Plasma Formate Is Greater in Fetal and Neonatal Rats Compared with Their Mothers.

BACKGROUND: Formate can be incorporated into 10-formyl-tetrahydrofolate (10-formyl-THF), which is a substrate for purine synthesis, and after further reduction of the one-carbon group, may be used as a substrate for thymidylate synthesis and for homocysteine remethylation. OBJECTIVE: We examined plasma formate concentrations and the expression of genes involved in the production and utilization of formate in fetal and neonatal rats and in pregnant and virgin female rats. METHODS: In 1 experiment, plasma formate was measured by GC-MS in rats aged 1-56 d. In a second experiment, virgin female (adult) rats, 19-d pregnant rats (P) and their male and female fetuses (F), and 3-d-old (N) and 7-d-old (J) offspring had plasma and amniotic fluid analyzed for formate by GC-MS, mRNA abundance in liver and placenta by qPCR, and several plasma amino acids by HPLC. RESULTS: The plasma formate concentration was significantly higher in fetuses at embryonic day 19 than in the mothers. It was also significantly higher in neonatal rats but slowly returned to adult concentrations by ∼3 wk. The abundance of mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase (Mthfd1l) mRNA was significantly higher in placenta (PP) and F liver than in liver of N or J. Expression of mitochondrial bifunctional NAD-dependent methylene-tetrahydrofolate dehydrogenase/methenyl-tetrahydrofolate cyclohydrolase (Mthfd2) was significantly enriched in PP and liver of P, intermediate in F liver, and much lower in liver of N and J, relative to PP. Serine hydroxymethyltransferase 2 (Shmt2), methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), and glycine decarboxylase protein of the glycine cleavage system (Gldc) mRNA expression was significantly lower in PP compared with other groups. Cytoplasmic NAD(P)-dependent 10-formyl-tetrahydrofolate dehydrogenase (Aldh1/1) and mitochondrial NAD(P)-dependent 10-formyl-tetrahydrofolate dehydrogenase (Aldh1/2) , genes responsible for the catabolism of 10-formylTHF, were very weakly expressed in PP, low in livers of F and N, and reached the significantly higher adult levels in J. Serine, glycine, and methionine concentrations in plasma of F were significantly higher than in plasma of P. CONCLUSIONS: Formate metabolism is highly active in fetuses and in placenta of pregnant rats.

Formate concentrations in maternal plasma during pregnancy and in cord blood in a cohort of pregnant Canadian women: relations to genetic polymorphisms and plasma metabolites.

BACKGROUND: One-carbon metabolism, responsible for purine and thymidylate synthesis and transmethylation reactions, plays a critical role in embryonic and fetal development. Formate is a key player in one-carbon metabolism. In contrast to other one-carbon metabolites, it is not linked to tetrahydrofolate, is present in plasma at appreciable concentrations, and may therefore be distributed to different tissues. OBJECTIVE: The study was designed to determine the concentration of formate in cord blood in comparison with maternal blood taken earlier in pregnancy and at delivery and to relate formate concentrations to potential precursors and key fetal genotypes. METHODS: Formate and amino acids were measured in plasma during early pregnancy (12-16 wk), at delivery (37-42 wk), and in cord blood samples from 215 mothers, of a prospective cohort study. Three fetal genetic variants in one-carbon metabolism were assessed for their association with cord plasma concentrations of formate. RESULTS: The formate concentration was ∼60% higher in the cord blood samples than in mothers' plasma. The maternal formate concentrations did not differ between the early pregnancy samples and those taken at delivery. Plasma concentrations of 4 formate precursors (serine, glycine, tryptophan, and methionine) were increased in cord blood compared with the maternal samples. Cord blood formate was influenced by fetal genotype, being ∼12% higher in infants harboring the MTHFR A1298C (rs1801131) AC or CC genotypes and 10% lower in infants harboring the MTHFD1 G1958A (rs2236225) GA or AA genotypes. CONCLUSIONS: The increased formate concentrations in cord blood may support the increased activity of one-carbon metabolism in infants. As such, it would support increased rates of purine and thymidylate synthesis and the provision of methionine for methylation reactions.

Enhancement of Cancer-Specific Protoporphyrin IX Fluorescence by Targeting Oncogenic Ras/MEK Pathway.

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells. Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted. Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model. Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Lifestyle, metabolite, and genetic determinants of formate concentrations in a cross-sectional study in young, healthy adults.

Background: Formate is an important metabolite that serves as a donor of one-carbon groups to the intracellular tetrahydrofolate pool. However, little is known of its circulating concentrations or of their determinants. Objective: This study aimed to define formate concentrations and their determinants in a healthy young population. Design: Serum formate was measured in 1701 participants from the Trinity Student Study. The participants were men and women, aged 18 to 28 y, enrolled at Trinity College, Dublin. Formate concentrations were compared with other one-carbon metabolites, vitamin status, potential formate precursors, genetic polymorphisms, and lifestyle factors. Results: Serum formate concentrations ranged from 8.7 to 96.5 µM, with a mean of 25.9 µM. Formate concentrations were significantly higher in women than in men; oral contraceptive use did not further affect them. There was no effect of smoking or of alcohol ingestion, but the TT genotype of the methylenetetrahydrofolate reductase (MTHFR) 677C→T (rs1801133) polymorphism was associated with a significantly decreased formate concentration. Formate was positively associated with potential metabolic precursors (serine, methionine, tryptophan, choline) but not with glycine. Formate concentrations were positively related to serum folate and negatively related to serum vitamin B-12. Conclusions: Formate concentrations were sensitive to the concentrations of metabolic precursors. In view of the increased susceptibility of women with the TT genotype of MTHFR to give birth to infants with neural tube defects as well as the effectiveness of formate supplementation in decreasing the incidence of folate-resistant neural tube defects in susceptible mice, it will be important to understand how this genotype decreases the serum formate concentration. This trial was registered at www.clinicaltrials.gov as NCT03305900.

Formate metabolism in fetal and neonatal sheep.

By virtue of its role in nucleotide synthesis, as well as the provision of methyl groups for vital methylation reactions, one-carbon metabolism plays a crucial role in growth and development. Formate, a critical albeit neglected component of one-carbon metabolism, occurs extracellularly and may provide insights into cellular events. We examined formate metabolism in chronically cannulated fetal sheep (gestation days 119-121, equivalent to mid-third trimester in humans) and in their mothers as well as in normal full-term lambs. Plasma formate levels were much higher in fetal lamb plasma and in amniotic fluid (191 ± 62 and 296 ± 154 µM, respectively) than in maternal plasma (33 ± 13 µM). Measurements of folate, vitamin B12, and homocysteine showed that these high formate levels could not be due to vitamin deficiencies. Elevated formate levels were also found in newborn lambs and persisted to about 8 wk of age. Formate was also found in sheep milk. Potential precursors of one-carbon groups were also measured in fetal and maternal plasma and in amniotic fluid. There were very high concentrations of serine in the fetus (∼1.6 mM in plasma and 3.5 mM in the amniotic fluid) compared with maternal plasma (0.19 mM), suggesting increased production of formate; however, we cannot rule out decreased formate utilization. Dimethylglycine, a choline metabolite, was also 30 times higher in the fetus than in the mother.

An isotope-dilution, GC-MS assay for formate and its application to human and animal metabolism.

Formate, a crucial component of one-carbon metabolism, is increasingly recognized as an important intermediate in production and transport of one-carbon units. Unlike tetrahydrofolate-linked intermediates, it is not restricted to the intracellular milieu so that circulating levels of formate can provide insight into cellular events. We report a novel isotope-dilution, GC-MS assay employing derivatization by 2,3,4,5,6-pentafluorobenzyl bromide for the determination of formate in biological samples. This assay is robust and sensitive; it may be applied to the measurement of formate in serum, plasma and urine. We demonstrate how this method may be applied by providing the first characterization of formate levels in a human population; formate levels were higher in males than in females. We also show how this procedure may be applied for the measurement of in vivo kinetics of endogenous formate production in experimental animals.

Suppression of IFN-induced transcription underlies IFN defects generated by activated Ras/MEK in human cancer cells.

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes.

Promotion of viral internal ribosomal entry site-mediated translation under amino acid starvation.

Cap-dependent and internal ribosomal entry site (IRES)-mediated translation are regulated differently within cells. Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. To investigate how cellular stresses influence the efficiency of viral IRES-mediated translation, we used a bicistronic luciferase reporter construct harbouring IRES elements from the following viruses: encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV) or human rhinovirus (HRV). NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was only observed under amino acid starvation when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we tested the involvement of eukaryotic initiation factor 4E-binding protein (4E-BP), general control non-depressed 2 (GCN2) and eukaryotic initiation factor 2B (eIF2B), as these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation, whereas GCN2 and eIF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide kinase-3 inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by the treatments was not sufficient to promote viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES-mediated translation under amino acid starvation, but not via its dephosphorylation.