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.

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.

[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).

Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation.

To explore the molecular mechanism underlying the translocation of cytochrome c from the mitochondrial inner membrane to the cytosol during apoptosis, we analyzed the molecular interaction between cytochrome c and cardiolipin (CL) by (1)H NMR spectroscopy. Bovine heart CL induced a drastic broadening of the linewidth of the downfield signals at 31.4 and 34.2 ppm assigned to the heme methyl group-3 and -8, respectively, of horse heart cytochrome c. In contrast, CL mono- and dihydroperoxides were less active in broadening the signals than CL, and CL trihydroperoxides induced almost no broadening of their linewidth. This finding suggests that the peroxidation of CL induces a release of cytochrome c from mitochondria into the cytosol, which release induces apoptosis in the cells.

Dissipation of mitochondrial membrane potential by exogenous phospholipid monohydroperoxide and protection against this effect by transfection of cells with phospholipid hydroperoxide glutathione peroxidase gene.

Two hours after its addition to cultures of a guinea pig cell line, 104C1, dilinoleoyl phosphatidylcholine monohydroperoxide (PCOOH) at concentrations of 5-160 microM induced a dissipation of the mitochondrial inner membrane potential (delta psi m), without any apparent morphological changes, in the cells. The PCOOH-induced loss of delta psi m was restored 4 hr after the replacement of the medium with PCOOH-free fresh medium. In contrast, 104C1/O4C cells, a stable clone from 104C1 cells transfected with the human phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene encoding a sequence including a signal peptide towards mitochondria, were resistant to the loss of delta psi m after a 2-hr exposure to PCOOH at concentrations up to 160 microM. Even after an 8-hr exposure to 80 microM PCOOH, the transfected cells retained their delta psi m intact, though the parent cells were killed by the same treatment. The present results strongly suggest that the expression of PHGPx protected the host cells from PCOOH-mediated injury at least by protecting their mitochondria from lipid hydroperoxide-induced loss of delta psi m.

Regulation of cell cycle-related genes in rat hepatocytes by transforming growth factor beta1.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of the proliferation of many cell types. We investigated the effects of TGF-beta1 on cyclin D1, cyclin A, p21, p27, and p53 mRNA expressions in primary cultured rat hepatocytes by the reverse-transcription polymerase chain reaction (RT-PCR) method. TGF-beta1 decreased the level of cyclin A mRNA in a dose-dependent manner, while it had little effect on the level of cyclin D1 mRNA. p21 mRNA expression was greatly induced by TGF-beta1 in a p53-independent mechanism, while p27 mRNA expression was not affected by TGF-beta1. These results suggest that TGF-beta1 may inhibit liver cell proliferation by regulating p21 and cyclin A mRNAs.

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.

Rapid loss in the mitochondrial membrane potential during geranylgeranoic acid-induced apoptosis.

A synthetic geranylgeranoic acid (GGA) induced apoptotic cell death in a human hepatoma cell line, HuH-7, but not in mouse primary cultured hepatocytes. Prior to chromatin condensation, GGA induced a dramatic loss of the mitochondrial membrane potential in 1 hour and in a dose dependent manner in HuH-7 cells, but not in the primary hepatocytes. Pretreatment with synthetic tetrapeptide cysteine protease inhibitor, either acetyl-Tyr-Val-Ala-Asp-chloromethylketone or acetyl-Asp-Glu-Val-Asp-aldehyde, blocked GGA-induced apoptosis without preventing a rapid loss of the mitochondrial membrane potential. alpha-Tocopherol prevented the cells from GGA-induced apoptosis as well as from a rapid loss of the membrane potential. The present study strongly suggests that GGA induces apoptosis in hepatoma cells through derangement of mitochondrial function and subsequent activation of the cysteine protease cascade.

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.

Limited degradation of retinoid X receptor by calpain.

Recently, we have found two major physiological forms of retinoid X receptor alpha (RXR alpha): the mature 54 kDa RXR alpha and the truncated 44 kDa RXR alpha lacking a portion of N-terminal A/B domain in human and rodent livers. In this communication, we show that m-calpain was active to digest 54 kDa RXR alpha in the human hepatoma-derived cell line, HuH7, nuclei to 44 kDa fragment through 47 kDa intermediate in vitro. Although both proteolytic fragments were revealed by anti-RXR alpha antibody against its E-domain, neither fragment reacted with anti-RXR alpha antibody specific for A/B domain. The profile of the calpain-induced proteolytic fragmentation of RXR alpha was almost identical to that endogenous RXR alpha in nonmalignant human and normal mouse liver nuclei. This is the first demonstration that of RXR alpha is a substrate for m-calpain, strongly suggesting that the enzyme might also be involved in post-translational modification of the receptor in hepatocytes.

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.

Impaired synthesis of retinol-binding protein and transthyretin in rat liver with bile duct obstruction.

To gain further insight into the protein metabolism in bile duct-obstruction, we examined the synthesis of retinol-binding protein (RBP) and transthyretin (TTR) in rats with common bile duct-ligation. In these rats, liver and plasma levels of RBP and TTR decreased markedly, whereas liver retinoid contents remained unchanged. Although there appeared no decrease in the total amount of RBP or TTR mRNA expressed in the liver, the subcellular distribution of these mRNAs changed from the membrane-bound polysome fraction to the membrane-unbound polysome fraction. This abnormal distribution recovered rapidly after biliary drainage, resulting in the subsequent recovery of the plasma RBP and TTR levels. These observations suggest that cholestasis inhibits the synthesis and secretion of RBP and TTR by disrupting the binding of their mRNAs to membrane-bound polysomes. Plasma levels of RBP and TTR might be sensitive indicators of the recovery of protein synthesis after biliary drainage in patients with obstructive biliary disorders.

Apoptosis in human hepatoma cell line induced by 4,5-didehydro geranylgeranoic acid (acyclic retinoid) via down-regulation of transforming growth factor-alpha.

Synthetic 4,5-didehydro GGA (geranylgeranoic acid), a potent ligand both for cellular retinoic acid-binding protein and for nuclear retinoid receptors, induced apoptosis in human hepatoma-derived cell line HuH-7 but not in primary hepatocytes, although all-trans or 9-cis retinoic acid did not induce any growth inhibition. Either exogenous transforming growth factor-alpha (TGF alpha) or epidermal growth factor(EGF) prevented the cells from apoptosis in the presence of 4,5-didehydro GGA, but hepatocyte growth factor, insulin-like growth factor-II, insulin or triiodothyronine was essentially inactive. 4,5-Didehydro GGA down-regulated the cellular levels of TGF alpha mRNA as early as 30 min after the treatment. Either anti-TGF alpha or anti-EGF receptor monoclonal antibody induced apoptosis in HuH-7 cells without using the acid. Taken together, the present study strongly suggests that 4,5-didehydro GGA induced apoptosis in HuH-7 cells through the destruction of autocrine loop consisting of TGF alpha and EGF receptor, due to the down regulation of TGF alpha gene expression.

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.

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.

Regulation of hepatic genes and liver transcription factors in rat hepatocytes by extracellular matrix.

Culturing hepatocytes on different extracellular matrix (ECM) substrata including tissue culture plastic, type I collagen, Engelbreth-Holm-Swarm (EHS) gel and poly-N-p-vinylbenzyl-D-lactonamide (PVLA) regulated levels of mRNAs for cytoskeleton and liver-specific genes. In hepatocytes on EHS gel, the ratio of albumin/beta-actin in mRNA levels was high and serially increased during the culture period, while the ratio was low and declined in cells on plastic substratum, collagen or PVLA. The changes in cellular levels of albumin mRNA which were regulated by ECM corresponded with those in two liver-specific transcription factors, hepatocyte nuclear factors-1 and -4, which control the transcription of liver-specific genes. These results suggest that cell-matrix interaction may determine and maintain the differentiated phenotype of hepatocytes by regulating liver-specific transcription factors.

Retinoid agonist activities of synthetic geranyl geranoic acid derivatives.

Micromolar concentrations of 4,5- didehydro geranyl geranoic acid (GGA) were able to induce up-regulation of retinoic acid receptor-beta gene expression in human hepatoma-derived cell line, HuH-7, to the same extent as all-trans RA. In chloramphenicol acetyl transferase (CAT) assay with retinoic acid response element-beta, GGA and 4,5-didehydro GGA were both positive, but 2,3-dihydro GGA was negative, even though these GGA derivatives have been reported to be all potent ligands for cellular retinoic-acid-binding protein(CRABP). However, 10,11,14,15- tetrahydro- 4,5- didehydro GGA, a compound without any affinity for CRABP, transactivated CAT gene expression. On the other hand, only GGA and 4,5-didehydro GGA were active to induce CAT gene expression through retinoid X response element of cellular retinol binding protein, type II gene. We show for the first time that chemically synthesized acyclic organic acids are potential agonists of natural retinoids.

Induction of apoptosis by acyclic retinoid in the human hepatoma-derived cell line, HuH-7.

HuH-7 cells, a human hepatoma-derived cell line, underwent apoptosis in response to all-trans 3, 7, 11, 15-tetramethyl- 2, 4, 6, 10, 14-hexadecapentaenoic acid, or acyclic retinoid. The retinoid-induced apoptosis was verified by a characteristic step-wise fragmentation of genomic DNA and chromatin condensation. The induction of apoptosis was detected as early as 8 hrs after the addition of the retinoid and was concentration dependent (0.1-10 microM). Neither the natural retinoid all-trans retinoic acid nor 9-cis retinoic acid induced apoptosis. These data strongly indicate that the antitumor activity of the acyclic retinoid may be partly explained by the induction of apoptosis in tumor cells.