Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Methylobacterium extorquens AM1 is a facultative methylotroph, which is a potential candidate to be used in commercial processes to convert simple one-carbon compounds to a variety of multicarbon chemicals and products. To better understand C(1) metabolism in M. extorquens AM1 at the systems level, metabolite profiling tools were developed and applied in this bacterium. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC x GC-TOFMS) was used to obtain metabolite profiles of M. extorquens AM1 (primarily organic acids) and to identify the metabolite differences between cells grown on methanol (C(1) substrate) and succinate (multicarbon substrate). In this study, a list of compounds that included amino acids and major intermediates of central C(1) and multicarbon metabolism were studied as target metabolites. For these, calibration curves were obtained for absolute quantification by spiking different amounts of standard mixtures to cell cultures. Parallel factor analysis (PARAFAC) was used for accurate peak quantification. Unknown chemical differences between cells grown on methanol and succinate were identified by applying Fisher ratio analysis at a selective mass channel (m/z 147). Thirty-six compounds were discovered to be statistically differentially expressed between C(1) and multicarbon metabolism. Among these, 13 were identified by matching to library mass spectra, and the rest were novel compounds that were not included in libraries. These differentially expressed compounds have provided clues to new pathways that are specifically linked to C(1) metabolism.     

十种一碳代谢关键产物的HPLC-MS/MS 定量检测方法

目的:利用HPLC-MS/MS方法对十种一碳代谢相关产物进行定量分析。方法:采用Aglient ZORBAX SB-AQ C18柱(2.1mm×100 mm,3.5 m)、电喷雾离子源(ESI),以多离子反应监测方式(MRM)进行正离子检测。对游离叶酸(FA)、5-甲酰四氢叶酸(5-FT)、5-甲基四氢叶酸(5-MT)、S-腺苷蛋氨酸(SAM)、S-腺苷同型半胱氨酸(SAH)、胱硫醚(CYSTA)、组氨酸(HIS)、丝氨酸(SER)、蛋氨酸(MET)、同型半胱氨酸(HCY)进行定量分析。结果:FA、5-FT、5-MT、SAM、SAH、CYSTA、HIS、SER、MET、HCY的检测限分别为0.1 ng.L-1、0.25 ng.L-1、0.1 ng.L-1、0.1 ng.L-1、0.25 ng.L-1、0.25 ng.L-1、0.1 ng.L-1、0.025 ng.L-1、0.1 ng.L-1、0.1 ng.L-1。FA、5-FT、5-MT、SAH、CYSTA浓度测定方法线性范围为2~50 ng.L-1,SER、SAM浓度测定方法线性范围20~500 ng.L-1,MET、HCY浓度测定方法线性范围200~5000 ng.L-1,HIS浓度测定方法线性范围为400~10000 ng.L-1,r均在0.993以上,全部涵盖了已报道的血清中指标的含量范围。结论:建立了HPLC-MS/MS方法,可同时分析十种一碳代谢通路的关键产物,所需样品量少,检测速度快,同时实现分项检测,可为多种代谢性疾病系统性地检测一碳代谢中间产物体液分析方法建立实验条件基础。     

Draft genome sequence of the chemolithoheterotrophic, halophilic methylotroph Methylophaga thiooxydans DMS010

Methylophaga thiooxydans is a mesophilic, obligately halophilic bacterium that is capable of methylotrophic growth on a range of one-carbon compounds as well as chemolithoheterotrophic growth at the expense of thiosulfate. Here we present the draft genome sequence of Methylophaga thiooxydans DMS010 (DSM 22068(T), VKM B2586(T)), the type strain of the species, which has allowed prediction of the genes involved in one-carbon metabolism, nitrogen metabolism, and other aspects of central metabolism.     

Analyses of copy number variation reveal putative susceptibility loci in MTX‐induced mouse neural tube defects

Copy number variations (CNVs) are thought to act as an important genetic mechanism underlying phenotypic heterogeneity. Impaired folate metabolism can result in neural tube defects (NTDs). However, the precise nature of the relationship between low folate status and NTDs remains unclear. Using an array‐comparative genomic hybridization (aCGH) assay, we investigated whether CNVs could be detected in the NTD embryonic neural tissues of methotrexate (MTX)‐induced folate dysmetabolism pregnant C57BL/6 mice and confirmed the findings with quantitative real‐time PCR (qPCR). The CNVs were then comprehensively investigated using bioinformatics methods to prioritize candidate genes. We measured dihydrofolate reductase (DHFR) activity and concentrations of folate and relevant metabolites in maternal serum using enzymologic method and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Three high confidence CNVs on XqA1.1, XqA1.1‐qA2, and XqE3 were found in the NTD embryonic neural tissues. Twelve putative genes and three microRNAs were identified as potential susceptibility candidates in MTX‐induced NTDs and possible roles in NTD pathogenesis. DHFR activity and 5‐methyltetrahydrofolate (5‐MeTHF), 5‐formyltetrahydrofolate (5‐FoTHF), and S‐adenosylmethionine (SAM) concentrations of maternal serum decreased significantly after MTX injection. These findings suggest that CNVs caused by defects in folate metabolism lead to NTD, and further support the hypothesis that folate dysmetabolism is a direct cause for CNVs in MTX‐induced NTDs. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 877–893, 2014     

Folate-mediated one-carbon metabolism and neural tube defects: balancing genome synthesis and gene expression

Neural tube defects (NTDs) refer to a cluster of neurodevelopmental conditions associated with failure of neural tube closure during embryonic development. Worldwide prevalence of NTDs ranges from approximately 0.5 to 60 per 10,000 births, with regional and population-specific variation in prevalence. Numerous environmental and genetic influences contribute to NTD etiology; accumulating evidence from population-based studies has demonstrated that folate status is a significant determinant of NTD risk. Folate-mediated one-carbon metabolism (OCM) is essential for de novo nucleotide biosynthesis, methionine biosynthesis, and cellular methylation reactions. Periconceptional maternal supplementation with folic acid can prevent occurrence of NTDs in the general population by up to 70%; currently several countries fortify their food supply with folic acid for the prevention of NTDs. Despite the unambiguous impact of folate status on NTD risk, the mechanism by which folic acid protects against NTDs remains unknown. Identification of the mechanism by which folate status affects neural tube closure will assist in developing more efficacious and better targeted preventative measures. In this review, we summarize current research on the relationship between folate status and NTDs, with an emphasis on linking genetic variation, folate nutriture, and specific metabolic and/or genomic pathways that intersect to determine NTD outcomes.     

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimer's disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC–MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC–MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).     

Human neural tube defects: genetic causes and prevention

Neural tube defects (NTDs) are severe congenital malformations affecting 1-2 in 1,000 live births, whose etiology is multifactorial, involving environmental and genetic factors. NTDs arise as consequence of the failure of fusion of the neural tube early during embryogenesis. NTDs' pathogenesis has been linked to genes involved in folate metabolism, consistent with an epidemiologic evidence that 70% of NTDs can be prevented by maternal periconceptional supplementation. However, polymorphisms in such genes are not linked in all populations, suggesting that other genetic factors and environmental factors could be involved. Animal models have provided crucial mechanistic information and possible candidate genes to explain susceptibility to NTDs. A crucial role has been assigned to the planar cell polarity (PCP) pathway, a highly conserved, non-canonical Wnt-frizzled-dishevelled signaling cascade that plays a key role in establishing and maintaining polarity in the plane of the epithelium and in the process of convergent extension during gastrulation and neurulation in vertebrates. The Loop-tail (Lp) mouse that develops craniorachischisis carry missense mutations in the PCP core gene Vangl2, that is the mammalian homolog of the Drosophila Strabismus/Van gogh (Stbm/Vang). The presence of mutations in human VANGL1 and VANGL2 genes encourages us to extend the investigation to other PCP genes that, with VANGL, play an essential role in neurulation during development.     

Alcohol Consumption,One-Carbon Metabolites,Liver Cancer and Liver Disease Mortality

Background

Excess alcohol consumption adversely affects one-carbon metabolism and increases the risk of liver disease and liver cancer. Conversely, higher folate levels have been inversely associated with liver damage. The current study investigated the effects of alcohol and one-carbon metabolite intake on liver cancer incidence and liver disease mortality within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study.

Methods

Cox proportional hazards modeling was used to calculate hazard ratios and 95% confidence intervals (CIs) in a population of 27,086 Finnish males with 194 incident liver cancers and 213 liver disease deaths. In a nested case-control subset (95 liver cancers, 103 controls), logistic regression was used to calculate odds ratios and 95% CIs for serum one-carbon metabolites in relation to liver cancer risk.

Results

Daily alcohol consumption of more than 20.44 g was associated with an increased risk of both liver cancer incidence (Hazard Ratio (HR) 1.52, 95%CI 1.06–2.18) and liver disease mortality (HR 6.68, 95%CI 4.16–10.71). These risks were unaffected by one-carbon metabolite intake. Similarly, in the case-control study, none of the serum one-carbon metabolites were associated with liver cancer.

Conclusions

The current study provided no convincing evidence for a protective association of one-carbon metabolite intake or serum level on the risk of liver cancer or liver disease mortality.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Gene expression profiles of the one-carbon metabolism pathway

One-carbon metabolism plays a critical role in both DNA methylation and DNA synthesis. Accumulating evidence has shown that interruptions of this pathway are associated with many disease outcomes including cardiovascular diseases and cancers. Mechanistic studies have been performed on genetic polymorphisms involved in one-carbon metabolism. However, expression profiles of these inter-related genes are not well-known. In this study, we examined the gene expression profiles of 11 one-carbon metabolizing genes by quantifying the mRNA level of the lymphocyte among 54 healthy individuals and explored the correlations of these genes. We found these genes were expressed in lymphocytes at moderate levels and showed significant inter-person variations, We also applied principle component analysis to explore potential patterns of expression. The components identified by the program agreed with existing knowledge about one-carbon metabolism. This study helps us better understand the biological functions of one-carbon metabolism.     

Influence of corrinoid antagonists on methanogen metabolism.

Iodopropane inhibited cell growth and methane production when Methanobacterium thermoautotrophicum, Methanobacterium formicicum, and Methanosarcina barkeri were cultured on H2-CO2. Iodopropane (40 microM) inhibited methanogenesis (30%) and growth (80%) when M. barkeri was cultured mixotrophically on H2-CO2-methanol. The addition of acetate to the medium prevented the observed iodopropane-dependent inhibition of growth. The concentrations of iodopropane that caused 50% inhibition of growth of M. barkeri on either H2-CO2, H2-CO2-methanol, methanol, and acetate were 112 +/- 6, 24 +/- 2, 63 +/- 11, and 4 +/- 1 microM, respectively. Acetate prevented the iodopropane-dependent inhibition of one-carbon metabolism. Cultivation of M. barkeri on H2-CO2-methanol in bright light also inhibited growth and methanogenesis to a greater extent in the absence than in the presence of acetate in the medium. Acetate was the only organic compound examined that prevented iodopropane-dependent inhibition of one-carbon metabolism in M. barkeri. The effect of iodopropane and acetate on the metabolic fates of methanol and carbon dioxide was determined with 14C tracers when M. barkeri was grown mixotrophically on H2-CO2-methanol. The addition of iodopropane decreased the contribution of methanol to methane and cell carbon while increasing the contribution of CO2 to cell carbon. Regardless of iodopropane, acetate addition decreased the contribution of methanol and CO2 to cell carbon without decreasing their contribution to methane. The corrinoid antagonists, light and iodopropane, appeared most specific for methanogen metabolic reactions involved in acetate synthesis from one-carbon compounds and acetate catabolism.     

Raltitrexed’s effect on the development of neural tube defects in mice is associated with DNA damage,apoptosis, and proliferation

The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H₂AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H₂AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.     

One-carbon metabolism in lectin-activated human lymphocytes

Serine is an essential amino acid for the lectin-mediated transformation of human peripheral blood lymphocytes due to the inability of this cell to synthesize sufficient quantities via either the phosphorylated pathway or by reversal of the serine hydroxymethyltransferase reaction to meet the metabolic demands. The level of intracellular serine is tightly regulated, and the culture medium concentration for optimum cellular transformation falls within a relatively narrow range. The three-carbon atom of serine is the major source of one-carbon units required for purine and pyrimidine nucleotide biosynthesis, but the key effect of both serine deprivation and of high medium serine levels would appear to be on protein synthesis. Although an alternative source of one-carbon units, as provided by high levels of formate in the culture medium, can partially reverse the effects of serine deprivation, the only other demonstrable source of one-carbon units, tryptophan, requires serine for its incorporation and subsequent metabolism. Methionine is also essential for lymphocyte transformation and is involved in the synthesis of a small amount of phosphatidylcholine, although most of this phospholipid is provided by choline and lysophosphatidylcholine from the serum-supplemented culture medium.     

Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.     

Application of Artificial Neural Networks to Investigate One-Carbon Metabolism in Alzheimer’s Disease and Healthy Matched Individuals

Folate metabolism, also known as one-carbon metabolism, is required for several cellular processes including DNA synthesis, repair and methylation. Impairments of this pathway have been often linked to Alzheimer’s disease (AD). In addition, increasing evidence from large scale case-control studies, genome-wide association studies, and meta-analyses of the literature suggest that polymorphisms of genes involved in one-carbon metabolism influence the levels of folate, homocysteine and vitamin B12, and might be among AD risk factors. We analyzed a dataset of 30 genetic and biochemical variables (folate, homocysteine, vitamin B12, and 27 genotypes generated by nine common biallelic polymorphisms of genes involved in folate metabolism) obtained from 40 late-onset AD patients and 40 matched controls to assess the predictive capacity of Artificial Neural Networks (ANNs) in distinguish consistently these two different conditions and to identify the variables expressing the maximal amount of relevant information to the condition of being affected by dementia of Alzheimer’s type. Moreover, we constructed a semantic connectivity map to offer some insight regarding the complex biological connections among the studied variables and the two conditions (being AD or control). TWIST system, an evolutionary algorithm able to remove redundant and noisy information from complex data sets, selected 16 variables that allowed specialized ANNs to discriminate between AD and control subjects with over 90% accuracy. The semantic connectivity map provided important information on the complex biological connections among one-carbon metabolic variables highlighting those most closely linked to the AD condition.     

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.     

Mono-2-ethylhexyl phthalate (MEHP) alters histiotrophic nutrition pathways and epigenetic processes in the developing conceptus

Histiotrophic nutrition pathways (HNPs) are processes by which the organogenesis-stage conceptus obtains nutrients, amino acids, vitamins and cofactors required for protein biosynthesis and metabolic activities. Nutrients are captured from the maternal milieu as whole proteins and cargoes via receptor-mediated endocytosis in the visceral yolk sac (VYS), degraded by lysosomal proteolysis and delivered to the developing embryo (EMB). Several nutrients obtained by HNPs are required substrates for one-carbon (C₁) metabolism and supply methyl groups required for epigenetic processes, including DNA and histone methylation. Increased availability of methyl donors has been associated with reduced risk for neural tube defects (NTDs). Here, we show that mono-2-ethylhexyl phthalate (MEHP) treatment (100 or 250 μM) alters HNPs, C₁ metabolism and epigenetic programming in the organogenesis-stage conceptus. Specifically, 3-h MEHP treatment of mouse EMBs in whole culture resulted in dose-dependent reduction of HNP activity in the conceptus. To observe nutrient consequences of decreased HNP function, C₁ components and substrates and epigenetic outcomes were quantified at 24 h. Treatment with 100-μM MEHP resulted in decreased dietary methyl donor concentrations, while treatment with 100- or 250-μM MEHP resulted in dose-dependent elevated C₁ products and substrates. In MEHP-treated EMBs with NTDs, H3K4 methylation was significantly increased, while no effects were seen in treated VYS. DNA methylation was reduced in MEHP-treated EMB with and without NTDs. This research suggests that environmental toxicants such as MEHP decrease embryonic nutrition in a time-dependent manner and that epigenetic consequences of HNP disruption may be exacerbated in EMB with NTDs.     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study

Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most "dietary sensitive" genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B 2 and B 6) were more "epigenetically active" as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention.