The cooperative interplay among inflammation, necroptosis and YAP pathway contributes to the folate deficiency-induced liver cells enlargement.

Change in cell size may bring in profound impact to cell function and survival, hence the integrity of the organs consisting of those cells. Nevertheless, how cell size is regulated remains incompletely understood. We used the fluorescent zebrafish transgenic line Tg-GGH/LR that displays inducible folate deficiency (FD) and hepatomegaly upon FD induction as in vivo model. We found that FD caused hepatocytes enlargement and increased liver stiffness, which could not be prevented by nucleotides supplementations. Both in vitro and in vivo studies indicated that RIPK3/MLKL-dependent necroptotic pathway and Hippo signaling interactively participated in this FD-induced hepatocytic enlargement in a dual chronological and cooperative manner. FD also induced hepatic inflammation, which convenes a dialog of positive feedback loop between necroptotic and Hippo pathways. The increased MMP13 expression in response to FD elevated TNFα level and further aggravated the hepatocyte enlargement. Meanwhile, F-actin was circumferentially re-allocated at the edge under cell membrane in response to FD. Our results substantiate the interplay among intracellular folate status, pathways regulation, inflammatory responses, actin cytoskeleton and cell volume control, which can be best observed with in vivo platform. Our data also support the use of this Tg-GGH/LR transgenic line for the mechanistical and therapeutic research for the pathologic conditions related to cell size alteration.

The Incoherent Fluctuation of Folate Pools and Differential Regulation of Folate Enzymes Prioritize Nucleotide Supply in the Zebrafish Model Displaying Folate Deficiency-Induced Microphthalmia and Visual Defects.

OBJECTIVE: Congenital eye diseases are multi-factorial and usually cannot be cured. Therefore, proper preventive strategy and understanding the pathomechanism underlying these diseases become important. Deficiency in folate, a water-soluble vitamin B, has been associated with microphthalmia, a congenital eye disease characterized by abnormally small and malformed eyes. However, the causal-link and the underlying mechanism between folate and microphthalmia remain incompletely understood. METHODS: We examined the eye size, optomotor response, intracellular folate distribution, and the expression of folate-requiring enzymes in zebrafish larvae displaying folate deficiency (FD) and ocular defects. RESULTS: FD caused microphthalmia and impeded visual ability in zebrafish larvae, which were rescued by folate and dNTP supplementation. Cell cycle analysis revealed cell accumulation at S-phase and sub-G1 phase. Decreased cell proliferation and increased apoptosis were found in FD larvae during embryogenesis in a developmental timing-specific manner. Lowered methylenetetrahydrofolate reductase (mthfr) expression and up-regulated methylenetetrahydrofolate dehydrogenase (NADP+-dependent)-1-like (mthfd1L) expression were found in FD larvae. Knocking-down mthfd1L expression worsened FD-induced ocular anomalies; whereas increasing mthfd1L expression provided a protective effect. 5-CH3-THF is the most sensitive folate pool, whose levels were the most significantly reduced in response to FD; whereas 10-CHO-THF levels were less affected. 5-CHO-THF is the most effective folate adduct for rescuing FD-induced microphthalmia and defective visual ability. CONCLUSION: FD impeded nucleotides formation, impaired cell proliferation and differentiation, caused apoptosis and interfered active vitamin A production, contributing to ocular defects. The developmental timing-specific and incoherent fluctuation among folate adducts and increased expression of mthfd1L in response to FD reflect the context-dependent regulation of folate-mediated one-carbon metabolism, endowing the larvae to prioritize the essential biochemical pathways for supporting the continuous growth in response to folate depletion.

Increased leptin-b expression and metalloprotease expression contributed to the pyridoxine-associated toxicity in zebrafish larvae displaying seizure-like behavior.

Epilepsy is a common neurological disorder affecting people of all ages, races and ethnic backgrounds world-wide. Vitamin B6 supplementation has been widely used as an adjuvant for treating epilepsy. However, the adverse effects, including nausea and peripheral sensory neuropathy, caused by long-term and high-dose consumption of vitamin B6 have undermined the usefulness of vitamin B6 supplementation, justifying additional experimental scrutiny of vitamin B6-associated toxicity. In the current study, we found that the presence of pyridoxine, the inactive form of B6 vitamer included in most nutrient supplements, increased the mortality of the larvae displaying chemical-induced epilepsy. The expression of leptin-b, one zebrafish ortholog of human leptin, was significantly increased in the larvae displaying seizures. Increased leptin-b expression alleviated larval seizure-like behavior when exposed to epilepsy inducer, but also increased larval mortality in the presence of pyridoxine. Meanwhile, elevated adam17 and mmp13 mRNA level were found in the larvae simultaneously exposed to epilepsy-inducer and pyridoxine. Adding TNF-α inhibitor and mmp13 inhibitor effectively improved the survival of larvae injected with leptin-b mRNA and exposed to pyridoxine subsequently. We conclude that increased leptin-b and metalloprotease expression contributed, at least partly, to the pyridoxine-associated toxicity observed in larvae displaying seizures.

An Open Pilot Study of the Effect and Tolerability of Add-On Multivitamin Therapy in Patients with Intractable Focal Epilepsy.

Observational studies have investigated the potential modulatory effect of neuronal excitability by vitamins in epilepsy. We aimed to investigate whether the addition of multivitamin therapy (B6/B9, D, E and Q) to regular antiepileptic drug therapy could ameliorate seizures in patients with refractory focal epilepsy. We conducted a prospective cohort open study to investigate the effect and tolerability of add-on multivitamin therapy (daily dose: B6 100 mg, B9 5 mg, D 1000 IU, E 400 IU and coenzyme Q10 100 mg) in patients with intractable focal epilepsy. All patients had effect and safety assessments at baseline and after one, three and six months of the supplementation. Thirty patients (11 men and 19 women) with a mean age of 42.37 ± 9.40 years were recruited and four patients discontinued. The seizure frequency significantly decreased after the six-month supplementation (9.04 ± 18.16/month and 2.06 ± 3.89/month, p = 0.045). At the final visit, 62.5% of the patients showed a ≥50% reduction in seizure frequency, and 12.5% were seizure-free. As to safety and tolerability, most patients did not experience significant adverse events, although three patients reported seizure worsening. In conclusion, this pilot study demonstrated the therapeutic potential and essentially good tolerability of add-on multivitamin therapy in patients with refractory focal epilepsy.

Pyridoxamine Supplementation Effectively Reverses the Abnormal Phenotypes of Zebrafish Larvae With PNPO Deficiency.

Neonatal epileptic encephalopathy (NEE), as a result of pyridoxine 5'-phosphate oxidase (PNPO) deficiency, is a rare neural disorder characterized by intractable seizures and usually leads to early infant death. The clinical phenotypes do not respond to antiepileptic drugs but are alleviated in most cases by giving large doses of pyridoxal 5'-phosphate (PLP). PLP is the active form of vitamin B6 participating in more than 100 enzymatic pathways. One of the causes of NEE is pathogenic mutations in the gene for human PNPO (hPNPO). PNPO is a key enzyme in converting pyridoxine (PN), the common dietary form of vitamin B6, and some other B6 vitamers to PLP. More than 25 different mutations in hPNPO, which result in reduced catalytic activity, have been described for PNPO-deficiency NEE. To date, no animal model is available to test new therapeutic strategies. In this report, we describe using zebrafish with reduced activity of Pnpo as an animal model. Knocking down zPnpo resulted in developmental anomalies including brain malformation and impaired locomotor activity, similar to the clinical features of PNPO-deficiency NEE. Other anomalies include a defective circulation system. These anomalies were significantly alleviated by co-injecting either zpnpo or hPNPO mRNAs. As expected from clinical observations in humans, supplementing with PLP improved the morphological and behavioral anomalies. PN only showed marginal positive effects, and only in a few anomalies. Remarkably, pyridoxamine (PM), another dietary form of vitamin B6, showed rescue effects even at a lower concentration than PLP, presenting a possible new therapeutic treatment for PNPO-deficiency NEE. Finally, GABA, a neurotransmitter whose biosynthesis depends on a PLP-dependent enzyme, showed some positive rescue effect. These results suggest zebrafish to be a promising PNPO-deficiency model for studying PLP homeostasis and drug therapy in vivo.

A novel zebrafish model to emulate lung injury by folate deficiency-induced swim bladder defectiveness and protease/antiprotease expression imbalance.

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

Supplementation with 5-formyltetrahydrofolate alleviates ultraviolet B-inflicted oxidative damage in folate-deficient zebrafish.

Ultraviolet (UV) is an omnipresent environmental carcinogen transmitted by sunlight. Excessive UV irradiation has been correlated to an increased risk of skin cancers. UVB, the most mutagenic component among the three UV constituents, causes damage mainly through inducing DNA damage and oxidative stress. Therefore, strategies or nutrients that strengthen an individual's resistance to UV-inflicted harmful effects shall be beneficial. Folate is a water-soluble B vitamin essential for nucleotides biosynthesis, and also a strong biological antioxidant, hence a micronutrient with potential of modulating individual's vulnerability to UV exposure. In this study, we investigated the impact of folate status on UV sensitivity and the protective activity of folate supplementation using a zebrafish model. Elevated reactive oxygen species (ROS) level and morphological injury were observed in the larvae exposed to UVB, which were readily rescued by supplementing with folic acid, 5-formyltetrahydrofolate (5-CHO-THF) and N-acetyl-L-cysteine (NAC). The UVB-inflicted abnormalities and mortality were worsened in Tg(hsp:EGFP-γGH) larvae displaying folate deficiency. Intriguingly, only supplementation with 5-CHO-THF, as opposed to folic acid, offered significant and consistent protection against UVB-inflicted oxidative damage in the folate-deficient larvae. We concluded that the intrinsic folate status correlates with the vulnerability to UVB-induced damage in zebrafish larvae. In addition, 5-CHO-THF surpassed both folic acid and NAC in preventing UVB-inflicted oxidative stress and injury in our current experimental zebrafish model.

One crisis, diverse impacts-Tissue-specificity of folate deficiency-induced circulation defects in zebrafish larvae.

Folate (vitamin B9) is an essential nutrient required for cell survival, proliferation, differentiation and therefore embryogenesis. Folate deficiency has been associated with many diseases, including congenital heart diseases and megaloblastic anemia, yet the mechanisms underlying these remains elusive. Here, we examine the impact of folate deficiency on the development of the circulation system using a zebrafish transgenic line which displays inducible folate deficiency. Impaired hematopoiesis includes decreased hemoglobin levels, decreased erythrocyte number, increased erythrocyte size and aberrant c-myb expression pattern were observed in folate deficient embryos. Cardiac defects, including smaller chamber size, aberrant cardiac function and cmlc2 expression pattern, were also apparent in folate deficient embryos. Characterization of intracellular folate content in folate deficiency revealed a differential fluctuation among the different folate derivatives that carry a single carbon group at different oxidation levels. Rescue attempts by folic acid and nucleotides resulted in differential responses among affected tissues, suggesting that different pathomechanisms are involved in folate deficiency-induced anomalies in a tissue-specific manner. The results of the current study provide an explanation for the inconsistent outcome observed clinically in patients suffering from folate deficiency and/or receiving folate supplementation. This study also supports the use of this model for further research on the defective cardiogenesis and hematopoiesis caused by folate deficiency.

Concentration effects of grape seed extracts in anti-oral cancer cells involving differential apoptosis, oxidative stress, and DNA damage.

BACKGROUND: Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer. METHODS: The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage. RESULTS: High concentrations (50-400 µg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 µg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations. CONCLUSIONS: Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.

Folate deficiency-induced oxidative stress contributes to neuropathy in young and aged zebrafish--implication in neural tube defects and Alzheimer's diseases.

Folate is a nutrient essential for the development, function and regeneration of nervous systems. Folate deficiency has been linked to many neurological disorders including neural tube defects in fetus and Alzheimer's diseases in the elderly. However, the etiology underlying these folate deficiency-associated diseases is not completely understood. In this study, zebrafish transgenic lines with timing and duration-controllable folate deficiency were developed by ectopically overexpressing a recombinant EGFP-γ-glutamyl hydrolase (γGH). Impeded neural crest cell migration was observed in the transgenic embryos when folate deficiency was induced in early stages, leading to defective neural tube closure and hematopoiesis. Adding reduced folate or N-acetylcysteine reversed the phenotypic anomalies, supporting the causal link between the increased oxidative stress and the folate deficiency-induced abnormalities. When folate deficiency was induced in aged fish accumulation of beta-amyloid and phosphorylated Tau protein were found in the fish brain cryo-sections. Increased autophagy and accumulation of acidic autolysosome were apparent in folate deficient neuroblastoma cells, which were reversed by reduced folate or N-acetylcysteine supplementation. Decreased expression of cathepsin B, a lysosomal protease, was also observed in cells and tissue with folate deficiency. We concluded that folate deficiency-induced oxidative stress contributed to the folate deficiency-associated neuropathogenesis in both early and late stages of life.

Methotrexate-induced decrease in embryonic 5-methyl-tetrahydrofolate is irreversible with leucovorin supplementation.

Folate is a nutrient crucial for rapidly growing tissues, including developing embryos and cancer cells. Folate participates in the biosynthesis of nucleic acids, proteins, amino acids, S-adenosylmethionine, many neurotransmitters, and some vitamins. The intracellular folate pool consists of different folate adducts, which carry one-carbon units at three different oxidative states and participate in distinct biochemical reactions. Therefore, the content and dynamics of folate adducts will affect the homeostasis of the metabolites generated in these folate-mediated reactions. Currently, the knowledge on the level of each individual folate adduct in developing embryos is limited. With an improved high-performance liquid chromatography protocol, we found that tetrahydrofolate (THF), the backbone of one-carbon carrier, gradually increased and became dominant in developing zebrafish embryos. 5-methyl-tetrahydrofolate (5-CH3-THF) was abundant in unfertilized eggs but decreased rapidly when embryos started to proliferate and differentiate. 10-formyltetrahydrofolate at first increased after fertilization, and then dropped dramatically before reaching a sustained level at later stages. Dihydrofolate (DHF) slightly decreased initially and remained low throughout embryogenesis. Exposure to methotrexate significantly decreased 5-CH3-THF levels and increased DHF pools, besides causing brain ventricle anomaly. Rescuing with leucovorin partly reversed the abnormal phenotype. Unexpectedly, the level of 5-CH3-THF remained low even when leucovorin was added for rescue. Our results show that different folate adducts fluctuated significantly and differentially in concert with the physiological requirement specific for the corresponding developmental stages. Furthermore, methotrexate lowered the level of 5-CH3-THF in developing embryos, which could not be reversed with folate supplementation and might be more substantial to cellular methylation potential and epigenetic control than to nucleotide synthesis.

Grape seed extract inhibits the growth and pathogenicity of Staphylococcus aureus by interfering with dihydrofolate reductase activity and folate-mediated one-carbon metabolism.

Staphylococcus aureus (S. aureus) is one of the most common pathogens that causes infectious and foodborne diseases worldwide. Searching for drug and chemical compounds against this bacterium is still in demand. We found that grape seed extract (GSE), a natural food product rich in polyphenols, inhibited the dihydrofolate reductase activity and growth of S. aureus. In addition, the intracellular content of tetrahydrofolate (THF), the major folate species identified in S. aureus, was significantly decreased when GSE was present in medium. The GSE-induced growth inhibition was reversed by adding, THF, 5,10-methylenetetrahydrofolate or methionine to the medium. The differential rescuing effects elicited by thymidine and methionine indicated that GSE-induced perturbation in folate-mediated one-carbon metabolism has more profound impact on methionine cycle than on thymidine monophosphate (TMP) synthesis. Significantly reduced inflammatory responses and mortality were observed in zebrafish infected with S. aureus pre-incubated with GSE. We conclude that GSE might serve as an effective natural alternative for the control of food poisoning caused by S. aureus with proper safety measure.

Characterization and comparative studies of zebrafish and human recombinant dihydrofolate reductases--inhibition by folic acid and polyphenols.

Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissue-specific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.

Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases.

Serine hydroxymethyltransferase (SHMT) provides activated one-carbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N(5),N(10)-methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N(5)-CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.