Using Differential Threshold Effects of Individual and Combined Periconceptional Methyl Donor Status on Maternal Genomic LINE-1 and Imprinted H19 DNA Methylation to Predict Birth Weight Variance in the Taiwan Pregnancy-Newborn Epigenetics (TPNE) Cohort Study.

BACKGROUND: Few studies have comprehensively examined the effect of methyl donor status on maternal DNA methylation and birth outcomes. OBJECTIVES: This study examined associations between periconceptional methyl donor status and genome-wide and specific imprinted gene methylation and fetal growth indices in the Taiwan Pregnancy-Newborn Epigenetics cohort. METHODS: Plasma folate, choline (free form), and betaine concentrations of the participants enrolled at 7-10 weeks of gestation were analyzed. DNA methylation at regulatory sequences of the imprinted H19 gene and genomic long interspersed nuclear element 1 (LINE-1) were measured in maternal lymphocytes using bisulfite/high-resolution melt polymerase chain reaction. Associations with birth weight (BW) were estimated through multiple regressions from 112 mother-newborn pairs. RESULTS: A nonlinear "L-shaped" relation and an inverse association between maternal plasma folate in T1 (mean ± SE: 17.6 ± 5.1 nmol/L) and lymphocytic LINE-1 methylation (ß: -0.49, P = 0.027) were characterized. After adjusting for LINE-1 methylation, individual maternal folate concentrations were positively associated with BW variance (ß = 0.24, P = 0.035), and the association was more pronounced in mothers with choline in T1 (mean ± SE: 5.4 ± 0.6 µmol/L; ß: 0.40, P = 0.039). Choline status of the mothers in T2 (mean ± SE: 7.2 ± 0.6 µmol/L) was inversely associated with LINE-1 methylation (ß: -0.43, P = 0.035), and a positive association was evident between T1 choline and H19 methylation (ß: 0.48, P = 0.011). After adjusting for epigenetic modification, maternal choline status predicted a positive association with BW (ß: 0.56, P = 0.005), but the effect was limited to mothers with high betaine concentrations in T3 (mean ± SE: 36.4 ± 8.8 µmol/L), depending on folate status. CONCLUSIONS: Our data highlight the differential threshold effects of periconceptional folate, choline, and betaine status on genomic LINE-1 and H19 DNA methylation and how their interplay has a long-term effect on BW variance.

The preventive effects of edible folic acid on cardiomyocyte apoptosis and survival in early onset triple-transgenic Alzheimer's disease model mice.

In recent years, neuropathological and epidemiological studies have indicated an association between Alzheimer's disease (AD) and several cardiovascular risk factors. In this study, the cardio-protective effects of folic acid (FA) in early stage AD was elucidated using a triple-transgenic (3xTg) Alzheimer's mouse model. Eleven-month-old C57BL/6 mice and 3xTg mice were assigned to five groups. During the four-month treatment period, the low-FA treatment group received FA through their diet, and the high-FA treatment groups received 3 mg/dl folate in drinking water and were also gastric-fed 1.2 mg/kg folate every day. In the C57B1/6J mice, treatment with high doses of FA (HFA) did not show any considerable effect compared to the control group or the low-dose dietary FA treatment group. However, Alzheimer's mice treated with HFA showed enhanced cardio-protection. Western blot analysis revealed that FA treatment restored SIRT1 expression, which was suppressed in 3xTg mice, through enhanced AMPK expression. FA significantly enhanced the IGF1 receptor survival mechanism in the hearts of the 3xTg mice and suppressed the expression-intrinsic and extrinsic apoptosis-associated proteins. The results suggest that FA intake may significantly alleviate cellular pathological events in the heart associated with AD.

PEMT rs7946 Polymorphism and Sex Modify the Effect of Adequate Dietary Choline Intake on the Risk of Hepatic Steatosis in Older Patients with Metabolic Disorders.

In humans, PEMT rs7946 polymorphism exerts sex-specific effects on choline requirement and hepatic steatosis (HS) risk. Few studies have explored the interaction effect of the PEMT rs7946 polymorphism and sex on the effect of adequate choline intake on HS risk. In this cross-sectional study, we investigated the association between PEMT polymorphism and adequate choline intake on HS risk. We enrolled 250 older patients with metabolic disorders with (n = 152) or without (n = 98; control) ultrasonically diagnosed HS. An elevated PEMT rs7946 A allele level was associated with a lower HS risk and body mass index in both men and women. Dietary choline intake-assessed using a semiquantitative food frequency questionnaire-was associated with reduced obesity in men only (p for trend < 0.05). ROC curve analysis revealed that the cutoff value of energy-adjusted choline intake for HS diagnosis was 448 mg/day in women (AUC: 0.62; 95% CI: 0.57-0.77) and 424 mg/day in men (AUC: 0.63, 95% CI: 0.57-0.76). In women, GG genotype and high choline intake (>448 mg/day) were associated with a 79% reduction in HS risk (adjusted OR: 0.21; 95% CI: 0.05-0.82); notably, GA or AA genotype was associated with a reduced HS risk regardless of choline intake (p < 0.05). In men, GG genotype and high choline intake (>424 mg/day) were associated with a 3.7-fold increase in HS risk (OR: 3.7; 95% CI: 1.19-11.9). Further adjustments for a high-density lipoprotein level and body mass index mitigated the effect of choline intake on HS risk. Current dietary choline intake may be inadequate for minimizing HS risk in postmenopausal Taiwanese women carrying the PEMT rs7946 GG genotype. Older men consuming more than the recommended amount of choline may have an increased risk of nonalcoholic fatty liver disease; this risk is mediated by a high-density lipoprotein level and obesity.

Dietary Folate Deficiency Promotes Lactate Metabolic Disorders to Sensitize Lung Cancer Metastasis through MTOR-Signaling-Mediated Druggable Oncotargets.

Lactate metabolism plays a pivotal role in cancers but is often overlooked in lung cancer (LC). Folate deficiency has been linked to lung cancer development, but its impact on lactate metabolism and cancer malignancy is unclear. To investigate this, mice were fed either a folate-deficient (FD) or control diet and intrapleurally implanted with lung cancer cells pre-exposed to FD growth medium. Results showed that FD promoted lactate over-production and the formation of tumor oncospheroids (LCSs) with increased metastatic, migration, and invasion potential. Mice implanted with these cells and fed an FD diet developed hyperlactatemia in blood and lungs. This coincided with increased expression of hexokinase 2 (HK2), lactate dehydrogenase (LDH), and decreased expression of pyruvate dehydrogenase (PDH). Pre-treatment of the FD-LCS-implanted mice with the mTORC1 inhibitor, rapamycin, and the anti-metabolic drug metformin abolished FD/LCS-activated mTORC1 and its targets including HIF1α, HK2, LDH, and monocarboxylate transporters (MCT1 and MCT4), which coincided with the reduction in lactate disorders and prevention of LC metastasis. The findings suggest that dietary FD promotes lactate metabolic disorders that sensitize lung cancer metastasis through mTOR-signaling-mediated targets.

Folic Acid and Folinic Acid Protect Hearts of Aging Triple-transgenic Alzheimer's Disease mice via IGF1R/PI3K/AKT and SIRT1/AMPK Pathways.

Patients with Alzheimer's disease have increased risk of developing heart disease, which therefore highlights the need for strategies aiming at reducing Alzheimer's disease-related cardiovascular disease. Folic acid and folinic acid are beneficial to the heart. We aimed to investigate the benefits of folic acid and folinic acid in heart of patients with late-stage Alzheimer's disease. Twelve 16-month-old mice of triple-transgenic late-stage Alzheimer's disease were divided into three groups: Alzheimer's disease group, Alzheimer's disease + folic acid group, and Alzheimer's disease + folinic acid group. The mice were administered 12 mg/kg folic acid or folinic acid once daily via oral gavage for 3 months. In the folic acid and folinic acid treatment groups, the intercellular space was reduced, compared with the Alzheimer's disease group. TUNEL assay and western blot images showed that the number of apoptotic cells and the apoptosis-related protein expression were higher in the Alzheimer's disease group than in other two treated groups. Folic acid and folinic acid induced the IGF1R/PI3K/AKT and SIRT1/ AMPK pathways in the hearts of mice with Alzheimer's disease. Our results showed that folic acid and folinic acid treatment increased survival and SIRT1 expression to reduce apoptotic proteins in the heart. The aging mice treated with folinic acid had more IGF1R and SIRT1/AMPK axes to limit myocardial cell apoptosis. In conclusion, folic acid and folinic acid promote cardiac cell survival and prevent apoptosis to inhibit heart damage in aging mice with triple-transgenic late-stage Alzheimer's disease. In particular, folinic acid provides a better curative effect than folic acid.

Folate deprivation enhances invasiveness of human colon cancer cells mediated by activation of sonic hedgehog signaling through promoter hypomethylation and cross action with transcription nuclear factor-kappa B pathway.

Low folate status is well recognized as one of the metabolic stressors for colorectal cancer carcinogenesis, but its role in colon cancer invasion remains unknown. Activation of the Sonic hedgehog (Shh) signal in interaction with the transcription nuclear factor-kappa B (NF-κB) pathway is crucial for cancer aggressiveness. The aims of this study were to investigate whether and how folate deprivation promotes invasion by colon cancer cells in relation to Shh signaling and NF-κB pathway activation. Cultivation of epithelial colon carcinoma-derived cells (HCT116) in folate-deficient (FD) medium enhanced cellular migration and invasion, in correlation with epithelial-mesenchymal transition (EMT) associated with Snail expression and E-cadherin suppression, increased production of ß1 integrin and increased proteolysis by matrix metalloproteinase 2. Blockade of Shh signaling by cyclopamine (CYC) or of NF-κB activation by BAY abolished FD-enhanced EMT and invasion by HCT116 cells. FD cells had 50-80% less intracellular folate, associated with aberrant hypomethylation of the Shh promoter, than control cells, and increased binding of nuclear NF-κB subunit p65 to the Shh promoter region, which coincided with increased Shh expression and protein production of Shh ligand; in addition, the FD-induced Shh signaling targeted Gli1 transcription activator as well as Ptch receptor. The FD-induced Shh induction and activated signaling were blocked by NF-κB inhibitor BAY. Blockade of Shh signaling abrogated FD-promoted NF-κB activation measured by IκBα degradation and by target gene TNFα expression. Taken together, these findings demonstrate that folate deprivation enhanced invasiveness of colon cancer cells mediated by activation of Shh signaling through promoter hypomethylation and cross actions with the NF-κB pathway.

Suboptimal folic acid exposure rewires oncogenic metabolism and proteomics signatures to mediate human breast cancer malignancy.

Whether treatment with folic acid (FA) affects human breast cancer positively or negatively remains unclear. We subjected human Michigan Cancer Foundation-7 cells, a human breast cancer cell line, to suboptimal FA at low levels (10 nM; LF) and high levels (50 µM; HF) and investigated the molecular mechanisms underlying their effects through metabolic flux and systematic proteomics analyses. The data indicated that LF induced and HF aggravated 2-fold higher mitochondrial toxicity in terms of suppressed oxidative respiration, increased fermented glycolysis, and enhanced anchorage-independent oncospheroid formation. Quantitative proteomics and Gene Ontology enrichment analysis were used to profile LF- and HF-altered proteins involved in metabolism, apoptosis, and malignancy pathways. Through STRING analysis, we identified a connection network between LF- and HF-altered proteins with mammalian target of rapamycin (mTOR). Rapamycin-induced blockage of mTOR complex 1 (mTORC1) signaling, which regulates metabolism, differentially inhibited LF- and HF-modulated protein signatures of mitochondrial NADH dehydrogenase ubiquinone flavoprotein 2, mitochondrial glutathione peroxidase 4, kynureninase, and alpha-crystallin B chain as well as programmed cell death 5 in transcript levels; it subsequently diminished apoptosis and oncospheroid formation in LF/HF-exposed cells. Taken together, our data indicate that suboptimal FA treatment rewired oncogenic metabolism and mTORC1-mediated proteomics signatures to promote breast cancer development.

Optimal Dietary Intake Composition of Choline and Betaine Is Associated with Minimized Visceral Obesity-Related Hepatic Steatosis in a Case-Control Study.

Few studies on humans have comprehensively evaluated the intake composition of methyl-donor nutrients (MDNs: choline, betaine, and folate) in relation to visceral obesity (VOB)-related hepatic steatosis (HS), the hallmark of non-alcoholic fatty liver diseases. In this case-control study, we recruited 105 patients with HS and 104 without HS (controls). HS was diagnosed through ultrasound examination. VOB was measured using a whole-body analyzer. MDN intake was assessed using a validated quantitative food frequency questionnaire. After adjustment for multiple HS risk factors, total choline intake was the most significant dietary determinant of HS in patients with VOB (Beta: -0.41, p = 0.01). Low intake of choline (<6.9 mg/kg body weight), betaine (<3.1 mg/kg body weight), and folate (<8.8 µg/kg body weight) predicted increased odds ratios (ORs) of VOB-related HS (choline: OR: 22, 95% confidence interval [CI]: 6.5-80; betaine: OR: 14, 95% CI: 4.4-50; and folate: OR: 19, 95% CI: 5.2-74). Combined high intake of choline and betaine, but not folate, was associated with an 81% reduction in VOB-related HS (OR: 0.19, 95% CI: 0.05-0.69). Our data suggest that the optimal intake of choline and betaine can minimize the risk of VOB-related HS in a threshold-dependent manner.

Probing Folate-Responsive and Stage-Sensitive Metabolomics and Transcriptional Co-Expression Network Markers to Predict Prognosis of Non-Small Cell Lung Cancer Patients.

Tumour metabolomics and transcriptomics co-expression network as related to biological folate alteration and cancer malignancy remains unexplored in human non-small cell lung cancers (NSCLC). To probe the diagnostic biomarkers, tumour and pair lung tissue samples (n = 56) from 97 NSCLC patients were profiled for ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS)-analysed metabolomics, targeted transcriptionomics, and clinical folate traits. Weighted Gene Co-expression Network Analysis (WGCNA) was performed. Tumour lactate was identified as the top VIP marker to predict advance NSCLC (AUC = 0.765, Sig = 0.017, CI 0.58-0.95). Low folate (LF)-tumours vs. adjacent lungs displayed higher glycolytic index of lactate and glutamine-associated amino acids in enriched biological pathways of amino sugar and glutathione metabolism specific to advance NSCLCs. WGCNA classified the green module for hub serine-navigated glutamine metabolites inversely associated with tumour and RBC folate, which module metabolites co-expressed with a predominant up-regulation of LF-responsive metabolic genes in glucose transport (GLUT1), de no serine synthesis (PHGDH, PSPH, and PSAT1), folate cycle (SHMT1/2 and PCFR), and down-regulation in glutaminolysis (SLC1A5, SLC7A5, GLS, and GLUD1). The LF-responsive WGCNA markers predicted poor survival rates in lung cancer patients, which could aid in optimizing folate intervention for better prognosis of NSCLCs susceptible to folate malnutrition.

HTDP-2, a new synthetic compound, inhibits glutamate release through reduction of voltage-dependent Ca²âº influx in rat cerebral cortex nerve terminals.

AIM: The present study was aimed at investigating the effect of trans-6-(4-chlorobutyl)-5-hydroxy-4-(phenylthio)-1-tosyl-5,6-dihydropyridine-2(1H)-one (HTDP-2), a novel synthetic compound, on the release of endogenous glutamate in rat cerebrocortical nerve terminals (synaptosomes) and exploring the possible mechanism. METHODS: The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an on-line enzyme-coupled fluorimetric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²âº indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²âº concentrations ([Ca²âº](c)). RESULTS: HTDP-2 inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by HTDP-2 was prevented by the chelating intraterminal Ca²âº ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-ß-benzyloxyaspartate. HTDP-2 did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in [Ca²âº](c). Furthermore, the inhibitory effect of HTDP-2 on the evoked glutamate release was abolished by the N-, and P/Q-type Ca²âº channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na⁺/Ca²âº exchanger blocker CGP37157. CONCLUSION: Based on these results, we suggest that, in rat cerebrocortical nerve terminals, HTDP-2 decreases voltage-dependent Ca²âº channel activity and, in so doing, inhibits the evoked glutamate release.

Mitochondrial DNA deletions of blood lymphocytes as genetic markers of low folate-related mitochondrial genotoxicity in peripheral tissues.

BACKGROUND: A low folate status and mitochondrial DNA (mtDNA) mutations are risk factors for various cancers and degenerative diseases. It is not known if lymphocytic mtDNA deletions can be used as genetic "markers" to reflect global mtDNA damage during folate deficiency. AIM OF THE STUDY: The aim of this study was to characterize folate-related mtDNA deletions in lymphocytes and their associations with mt genotoxicity in peripheral tissues. METHODS: Weaning Wistar rats were fed folate-deficient and folate-replete (control) diets for 2 and 4 weeks. Folate levels of blood lymphocytes and various tissues were assayed by the Lactobacillus casei method. mtDNA deletions were measured by a real-time polymerase chain reaction analysis of whole DNA extracts. RESULTS: Compared to the control counterparts, mtDNA deletions of lymphocytes increased by 3.5-fold (P < 0.05) after 4 weeks of folate deficiency. Lymphocytic mtDNA deletions were inversely associated with plasma (r = -0.619, P = 0.018), red blood cell (r = -0.668, P = 0.009), and lymphocytic folate levels (r = -0.536, P = 0.048). Frequencies of lymphatic mtDNA deletions were positively correlated with mtDNA deletions in tissues including the lungs (r = 0.803, P = 0.0005), muscles (r = 0.755, P = 0.001), heart (r = 0.633, P = 0.015), liver (r = 0.722, P = 0.003), kidneys (r = 0.737, P = 0.006), pancreas (r = 0.666, P = 0.009), and brain (r = 0.917, P < 0.0001). CONCLUSIONS: Our data demonstrate that accumulated mtDNA deletions of lymphocytes depended upon dietary folate deprivation. The accumulated mt deletions in lymphocytes closely reflected the mt genotoxicity in the peripheral tissues during folate deficiency.

A new synthetic compound, 2-OH, enhances interleukin-2 and interferon-gamma gene expression in human peripheral blood mononuclear cells.

A new synthetic compound, 6-hydroxy-2-tosylisoquinolin-1(2H)-one (2-OH), was selected for immunopharmacological activity tests. The effects of 2-OH on human peripheral blood mononuclear cell (PBMC) proliferation were determined by tritiated thymidine uptake. Compared to phytohemagglutinin (PHA; 5 microg/mL) stimulation, 2-OH significantly enhanced PBMC proliferation in a dose-dependent manner. The 50% enhancement activity (EC(50)) for 2-OH was 4.4+/-0.1 microM. In addition, effects of 2-OH on interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in PBMC were determined by enzyme immunoassay. Results demonstrated that 2-OH stimulated IL-2 and IFN-gamma production in PBMC. Data from reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR indicated that IL-2 and IFN-gamma mRNA expression in PBMC could be induced by 2-OH. Therefore, 2-OH enhanced IL-2 and IFN-gamma production in PBMC by modulation their gene expression. We suggest that 2-OH may be an immunomodulatory agent.

HDT-1, a new synthetic compound, inhibits glutamate release in rat cerebral cortex nerve terminals (synaptosomes).

AIM: Excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. In this study, we investigated the effect of HDT-1 (3, 4, 4a, 5, 8, 8a-hexahydro-6,7-dimethyl-4a-(phenylsulfonyl)- 2-tosylisoquinolin-1(2H)-one), a novel synthetic compound, on glutamate release in rat cerebrocortical nerve terminals and explored the possible mechanism. METHODS: The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) or the high external [K+] and measured by one-line enzyme-coupled fluorometric assay. We also determined the loci at which HDT-1 impinges on cerebrocortical nerve terminals by using membrane potentialsensitive dye to assay nerve terminal excitability and depolarization, and Ca2+ indicator Fura-2 to monitor Ca2+ influx. RESULTS: HDT-1 inhibited the release of glutamate evoked by 4-AP and KCl in a concentration-dependent manner. HDT-1 did not alter the resting synaptosomal membrane potential or 4-APevoked depolarization. Examination of the effect of HDT-1 on cytosolic [Ca2+] revealed that the diminution of glutamate release could be attributed to reduction in voltage-dependent Ca2+ influx. Consistent with this, the HDT-1-mediated inhibition of glutamate release was significantly prevented in synaptosomes pretreated with the N- and P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC. CONCLUSION: In rat cerebrocortical nerve terminals, HDT-1 inhibits glutamate release through a reduction of voltage-dependent Ca2+ channel activity and subsequent decrease of Ca2+ influx into nerve terminals, rather than any upstream effect on nerve terminal excitability.

Low-folate stress reprograms cancer stem cell-like potentials and bioenergetics metabolism through activation of mTOR signaling pathway to promote in vitro invasion and in vivo tumorigenicity of lung cancers.

Low-folate (LF) nutritional status is associated with increased risks of lung cancer. It has unexplored effects on lung cancer malignancy, a cancer stem cell (CSC) disease. We hypothesized that LF may reprogram CSC-like potential and bioenergetics metabolism to increase metastasis potential of lung cancers. Cultivation of human non-small-cell lung cancer cells (H23) in an LF medium enhanced CSC-like properties signified by increased expressions of the CSC surface marker CD44 and pluripotency markers Sox2, Oct4 and ALDH1A1, and promoted self-renewal ability of anchorage-independent oncospheroid formation. The CSC-like phenotype of LF-treated H23 cells coupled with the metabolic reprogramming to aerobic glycolysis evident by elevated lactate release and medium acidification suppressed expressions of pyruvate dehydrogenase E1-α, and elevated redox status of the NADH/NAD+ and NADPH/NADP+ ratios. The LF-induced metabostemness phenotype of H23 cells was modified by DNA methylation inhibitor 5-AdC and histone acetylation inhibitor EX. Treatment of H23 cells with mTOR siRNA or the mTOR inhibitor rapamycin abrogated LF-activated Akt-mTOR-Hif1-Foxo signaling and stemness-associated sonic hedgehog pathway, reversed Warburg metabolic switch and diminished invasion of H23 cells. Intrapleural injection of LF-induced lung oncospheres into the LF recipient mice, but not the control recipient mice, caused metastasis xenograft lung tumors. The in vitro and in vivo data corroboratively demonstrate that LF stress reprograms metabostemness signatures through activated mTOR signaling pathway to promote metastasis tumorigenicity of lung cancers.

Dietary choline and folate relationships with serum hepatic inflammatory injury markers in Taiwanese adults.

BACKGROUND AND OBJECTIVES: The relationships of dietary choline and folate intake with hepatic function have yet to be established in the Taiwanese population. We investigated the associations of choline and folate intake with hepatic inflammatory injury in Taiwanese adults. METHODS AND STUDY DESIGN: Blood samples and data on dietary choline components and folate intake from 548 Taiwanese adults without pathological liver disease were collected. Dietary intake was derived using a semiquantitative food-frequency questionnaire. Serum liver injury markers of alanine transaminase, aspartate transaminase, and hepatitis viral infection were measured. RESULTS: Elevated serum hepatic injury markers (>40 U/L) were associated with low folate and free choline intake (p<0.05). Folate intake was the most significant dietary determinant of serum aspartate transaminase concentration (beta=-0.05, p=0.04), followed by free choline intake (beta=-0.249, p=0.055). Folate intake exceeding the median level (268 µg/d) was correlated with a reduced rate of hepatitis viral infection (p=0.032) and with normalized serum aspartate transaminase (odds ratio [OR]=0.998, 95% confidence interval [CI]=0.996-1, p=0.042) and alanine transaminase (OR=0.998, 95% CI=0.007-1, p=0.019). Total choline intake exceeding the median level (233 mg/d) was associated with normalized serum aspartate transaminase (OR=0.518, 95% CI=0.360-0.745, p=0.018). CONCLUSIONS: The newly established relationships of dietary intake of total choline and folate with normalized hepatic inflammatory markers can guide the development of dietary choline and folate intake recommendations for Taiwanese adults.

Folate deprivation and copper exposure potentiate reactive oxygen species generation and chromosomal DNA loss but not mitochondrial DNA deletions in rat hepatocytes.

Both increased copper and reduced folate levels are commonly found in patients with liver diseases. To better understand the mechanisms by which folate deprivation interacts with copper to contribute to hepatocellular toxicity, rat primary hepatocytes were isolated, cultured in folate-deprived (FD) RPMI medium, and assayed for cytotoxicity after copper sulfate (CuSO4) exposure. MTT measurement and trypan blue assay showed that elevated CuSO4 levels aggravated cell death of folate-deprived but not folate-sufficient hepatocytes. CuSO4 treatment increased the levels of intracellular reactive oxygen species (ROS) by 3 times in FD hepatocytes and tripled the proportion of FD hepatocytes with hypodiploid DNA contents. Measurement of membrane phosphatidylserine exposure indicated that the CuSO4-mediated toxicity in FD hepatocytes was not mediated by the apoptotic pathway. Real-time polymerase chain reaction (PCR) analysis revealed that CuSO4 treatment did not increase the occurrence of a 4834-bp mtDNA (mtDNA4834) deletion in FD hepatocytes. Preincubation of FD hepatocytes with various concentrations of folate prior to CuSO4 treatment did not modulate the mtDNA4834 deletion. Taken together, the data suggest that elevated copper levels potentiate cell death of folate-deprived hepatocytes, which is primarily associated with increased ROS generation and chromosomal DNA loss. The cytotoxicity exerted by folate depletion and elevated copper levels, however, is not due to apoptosis or accumulated mtDNA4834 deletions in primary hepatocytes.

Inhibition of colon cancer cell growth by nanoemulsion carrying gold nanoparticles and lycopene.

Lycopene (LP), an important functional compound in tomatoes, and gold nanoparticles (AN), have received considerable attention as potential candidates for cancer therapy. However, the extreme instability and poor bioavailability of LP limits its in vivo application. This study intends to develop a nanoemulsion system incorporating both LP and AN, and to study the possible synergistic effects on the inhibition of the HT-29 colon cancer cell line. LP-nanogold nanoemulsion containing Tween 80 as an emulsifier was prepared, followed by characterization using transmission electron microscopy (TEM), dynamic light scattering (DLS) analysis, ultraviolet spectroscopy, and zeta potential analysis. The particle size as determined by TEM and DLS was 21.3±3.7 nm and 25.0±4.2 nm for nanoemulsion and 4.7±1.1 nm and 3.3±0.6 nm for AN, while the zeta potential of nanoemulsion and AN was -32.2±1.8 mV and -48.5±2.7 mV, respectively. Compared with the control treatment, both the combo (AN 10 ppm plus LP 12 µM) and nanoemulsion (AN 0.16 ppm plus LP 0.4 µM) treatments resulted in a five- and 15-fold rise in early apoptotic cells of HT-29, respectively. Also, the nanoemulsion significantly reduced the expressions of procaspases 8, 3, and 9, as well as PARP-1 and Bcl-2, while Bax expression was enhanced. A fivefold decline in the migration capability of HT-29 cells was observed for this nanoemulsion when compared to control, with the invasion-associated markers being significantly reversed through the upregulation of the epithelial marker E-cadherin and downregulation of Akt, nuclear factor kappa B, pro-matrix metalloproteinase (MMP)-2, and active MMP-9 expressions. The TEM images revealed that numerous nanoemulsion-filled vacuoles invaded cytosol and converged into the mitochondria, resulting in an abnormally elongated morphology with reduced cristae and matrix contents, demonstrating a possible passive targeting effect. The nanoemulsion containing vacuoles were engulfed and internalized by the nuclear membrane envelop for subsequent invasion into the nucleoli. Taken together, LP-nanogold nanoemulsion could provide synergistic effects at AN and LP doses 250 and 120 times lower than that in the combo treatment, respectively, demonstrating the potential of nanoemulsion developed in this study for a possible application in colon cancer therapy.

Effect of genistein supplementation on tissue genistein and lipid peroxidation of serum, liver and low-density lipoprotein in hamsters.

The aim of this study was to investigate the effects of genistein supplementation in a vitamin E-deficient diet on the genistein concentrations and the lipid oxidation of serum, liver and low-density lipoprotein (LDL) of hamsters. Thirty-six male hamsters were randomly divided into three groups and fed a vitamin E-deficient semisynthetic diet (AIN-76) containing different levels of genistein, i.e., G0 (control group, genistein-free diet), G50 (50 mg genistein/kg diet) and G200 (200 mg genistein/kg diet) for 5 weeks. The concentrations of genistein in serum and liver significantly increased with the increase of genistein supplementation. The vitamin E contents in LDL were higher in hamsters fed G50 or G200 diets than in hamsters fed genistein-free diet. Genistein supplementation to hamsters significantly reduced the propagation rate during conjugated diene formation of LDL oxidation, and the lag time of LDL oxidation in hamsters fed G200 diets was significantly lower than that of G0 diets. In addition, genistein supplementation significantly raised serum total antioxidant capacity and decreased the thiobarbituric acid-reactive substances (TBARS) of LDL and liver in hamsters. However, no significant differences in TBARS were found in serum, irrespective of genistein addition. On the other hand, the relative contents of polyunsaturated fatty acids in LDL were decreased after genistein supplementation. There was a negative correlation between lag time and P/S ratio, and a positive correlation between lag time and vitamin E contents. These data demonstrate that genistein supplementation markedly increased its concentrations in body tissues and reduced oxidative stress of lipid oxidation of serum, liver and LDL.

Depleted folate pool and dysfunctional mitochondria associated with defective mitochondrial folate proteins sensitize Chinese ovary cell mutants to tert-butylhydroperoxide-induced oxidative stress and apoptosis.

The functional role of mitochondrial (mt) folate-associated proteins in mammalian cells is not clearly understood. We investigated the respiratory function and apoptosis phenotype of Chinese hamster ovary (CHO) mutant cells with defective mt serine hydroxymethyltransferase (SHMT) activities (glyA) or with defective mt folate transporter (glyB) in the absence/presence of oxidant challenge. The mechanisms underlying their aberrant phenotypes were explored. Compared with CHOK1 wild-type cells, both mutants carried dysfunctional mitochondria with reduced respiratory complex IV activity and depolarized mt membrane potential (P<.05). Elevated superoxide levels and accumulated mtDNA large deletions were observed in glyB in association with a depleted compartmental folate pool (P<.05). tert-Butylhydroperoxide (tBH) treatment at 50 microM for 72 h significantly depleted mt and cytosolic folate levels, impaired antioxidant defenses, and aggravated mt oxidative dysfunction in both mutants (P<.05), more severely in glyB. Only tBH-treated glyB cells displayed an elevated ratio of mt Bax/Bcl-2, activation of procaspases 9 and 3, and apoptosis promotion. The apoptotic phenotype of tBH-treated glyB could be partially corrected by folate supplementation (10-1000 microM), which enriched compartmental folate levels, restored antioxidant defenses, eliminated mt oxidative injuries, and normalized mt membrane function. Our data identify previously unrecognized roles of mt folate-associated proteins in the protection of mitochondria against oxidative insults. Defective mt folate transporter sensitized glyB cells to elevated oxidative stress and tBH-induced apoptosis, partly mediated by depleted compartmental folate and mt dysfunction. Defective mt SHMT sensitized glyA to respiratory dysfunction and tBH-induced oxidative injury without apoptosis promotion.

Folic Acid potentiates the effect of memantine on spatial learning and neuronal protection in an Alzheimer's disease transgenic model.

Folic acid deficiency and hyperhomocysteinemia potentiate amyloid-beta (Abeta) neuron toxicity. Memantine, an NMDA antagonist used in moderate to severe AD, is considered to be neuroprotective. We propose that folic acid might have a synergistic effect for memantine in protecting neurons from Abeta accumulation. We treated 8-month-old Tg2576 transgenic mice with memantine (30 mg/kg/day) with or without folic acid (8 mg/kg/day) for 4 months. Escape latencies in the Morris water maze were significantly shorter in the folic acid-memantine treatment group Tg(+)_M+F compared to both the non-treatment transgenic controls Tg(+) and the memantine-treatment group Tg(+)_M (both p < 0.05). Analysis of Abeta40 and Abeta42 showed lower brain loads in both treatment groups but this did not reach statistical significance. Histopathology analysis showed that Tg(+)_M+F had lower ratios of neuronal damage than Tg(+) (p < 0.001) and Tg(+)_M (p< 0.005). DNA analysis revealed that in the Tg(+)M_+F group, transcription was upregulated in 72 brain genes involved in neurogenesis, neural differentiation, memory, and neurotransmission compared to the Tg(+)_M group. In conclusion, we found that folic acid may potentiate the effect of memantine on spatial learning and neuronal protection. The benefit of combination therapy may be through co-action on the methylation-controlled Abeta production, and modification of brain gene expression.