Associations of Buccal Cell Telomere Length with Daily Intake of ß-Carotene or α-Tocopherol Are Dependent on Carotenoid Metabolism-related Gene Polymorphisms in Healthy Japanese Adults.

OBJECTIVE: Telomere length shortening is modulated not only by aging, but also by both genetic and environmental factors. The aim of this study was to investigate the interactions between antioxidant nutrient metabolism-related gene single nucleotide polymorphisms (the genetic factors) and nutrient intake (the environmental factors) in their effects on telomere length shortening. SETTING AND PARTICIPANTS: Data were collected on the relative telomere lengths (RTLs) of buccal cells and the habitual food intake of 70 healthy Japanese adults. MEASUREMENTS: All subjects were genotyped for two common single nucleotide polymorphisms: rs6564851 in the ß-carotene-15,15'-mono-oxygenase 1 (BCMO1) gene and rs362090 in the intestine-specific homeobox (ISX) gene. RESULTS: Univariate analysis revealed that buccal RTL was not significantly modulated by either age or gender. Then, we subdivided the study population into four groups based on combinations of the rs6564851 and rs362090 genotypes. After this subdivision, we showed a positive effect of daily α- or ß-carotene intake on buccal RTL in the ISX rs362090 G-allele carrier + BCMO1 rs6564851 GG-genotype group (p = 0.026). Additionally, daily intake of another antioxidative fat-soluble vitamin, α-tocopherol, was positively associated with buccal RTL in the ISX rs362090 AA-homozygote + BCMO1 rs6564851 T-allele carrier group (p = 0.037). CONCLUSION: Our study clearly indicates that high dietary intake of the antioxidants α, ß-carotene and α-tocopherol protects buccal cells from RTL shortening, depending on the genetic background of antioxidant vitamin-related genes.

Growth retardation in human cervical dysplasia-derived cell lines by beta-carotene through down-regulation of epidermal growth factor receptor.

We used newly established cervical dysplasia-derived cell lines to elucidate a molecular mechanism of the preventive action of beta-carotene in cervical multi-step carcinogenesis. Liposomal beta-carotene was added to the culture medium for human cervical dysplasia cell lines, CICCN-2 from cervical intraepithelial neoplasia grade I (CIN I), CICCN-3 from CIN II, and CICCN-4 from CIN III, and human cervical carcinoma-derived cell lines such as CICCN-6, CICCN-18, and HeLa cells. beta-Carotene (10 mumol/L) induced significant growth retardation in three cervical dysplasia cell lines but not in three cervical carcinoma-derived cell lines. Binding activities of epidermal growth factor (EGF) and cellular amounts of either messenger RNA for EGF receptor gene or EGF receptor protein were all highest in CICCN-4 cells. Cell surface binding, as well as internalization, of 125I-labeled EGF was rapidly reduced after beta-carotene treatment in dysplasia cell lines and 170-kD protein bands of EGF receptor disappeared from protein immunoblots at day 3 of the treatment. Cellular amounts of EGF receptor messenger RNA remained constant until day 3 of the treatment and were substantially reduced after day 7. Chromatin condensations, morphologic evidence for apoptotic cell death, were observed at day 1 by staining. From these results, we contend that prevention of cervical carcinogenesis by beta-carotene is due to induction of apoptosis in cervical dysplastic cells, which are premalignant cells in cervical multi-step carcinogenesis, via down-regulation of EGF receptor protein.

Aberrant metabolism of retinoid X receptor proteins in human hepatocellular carcinoma.

A polyclonal antibody was raised against a recombinant ligand binding domain construct of the human retinoid X receptor (RXR) alpha. This antibody reacted with an endogenous 54 kDa nuclear protein from human hepatoma-derived HuH7 cells in immunoblot analyses. Immunoblotting of nuclear proteins from human hepatocellular carcinomas (HCCs) and their surrounding tissues revealed the presence of a 44 kDa RXR distinct from the 54 kDa RXR and a dramatic decrease in the relative amounts of 44 kDa RXR to 54 kDa RXR in all HCCs compared with normal tissue. In vitro shift and intracellular conversion from 54 kDa RXR to 44 kDa species were observed with the nuclear extracts of HuH7 cells. Furthermore, transfection of hRXR alpha cDNA into HuH7 cells resulted in the increase of 54 kDa RXR, whereas transfected mouse hepatocytes accumulated 44 kDa RXR. These results strongly indicated that 44 kDa RXR was a physiological proteolytic fragment of 54 kDa RXR and that post-translational metabolism of RXR was impaired in HCC and the HuH7 hepatoma-derived cell line.

Urinary excretion of retinol-binding protein (RBP) in rabbit chronically poisoned with cadmium. Isolation and partial characterization of rabbit RBP.

Studies were conducted to induce urinary excretion of retinol-binding protein (RBP) in the rabbit by long-term administration of cadmium, and to isolate and characterize rabbit RBP. Two rabbits were exposed to Cd at a dose of 0.8 and/or 1.5mg/kg of body weight by means of subcutaneous injections five times per week. One rabbit excreted large amounts of protein (0.8 to 1.6 g/day) from days 80 to 118. The electrophoretic patterns of the urinary protein showed two fluorescent bands specific for protein-bound retinol in the alpha-region; these were presumed to be RBP. Rabbit RBP was isolated from the pooled urine (10.3 liters) by a sequence of procedures which included gel filtration on Sephadex G-100 and chromatography on DEAE- and SP-Sephadex. Purified RBP (20 mg) was found to be homogeneous by physical and immunological criteria. The RBP had alpha-mobility, with a molecular weight of approximately 20,000. The properties of rabbit RBP resembled those of human RBP simultaneously isolated from the urine of patients with "Itai-Itai" disease in many ways: ultraviolet and fluorescence spectra, and amino acid compositions. A monospecific anti-rabbit RBP antiserum was raised in a goat. There was no immunological cross-reactivity between rabbit and human RBP. The molecular size of the retinol-containing protein in fresh rabbit serum was estimated to be about 60,000 to 70,000 by gel filtration on Sephadex G-200. Rabbit RBP in the serum was also shown to be immunologically identical with purified RBP from the urine.

Recent studies on the involvement of retinyl phosphate as a carrier of mannose in biological membranes.

Rat liver microsomes synthesized [14C]mannosylretinylphosphate and dolichyl [14C]mannosylphosphate from guanosinedisphosphate [14C]mannose, retinylphosphate and dolichylphosphate. Two distinct enzyme activities were shown to be responsible for the biosynthesis of the two mannolipids. A higher affinity mannosyl transferase (EA I), responsible for dolichylmannosylphosphate synthesis, displayed a Km for GDP-mannose of 1.7 microM; while a lower affinity enzyme (EA II), responsible for mannosylretinylphosphate synthesis, displayed a Km for GDP-mannose of 12.5 microM. These Km values were unaffected by the addition of either dolichylphosphate for EA II, or retinylphosphate for EA I. The same Km values were found before and after solubilization of the enzyme activity with 1% Triton X-100. Differential solubilization of EA I and EA II was demonstrated, utilizing different concentrations of Triton X-100. Triple-labeled mannosylretinylphosphate was prepared from [3H]retinylphosphate, retinyl[32P]phosphate and GDP-[14C]mannose from incubations containing rat liver microsomes. This compound was shown to donate [14C]mannose to endogenous acceptors of rat liver microsomes.

Growth suppression and induction of heat-shock protein-70 by 9-cis beta-carotene in cervical dysplasia-derived cells.

The aim of the present study was to determine to what extent 9-cis beta-carotene, one of the most abundant naturally-occurring cis-isomers of beta-carotene, can inhibit the growth of cervical dysplasia-derived cells in comparison with all-trans beta-carotene. We found that 9-cis beta-carotene was dose-dependently more effective than all-trans beta-carotene. Both carotenes induced the intracellular accumulation of heat-shock protein-70 (HSP70), and the treated cells showed morphological changes indicative of apoptosis. The results of the present study strongly suggest that the induction of HSP70 by beta-carotene might be involved in beta-carotene-mediated suppression of the cell growth through apoptosis.

Suppression by carotenoids of microcystin-induced morphological changes in mouse hepatocytes.

Microcystin-LR is a liver tumor promoter in the okadaic acid class, a group of potent inhibitors of protein phosphatases 1 and 2A. Because of inhibition of protein phosphatases, microcystin-LR induces hyperphosphorylation of cellular proteins, including cytoskeletal proteins--cytokeratins 8 and 18--and causes morphological changes in mouse hepatocytes in primary culture. We studied the effects of carotenoids to antagonize microcystin-LR-induced morphological changes in hepatocytes. beta-carotene (100 nM to 100 microns) suppressed the morphological changes induced by 100 nM microcystin-LR in a dose-dependent manner. Other carotenoids tested exerted similar suppressive effects, although retinoids, such as all-trans retinol, all-trans retinoic acid, and 9-cis retinoic acid, were only weakly suppressive. The relative potency of the suppression correlated significantly with the number of conjugated double bonds in the trans configuration. beta-carotene strongly suppressed the hyperphosphorylation of cellular proteins induced by microcystin-LR without significant changes in the basal phosphorylation level. Other antioxidants, such as alpha-tocopherol, did not protect the cells against microcystin-LR. Taken together, the antagonistic effects of carotenoids against microcystin-LR are difficult to explain by their antioxidant or provitamin A activities. Suppression of the hyperphosphorylation of cellular proteins may be a novel mechanism by which carotenoids inhibit tumor promotion.

A new aspect on the mechanism of intestinal cholesterol absorption in rat.

In order to study the mechanism of intestinal cholesterol absorption, the relationship between the amount of cholesterol administered and the rate of absorption was investigated by the dual isotope plasma ratio method in vivo and the ligated-loop method in situ. The energy requirement of cholesterol absorption was also observed by means of the ligated-loop method. The results obtained are summarized below. 1) Tri-phase absorption was observed by the dual isotope plasma ratio method. When less than 300 microgram of cholesterol was administered, absorption increased linearly, with the coefficient of absorption being more than 80%. When the amount administered was between 300 and 500 microgram, the absorption was constant. With the administration of more than 500 microgram, absorption increased linearly, but the coefficient of absorption decreased to approximately 55%. 2) With the ligated-loop method, a second saturation profile was obtained when between 250 and 400 microgram of cholesterol was administered to a segment. When 50 to 250 microgram of cholesterol were administered, absorption increased in proportion to the increase in cholesterol dosage. 3) The mucosal uptake of cholesterol decreased to 40-60% of the control with the addition of metabolic inhibitors such as NaN3, KCN, 2,4-DNP and ouabain, whereas the uptake of palmitate showed no significant decrease. In addition, the uptake of cholesterol decreased remarkably to 25% of the control with the lowering of body temperature from 37 degrees C to 27 C. These results suggest the existence of an active transport system which has a limited capacity for cholesterol absorption and which requires energy for its operation in the physiological state.

Inhibition of beta-sitosterol on intestinal cholesterol absorption in rat using in vivo dual isotope ratio method.

The inhibitory effects of beta-sitosterol on intestinal cholesterol absorption were studied by means of a dual isotope plasma ratio method (in vivo), which is a new technique for the measurement of cholesterol absorption, as well as a ligated-loop method (in situ). The results obtained were as follows: 1. The absorption of beta-sitosterol itself was significantly less than cholesterol. Cholesterol was selectively absorbed from rat intestine. 2. When 100 to 1,000 microgram of beta-sitosterol were added to the dose solution containing 10 microgram of cholesterol, cholesterol absorption by the in vivo experiment decreased with the increae of additional beta-sitosterol. 3. A similar inhibitory effect of beta-sitosterol was observed by the in situ ligated-loop method. These results suggest that beta-sitosterol actually inhibits cholesterol absorption in the physiological state of an animal.

Antioxidative mechanism and apoptosis induction by 3-hydroxyanthranilic acid, an antioxidant in Indonesian food Tempeh, in the human hepatoma-derived cell line, HuH-7.

Recently, a new potent antioxidant was isolated from Tempeh (a traditional fermented soybean food in Indonesia) and was identified as 3-hydroxyanthranilic acid (HAA). This study deals with the antioxidant mechanism of HAA under biological systems and the cytokilling function of HAA to human malignant cells. HAA eliminated free radicals and inhibited the formation of fatty acid hydroperoxide in vitro, suggesting that HAA would serve as an antioxidant in the initial reaction in lipid oxidation systems. Actually, HAA inhibited the formation of the dominant product of membrane lipids, 12-hydroxyeicosatetraenoic acid (12-HETE) at a high concentration, while HAA accelerated 12-HETE formation at a low concentration in mammalian tissue. HAA oxidized glutathione and inhibited superoxide dismutase in vitro. Furthermore, HAA inhibited cell growth and induced apoptosis to HuH-7, a human hepatoma-derived cell line. As long as HAA is taken as a component of Tempeh, and not in large doses as a chemical, it may possibly act as a prooxidant rather than an antioxidant in vivo.

Minerals and vitamin-A status in relation to gustatory functions for salt in healthy female college students.

Twenty-five healthy female college students were studied for the gustatory function tests for salt (NaCl) and some selected biochemical parameters including plasma retinol and plasma retinol-binding protein (PRBP). Plasma zinc (PZn) and retinol levels were comparable with those of good responders in tests of discrimination between two levels of NaCl in the previous report, but PRBP was lower in the present subjects. From the results of correlation analysis and stepwise multiple regression analysis, the individual student's discriminability of NaCl concentrations was related to the parameters regarding metabolic status of calcium (Ca), magnesium (Mg), sodium (Na), and selenium (Se). The detection threshold of NaCl concentration ranged from 1 to 60 mmol/liter and was related to PRBP. Plasma Zn was significantly positively correlated with both plasma retinol and PRBP, but significantly negatively correlated only for the detection threshold of NaCl concentration. On the basis of these results, the importance of vitamin-A nutrition and the relation of minerals such as Na, Ca, Mg, and Zn to the gustatory functions of NaCl was confirmed and a possible participation of Se to the functions was suggested.

Interrelationship of some selected nutritional parameters relevant to taste for salt in a group of college-aged women.

Students in a women's college were investigated for taste acuity for salt, discrimination of salt concentrations in food, and anthropometrical (the body mass index, and systolic and diastolic blood pressures) and biochemical nutritional parameters (blood hemoglobin, plasma zinc, plasma copper, plasma vitamin A, plasma retinol-binding protein, urinary sodium, urinary potassium, urinary magnesium, urinary calcium and urinary zinc). Among 95 students who participated in the test for discrimination of salt concentrations, which was repeated 6 times with 5 different test samples, only 43 (45.3%) committed no mistakes. The detection threshold for taste of salt was significantly associated with neither the discriminability of salt concentrations nor any biochemical parameters. Levels of plasma zinc (PZn), urinary zinc (UZn) and plasma vitamin A (VA) were lower in the present subjects than in those reported previously. The rate of correct discrimination (RCD) was significantly correlated with PZn and VA positively, and with urinary sodium (UNa) and urinary potassium (UK) negatively. In the factor analysis to investigate the interrelationship of nutritional parameters, 6 factors with significance were extracted, among which factors 3 and 4 were related to RCD. Factor 3 had large loadings on VA, plasma retinol-binding protein (RBP) and RCD, and factor 4 was positively loaded on UNa and UK and negatively on UZn and RCD. In the stepwise multiple regression analysis (RCD being the dependent variable), significant independent variables selected were VA, UK, PZn, systolic blood pressure and UNa. From these results, the college-aged women's failure in discriminating salt concentrations in food was likely to be related to vitamin A inadequacy, mild Zn deficiency and excessive intakes of Na and K.

[Basic aspects of vitamin A--chemical structure, physiological functions and metabolism].

Vitamin A research has been facing the third wave which was initiated both by a cloning of nuclear retinoid receptors and therapeutic application of retinoic acid for acute promyelocytic leukemia. The third wave also gives rise to confusion between vitamin A, retinoids, and retinoate analogues. Physiological functions of vitamin A still remain almost unresolved at a molecular level except a visual cycle. Future study may unravel a molecular involvement of vitamin A in sensation such as smelling, hearing and tasting, and a specific role of vitamin A in dopaminergic neuron will be presented in the near future. A refined control mechanism for intracellular level of retinoic acid is also discussed with retinal dehydrogenase II and cytochrome P450 RAI (CYP26).

Association and interaction effect between VEGF receptor-2 (VEGFR-2) gene polymorphisms and dietary pattern on blood uric acid in Malays and Indians.

INTRODUCTION: Gout and hyperuricaemia attributed to genetic and lifestyle factors have been associated with several chronic diseases. This study aimed to determine the association and interaction effects between vascular endothelial growth factor receptor-2 (VEGFR-2) gene polymorphisms (rs1870377 and rs2071559) and dietary patterns on blood uric acid in Malay and Indian adults. METHODS: Dietary intakes of 153 Malays and 177 Indians were obtained using a food frequency questionnaire for the construction of dietary patterns using factor analysis. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Anthropometric measurements, body mass index (BMI) and blood pressure and biomarkers, uric acid, glycated haemoglobin A1c (HbA1c), and blood lipids were determined. RESULTS: There were significant differences in the mean values for HbA1c (41 +/- 12 vs 45 +/- 8 mmol/mol, p < 0.001) and blood lipids levels (p < 0.05) between Malays and Indians. Significant correlations were obtained between uric acid with selected blood lipids (p < 0.05) and BMI in Malays (r = 0.362, p < 0.001) and Indians (r = 0.212, p < 0.01). Four dietary patterns were extracted from dietary intakes of all subjects: 'Vegetables diet'; 'Fruits diet' (FD); 'Animal protein and rice diet'; and 'Fast foods and preserved foods diet'. There were no significant associations between dietary patterns (p = 0.054-0.609) and VEGFR-2 gene polymorphisms (p = 0.348-0.778) with uric acid. In Malay subjects, the interaction of rs2071559 and FD had a borderline effect (p = 0.05) on blood uric acid after adjusting for potential confounders. CONCLUSION: The associations and gene-diet interactions involving VEGFR-2 gene polymorphisms and FD on uric acid provide new information on gout and hyperuricaemia risks in Malays.

Association and Interaction Effect between VEGF Receptor- 2 (VEGFR-2) Gene Polymorphisms and Dietary Pattern on Blood Uric Acid in Malays and Indians

Introduction: Gout and hyperuricaemia attributed to genetic and lifestyle factors have been associated with several chronic diseases. This study aimed to determine the association and interaction effects between vascular endothelial growth factor receptor-2 (VEGFR-2) gene polymorphisms (rs1870377 and rs2071559) and dietary patterns on blood uric acid in Malay and Indian adults. Methods: Dietary intakes of 153 Malays and 177 Indians were obtained using a food frequency questionnaire for the construction of dietary patterns using factor analysis. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Anthropometric measurements, body mass index (BMI) and blood pressure and biomarkers, uric acid, glycated haemoglobin A1c (HbA1c), and blood lipids were determined. Results: There were significant differences in the mean values for HbA1c (41±-12 vs 45±-8 mmol/mol, p<0.001) and blood lipids levels (p<0.05) between Malays and Indians. Significant correlations were obtained between uric acid with selected blood lipids (p<0.05) and BMI in Malays (r=0.362, p<0.001) and Indians (r=0.212, p<0.01). Four dietary patterns were extracted from dietary intakes of all subjects: ‘Vegetables diet’; ‘Fruits diet’ (FD); ‘Animal protein and rice diet’; and ‘Fast foods and preserved foods diet’. There were no significant associations between dietary patterns (p=0.054-0.609) and VEGFR-2 gene polymorphisms (p=0.348-0.778) with uric acid. In Malay subjects, the interaction of rs2071559 and FD had a borderline effect (p=0.05) on blood uric acid after adjusting for potential confounders. Conclusion: The associations and gene-diet interactions involving VEGFR-2 gene polymorphisms and FD on uric acid provide new information on gout and hyperuricaemia risks in Malays.