Folate deficiency induces genomic uracil misincorporation and hypomethylation but does not increase DNA point mutations.

BACKGROUND AND AIMS: Epidemiologic studies have linked nutritional folate deficiency to an increased risk of cancer, but recent trials suggest that folate supplementation does not protect against tumor formation. Our aim was to analyze the genetic and epigenetic consequences of folate deficiency and to investigate whether impairment of the uracil base excision repair pathway can enhance its effects. METHODS: Wild-type mice and those deficient in uracil DNA glycosylase (Ung(-/-)) were placed on a folate-deficient diet for 8 months. We measured tumor incidence in major organs, DNA mutation rates, DNA mutation spectra, local DNA methylation, and global DNA methylation in colon epithelial cells. RESULTS: The experimental diet increased plasma homocysteine (60%, P< .001) and DNA uracil content (24%, P< .05) but not tumor formation. Global DNA methylation was slightly decreased in splenocytes (9.1%) and small intestinal epithelial cells (4.2%), and significantly reduced in colon epithelial cells (7.2%, P< .04). No gene-specific changes in methylation were detected at the mouse B1 element, the H19 DMR, or the Oct4 gene. By lambda CII assay and sequencing analysis of 730 mutants, we found that Ung(-/-) mice had a higher frequency of point mutations and increased C:G to T:A transitions at non-CpG sites. However, folate deficiency had no additional effect on the DNA mutation frequency or spectrum in Ung(-/-) or wild-type mice. CONCLUSIONS: Contradicting current concepts, these findings indicate that the effects of a low-folate diet on DNA methylation and point mutations are insufficient to promote tumor development, even in the presence of Ung deficiency.

Cognitive impairment in folate-deficient rats corresponds to depleted brain phosphatidylcholine and is prevented by dietary methionine without lowering plasma homocysteine.

Poor folate status is associated with cognitive decline and dementia in older adults. Although impaired brain methylation activity and homocysteine toxicity are widely thought to account for this association, how folate deficiency impairs cognition is uncertain. To better define the role of folate deficiency in cognitive dysfunction, we fed rats folate-deficient diets (0 mg FA/kg diet) with or without supplemental L-methionine for 10 wk, followed by cognitive testing and tissue collection for hematological and biochemical analysis. Folate deficiency with normal methionine impaired spatial memory and learning; however, this impairment was prevented when the folate-deficient diet was supplemented with methionine. Under conditions of folate deficiency, brain membrane content of the methylated phospholipid phosphatidylcholine was significantly depleted, which was reversed with supplemental methionine. In contrast, neither elevated plasma homocysteine nor brain S-adenosylmethionine and S-adenosylhomocysteine concentrations predicted cognitive impairment and its prevention by methionine. The correspondence of cognitive outcomes to changes in brain membrane phosphatidylcholine content suggests that altered phosphatidylcholine and possibly choline metabolism might contribute to the manifestation of folate deficiency-related cognitive dysfunction.

A Ketoconazole Susceptibility Test for Malassezia pachydermatis Using Modified Leeming⁻Notman Agar.

The aim of this study was to establish a ketoconazole susceptibility test for Malassezia pachydermatis using modified Leeming⁻Notman agar (mLNA). The susceptibilities of 33 M. pachydermatis isolates obtained by modified CLSI M27-A3 method were compared with the results by disk diffusion method, which used different concentrations of ketoconazole on 6 mm diameter paper disks. Results showed that 93.9% (31/33) of the minimum inhibitory concentration (MIC) values obtained from both methods were similar (consistent with two methods within 2 dilutions). M. pachydermatis BCRC 21676 and Candida parapsilosis ATCC 22019 were used to verify the results obtained from the disk diffusion and modified CLSI M27-A3 tests, and they were found to be consistent. Therefore, the current study concludes that this new novel test-using different concentrations of reagents on cartridge disks to detect MIC values against ketoconazole-can be a cost-effective, time-efficient, and less technically demanding alternative to existing methods.

The basic reproductive number estimated from a Mycoplasma conjunctivae outbreak in a dairy goat farm.

Clinicians can evaluate the relevance of an outbreak based on its basic reproductive number (R0). So far there has been no report on the R0 of Mycoplasma conjunctivae which is a major cause of goats' conjunctivitis in Taiwan. The present study sought to investigate an outbreak of infectious keratoconjunctivitis (IKC) by Mycoplasma conjunctivae (MC) in an indoor dairy goat barn. The epidemiological curve was recorded to build a susceptible-infected-recovered model and to estimate the R0 by three methods In the investigated goat barn, 60% (31/55) goats showed degrees of IKC signs. The number of infected animals increased quickly after 15days, but slowed down after 41days. The sick goats began to recover after 30days. The epidemic fully stopped after 81days. The estimated R0 ranged from 1.35 to 4.46. In summary, this is the first MC report in Taiwan, and the first one to estimate the R0 of MC.

The cognitive impact of nutritional homocysteinemia in apolipoprotein-E deficient mice.

Homocysteinemia is associated with cognitive dysfunction in the elderly ranging from subtle cognitive decline to dementia. Homocysteine is generated from methionine as a product of biological methylation reactions and is disposed of through reactions that require folate and vitamins B12 and B6. While different disruptions in these reactions can result in homocysteinemia, it is unclear if they will also result in homocysteine-mediated cognitive dysfunction. Young ApoE-deficient mice were fed one of four diets with differing methionine and B-vitamin content for eight weeks, before undergoing psychomotor tests, the Morris Water Maze test of spatial memory and learning, and measurement of home-cage activity. B-vitamin deficiency induced homocysteinemia and selectively impaired Morris Water Maze performance without affecting other behavioral measures. The cognitive deficits occurred in the absence of overt histologic neurodegeneration but in association with moderate impairments of brain methylation potential. Diets that yielded cognitive deficits were different from those that exacerbated aortic pathology. These findings are inconsistent with a single mechanism linking homocysteinemia to neurological dysfunctions mediated by homocysteine vasotoxicity. Instead, they indicate that different "types" of homocysteinemia, or in other words different impairments of nutritional metabolism affecting homocysteine levels, may lead to different end organ dysfunctions and/or diseases.

Uracil misincorporation into DNA and folic acid supplementation.

BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different tissues and the effect of folate supplementation on UrMis. DESIGN: We analyzed UrMis concentrations in rectal mucosa (n = 92) and white blood cells (WBCs; n = 60) among individuals randomly assigned to receive supplementation with 1 mg folate/d or placebo, who were then evaluated for colorectal adenoma recurrence. RESULTS: As expected, total homocysteine was significantly lower among the study participants who received active folate treatment (Wilcoxon's P = 0.003) than among those in the placebo group. The median UrMis concentration in rectal mucosa and WBCs among individuals treated with folate was not significantly lower than that in those who received placebo (Wilcoxon's P = 0.17). UrMis concentrations in both rectal mucosa and WBCs did not correlate significantly with folate measured in plasma and red blood cells. UrMis in rectal mucosa was marginally associated with an increased risk of adenoma recurrence (odds ratio per SD: 1.43; 95% CI: 0.91, 2.25). CONCLUSIONS: UrMis measurements in WBCs are not a robust surrogate for UrMis measurements in the rectal mucosa (Spearman correlation coefficient = 0.23, P = 0.08). Furthermore, folate supplementation in an already replete population (half treated with folic acid supplements and all exposed to folic acid fortification of the food supply) was not significantly associated with reduced UrMis in rectal mucosa cells or WBCs. Large-scale studies are needed to evaluate whether excessive UrMis concentrations are an important risk factor for colorectal neoplasia. This trial was registered at clinicaltrials.gov as NCT00272324.

Taurine-deficient diet up-regulated cystathionine beta-synthase monoallele in hemizygous cystathionine beta-synthase knockout mice.

Impaired cystathionine beta-synthase (CBS) causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Reduced CBS activity could decrease cysteine and taurine biosyntheses (metabolites of homocysteine degradation) and lead to less taurocholic acid production with a resultant increased cholesterol content. We hypothesized that a deficiency in CBS genetic material and enzyme activity would reduce taurine synthesis, which would lead to an elevated cholesterol concentration. Both sexes of hemizygous C57BL/6J-Cbs(tm1Unc) [CBS (+/-)] and wild-type C57BL/6J mice [CBS (+/+)] were divided into 2 groups. One group of CBS (+/-) and CBS (+/+) mice was fed a cysteine- and taurine-deficient diet for 8 weeks, and the other group was fed a cysteine, taurine, and vitamin B6-deficient diet for 8 weeks. Significantly higher plasma total homocysteine concentrations occurred in the CBS (+/-) mice than their CBS (+/+) cohorts. Female mice of both genotypes had significantly higher plasma total homocysteine concentrations and significantly lower relative CBS mRNA levels than did male mice. During vitamin B(6) deficiency, plasma total homocysteine concentrations were significantly elevated. Three important findings were a differential sex response of CBS mRNA to feeding the vitamin B(6) diet; CBS (+/-) mice had a significantly lower plasma cholesterol concentration, contrary to what was anticipated; and during feeding, the taurine- and cysteine-deficient diet, CBS mRNA levels in CBS (+/-) mice were reduced only 13% rather than the expected 50%. We conclude that the remaining CBS monoallele is up-regulated in mice when fed a taurine-deficient diet to produce additional CBS mRNA.

Total homocysteine, diet, and lipid profiles in type 1 and type 2 diabetic and nondiabetic adolescents.

BACKGROUND AND RESEARCH OBJECTIVE: Limited research is available on the possible differences in the cardiovascular risk factors of total homocysteine (tHcy), dietary energy, and lipids among adolescents with type 1 diabetes mellitus (DM), type 2 DM, or healthy controls. This study's primary aim was to compare the dietary energy and the intake of macronutrients and micronutrients of folate, and vitamins B6 and B12, as well as lipids and tHcy for adolescents with type 1 DM, type 2 DM, and healthy non-DM controls. SUBJECTS AND METHODS: This secondary analysis of the merging of 2 datasets included the following adolescents: 50 with type 1 DM, 14 with type 2 DM, and 53 controls. Mean ages for those with type 1 versus type 2 DM were 15.2 +/- 1.9 versus 16.1 +/- 1.9 years, respectively. Mean age for the controls was 16.5 +/- 1.0 years. Variables included fasting tHcy and lipids, and 24-hour dietary recalls for macronutrients and micronutrients. Hemoglobin A1c was obtained for those with DM. Statistical analyses included one-way analyses of variance, Pearson correlations, and stepwise regression. RESULTS AND CONCLUSIONS: Adolescents with type 1 DM had the lowest tHcy values (P <.05), which were reflective of the limited extant research with this population. Lipid profiles and dietary energy did not differ significantly among the 3 groups. Hemoglobin A1c was related to total cholesterol and triglycerides in those with type 1 DM, confirming the importance of promoting better metabolic control in lipid management for these youth. Future research should continue to explore the validity of tHcy and lipids as predictors of CV risks for youth with type 1 and type 2 DM.