Disruption Of Folate Metabolism Research Articles

Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations.

Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrr gt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrr gt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrr gt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrr gt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrr gt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrr gt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrr gt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr +/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrr gt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.

Disruption of Cytosolic Folate Integrity Aggravates Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors and Modulates Metastatic Properties in Non-Small-Cell Lung Cancer Cells

Patients with advanced-stage non-small-cell lung cancer (NSCLC) are susceptible to malnutrition and develop folate deficiency (FD). We previously found that folate deprivation induces drug resistance in hepatocellular carcinoma; here, we assessed whether disrupted cytoplasmic folate metabolism could mimic FD-induced metastasis and affect the sensitivity of NSCLC cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We examined whether cytosolic folate metabolism in NSCLC cells was disrupted by FD or the folate metabolism blocker pemetrexed for 1–4 weeks. Our results revealed an increase in NF-κB overexpression–mediated epithelial-mesenchymal transition biomarkers: N-cadherin, vimentin, matrix metalloproteinases (MMPs), SOX9, and SLUG. This finding suggests that the disruption of folate metabolism can drastically enhance the metastatic properties of NSCLC cells. Cytosolic FD also affected EGFR-TKI cytotoxicity toward NSCLC cells. Because SLUG and N-cadherin are resistance effectors against gefitinib, the effects of SLUG knockdown in folate antagonist–treated CL1-0 cells were evaluated. SLUG knockdown prevented SLUG/NF-κB/SOX9-mediated invasiveness and erlotinib resistance acquisition and significantly reduced pemetrexed-induced gelatinase activity and MMP gene expression. To summarize, our data reveal two unprecedented adverse effects of folate metabolism disruption in NSCLC cells. Thus, the folic acid status of patients with NSCLC under treatment can considerably influence their prognosis.

Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

Methotrexate induces astrocyte apoptosis by disrupting folate metabolism in the mouse juvenile central nervous system

Methotrexate (MTX) is a folic acid antagonist and widely used for acute lymphoblastic leukemia (ALL) in children. MTX is associated with acute and chronic neurotoxicity during treatment, however the underlying mechanism is still poorly understood. In this study we investigate whether MTX is neurovirulent to astrocytes in the Central Nervous System (CNS) of adolescent mice. We demonstrated that MTX induced severe cytotoxicity in C6 astrocyte-like cell line and rat primary cultures of astrocytes in a dose-dependent manner. Moreover, GFAP-labeled astrocyte cells significantly decreased in the mouse spinal cord and brain. Furthermore, protein levels of PARP and pro-Caspase-3 were reduced by MTX, indicating MTX-induced apoptosis leads to the astrocytes loss. Notably, overexpression of dihydrofolate reductase (DHFR) or exogenous addition of folate markedly reversed the astrocytes toxicity induced by MTX through activating folate metabolism pathway. Taken together, our study provides evidence for neurotoxic effect of MTX-induced astrocytes apoptosis both in vitro and in vivo with disruption of folate metabolism, and additional supplement of folate may provide novel approaches for alleviating the astrocytes toxicity induced by MTX in the clinic.

The epidemiology and possible mechanisms of disinfection by-products in drinking water

This paper summarizes the epidemiological evidence for adverse health effects associated with disinfection by-products (DBPs) in drinking water and describes the potential mechanism of action. There appears to be good epidemiological evidence for a relationship between exposure to DBPs, as measured by trihalomethanes (THMs), in drinking water and bladder cancer, but the evidence for other cancers including colorectal cancer is inconclusive and inconsistent. There appears to be some evidence for an association between exposure to DBPs, specifically THMs, and little for gestational age/intrauterine growth retardation and, to a lesser extent, pre-term delivery, but evidence for relationships with other outcomes such as low birth weight, stillbirth, congenital anomalies and semen quality is inconclusive and inconsistent. Major limitations in exposure assessment, small sample sizes and potential biases may account for the inconclusive and inconsistent results in epidemiological studies. Moreover, most studies have focused on total THMs as the exposure metric, whereas other DBPs appear to be more toxic than the THMs, albeit generally occurring at lower levels in the water. The mechanisms through which DBPs may cause adverse health effects including cancer and adverse reproductive effects have not been well investigated. Several mechanisms have been suggested, including genotoxicity, oxidative stress, disruption of folate metabolism, disruption of the synthesis and/or secretion of placental syncytiotrophoblast-derived chorionic gonadotropin and lowering of testosterone levels, but further work is required in this area.

Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus

Folate metabolism is necessary for the biosyntheses of purine nucleotides and thymidylate and for the synthesis of S-adenosylmethionine, a cofactor required for cellular methylation reactions and a precursor of spermidine and spermine syntheses. Disruption of folate metabolism is associated with several pathologies and developmental anomalies including cancer and neural tube defects. The enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, and has been shown to affect intracellular folate concentrations by accelerating folate degradation. Mammalian MTHFS proteins described to date are not stable and no recombinant mammalian MTHFS protein has been successfully expressed in Escherichia coli. The three-dimensional structure of MTHFS has not been solved. The cDNA coding for Mus musculus MTHFS was isolated and expressed in E. coli with a hexa-histidine tag. Milligram quantities of recombinant mouse MTHFS were purified using metal affinity chromatography and the protein was stabilized with Tween 20. Mouse MTHFS has a molecular mass of 23 kDa and is 84% identical in amino acid sequence to the human enzyme. Activity assays confirmed the functionality of the recombinant protein, with K m=5 μM for (6 S)-5-formyltetrahydrofolate and K m=769 μM for Mg–ATP. This is the first example of a mammalian form of MTHFS expressed in E. coli that yielded sufficient quantities of stable purified protein to allow for detailed characterization of its three-dimensional structure and kinetic properties.

Disruption of Folate and Vitamin B12Metabolism in Aged Rats Following Exposure to Nitrous Oxide

The ability of nitrous oxide (N2O) to disrupt folate and vitamin B12 metabolism was examined in young (2-month), middle-aged (12-month), and elderly (24-month) Fischer 344 rats. Abnormalities in folate metabolism were assessed in a noninvasive manner by measuring the urinary excretion of formic acid and formiminoglutamic acid (FIGLU), compounds that are elevated in the urine of mammals with a deficiency in folate. After a 6-h exposure to 60% N2O/40% O2, urinary formic acid excretion increased 3- to 25-fold the first day following N2O exposure and returned to background levels by the second day after exposure in all age groups. Urinary FIGLU excretion increased 100- to 300-fold in the first day following N2O exposure, with the highest FIGLU excretion rates found in the elderly rats and the lowest in the young rats. By the second day after N2O exposure, FIGLU excretion rates returned to baseline levels in all age groups. Plasma folate progressively decreased with increasing age, whereas no age-dependent changes were observed in red cell folate, liver folate, or plasma vitamin B12 levels. The elderly rats demonstrated the highest vitamin B12 content in the liver and the lowest vitamin B12 content in the kidney compared to the other age groups. Hepatic methionine synthase activities (measured 16-21 days after N2O exposure) were elevated in the elderly compared to the middle-aged or young rats, but methionine synthase activities in kidney and brain were not different among the three different age groups. It was concluded that in rats, aging per se only slightly influences the disruption of folate metabolism produced by exposure to N2O.

Characterization of the function of mammalian folylpolyglutamate synthetase (FPGS).

The function and characteristics of mouse folylpolyglutamate synthetase have been examined. Folate polyglutamates were poor substrates for the efflux mechanism for monoglutamates in L1210 mouse leukemia cells, with negligible loss of preformed folate polyglutamates to the medium over four hours. Disruption of folate metabolism with methotrexate did not augment efflux of folate polyglutamates. Folylpolyglutamate synthetase, partially purified from mouse liver, was found to accept a variety of folate derivatives as substrates, including pteroic acid and methotrexate; however, the concentration of these substrates that saturated the reaction varied considerably. The enzyme that catalyzed the addition of glutamic acid to methotrexate and to the naturally-occurring folate monoglutamates appeared to be the same. The cytotoxicity of folylpolyglutamate synthetase inhibitors was predicted to require continued cell division since their effects would be based upon a decreased rate of synthesis of folate cofactors capable of retention by the cell membrane. Hence folylpolyglutamate synthetase inhibitors should have low toxicity to nonproliferative cell populations.