Folate Deficiency and/or the Genetic Variant Mthfr677C >T Can Drive Hepatic Fibrosis or Steatosis in Mice, in a Sex-Specific Manner.

SCOPE: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C >T variant on NAFLD. METHODS AND RESULTS: This study uses the new Mthfr677C >T mouse model for the human MTHFR677C >T variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. CONCLUSION: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Sex-Specific Gene Expression in Brain of Young Mice and Embryos.

Food fortification and increased vitamin intake have led to higher folic acid (FA) consumption by many pregnant women. We showed that FA-supplemented diet in pregnant mice (fivefold higher FA than the recommended level (5xFASD)) led to hyperactivity-like behavior and memory impairment in pups. Disturbed choline/methyl metabolism and altered placental gene expression were identified. The aim of this study was to examine the impact of 5xFASD on the brain at two developmental stages, postnatal day (P) 30 and embryonic day (E) 17.5. Female C57BL/6 mice were fed a control diet or 5xFASD for 1 month before mating. Diets were maintained throughout the pregnancy and lactation until P30 or during pregnancy until E17.5. The 5xFASD led to sex-specific transcription changes in a P30 cerebral cortex and E17.5 cerebrum, with microarrays showing a total of 1003 and 623 changes, respectively. Enhanced mRNA degradation was observed in E17.5 cerebrum. Expression changes of genes involved in neurotransmission, neuronal growth and development, and angiogenesis were verified by qRT-PCR; 12 and 15 genes were verified at P30 and E17.5, respectively. Hippocampal collagen staining suggested decreased vessel density in FASD male embryos. This study provides insight into the mechanisms of neurobehavioral alterations and highlights potential deleterious consequences of moderate folate oversupplementation during pregnancy.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Methyl Metabolism and in Sex-Specific Placental Transcription Changes.

SCOPE: Many pregnant women have higher folic acid (FA) intake due to food fortification and increased vitamin use. It is reported that diets containing five-fold higher FA than recommended for mice (5xFASD) during pregnancy resulted in methylenetetrahydrofolate reductase (MTHFR) deficiency and altered choline/methyl metabolism, with neurobehavioral abnormalities in newborns. The goal is to determine whether these changes have their origins in the placenta during embryonic development. METHODS AND RESULTS: Female mice are fed control diet or 5xFASD for a month before mating and maintained on these diets until embryonic day 17.5. 5xFASD led to pseudo-MTHFR deficiency in maternal liver and altered choline/methyl metabolites in maternal plasma (increased methyltetrahydrofolate and decreased betaine). Methylation potential (S-adenosylmethionine:S-adenosylhomocysteine ratio) and glycerophosphocholine are decreased in placenta and embryonic liver. Folic acid supplemented diet results in sex-specific transcriptome profiles in placenta, with validation of dietary expression changes of 29 genes involved in angiogenesis, receptor biology or neurodevelopment, and altered methylation of the serotonin receptor 2A gene. CONCLUSION: Moderate increases in folate intake during pregnancy result in placental metabolic and gene expression changes, particularly in angiogenesis, which may contribute to abnormal behavior in pups. These results are relevant for determining a safe upper limit for folate intake during pregnancy.

High folic acid intake increases methylation-dependent expression of Lsr and dysregulates hepatic cholesterol homeostasis.

Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/- mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism.

Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.

Early Manifestations of Brain Aging in Mice Due to Low Dietary Folate and Mild MTHFR Deficiency.

Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer's disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/- mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/- genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.

Mild Methylenetetrahydrofolate Reductase Deficiency Alters Inflammatory and Lipid Pathways in Liver.

SCOPE: Dietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677C→T) causes mild MTHFR deficiency with lower 5-methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD-related effects. METHODS AND RESULTS: Wild-type and Mthfr+/- mice, a model for the human variant, are fed control (CD) or high-fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S-adenosylmethionine, increased S-adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti-inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro-inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism. CONCLUSION: Individuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high-fat diets.

Testicular MTHFR deficiency may explain sperm DNA hypomethylation associated with high dose folic acid supplementation.

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.

Murine diet/tissue and human brain tumorigenesis alter Mthfr/MTHFR 5'-end methylation.

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5' to the upstream translational start site or in another region 3' to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.

Quantitative proteomics reveals differentially expressed proteins in murine preneoplastic intestine in a model of intestinal tumorigenesis induced by low dietary folate and MTHFR deficiency.

Colorectal cancer risk is increased when dietary folate intake is low, with or without a deficiency in methylenetetrahydrofolate reductase (MTHFR). We have observed that intestinal tumors are induced in mice fed low-folate diets, and that tumor incidence is increased when these mice also have MTHFR deficiency. This study was undertaken to identify differentially expressed proteins in conditions favoring initial steps of murine carcinogenesis in normal preneoplastic intestine. We compared the proteome of BALB/c normal intestine in Mthfr(+/+) mice fed control diets for 1 year (low susceptibility to tumorigenesis) with the proteome of Mthfr(+/-) animals fed low folate diets (higher tumor susceptibility). Our data suggest that the NuRD complex, KRAS-related proteins, the protein synthetic machinery, and fatty acid-related metabolic proteins are upregulated in the early stages of tumorigenesis. These proteins may serve as biomarkers or targets for colorectal cancer diagnosis or therapy.

Severe methylenetetrahydrofolate reductase deficiency: clinical clues to a potentially treatable cause of adult-onset hereditary spastic paraplegia.

IMPORTANCE: Hereditary spastic paraplegia is a highly heterogeneous group of neurogenetic disorders with pure and complicated clinical phenotypes. No treatment is available for these disorders. We identified 2 unrelated families, each with 2 siblings with severe methylenetetrahydrofolate reductase (MTHFR) deficiency manifesting a complicated form of adult-onset hereditary spastic paraparesis partially responsive to betaine therapy. OBSERVATIONS: Both pairs of siblings presented with a similar combination of progressive spastic paraparesis and polyneuropathy, variably associated with behavioral changes, cognitive impairment, psychosis, seizures, and leukoencephalopathy, beginning between the ages of 29 and 50 years. By the time of diagnosis a decade later, 3 patients were ambulatory and 1 was bedridden. Investigations have revealed severe hyperhomocysteinemia and hypomethioninemia, reduced fibroblast MTHFR enzymatic activity (18%-52% of control participants), and 3 novel pathogenic MTHFR mutations, 2 as compound heterozygotes in one family and 1 as a homozygous mutation in the other family. Treatment with betaine produced a rapid decline of homocysteine by 50% to 70% in all 4 patients and, over 9 to 15 years, improved the conditions of the 3 ambulatory patients. CONCLUSIONS AND RELEVANCE: Although severe MTHFR deficiency is a rare cause of complicated spastic paraparesis in adults, it should be considered in select patients because of the potential therapeutic benefit of betaine supplementation.

Genes with aberrant expression in murine preneoplastic intestine show epigenetic and expression changes in normal mucosa of colon cancer patients.

An understanding of early genetic/epigenetic changes in colorectal cancer would aid in diagnosis and prognosis. To identify these changes in human preneoplastic tissue, we first studied our mouse model in which Mthfr⁺/⁻ BALB/c mice fed folate-deficient diets develop intestinal tumors in contrast to Mthfr⁺/⁺ BALB/c mice fed control diets. Transcriptome profiling was performed in normal intestine from mice with low or high tumor susceptibility. We identified 12 upregulated and 51 downregulated genes in tumor-prone mice. Affected pathways included retinoid acid synthesis, lipid and glucose metabolism, apoptosis and inflammation. We compared murine candidates from this microarray analysis, and murine candidates from an earlier strain-based comparison, with a set of human genes that we had identified in previous methylome profiling of normal human colonic mucosa, from colorectal cancer patients and controls. From the extensive list of human methylome candidates, our approach uncovered five orthologous genes that had shown changes in murine expression profiles (PDK4, SPRR1A, SPRR2A, NR1H4, and PYCARD). The human orthologs were assayed by bisulfite-pyrosequencing for methylation at 14 CpGs. All CpGs exhibited significant methylation differences in normal mucosa between colorectal cancer patients and controls; expression differences for these genes were also observed. PYCARD and NR1H4 methylation differences showed promise as markers for presence of polyps in controls. We conclude that common pathways are disturbed in preneoplastic intestine in our animal model and morphologically normal mucosa of patients with colorectal cancer, and present an initial version of a DNA methylation-based signature for human preneoplastic colon.

ß,ß-carotene 15,15'-monooxygenase and its substrate ß-carotene modulate migration and invasion in colorectal carcinoma cells.

BACKGROUND: ß,ß-Carotene 15,15'-monooxygenase (BCMO1) converts ß-carotene to retinaldehyde. Increased ß-carotene consumption is linked to antitumor effects. Retinoic acid reduces the invasiveness in cancer, through inhibition of matrix metalloproteinases (MMPs). In our studies of a mouse model that develops intestinal tumors after low dietary folate, we found reduced BCMO1 expression in normal preneoplastic intestine of folate-deficient tumor-prone mice. OBJECTIVE: Our goal was to determine whether BCMO1 expression could influence transformation potential in human colorectal carcinoma cells, by examining the effect of BCMO1 modulation on cellular migration and invasion, and on expression of MMPs. DESIGN: LoVo colon carcinoma cells were transfected with BCMO1 small interfering RNA (siRNA) or scrambled siRNA. Migration and invasion were measured, and the expression of BCMO1, MMP7, and MMP28 was assessed by quantitative reverse-transcriptase polymerase chain reaction. These variables were also measured after treatment of cells with retinoic acid, 5-aza-2'-deoxycytidine, folate-depleted/high-methionine medium, and ß-carotene. RESULTS: Retinoic acid decreased the migration, invasion, and expression of MMP28 mRNA. Transfection of cells with BCMO1 siRNA inhibited BCMO1 expression, enhanced migration and invasion, and increased expression of MMP7 and MMP28. 5-Aza-2'-deoxycytidine decreased, whereas folate-depleted/high-methionine medium increased invasiveness. ß-Carotene increased BCMO1 expression and reduced invasiveness with a decrease in expression of MMP7 and MMP28. CONCLUSIONS: Inhibition of BCMO1 expression is associated with increased invasiveness of colon cancer cells and increased expression of MMP7 and MMP28. ß-Carotene can upregulate BCMO1 and reverse these effects. These novel associations suggest a critical role for BCMO1 in cancer and provide a mechanism for the proposed antitumor effects of ß-carotene.

Differential gene expression and methylation in the retinoid/PPARA pathway and of tumor suppressors may modify intestinal tumorigenesis induced by low folate in mice.

SCOPE: Inadequate folate intake increases risk for colorectal cancer. We previously showed that low-folate diets induced intestinal tumors in BALB/c mice, but not in C57BL/6 mice. We suggested that DNA damage, altered methylation, and reduced apoptosis could contribute to tumorigenesis in this model. METHODS AND RESULTS: To identify genes involved in tumorigenesis, we compared gene expression profiles in preneoplastic intestine of BALB/c and C57BL/6 mice-fed low folate. We identified 74 upregulated and 90 downregulated genes in BALB/c compared to C57BL/6 mice. We validated decreased expression of Bcmo1 and increased expression of Aldh1a, which would be expected to upregulate the peroxisome proliferator-activated receptor alpha (PPARA) pathway, and confirmed the expected upregulation of several Ppara downstream genes. We verified, in BALB/c mice, reduced expression of Sprr2a, a gene that increases resistance to oxidative damage, and of two oncosuppressors (Bmp5 and Arntl). Low folate increased Ppara and Aldh1a1 expression, and decreased Bcmo1, Sprr2a, and Bmp5 expression in BALB/c, compared to BALB/c on control diets. Bcmo1, Ppara, and Bmp5 showed differential DNA methylation related to strain, diet, and/or Mthfr genotype. CONCLUSION: Disturbed regulation of the retinoid/PPARA pathway, which influences oxidative damage, and altered expression of tumor suppressors may contribute to intestinal tumorigenesis induced by low-folate intake.

Severe methylenetetrahydrofolate reductase deficiency in mice results in behavioral anomalies with morphological and biochemical changes in hippocampus.

The brain is particularly sensitive to folate metabolic disturbances, since methyl groups are critical for its functions. Methylenetetrahydrofolate reductase (MTHFR) generates the primary circulatory form of folate required for homocysteine remethylation to methionine. Neurological disturbances have been described in homocystinuria caused by severe MTHFR deficiency. The goal of this study was to determine if behavioral anomalies are present in severe Mthfr-deficient (Mthfr(-/-)) mice and to identify neurobiological changes that could contribute to these anomalies. Adult male mice of 3 Mthfr genotypes (+/+, +/-, -/-) were tested on motor, anxiety, exploratory and cognitive tasks. Volumes (whole brain and hippocampus) and morphology, global DNA methylation, apoptosis, expression of choline acetyltransferase (ChAT) and glucocorticoid receptor (GR), and concentrations of choline metabolites were assessed in hippocampus. Mthfr(-/-) mice had impairments in motor function and in short- and long-term memory, increased exploratory behavior and decreased anxiety. They showed decreased whole brain and hippocampal volumes, reduced thickness of the pyramidal cell layer of CA1 and CA3, and increased apoptosis in hippocampus. There was a disturbance in choline metabolism as manifested by differences in acetylcholine, betaine or glycerophosphocholine concentrations, and by increased ChAT levels. Mthfr(-/-) mice also had increased GR mRNA and protein. Our study has revealed significant anomalies in affective behavior and impairments in memory of Mthfr(-/-) mice. We identified structural changes, increased apoptosis, altered choline metabolism and GR dysregulation in hippocampus. These findings, as well as some similar observations in cerebellum, could contribute to the behavioral changes and suggest that choline is a critical metabolite in homocystinuria.

Targeted insertion of two Mthfr promoters in mice reveals temporal- and tissue-specific regulation.

Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, synthesizes 5-methyltetrahydrofolate, the main circulatory form of folate which is required for maintaining nontoxic levels of homocysteine and providing one-carbon units for methylation. A common 677C â†’ T variant in MTHFR confers mild MTHFR deficiency and has been associated with a number of human disorders, including neural tube defects and vascular disease. Two promoters of Mthfr, designated as upstream and downstream promoters, are located upstream of a transcription start site cluster and have previously demonstrated cell-specific activities. In this study we used a unique approach for targeted, single-copy transgene insertion to generate transgenic mice carrying a Mthfr upstream or Mthfr downstream promoter-reporter construct located 5' to the endogenous Hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The Mthfr downstream promoter demonstrated activity in the neural tube, neural crest cells, dorsal root ganglia, heart, and endothelial cells of blood vessels in 10.5-days post coitum embryos and placentas. Upstream promoter activity was absent at this developmental stage. Postnatally, both promoters demonstrated activity in the brain stem, hippocampus, and thalamus of 1-week-old brain that became stronger in the adult. The Mthfr upstream promoter also showed activity in the cerebellum and cerebral cortex. Both promoters were active in male reproductive tissues, including 1-week-old epididymides, and there was upstream promoter-specific activity in the adult testis. Our investigation of Mthfr regulation in an in vivo mouse model revealed temporal- and tissue-specific regulation that supports important roles for MTHFR in the developing embryo, and in postnatal brain and male reproductive tissues.

Susceptibility to intestinal tumorigenesis in folate-deficient mice may be influenced by variation in one-carbon metabolism and DNA repair.

Low dietary folate is associated with increased risk of colorectal cancer. In earlier work, we showed that folate deficiency induced intestinal tumors in BALB/c but not C57Bl/6 mice through increased dUTP incorporation into DNA with consequent DNA damage. To determine whether strain differences between one-carbon metabolism and DNA repair pathways could contribute to increased tumorigenesis in BALB/c mice, we measured amino acids and folate in the normal intestinal tissue of both strains fed a control diet or a folate-deficient diet. We also determined the expression of critical folate-metabolizing enzymes and several DNA repair enzymes. BALB/c mice had lower intestinal serine (major cellular one-carbon donor), methionine and total folate than C57Bl/6 mice under both dietary conditions. BALB/c mice had higher messenger RNA and protein levels of three folate-interconverting enzymes: trifunctional methyleneTHF (5,10-methylenetetrahydrofolate) dehydrogenase-methenylTHF cyclohydrolase-formylTHF (10-formyltetrahydrofolate) synthetase 1, bifunctional methyleneTHF dehydrogenase-methenylTHF cyclohydrolase and methylenetetrahydrofolate reductase. This pattern of expression could limit the availability of methyleneTHF for conversion of dUMP to dTMP. BALB/c mice also had higher levels of uracil DNA glycosylase 2 protein without an increase in the rate-limiting DNA polymerase ß enzyme, compared with C57Bl/6 mice. We conclude that BALB/c mice may be more prone to DNA damage through decreased amounts of one-carbon donors and the diversion of methyleneTHF away from the conversion of dUMP to dTMP. In addition, incomplete excision repair of uracil in DNA could lead to accumulation of toxic repair intermediates and promotion of tumorigenesis in this tumor-susceptible strain.

Valproic acid increases expression of methylenetetrahydrofolate reductase (MTHFR) and induces lower teratogenicity in MTHFR deficiency.

Valproate (VPA) treatment in pregnancy leads to congenital anomalies, possibly by disrupting folate or homocysteine metabolism. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate interconversion and homocysteine metabolism, we addressed the possibility that VPA might have different teratogenicity in Mthfr(+/+) and Mthfr(+/-) mice and that VPA might interfere with folate metabolism through MTHFR modulation. Mthfr(+/+) and Mthfr(+/-) pregnant mice were injected with VPA on gestational day 8.5; resorption rates and occurrence of neural tube defects (NTDs) were examined on gestational day 14.5. We also examined the effects of VPA on MTHFR expression in HepG2 cells and on MTHFR activity and homocysteine levels in mice. Mthfr(+/+) mice had increased resorption rates (36%) after VPA treatment, compared to saline treatment (10%), whereas resorption rates were similar in Mthfr(+/-) mice with the two treatments (25-27%). NTDs were only observed in one group (VPA-treated Mthfr(+/+)). In HepG2 cells, VPA increased MTHFR promoter activity and MTHFR mRNA and protein (2.5- and 3.7-fold, respectively). Consistent with cellular MTHFR upregulation by VPA, brain MTHFR enzyme activity was increased and plasma homocysteine was decreased in VPA-treated pregnant mice compared to saline-treated animals. These results underscore the importance of folate interconversion in VPA-induced teratogenicity, since VPA increases MTHFR expression and has lower teratogenic potential in MTHFR deficiency.

Reduced folate carrier 80A-->G polymorphism, plasma folate, and risk of placental abruption.

Folate deficiency and maternal smoking are strong risk factors for placental abruption. We assessed whether the reduced folate carrier [NM_194255.1: c.80A-->G (i.e., p.His27Arg)] (RFC-1) polymorphism was associated with placental abruption, and evaluated if maternal smoking modified the association between plasma folate and abruption. Data were derived from the New Jersey-Placental Abruption Study--a multicenter, case-control study of placental abruption (2002-2007). Maternal DNA was assayed for the RFC-1 c.80A-->G polymorphism using a PCR-dependent diagnostic test. Maternal folate (nmol/l) was assessed from maternal plasma, collected immediately following delivery. Due to assay limitations, folate levels at > or =60 nmol/l were truncated at 60 nmol/l. Therefore, case-control differences in folate were assessed from censored log-normal regression models following adjustment for potential confounders. Distribution of the mutant allele (G) of the RFC-1 c.80A-->G polymorphism was similar between cases (52.3%; n = 196) and controls (50.5%; n = 191), as was the homozygous mutant (G/G) genotype (OR 1.1, 95% CI 0.6-2.2). In a sub-sample of 136 cases and 140 controls, maternal plasma folate levels (mean +/- standard error) corrected for assay detection limits were similar between placental abruption cases (63.6 +/- 5.1 nmol/l) and controls (58.3 +/- 4.7 nmol/l; P = 0.270), and maternal smoking did not modify this relationship (interaction P = 0.169). We did not detect any association between the RFC-1 c.80A-->G polymorphism and placental abruption, nor was an association between plasma folate and abruption risk evident. These findings may be the consequence of high prevalence of prenatal multivitamin and folate supplementation in this population (over 80%). It is therefore not surprising that folate deficiency may be rare and that the RFC-1 c.80A-->G polymorphism is less biologically significant for placental abruption.

Altered expression of methylenetetrahydrofolate reductase modifies response to methotrexate in mice.

OBJECTIVE: Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism (677C-->T) in methylenetetrahydrofolate reductase (MTHFR) encodes an enzyme with reduced activity. Response to the antifolate methotrexate (MTX) may be modified in 677TT individuals because MTHFR converts nonmethylated folates, used for thymidine and purine synthesis, to 5-methyltetrahydrofolate, used in homocysteine remethylation to methionine. To study potential interactions between MTHFR activity and MTX, we examined the impact of decreased and increased MTHFR expression on MTX response in mice. METHODS: Mthfr-deficient (Mthfr and Mthfr) and wild-type (Mthfr) mice were injected with MTX or saline and assessed for hematological parameters (hematocrit, hemoglobin, red, and white blood cell numbers), plasma homocysteine, nephrotoxicity, hepatotoxicity, and splenic 2'-deoxyuridine 5'-triphosphate/2'-deoxythymidine 5'-triphosphate ratios. MTHFR-overexpressing transgenic mice (MTHFR-Tg) were generated, metabolites and folate distributions were measured, and response to MTX was assessed. RESULTS: MTX-treated Mthfr and Mthfr mice displayed hyperhomocysteinemia and decreased hematocrit, hemoglobin, and red blood cell numbers compared with wild-type animals. Mthfr mice also showed increased nephrotoxicity and hepatotoxicity. MTHFR-Tg mice were generated and confirmed to have increased levels of MTHFR with altered distributions of folate and thiols in a tissue-specific manner. After MTX treatment, MTHFR-Tg mice exhibited the same decreases in hematological parameters as Mthfr-deficient mice, and significantly decreased thymidine synthesis (higher 2'-deoxyuridine 5'-triphosphate/2'-deoxythymidine 5'-triphosphate ratios) compared with wild-type mice, but they were protected from MTX-induced hyperhomocysteinemia. CONCLUSION: Underexpression and overexpression of Mthfr/MTHFR increase MTX-induced myelosuppression but have distinct effects on plasma homocysteine and nephrotoxicity. Pharmacogenetic analysis of polymorphisms in folate-dependent enzymes may be useful in optimization of MTX therapy.