with ADR.

Degeneration of pontine mossy fibres during cerebellar development in weaver mutant mice.

In weaver mutant mice, substitution of an amino acid residue in the pore region of GIRK2, a subtype of the G-protein-coupled inwardly rectifying K+ channel, changes the properties of the homomeric channel to produce a lethal depolarized state in cerebellar granule cells and dopaminergic neurons in substantia nigra. Degeneration of these types of neurons causes strong ataxia and Parkinsonian phenomena in the mutant mice, respectively. On the other hand, the mutant gene is also expressed in various other brain regions, in which the mutant may have effects on neuronal survival. Among these regions, we focused on the pontine nuclei, the origin of the pontocerebellar mossy fibres, projecting mainly into the central region of the cerebellar cortex. The results of histological analysis showed that by P9 the number of neurons in the nuclei was reduced in the mutant to about one half and by P18 to one third of those in the wild type, whereas until P7 the number were about the same in wild-type and weaver mutant mice. Three-dimensional reconstruction of the nuclei showed a marked reduction in volume and shape of the mutant nuclei, correlating well with the decrease in neuronal number. In addition, DiI (a lipophilic tracer dye) tracing experiments revealed retraction of pontocerebellar mossy fibres from the cerebellar cortex after P5. From these results, we conclude that projecting neurons in the pontine nuclei, as well as cerebellar granule cells and dopaminergic neurons in substantia nigra, strongly degenerate in weaver mutant mice, resulting in elimination of pontocerebellar mossy fibres during cerebellar development.

Prostaglandin E2 reinforces the activation of Ras signal pathway in lung adenocarcinoma cells via EP3.

Prostaglandin E2 (PGE2)-dependent effects on various cell responses are regulated by respective PGE2 receptors (EP1, EP2, EP3, EP4) expressing in target cells. Alveolar type II cell (a main progenitor cell of lung adenocarcinoma) expressed only EP4, while human lung adenocarcinoma cells (A549) expressed EP3 as well as EP4. An antagonistic effect of EP3 against EP4 through the modulation of cyclic AMP level is required for PGE2-mediated activation of Ras signal pathway in A549 cells. These results suggest that the expression of EP3 may be a critical factor for the PGE2-mediated activation of Ras signal pathway in A549 cells.

Isolation and structure of the mouse 14-3-3 eta chain gene and the distribution of 14-3-3 eta mRNA in the mouse brain.

14-3-3 protein is a brain-specific protein discovered by Moore and Perez, but at present is thought to be a multifunctional protein. To clarify the brain-specific function of the protein, we intend constructing a 14-3-3 eta gene knock-out mouse. As the first step of this process, we isolated the mouse 14-3-3 eta chain gene and determined its structure. The mouse gene is about 10 kb long and composed of two exons separated by a long intron. The transcription start site was identified and the polyadenylation signals (AATAAA) were found in exon 2 of the mouse gene. In the 5'-upstream sequence, we found several cis elements including a CRE sequence, a TATA box-like sequence, and a C/EBP element. Furthermore, the distribution of 14-3-3 eta mRNA in the mouse brain was examined by in situ hybridization histochemistry. The highest signals were found in the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and the olfactory bulb neurons of the adult mouse. Neuronal expression of 14-3-3 eta in these regions mRNA may generally increase during postnatal brain development. The distribution of protein kinase C gamma in the mouse brain was also examined by immunohistochemistry. From the distribution of 14-3-3 eta mRNA and protein kinase C gamma in the mouse brain, the involvement of these compounds in the induction and maintenance of LTP was discussed.

Immunohistochemical Changes of the Transcription Regulatory Factors in Rat Striatum after Methamphetamine Administration.

We have found evidence for cyclic adenosine monophosphate (cAMP) response element (CRE) in the 5'-upstream region of the human 14.3.3 η chain gene during studies on isolation and structure of animal brain 14.3.3 cDNA and the human 14.3.3 h chain gene. cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) may bind to this CRE. Since it was considered that these CREBs may play an important role in molecular mechanisms in the brain of animals treated with methamphetamine, we examined the expression of CREB and pCREB in rat brain after acute and chronic methamphetamine administrations using antibodies against CREB and pCREB. We observed findings for change in the expression of these factors. Our findings should be discussed in relation to the data of other authors.