High Folic Acid Diet Research Articles

High 5-Methyletetrahydrofolate and Folic Acid Gestational Diets Differ on Leptin-Dependent Hypothalamic Genes Regulating Food Intake in Female Offspring

ObjectivesWhile the replacement of folic acid (FA) with the bioactive folate form 5-methyltetrahydrofolate (MTHF) is gaining popularity, a comparison of their effects during pregnancy is limited. In a comparison of maternal intakes of FA and MTHF at recommended or high (5X) doses in Wistar rat dams, we found that dams fed 5X MTHF gained > 70% more weight and ate 8% more food to 19-weeks post weaning (PW) than those fed the high FA diet (Nutrients. 2021;13:48). However, both high dose diets resulted in dysregulation of leptin and central energy regulatory systems. The objective of this study was to compare the effect of gestational FA and MTHF diets on metabolic response and leptin-dependent hypothalamic energy-regulatory genes in the brain of their female offspring. MethodsPregnant Wistar rats were fed an AIN-93G diet with 1X recommended (2mg/kg diet) or 5X FA or equimolar MTHF. At weaning, female offspring were fed a high-fat diet until 19-weeks PW. Body weight, food intake, adiposity, plasma leptin, and gene expression of leptin signaling and related candidate genes in the hypothalamus were measured. ResultsSimilar to the mothers, the 5X-MTHF gestational diet resulted in offspring with higher weight (>15%, P < 0.01) independent of visceral adiposity, and higher food intake (8% P < 0.01) compared to those born to dams fed the 5X-FA, but not from the 1X folate diets. Both 5X diets led to higher plasma leptin at birth (60%, P < 0.05) and at 19-weeks PW (40%, P < 0.01) and up-regulated hypothalamic mRNA of the downstream leptin signalling gene, signal transducer and activator of transcription-3 (Stat3). However, only 5X-MTHF offspring had lower expression of leptin receptor (Ob-rB) and higher expression of suppressor of cytokine signaling-3 (Socs-3), an inhibitor of leptin signalling and indicator of central leptin resistance. In contrast, 5X-FA offspring had higher expression (>1.5-fold, P < 0.01) of dopamine and GABA- receptors which are targets of leptin and associated with feeding inhibition and hyperactivity. ConclusionsFolate form and dose during pregnancy affects long-term programming of leptin dependent hypothalamic regulatory pathways in female offspring. Unfavorable differences in response to FA and MTHF were seen at the higher doses, thus neither should be consumed at high intakes. Funding SourcesCIHR-INMD; EP by NSERC-CGS.

Choline and Folic Acid in Diets Consumed during Pregnancy Interact to Program Food Intake and Metabolic Regulation of Male Wistar Rat Offspring

North American women consume high folic acid (FA), but most are not meeting the adequate intakes for choline. High-FA gestational diets induce an obesogenic phenotype in rat offspring. It is unclear if imbalances between FA and other methyl-nutrients (i.e., choline) account for these effects. This study investigated the interaction of choline and FA in gestational diets on food intake, body weight, one-carbon metabolism, and hypothalamic gene expression in male Wistar rat offspring. Pregnant Wistar rats were fed an AIN-93G diet with recommended choline and FA [RCRF; 1-fold, control] or high (5-fold) FA with choline at 0.5-fold [low choline and high folic acid (LCHF)], 1-fold [recommended choline and high folic acid (RCHF)], or 2.5-fold [high choline and high folic acid (HCHF)]. Male offspring were weaned to an RCRF diet for 20 wk. Food intake, weight gain, plasma energy-regulatory hormones, brain and plasma one-carbon metabolites, and RNA sequencing (RNA-seq) in pup hypothalamuses were assessed. Adult offspring from LCHF and RCHF, but not HCHF, gestational diets had 10% higher food intake and weight gain than controls (P<0.01). HCHF newborn pups had lower plasma insulin and leptin compared with LCHF and RCHF pups (P<0.05), respectively. Pup brain choline (P<0.05) and betaine (P<0.01) were 22-33% higher in HCHF pups compared with LCHF pups; methionine was ∼23% lower after all high FA diets compared with RCRF (P<0.01). LCHF adult offspring had lower brain choline (P<0.05) than all groups and lower plasma 5-methyltetrahydrofolate (P<0.05) than RCRF and RCHF groups. HCHF adult offspring had lower plasma cystathionine (P<0.05) than LCHF adult offspring and lower homocysteine (P<0.01) than RCHF and RCRF adult offspring. RNA-seq identified 144 differentially expressed genes in the hypothalamus of HCHF newborns compared with controls. Increased choline in gestational diets modified the programming effects of high FA on long-term food intake regulation, plasma energy-regulatory hormones, one-carbon metabolism, and hypothalamic gene expression in male Wistar rat offspring, emphasizing a need for more attention to the choline and FA balance in maternal diets.

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.

The Effect of Folic Acid Supplementation in the Ovary and Upon Embryo Development

Altered maternal nutrition around conception can affect oocyte and embryo development which influences later-life health outcomes. Low folate is related to poor reproductive outcomes. Folic acid (FA) supplementation and fortification have been effective strategies to avoid NTD. On the other hand, the FA levels in women of reproductive age are increased more than recommended, with unknown consequences. This project aims to determine the effect of high FA diet on the ovary and embryo development. C57BL/6 female mice at PND74 were fed with control (1mg FA/kg food) or high (5mg FA/kg food) FA diet for four weeks and culled at diestrus stage (PND102). In parallel, a group of animals were maintained on the control diet for another four weeks (PND130) or either mated and culled at 3.5 days post coitum (dpc). Ovaries and embryos were collected, RNA extracted and analysed by qPCR. Morphological and immunostaining analyses were also performed to determine the effect of FA in the ovary and blastocyst. High FA diet reduced expression of follicle developmental control genes at PND102 such as Fshr and Oct4, but also epigenetic writers like Ezh2 and Bmi1. In contrast, four weeks after FA diet release, the same genes were upregulated in the ovary. Females with a preconceptional high FA diet showed an increased mating period compared to the control group. The embryos of these mice showed reduced TE cells and lower expression of CDX2. In parallel, embryos exposed to high FA diet exclusively during preimplantation showed delayed development with decreased total cell number and lower expression of lineage markers (Oct4, NANOG and Gata6). High FA diet not only altered follicle growth factors during and after supplementation in a different pattern but also affected blastocyst biogenesis. This could impact on later-life health outcomes of the offspring.

Choline and Folic Acid Balance in Diets During Pregnancy Programs Food Intake Regulation in Wistar Rat Offspring (FS08-05-19)

ObjectivesHigh gestational folic acid (FA) induces an obesogenic phenotype in male Wistar rat offspring. Imbalances between FA and other methyl-nutrients (i.e., choline) leading to perturbations in the 1-carbon cycle may account for the effects of high FA diets. Canadian women consume high (2–7-fold) intakes of FA, but most are not meeting recommended adequate intakes for choline. Choline is also absent from Canadian prenatal supplements. The objective of this study is to evaluate the effects of the interaction between choline and FA in maternal diets of rats on the 1-carbon cycle, and the programming of food intake, body weight gain and biomarkers of obesity in the offspring later in life. MethodsPregnant Wistar rat dams were fed the AIN-93 G diet with recommended (1X) choline and FA (RCRF, control), or a 5X FA diet with either 0.5X choline (LCHF), 1X choline (RCHF), or 2.5X choline (HCHF). Brain and blood were collected at birth. At weaning one male pup/dam from all groups was maintained on the control diet for 20 weeks then terminated. Dependent measures include weekly body weight-gain and food intake, plasma glucoregulatory hormones and 1-carbon metabolites at birth and post-weaning. ResultsIncreasing choline content to 2.5-fold in a high (5-fold) gestational FA diet (HCHF) led to lower plasma insulin and leptin levels at birth compared to the LCHF and RCHF diets, respectively (P < 0.05). It also led to lower (25%, P = 0.03) plasma 5-methyltetrahydrofolate concentrations at birth compared to the RCHF diet, suggesting more efficient utilization of FA. Offspring born to dams maintained on a high folic acid diet with either low or recommended choline had higher weekly food intake (6%, P < 0.05) and body weight-gain (9%, P < 0.01). In contrast, offspring from dams fed the HCHF gestational diet were not different from those born to dams fed the RCRF (control) diet, highlighting the mitigating effects of a balanced choline and FA gestational diet. ConclusionsIncreased intakes of choline mitigate the effects of high FA diets. Maternal dietary choline interacts with FA on the long-term programming of food intake regulation in the offspring; emphasizing a need for more attention to improving choline intakes by women of child-bearing age. Funding SourcesThis research was funded by the Canadian Institute of Health Research, Institute of Nutrition, Metabolism and Diabetes (CIHR-INMD).

High folic acid diet enhances tumour growth in PyMT-induced breast cancer

Background:The B-vitamin folate is among the most studied bioactive food compound, and a dietary intake meeting the daily requirements has been found to reduce the risk of cancer and cardiovascular diseases as well as preventing neural tube defects during fetal development. Several countries have therefore introduced dietary fortification with folic acid. However, clinical and animal studies suggest that folic acid has a dual role in cancer development.Methods:During the period of initial tumour progression, MMTV-PyMT (MMTV-polyoma virus middle T) transgenic mice were fed with normal diet and high folic acid diet.Results:We found that PyMT-induced breast tumours highly express the cancer-specific folate receptor (FR), a feature they share with several human epithelial cancers in which expression of FRα correlates with tumour grade. Mice receiving a high folic acid diet displayed a significantly increased tumour volume compared with mice receiving normal diet. In the largest tumours, only found in mice on high folic acid diet, STAT3 was activated. In primary cells from PyMT tumours, STAT3 was activated upon treatment with folic acid in culture.Conclusions:Our results offer a novel molecular explanation for folic acid-induced growth of existing tumours.

Metabolite profiling of whole murine embryos reveals metabolic perturbations associated with maternal valproate-induced neural tube closure defects.

Valproic acid (VPA) is prescribed therapeutically for multiple conditions, including epilepsy. When taken during pregnancy, VPA is teratogenic, increasing the risk of several birth and developmental defects including neural tube defects (NTDs). The mechanism by which VPA causes NTDs remains controversial and how VPA interacts with folic acid (FA), a vitamin commonly recommended for the prevention of NTDs, remains uncertain. We sought to address both questions by applying untargeted metabolite profiling analysis to neural tube closure (NTC) stage mouse embryos. Pregnant SWV dams on either a 2 ppm or 10 ppm FA supplemented diet were injected with a single dose of VPA on gestational day E8.5. On day E9.5, the mouse embryos were collected and evaluated for NTC status. Liquid chromatography coupled to mass spectrometry metabolomics analysis was performed to compare metabolite profiles of NTD-affected VPA-exposed whole mouse embryos with profiles from embryos that underwent normal NTC from control dams. NTDs were observed in all embryos from VPA-treated dams and penetrance was not diminished by dietary FA supplementation. The most profound metabolic perturbations were found in the 10ppm FA VPA-exposed mouse embryos, compared with the other three treatment groups. Affected metabolites included amino acids, nucleobases and related phosphorylated nucleotides, lipids, and carnitines. Maternal VPA treatment markedly perturbed purine and pyrimidine metabolism in E9.5 embryos. In combination with a high FA diet, VPA treatment resulted in gross metabolic changes, likely caused by a multiplicity of mechanisms, including an apparent disruption of mitochondrial beta-oxidation. Birth Defects Research 109:106-119, 2017. © 2016 Wiley Periodicals, Inc.

Acute alcohol exposure during mouse gastrulation alters lipid metabolism in placental and heart development: Folate prevention.

BackgroundEmbryonic acute exposure to ethanol (EtOH), lithium, and homocysteine (HCy) induces cardiac defects at the time of exposure; folic acid (FA) supplementation protects normal cardiogenesis (Han et al., 2009, 2012; Serrano et al., 2010). Our hypothesis is that EtOH exposure and FA protection relate to lipid and FA metabolism during mouse cardiogenesis and placentation.MethodsOn the morning of conception, pregnant C57BL/6J mice were placed on either of two FA‐containing diets: a 3.3 mg health maintenance diet or a high FA diet of 10.5 mg/kg. Mice were injected a binge level of EtOH, HCy, or saline on embryonic day (E) 6.75, targeting gastrulation. On E15.5, cardiac and umbilical blood flow were examined by ultrasound. Embryonic cardiac tissues were processed for gene expression of lipid and FA metabolism; the placenta and heart tissues for neutral lipid droplets, or for medium chain acyl‐dehydrogenase (MCAD) protein.ResultsEtOH exposure altered lipid‐related gene expression on E7.5 in comparison to control or FA‐supplemented groups and remained altered on E15.5 similarly to changes with HCy, signifying FA deficiency. In comparison to control tissues, the lipid‐related acyl CoA dehydrogenase medium length chain gene and its protein MCAD were altered with EtOH exposure, as were neutral lipid droplet localization in the heart and placenta.ConclusionEtOH altered gene expression associated with lipid and folate metabolism, as well as neutral lipids, in the E15.5 abnormally functioning heart and placenta. In comparison to controls, the high FA diet protected the embryo and placenta from these effects allowing normal development. Birth Defects Research (Part A) 106:749–760, 2016. © 2016 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc.

Effect of High Folic Acid Diet on Mitochondrial DNA Content in Young Adult and Aged Mice

Mitochondrial DNA (mtDNA) codes for proteins of oxidative phosphorylation system and maintaining adequate mtDNA copy number is necessary for cell survival. Studies in animal models have shown that folate deficiency can affect mtDNA content. Current data suggests excess folic acid intake can result in conditions that resemble folate deficiency. We tested the hypothesis that excess folic acid in diet can influence mtDNA gene content in old and young mice. Young (3 months) and old mice (16 months) mice were fed a control (1xRDA) or high folic acid (20 x RDA) diet for 3 months (n=8–11/ group). The colonic mucosal mtDNA content was determined by real‐time PCR using primers specific for mtDNA and normalized to an endogenous reference gene, mapK. The data were analyzed using student's t‐test. Among mice on control diet, mtDNA content of old mice was 0.4 fold that of the young mice (p=0.004). There was no difference in mtDNA content of young mice on control vs high folic acid diet. MtDNA content of the old mice fed a high folic acid diet was 7‐fold higher than those fed a control diet (p=0.001). While higher mtDNA copy number is considered to have beneficial effects on survival, very high mtDNA copy number has been shown to be accompanied by nucleoid enlargement, defective transcription and mtDNA deletions in old mice. Thus the dramatic increase in mtDNA content due to excess folic acid diet may be detrimental. Further research is required to establish if the high mtDNA content due a high folic acid diet can result in mitochondrial dysfunction in aged mice.Support or Funding InformationSupport from USDA cooperative agreement 51520‐008‐04S and pilot grant from JM USDA HNRCA on Aging at Tufts University

High folic acid intake reduces natural killer cell cytotoxicity in aged mice

Presence of unmetabolized folic acid in plasma, which is indicative of folic acid intake beyond the metabolic capacity of the body, is associated with reduced natural killer (NK) cell cytotoxicity in postmenopausal women ≥50years. NK cells are cytotoxic lymphocytes that are part of the innate immune system critical for surveillance and defense against virus-infected and cancer cells. We determined if a high folic acid diet can result in reduced NK cell cytotoxicity in an aged mouse model. Female C57BL/6 mice (16-month-old) were fed an AIN-93M diet with the recommended daily allowance (1× RDA, control) or 20× RDA (high) folic acid for 3months. NK cytotoxicity was lower in splenocytes from mice fed a high folic acid diet when compared to mice on control diet (P<.04). The lower NK cell cytotoxicity in high folic acid fed mice could be due to their lower mature cytotoxic/naïve NK cell ratio (P=.03) when compared to the control mice. Splenocytes from mice on high folic acid diet produced less interleukin (IL)-10 when stimulated with lipopolysaccharide (P<.05). The difference in NK cell cytotoxicity between dietary groups was abolished when the splenocytes were supplemented with exogenous IL-10 prior to assessment of the NK cytotoxicity, suggesting that the reduced NK cell cytotoxicity of the high folic acid group was at least partially due to reduced IL-10 production. This study demonstrates a causal relationship between high folic acid intake and reduced NK cell cytotoxicity and provides some insights into the potential mechanisms behind this relationship.

Abstract 13595: Folic Acid Protects Embryo From Environmental Effects on Lipid Metabolism During Cardiac and Placental Development

Introduction: Congenital heart defects (CHDs) are the most prevalent of human birth defects with males exhibiting more severe types of CHDs. Cardiac cell and placental trophoblast differentiation are two embryonic processes occurring early between E6.5-E8.0 of mouse gestation (16-19 days of human pregnancy) that can be interfered with by environmental factors to induce anomalies. Objective: To determine the predominant pathway(s) targeted by exposures to lithium (Li+), homocysteine (HCys) , or alcohol (ethanol, EtOH) that result in CHDs and that can be protected by high dose folic acid (FA) supplementation. Method: On morning of conception, pregnant mice were placed on a protective, high FA diet (10 mg/kg) or on a health maintenance FA diet (3.3 mg/kg) that did not prevent CHDs. At gastrulation (E6.75) the mice received an i.p. injection of Li, HCys, or EtOH. Pregnant control groups received either the protective high FA or the normal FA maintenance diet and an i.p. injection of physiological saline only. Echocardiography on E15.5 determined embryos with abnormal cardiac function. All abnormal hearts and normal control hearts were removed, total RNA extracted. Microarrays were used to evaluate gene changes. Results: Significant expression changes among 12 treatment groups with Li and HCys exposures indicated that Gene Ontology (GO) categories involving lipid metabolism were chiefly altered. When the GO sets were sorted according to gender, male embryos displayed greater changes in lipid-related categories than the female. Validation studies demonstrated that Acyl CoA dehydrogenase medium length chain (Acadm) and its protein product important in fatty acid oxidation were modulated by the exposures, including by alcohol. Oil Red O (ORO) localization of neutral lipids in the heart and placenta demonstrated that lipids are highly altered possibly related to altered umbilical blood flow seen with embryos showing abnormal cardiac function. High FA restored normal ORO patterns, placental blood flow, and heart function. Conclusions: Li, HCys and alcohol exposed embryos display altered lipid metabolism. Early gestational supplementation with high-dose FA protects lipid metabolism at control levels, maintains placental physiology, and prevents CHDs.

Effect of a High Folic Acid Diet on Telomere Length of Colonic Mucosa in Mice

In vitro studies and mathematical modeling suggest that excess folate can inhibit folate cycle and mimic folate deficiency. Under low folate conditions telomeres may shorten due to DNA damage or become abnormally long possibly due to loss of epigenetic control from DNA hypomethylation, both of which are associated with chromosomal instability. Our prior work has shown that high plasma folate is associated with shorter leukocyte telomere length in Framingham Offspring cohort. We investigated the effect of high folic acid diet on telomere length of colonic mucosa in mice. Young (3 months) and old mice (16 months) mice were fed a control (1xRDA) or high folic acid (20 x RDA) diet for 3 months (n=8-10/ group). Folate was determined using a microbial assay and telomere length was determined using a real-time PCR method as the ratio of telomeric repeats to a single copy gene. The data were analyzed using students t-test. Both young and old mice on high folic acid diet had significantly higher folate in colonic ...

Reduced IL‐10 production contributes to impaired NK cytotoxicity in old mice fed a high folic acid diet (638.3)

Natural killer (NK) cells are important for immunity against malignant and virally infected cells. High folic acid intake, which may be linked with higher risk of colon cancer, was associated with lower NK activity in older women. To study the underlying mechanisms, we created a mouse model and found that aged mice fed a high folic acid (HFA) diet had reduced NK activity. In this study, we determined if expression of cytokines that are known to activate NK activity (IL‐10, IFN‐γ, TNF‐α) are affected by a HFA diet in aged mice.Female C57BL/6 mice (16 mo) were fed a control (1x RDA folic acid) or a HFA diet (20x RDA folic acid) for 3 months. Splenocytes were collected and stimulated with LPS for 24 h for ex vivo production of IL‐10, IFN‐γ, and TNF‐α determined by cytometric bead array.In HFA mice, IL‐10 production was 50.7% less than that of the control mice (P=0.028) but no differences were found in IFN‐γ and TNF‐α production. Since IL‐10 activates NK cells, we examined whether addition of IL‐10 to the splenocyte cultures would restore NK activity in the HFA group. We found that NK activity increased for both groups (P&lt;0.01), but the HFA group had larger percent increase in NK activity as compared to control mice (P=0.058). These results indicate that IL‐10 restored the lower NK activity in aged mice fed a HFA diet.These findings suggest the decreased NK activity due to a high folic acid diet may in part be due to impaired IL‐10 secretion.Grant Funding Source: Supported by USDA contract # 51520‐008‐045 and HNRCA Pilot grant

Natural killer cell cytotoxicity is reduced in aged female mice fed a high folic acid diet

Unmetabolized folic acid in plasma, which is indicative of high folic acid intake, is associated with reduced natural killer (NK) cell‐mediated cytotoxicity in elderly (&gt;;60y) women. In this study, we determined if high folic acid intake affects NK cytotoxicity in aged female mice. Aged (16 months) female C57BL/6 mice were fed a control diet (1× RDA folic acid; 2mg/kg) or a high folic acid (HF) diet (20× RDA folic acid;HF 40mg/kg) for 3 months. Folate concentration and splenic NK activity were evaluated. Mice on HF diet had significantly higher unmetabolized folic acid (29.0 pmol/mL versus 0.6 pmol/mL) and formylated folate (19.0 pmol/mL versus 3.1 pmol/mL) (P&lt;0.01). Spleen NK activity was determined as percent dead target cells at multiple effector: target (E:T) cell ratios, where the dead (not lysed) target cells were identified by flow cytometry. In HF mice, percent dead target cells were lower by 14% and 23% for (E:T) cell ratio 25:1 (P=0.03) and 12.5:1 respectively (P=0.036) than the control mice. Percent NK cell was lower by 21% in the spleen of the HF mice when compared to the control mice (P&lt;0.01), but not percent of NKT cells, suggesting that the decrease in cytotoxicity of HF mice might be due to the reduction in the number of spleen NK cells. Further studies are required to understand the mechanism that underlies the HF‐induced reduction in NK cell number and cytotoxicity.Grant Funding Source: HNRCA Pilot grant

Folate Deficiency and Supplementation Result in DNA Methylation Defects in Sperm.

Folate is a nutrient that is essential for cell proliferation and involved in the biosynthesis of nucleotides and S-adenosylmethionine (SAM). SAM is the methyl donor for numerous cellular biochemical reactions including DNA methylation, an important epigenetic modification implicated in the control of gene expression, genomic imprinting and normal fertility. Although folate supplementation is used as a treatment for human male infertility, little is known about the effects of low or high dietary folate concentrations on sperm DNA methylation patterns. Our goal was to use a mouse model to examine the effects of folic acid deficiency and supplementation on epigenetic patterns in male germ cells. Male Balb/c strain mice were fed, from weaning age (day 21) to one year of age, either a control diet containing an adequate amount of folic acid (2 mg/kg diet), a 7X low folic acid diet (0.3 mg/kg diet) or a 20X high folic acid diet (40 mg/kg diet). The mice were sacrificed at 12 months and reproductive organs (testes, epididymides and seminal vesicles) collected and weighed. Sperm were collected from the cauda epididymides and genomic DNA extracted. Restriction landmark genomic scanning (RLGS) was used to assess genome-wide levels and patterns of DNA methylation (n=4 samples/group). Although weight gain and reproductive organ weights were not affected by the folate deficiency or supplementation, for both treatment groups, RLGS demonstrated evidence of epigenetic abnormalities in sperm. Mice in the folic acid supplemented group exhibited six consistent changes in their methylation profiles, all of which were hypermethylation. Interestingly, among the 14 loci showing methylation alterations in all of the folic acid deficient mice, 10 were hypermethylated and four were hypomethylated. The loci with altered methylation are in the process of being identified using a virtual RLGS profile. Together, the results indicate that DNA methylation patterns in mouse sperm can be altered by long term exposure to both low and high dietary folate. (Supported by CIHR) (poster)

Low folate status increases chromosomal damage by X-ray irradiation

Purpose: To examine how folate status influences chromosomal damage following X-ray irradiation.Material and methods: In an animal study, mice were fed either a low, basal, or high folic acid diet (0, 2, or 40 mg/kg diet, respectively) for 4 weeks, and then given total body irradiation (TBI) at 0.5 Gy. In a human study, subjects were supplemented with folic acid (800 μg/day) for 2 weeks and their peripheral blood was irradiated at 0.5 Gy in vitro. Chromosomal damage was determined by micronucleus assay.Results: In an animal study, TBI-induced chromosomal damage was higher and folate concentration was lower in the bone marrow of the low folic acid group compared to the other two diet groups. The chromosomal damage and folate concentration were comparable between the basal and high folic acid groups. TBI administered to mice decreased folate in the plasma, erythrocyte and bone marrow. In a human study, supplementation with folic acid increased plasma folate, but did not influence either plasma homocysteine or X-ray-induced chromosomal damage in lymphocytes.Conclusion: Low folate status increases susceptibility to X-ray-induced chromosomal damage, but excessive folic acid supplementation under normal conditions yields no further protection due to folate saturation in the target tissue.

931 Oral folic acid improves lometrexol toxicity profile: A phase I study

Lometrexol, the antipurine antifolate, has an MTD of 12 mg/m 2 when given as a single agent every 4 weeks. The dose limiting toxicities, myelosuppression and diarrhea, were substantially reduced in mice given a high folic acid diet. In an ongoing phase I dose escalation study of lometrexol coadministered with folic acid (5 mg/day for 14 days), 32 patients received lometrexol at doses of 12 mg/m 2 (3 pts), 16 mg/m 2 (4 pts), 30 mg/m 2 (5 pts), 45 mg/m 2 (11 pts), 60 mg/m 2 (6 pts) and 78 mg/m 2 (3 pts). Tumour types were: 6 breast, 5 ovary, 5 melanoma, 1 renal, 7 colorectal, 2 NSCLC, 2 pancreas, and 4 primary unknown. Initial doses were given at 28 day intervals with subsequent reduction to a 21 day cycle after 8 patients had been treated at 45 mg/m 2 . A median of 2 courses was given (range 1–5). Haematological toxicity was mild. One patient experienced a grade 3 neutropenia and 2 patients grade 3/4 thrombocytopenia (toxicities at 45 mg/m 2 ). No grade 3/4 haematological toxicity was seen at 60 mg/m 2 or 78 mg/m 2 . Grade 3 diarrhea (45 mg/m 2 ), nausea (45 mg/m 2 ) and mucositis (60 mg/m 2 ) were seen in 3 separate patients. We previously reported a 21–50% fall in GFR with repeated dosing. At 60 mg/m 2 the change in pretreatment GFR measured by 51Cr-EDTA was between −11.9% and +23.8%. No alteration in serum creatinine was seen at any dose level. One patient (30 mg/m 2 , breast cancer) had a partial response of 6 weeks duration. The MTD has not been reached and the next dose escalation will be 100 mg/m 2 . It is clear that coadministration of folic acid ameliorates the clinical toxicities seen with lometrexol.