In nature, eight substances have been found to have vitamin E activity: a-, b-, g-, and d-tocopherol; and a-, b-, g-, and d-tocotorienol.1) Tocotrienols possess powerful antithrombotic, neuroprotective, anti-cancer, and cholesterollowering properties that are often not exhibited by tocopherols. [2][3][4][5][6][7][8][9][10][11][12] Melanin is synthesized by tyrosinase and other enzymes in the tyrosinase family, such as tyrosinase-related protein (TRP)-1 and TRP-2, in melanosomes. We previously reported that the decrease (35%) in the melanin content of mouse melanoma B16 cells treated with 50 mM d-tocotrienol for 24 h was the result of a decrease in the level of tyrosinase.13) d-Tocotrienol might also be useful as a therapeutic or preventive drug for hyperpigmentation and as a component of whitening and/or lightening cosmetics not causing severe side effect (reduction of cholesterol content and release of lysosomes/melanosomes), although 50 mM dtocotrienol for 24 h caused a decrease in the level of mevalonate pyrophosphate decarboxylases, an enzyme involved in cholesterol biosynthesis.14) Kamei et al. reported that d-tocopherol has cytotoxicity, although it shows antimelanogenic activity at a low concentration long term (72 h).15) Although in number and position of methyl groups on the chromanol ring, of d-tocotrienol is similar to d-tocopherol, d-tocotrienol possesses a farnesyl instead of a saturated phytyl side chain. Therefore, the antimelanogenic activity of d-tocotrienol and d-tocopherol seems to relate to the number and position of the methyl groups on the chromanol ring. Treatment with d-tocotrienol long term may also show cytotoxicity.Thus, we investigated whether treatment with d-tocotrienol long term causes cytotoxicity, and also the dose-dependent effect of d-tocotrienol on melanin levels in cells. Furthermore, we examined whether other enzymes producing melanin, tyrosinase related protein-1 (TRP-1) and -2 (TRP-2), are involved in the decrease in melanin content. MATERIALS AND METHODS MaterialsThe d-tocotrienol (98.7%) was obtained from Eisai Food Chemical (Tokyo, Japan). The cholesterol E-test Wako kits were from Wako (Osaka, Japan), Dulbecco's modified Eagle's medium (D-MEM) from Gibco (Tokyo, Japan), B16 cells from RIKEN (Ibaraki, Japan), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoglobulin G (IgG), rabbit anti-TRP-2 IgG, goat anti-tyrosinase IgG, goat anti-TRP-1 IgG, and goat anti-lactate dehydrogenase (LDH: C-17) IgG from Santa Crutz Biotechnology, Inc. Melanoma Cell Line B16 cells were diluted to 1.5ϫ10 6 per 35-mm tissue culture dish with D-MEM containing 10% fetal bovine serum (FBS), and then incubated in humidified air containing 5% CO 2 at 37°C for 24 h. In some experiments, the cells were shifted to 1 ml of D-MEM containing 10% FBS in the presence of 25 or 50 mM d-tocotrienol (dissolved in 1 ml dimethyl sulfoxide (DMSO)) for 48 or 72 h. One milliliter of D-MEM in the absence or presence of d-tocotrienol was exchanged at intervals of 1 d. Preparation of Samples B16 cell...

Spontaneously hypertensive rat (SHR)/NDmcr-cp (cp/cp) rat (SHR-cp) is a congenic SHR rat with nonsense mutation introduced into the leptin receptor.1) Metabolic syndrome, characterized by the concomitant presence of obesity, hypertension, hyperlipidemia and diabetes, develops spontaneously in the SHR-cp, 2-4) along with age-related histologically evident glomerular injury and tubulointerstitial damage, including mesangial activation, podocyte injury, and inflammatory cell infiltration in the tubulointerstitium. 5) SHR-cp was reported to show hypercholesteremia and hyperglyceridemia, as compared with lean littermates (SHR-ln) that have hypertension.2) The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of Wistar Kyoto rat (WKY), 6) but that in SHR-cp at 10 weeks of age is the same.7) It remains unclear whether there are differences in the system regulating serum cholesterol levels between SHRcp and WKY at 10 weeks of age. Thus, we compared cholesterol levels in serum and liver between SHR-cp and WKY. Furthermore, mRNA levels of receptors or enzymes involved in uptake from serum to liver, cholesterol catabolism, or cholesterol biosynthesis in liver were compared between SHR-cp and WKY. MATERIALS AND METHODS MaterialsThe Cholesterol E-test Wako was obtained from Wako (Osaka, Japan). The high density lipoprotein (HDL) and very low density lipoprotein (VLDL)/low density lipoprotein (LDL) Quantification Kit was from BioVision, Inc. The Biomasher was obtained from Assist (Tokyo, Japan), QuickGene RNA tissue kit SII from Fujifilm (Tokyo, Japan), and SYBR Ex Script reverse transcription-polymerase chain reaction (RT-PCR) kit from TaKaRa (Shiga, Japan). All other chemicals were of reagent grade and purchased commercially.Animals Male (6-week-old) WKY and SHR-cp were obtained from the Disease Model Co-operative Research Association, Japan. The rats were fed standard chow for 4 weeks and used at 10 weeks of age. The experimental protocol was reviewed and approved by the Animal Care and Use Committee of Fukuyama University.Cholesterol Levels in Liver and Serum One hundred milligrams of liver was homogenized in 500 ml of homogenization buffer (50 mM Tris-HCl, pH 7.5 containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM 2-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitors [1 mM leupeptin, 1 mM pepstatin A, 1 mM chymostatin, and 1 mM antipain]) and centrifuged at 1000ϫg for 10 min. Forty microliters of postnuclear supernatant (PNS) was mixed with 5 ml of Folch extract (chloroformmethanol, 2 : 1), and the mixture was incubated for 10 min at 37°C with shaking. After the mixture was centrifuged at 3000ϫg for 10 min, 3 ml of the supernatant was evaporated dry by boiling at 100°C, and then dissolved in 200 ml of isopropyl alcohol containing 1% Triton-X-100. The cholesterol content of the solution or serum (20 ml) was determined using the Cholesterol E-test Wako (optical density (OD) 600 nm). Isolation of HDL and VLDL/LDL Fractions from SerumThe HDL and VLDL/LD...