Metabolism Methylation Research Articles

Methyl group metabolism in maternal and fetal hepatic tissues is altered by diabetes and retinoic acid treatment

Adequate methyl group supply is vital for numerous biological reactions, especially during development. A lack of methyl groups, B‐vitamins, and changes in relevant enzymes, can result in a host of pathologies, including neural tube defects. If inappropriately activated, hepatic glycine N‐methyltransferase (GNMT) a regulatory enzyme in folate and methyl group metabolism, leads to methyl deficiency. We have shown that both a diabetic state and retinoic acid (RA) treatment result in activation of GNMT. The goal of this pilot study was to determine if induction of GNMT during pregnancy alters methyl group metabolism in the fetus. Non‐diabetic and diabetic (STZ) pregnant rats treated with and without RA, were sacrificed on d 18 of gestation for collection of blood and liver tissue. Two hepatic immunoreactive proteins were clearly detected (32 and ~36 kD) in liver tissue. The lighter form (L‐GNMT) was more responsive in maternal liver, whereas the heavier protein (H‐GNMT) dominated in fetal liver. Rats receiving both RA and STZ exhibited elevated maternal hepatic GNMT activity, L‐GNMT abundance, and DNA hypomethylation, and reduced circulating homocysteine levels. Fetal liver expressed only H‐GNMT which was slightly responsive to treatment; however, L‐GNMT expression was evident in the combined treatment group. Fetal hepatic tissue did not exhibit changes in DNA methylation, due to the lack of L‐GNMT expression.

Phosphocreatine and genistein supplementation attenuate plasma homocysteine concentrations in rats

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. S‐adenosylmethionine (SAM) is the methyl group donor for most biological methylations. The product of this reaction, S‐adenosylhomocysteine, is converted to homocysteine (Hcy), which is remethylated back to methionine or irreversibly catabolized. Thus, Hcy pools reflect a balance between production and metabolism. Biosynthesis of phosphocreatine (PCr) from guanidinoacetate (GAA) is a transmethylation reaction that requires SAM‐derived methyl groups, contributing significantly to Hcy production. Therefore, dietary supplementation with PCr should decrease the demand on SAM for GAA synthesis. The isolflavone, genistein (G), may promote homeostatic methyl group metabolism. However, mechanisms underlying the effects of G are not well understood. We examined the effects of PCr and G supplementation on elevated Hcy during moderate folate deficiency. Half of the rats received a FD diet for 5 and ½ wk, followed by supplementation with either PCr (4g/kg/diet) or G (300mg/kg/diet) for 10 d. As expected, FD increased total plasma Hcy concentrations by 95%. PCr significantly reduced Hcy during FD (p = 0.049). However, Hcy was attenuated in all control and FD groups receiving PCr and G. Taken together, PCr and G may represent a viable dietary strategy to reduce plasma Hcy.

THE QUEENSLAND PANCREATIC CANCER STUDY-IDENTIFYING RISK FACTORS FOR PANCREATIC CANCER

Apart from cigarette smoking, family history and diabetes, there are few known causes of pancreatic cancer and there is a paucity of information about interactions between environmental factors and common polymorphisms in relevant genes. Synthesis of current research evidence suggests that there are at least three biologically plausible aetiological pathways which warrant further investigation in the short-to-medium term. These include the `insulin hypothesis' (that insulin resistance is a major risk factor), the methyl group theory (that impaired methyl group metabolism can influence cellular differentiation in the pancreas), and the possibility that infection with Helicobacter pylori is associated with risk. Exploration of these theories requires a hypothesis-driven approach in large studies integrating genetic and environmental components of each pathway. The Queensland Pancreatic Cancer Study is a case-control study, funded by the National Health and Medical Research Council for five years, endeavouring to test some of these hypotheses. We are aiming to recruit all people diagnosed with pancreatic cancer in Queensland, through a network of surgeons, gastroenterologists, oncologists, palliative care consultants and general physicians. Patients undergo an interview about their medical (including medication use) and family history and lifestyle and are asked to donate a blood sample. Controls will be selected from the electoral roll, matched to the cases by age and sex. Since the study was launched in June 2007, we have interviewed nearly 70 patients who had a provisional or confirmed diagnosis of pancreatic cancer at the time of recruitment. Two thirds of these are male and the average age is 66. Approximately 70% are past or current smokers. The Queensland Pancreatic Cancer Study is building an invaluable resource which, in addition to studies of aetiology, can be used to answer an assortment of additional questions. The resource will be complete in 2012.

Tissue‐specific regulation of glycine N‐methyltransferase (GNMT) activity and abundance in the prediabetic ZDF (type 2) rat: evidence for two regulated GNMT isoforms

Perturbations of methyl group metabolism have been demonstrated in diabetic rat models. Altered expression and activity of key enzymes were evident at 5 and 11 wk of age in the Zucker diabetic fatty (ZDF) rat liver. We have shown that hepatic GNMT is induced in type 1 diabetic rats, concomitant with DNA hypomethylation. The objective of this study was to determine how these alterations, particularly in GNMT, in the ZDF rat might be related to diabetic complications, such as cardiovascular disease. Blood and tissues were collected from ZDF (fa/fa) and lean controls (+/?) at 8 wk of age. Blood glucose was elevated 100% and plasma homocysteine was decreased 51% in ZDF rats compared to controls. Using a GNMT peptide antibody, two immunoreactive proteins (32 and 36 kDa) were clearly detected. Interestingly, both the lighter (L) and heavier (H) forms of GNMT were observed in hepatic and pancreatic samples, whereas only H‐GNMT was present in renal and cardiac tissue. Measurable GNMT enzymatic activity was detected in all tissues except heart. There were no differences between the ZDF rats and controls in the kidney, but both pancreatic activity and abundance of L‐GNMT were lower in the diabetic animals, whereas H‐, but not L‐GNMT, was greater in the diabetic rat liver. These results suggest there is tissue‐specific regulation of both L‐ and H‐GNMT during the progression of type 2 diabetes in the prediabetic ZDF rat. (Am Heart Assoc)

Choline metabolism and risk of breast cancer in a population‐based study

Choline is an essential nutrient required for methyl group metabolism, but its role in carcinogenesis and tumor progression is not well understood. By utilizing a population-based study of 1508 cases and 1556 controls, we investigated the associations of dietary intake of choline and two related micronutrients, methionine and betaine, and risk of breast cancer. The highest quintile of choline consumption was associated with a lower risk of breast cancer [odds ratio (OR): 0.76; 95% confidence interval (CI): 0.58-1.00] compared with the lowest quintile. Two putatively functional single nucleotide polymorphisms of choline-metabolizing genes, PEMT -774G>C (rs12325817) and CHDH +432G>T (rs12676), were also found be related to breast cancer risk. Compared with the PEMT GG genotype, the variant CC genotype was associated with an increased risk of breast cancer (OR: 1.30; 95% CI: 1.01-1.67). The CHDH minor T allele was also associated with an increased risk (OR: 1.19; 95% CI: 1.00-1.41) compared with the major G allele. The BHMT rs3733890 polymorphism was also examined but was found not to be associated with breast cancer risk. We observed a significant interaction between dietary betaine intake and the PEMT rs7926 polymorphism (P(interaction)=0.04). Our findings suggest that choline metabolism may play an important role in breast cancer etiology.

Alterations in Homocysteine Metabolism Among Alcohol Dependent Patients - Clinical, Pathobiochemical and Genetic Aspects

Addiction research focusing on homocysteine metabolism and its association with aspects of alcohol dependence has revealed important findings. Recent literature on this topic has been taken into account for the review provided. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the homocysteine metabolism. Plasma homocysteine levels are influenced by the single-nucleotide polymorphism (SNP) MTHFR C677T. Besides genetic factors, environmental factors have an impact on homocysteine plasma levels too. Thus, chronic alcohol intake is associated with elevated homocysteine plasma concentrations. Elevation of plasma homocysteine concentration is considered as a predictor for the occurrence of alcohol withdrawal seizures and--as homocysteine is a cardiovascular risk factor--might contribute to the higher risk for myocardial infarction among alcohol dependent patients. Homocysteine acts as an N-methyl-D-aspartate (NMDA) receptor agonist and has excitotoxic effects. Furthermore, it has been demonstrated that homocysteine has neurotoxic effects especially on dopaminergic neurons. As the rewarding effects of alcohol are mediated by the dopaminergic system, a homocysteine-dependent impairment of the reward system possibly leads to an altered drinking behaviour according to the deficit hypothesis of addiction. Homocysteine is involved in the metabolism of methyl groups and DNA-methylation plays a role in regulation of gene expression. Therefore it has been suggested that homocysteine is an important epigenetic factor. It remains to be determined whether alcohol dependent patients benefit from homocysteine lowering strategies, e.g., via supplementation of folate, vitamin B6 and B12. In this respect it is not clear yet, if a supplementation therapy can reduce the risk for the occurrence of alcohol withdrawal seizures.

Rumen-protected choline and vitamin E supplementation in periparturient dairy goats: effects on milk production and folate, vitamin B12 and vitamin E status

We investigated the effects of rumen-protected choline (RPC) and vitamin E (VITE) administration on milk production and status of folate, vitamin B12 and vitamin E during the periparturient period of dairy goats. Forty-eight Saanen multiparous goats were selected for the 72-day experiment, being moved to a maternity pen 30 days before expected parturition and assigned to one of the four experimental groups: control (CTR), no choline or vitamin E supplementation; choline (RPC), supplemented with 4 g/day choline chloride in rumen-protected form; vitamin E (VITE), supplemented with 200 IU/day vitamin E in rumen-protected form; and choline and vitamin E (RPCE), supplemented with 4 g/day RPC chloride and 200 IU/day vitamin E. Supplements were administered individually before the morning feed to ensure complete consumption, starting 30 days before kidding and continuing for 35 days after. During the experiment, milk yield and 4% fat-corrected milk (FCM) yield were, respectively, 210 and 350 g/day higher in RPC-supplemented goats than in non-supplemented goats. Milk fat concentration and fat yield were also increased by RPC treatment. Milk yield and composition were unaffected by vitamin E supplementation. There were no significant interactions between RPC and VITE for any of the variables measured. Plasma metabolites did not differ between treatments before and after kidding except that plasma folate at parturition was higher in RPC-supplemented goats. Neither choline nor vitamin E affected vitamin B12 plasma concentrations, while a time effect was evident after the second week of lactation, when B12 levels in each treatment group started to increase. Vitamin E administration resulted in plasma α-tocopherol levels that were 2 to 2.5 times higher than in non-supplemented goats. Overall, these results suggest that greater choline availability can improve milk production and methyl group metabolism in transition dairy goats.

Genetic polymorphisms in methyl-group metabolism and epigenetics: Lessons from humans and mouse models

Genetic polymorphisms in methyl-group metabolism and epigenetics: Lessons from humans and mouse models

Relationship of Dimethylglycine, Choline, and Betaine with Oxoproline in Plasma of Pregnant Women and Their Newborn Infants ,

Choline and glycine are inter-related through their roles in methyl metabolism. Choline is metabolized to betaine, which donates a methyl group to homocysteine to form methionine, also generating dimethylglycine, which is further metabolized to glycine. Choline is transported across the placenta and is higher in fetal than maternal plasma. Placental glycine transfer, however, is limited and poor glycine status has been suggested in preterm infants. Insufficient glycine for glutathione (GSH) synthesis results in increased metabolism of γ-glutamyl cysteine to 5-oxoproline. We measured plasma 5-oxoproline as a metabolic indicator to address whether choline, via dimethylglycine, contributes physiologically relevant amounts of glycine in pregnancy. Blood was collected from healthy term pregnant women and their newborn infants at delivery (n = 46) and nonpregnant healthy women (n = 19) as a reference group. Plasma choline, betaine, dimethylglycine, homocysteine, methionine, and 5-oxoproline were quantified by HPLC-tandem MS. Plasma choline was 45% higher, but betaine was 63% lower and dimethylglycine was 28% lower in pregnant than nonpregnant women (P < 0.01). Higher white blood cell choline dehydrogenase messenger RNA levels in a random subset of pregnant (n = 8) than nonpregnant women (n = 7) (P < 0.01) suggest increased betaine and dimethylglycine turnover rather than decreased synthesis. Plasma choline, betaine, and dimethylglycine were higher (P < 0.001) in fetal plasma (36.4 ± 13, 29.4 ± 1.0, and 2.44 ± 0.12 μmol/L, respectively) than maternal plasma (15.3 ± 0.42, 14.1 ± 0.6 and 1.81 ± 0.12 μmol/L, respectively). Concentrations of 5-oxoproline and dimethylglycine were inversely (P < 0.05) correlated in maternal (Spearman rho = −0.35) and fetal plasma (Spearman rho = −0.32), suggesting that choline, via dimethylglycine, contributes glycine for GSH synthesis in human development.

Colorectal Carcinomas with Microsatellite Instability Display Increased Thymidylate Synthase Gene Expression Levels

This study investigated whether patients with colorectal cancer (CRC) who have tumors with high microsatellite instability (MSI; MSI-H) had an altered expression of the folate and methyl-group metabolism. The gene expression levels of thymidylate synthase (TS), reduced folate carrier (RFC-1), folylpolyglutamate synthase (FPGS), and methylenetetrahydrofolate reductase (MTHFR) in mucosa and tumor were compared with patients with MSS. Furthermore, the influence of TS polymorphisms on TS gene expression levels and MSI-H was studied. The microsatellite status (MSI-H, low instability [MSI-L], or stable [MSS]) and TS polymorphisms were analyzed in genomic DNA from 181 patients with CRC. Gene expression levels of TS, RFC-1, FPGS, and MTHFR in mucosa and tumors were quantified and the difference in TS expression between tumor and mucosa was designated DeltaTS. Significantly higher gene expression levels of TS (P < .0001) were detected in patients with CRC with MSI-H compared with MSS/MSI-L tumors. Gene expression of TS and FPGS were significantly higher in right-sided MSI-H tumors compared with right-sided MSS/MSI-L tumors (P < .0001, P = .041, respectively). A significant correlation between DeltaTS and the number of unstable markers was found (P < .0001). An inverse association between age and TS expression was found in MSI tumors (r = -0.57; P = .0004) and also in right-sided tumors (r = -0.25, P = .011) regardless of MSI status. No relation was detected between MSI status and the TS polymorphisms or between the TS polymorphisms and TS expression. This study has revealed, for the first time, that age and the frequency of unstable MSI markers were factors that were linked to the variability in TS gene expression in tumors.

Are genetic variants of the methyl group metabolism enzymes risk factors predisposing to obesity?

Obesity, due to the combination of inherited genes and environmental factors, is continually increasing. We evaluated the relationship between polymorphisms of methylene-tetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G), betaine:homocysteine methyltransferase (BHMT G742A) and cystathionine beta-synthase (CBS 68-bp ins) genes and the risk of obesity. We studied these polymorphic variants in 54 normal and 82 obese subjects [body mass index (BMI)=22.4+/-1.8, 34.1+/-7.1; ages 35.2+/-10.7, 43.3+/-10.6 respectively]. Levels of total plasma homocysteine (t-Hcy), folates, and vitamins B6 and B12 were not significantly different, while leptin concentration was significantly higher (p=0.005) in the obese patients compared to the lean controls. The frequency of only (a) MTHFR (AC), (b) MTR (AG), and (c) MTRR (AG) heterozygous genotypes was statistically different in the obese compared to the control group (p=0.03, p=0.007, and p=0.01). Single (a), (b), and (c) heterozygous genotypes had a significant risk of developing obesity [p=0.02, 0.01, and 0.03; odds ratio (OR)=2.5, 3.0, and 2.4; 95% confidence interval (CI)=1.2-5.3, 1.3-7.1, and 1.2-5.1 respectively] and the risk remarkably increased for combined genotypes a+b, a+c, b+c, and a+b+c (p=0.002, 0.002, 0.016, 0.006; OR=7.7, 5.4, 5.8, 15.4; 95% CI=1.9-30.4, 1.7-16.8, 1.4-23.2, 1.6- 152.3). These findings suggest that in obese subjects, Hcy cycle efficiency is impaired by MTHFR, MTR, and MTRR inability to supply methyl-group donors, providing evidence that MTHFR, MTR, and MTRR gene polymorphisms are genetic risk factors for obesity.

Hyperhomocysteinemia-mediated DNA Hypomethylation and its Potential Epigenetic Role in Rats

Hyperhomocysteinemia (HHcy), which is an independent risk factor for atherosclerosis, might cause dysregulation of gene expression, but the characteristics and key links involved in its pathogenic mechanisms are still poorly understood. The objective of the present study was to investigate the effect of HHcy on DNA methylation and the underlying mechanism of homocysteine (Hcy)-induced DNA methylation. HHcy was induced in Sprague-Dawley rats after 4 weeks of a low, medium or high methionine diet. The levels of total homocysteine, S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) were detected by high-performance liquid chromatography. The expression levels of genes and proteins of S-adenosylhomocysteine hydrolase, DNA methyltransferase and methyl-CpG-binding domain 2 were detected by real-time reverse transcription-polymerase chain reaction and Western blot analysis. A high-throughput quantitative methylation assay using fluorescence-based real-time polymerase chain reaction was employed to determine the levels of DNA methylation. The results indicated that HHcy induced the elevation of AdoHcy concentration, the decline of AdoMet concentration, the ratios of AdoMet/AdoHcy and the RNA and protein expression of S-adenosylhomocysteine hydrolase and methyl-CpG-binding domain 2, as well as an increase of DNA methyltransferase activity. With different methylation-dependent restriction endonucleases, the aberrant demethylation was found to prefer CCGG sequences to CpG islands. Increasing levels of HHcy significantly increased genome hypomethylation in B1 repetitive elements. The impacts of different levels of HHcy showed that the varied detrimental effects of HHcy could be attributed to different concentrations through different mechanisms. In mild and moderate HHcy, the Hcy might primarily influence the epigenetic regulation of gene expression through the interference of transferring methyl-group metabolism. However, at high Hcy concentrations, the impacts might be more injurious through oxidative stress, apoptosis and inflammation.

Genetic polymorphisms of methylenetetrahydrofolate reductase and promoter methylation of MGMT and FHIT genes in diffuse large B cell lymphoma risk in Middle East

Diffuse large B cell lymphoma (DLBCL) is one of the most common non-Hodgkin's lymphoma types. Methylenetetrahydrofolate reductase (MTHFR) balances the pool of folate coenzymes in one carbon metabolism of deoxyribonucleic acid (DNA) synthesis and methylation; both are implicated in carcinogenesis of many types of cancer including lymphoma. Two common variants in the MTHFR gene (C677T and A1298C) have been associated with reduced enzyme activity, thereby making MTHFR polymorphisms a potential candidate as a cancer-predisposing factor. The O6 methylguanine DNA methyltransferase (MGMT) and fragile histidine triad (FHIT) genes are transcriptionally silenced by promoter hypermethylation in DLBCL. These genetic differences are highly race specific and have never been screened in the Saudi DLBCL patients. We conducted a hospital-based case-control study including 160 DLBCL cases and 511 Saudi control samples analyzing the MTHFR C677T and A1298C functional polymorphisms by the restriction fragment length polymorphism method and their association with MGMT and FHIT genes promoter hypermethylation. Our data demonstrated that Saudi individuals carrying MTHFR genotype 1298CC (p < 0.001) and the 1298C allele (p = 0.012) had 4.23 and 1.73-fold higher risk of developing DLBCL, respectively. Additionally, combined genotype CCCC (MTHFR 677CC + MTHFR 1298CC) was associated with 3.489-fold, and CTCC (MTHFR 677 CT + 1298CC) was related to 9.515-fold higher risk, compared with full MTHFR enzyme activity. No significant association between MTHFR variant genotypes and methylation of MGMT and FHIT genes were observed. Our findings suggested that polymorphisms of MTHFR enzyme genes might be associated with the individual susceptibility to develop DLBCL. Additionally, the results indicated that MTHFR variants were not related to MGMT or FHIT hypermethylation in DLBCL.

Dietary Choline and Betaine and the Risk of Distal Colorectal Adenoma in Women

Choline and betaine are involved in methyl-group metabolism as methyl-group donors; thus, like folate, another methyl-group donor, they may be associated with a reduced risk of colorectal adenomas. No epidemiologic study has examined the association of intake of these nutrients and colorectal adenoma risk. We investigated the relationship between intakes of choline and betaine and risk of colorectal adenoma in US women enrolled in the Nurses' Health Study. Dietary intake was measured by food-frequency questionnaires, and individual intakes of choline and betaine were calculated by multiplying the frequency of consumption of each food item by its choline and betaine content and summing the nutrient contributions of all foods. Logistic regression models were used to calculate adjusted odds ratios (as approximations for relative risks) and 95% confidence intervals (CIs) of colorectal adenoma. All statistical tests were two-sided. Among 39246 women who were initially free of cancer or polyps and who had at least one endoscopy from 1984 through 2002, 2408 adenoma cases were documented. Increasing choline intake was associated with an elevated risk of colorectal adenoma; the multivariable relative risks (95% CIs) for increasing quintiles of intake, relative to the lowest quintile, were 1.03 (0.90 to 1.18), 1.01 (0.88 to 1.16), 1.23 (1.07 to 1.41), and 1.45 (1.27 to 1.67; P(trend)<.001). Betaine intake had a nonlinear inverse association with colorectal adenoma; the multivariable relative risks (95% CIs) for increasing quintiles of intake were 0.94 (0.83 to 1.07), 0.85 (0.75 to 0.97), 0.86 (0.75 to 0.98), and 0.90 (95% CI = 0.78 to 1.04; P(trend) = .09). Among individual sources of choline, choline from phosphatidylcholine and from sphingomyelin were each positively related to adenoma risk. Our findings do not support an inverse association between choline intake and risk of colorectal adenoma. The positive association between choline intake and colorectal adenoma that we observed could represent effects of other components in the foods from which choline was derived and should be investigated further.

Methylation levels of LINE‐1 repeats and CpG island loci are inversely related in normal colonic mucosa

Hypermethylation of CpG island loci within gene promoter regions is a frequent event in colorectal cancer that is often associated with transcriptional silencing and has been referred to as CIMP+. DNA hypomethylation can occur in concert with CIMP+, although these two phenomena appear not to be related in colorectal cancer. The authors investigated here whether the methylation level of LINE-1 repeats, a surrogate marker for genomic methylation, was associated with the level of CpG island methylation in colorectal cancers and in matching normal colonic mucosa from 178 patients. The MethyLight assay was used to quantitate the methylation of CpG islands within the MLH1, P16(INK4A), TIMP3, DAPK, APC, ER and MYOD genes. A real-time, methylation-specific polymerase chain reaction assay was also used to quantitate the methylation of LINE-1 repeats. In colorectal cancer, no associations were seen between methylation levels in LINE-1 repeats and CpG island loci, including a new CpG island panel that was recently proposed for CIMP+. In normal colonic mucosa, however, the methylation level of LINE-1 repeats was inversely correlated with CpG-island methylation of the MLH1, P16, TIMP3, APC, ER and MYOD genes. The methylation level of LINE-1 repeats in normal colonic mucosa also showed significant associations with common polymorphisms in the methylene tetrahydrofolate reductase and methylene tetrahydrofolate dehydrogenase genes involved in methyl group metabolism. Further investigation of genomic and CpG island methylation in normal colonic mucosa and the possible influences of environmental and genetic factors may provide new insights into the development of CIMP+ colorectal cancer.

Folate deficiency during pregnancy impacts on methyl metabolism without affecting global DNA methylation in the rat fetus

The methionine cycle and methyl group metabolism are implicated in the long-term programming of metabolism. Diets deficient in folic acid, methionine and choline have been fed to pregnant rats to examine the effects on amino acid metabolism, choline reserves and DNA methylation in dam and fetuses. Animals were fed folate-deficient, folate-deficient with low methionine, folate-deficient with low choline and folate-deficient, low-methionine, low-choline diets starting 2 weeks before mating. The dams and their fetuses were subsequently killed on day 21 of gestation for analysis. Diets low in methionine reduced fetal and maternal weight. Folate deficiency increased the concentrations of homocysteine, glycine, serine and threonine in the maternal plasma, and this was exacerbated by the low-methionine diets. The changes in the amino acid profile in the fetal serum were similar but less pronounced. This result suggests that fetal metabolism was less perturbed. Folate deficiency increased free choline in the maternal liver at the expense of phosphocholine stores. It has been suggested that a deficiency in methyl donors in the diet during pregnancy may impact on key methylation reactions, including the methylation of DNA. Despite widespread changes in the metabolism of choline and amino acids, there was no change in the global methylation of cytosine in DNA from either maternal or fetal livers. This suggests a more indirect mechanism in which gene-nutrient interactions modify the process of differential methylation during development.

Folate and Methylation Status in Relation to Phosphorylated Tau Protein(181P) and β-Amyloid(1–42) in Cerebrospinal Fluid

Increased plasma total homocysteine (tHcy) is a risk factor for neurological diseases, but the underlying pathophysiology has not been adequately explained. We evaluated concentrations of tHcy, S-adenosyl homocysteine (SAH), S-adenosyl methionine (SAM), folate, and vitamin B(12) in cerebrospinal fluid (CSF) and plasma or serum from 182 patients with different neurological disorders. We measured concentrations of phosphorylated tau protein (P-tau)((181P)) and beta-amyloid(1-42) in the CSF. Aging was associated with higher concentrations of tHcy and SAH in the CSF, in addition to lower concentrations of CSF folate and lower SAM:SAH ratio. Concentrations of CSF SAH and CSF folate correlated significantly with those of P-tau (r = 0.46 and r = -0.28, respectively). Moreover, P-tau correlated negatively with SAM:SAH ratio (r = -0.40, P <0.001). The association between SAH and higher P-tau was observed in 3 age groups (<41, 41-60, and >60 years). CSF tHcy was predicted by concentrations of CSF cystathionine (beta = 0.478), folate (beta = -0.403), albumin (beta = 0.349), and age (beta = 0.298). tHcy concentration in the brain is related to age, B vitamins, and CSF albumin. Increase of CSF SAH is related to increased CSF P-tau; decreased degradation of P-tau might be a plausible explanation. Disturbed methyl group metabolism may be the link between hyperhomocysteinemia and neurodegeneration. Lowering tHcy and SAH might protect the brain by preventing P-tau accumulation.

γ-Secretase is Differentially Modulated by Alterations of Homocysteine Cycle in Neuroblastoma and Glioblastoma Cells

Multiple aspects of homocysteine metabolism were studied to understand the mechanism responsible for hyperhomocysteinemia toxicity in Alzheimer disease. Besides oxidative stress and vascular damage, homocysteine has also a great importance in regulating DNA methylation through S-adenosylmethionine, the main methyl donor in eukaryotes. Alterations of S-adenosylmethionine and methylation were evidenced in Alzheimer disease and in elderly. In order to clarify whether DNA methylation can provide the basis for amyloid-beta overproduction, we used human SK-N-BE neuroblastoma and A172 glioblastoma cell lines. We tested the effects of folate, B12 and B6 deprivation and S-adenosylmethionine addition on methylation metabolism. Our results indicate that homocysteine accumulation induced through vitamin B deprivation could impair the "Methylation Potential" with consequent presenilin 1, BACE and amyloid-beta upregulation. Moreover, we found that homocysteine alterations had an effect on neuroblastoma but not on glioblastoma cells; this suggests a possible differential role of the two cell types in Alzheimer disease.

Folate status and S-adenosylmethionine/S-adenosylhomocysteine ratio in colorectal adenocarcinoma in humans

This study reports the influence of colorectal neoplasia on methylation intermediates and folate concentrations in human colonic mucosa, as well as systemic measures of folate status, to examine biomarkers and possible mechanisms of folate-related carcinogenesis. A total of 47 patients were selected from those previously diagnosed with adenocarcinoma of the colorectum undergoing surgery. For each individual, we obtained a biopsy of the adenocarcinoma and a biopsy of normal appearing mucosa, to perform an intra-individual comparison. The 'methylation' ratio (S-adenosylmethionine/S-adenosylhomocysteine (SAH)) was lower in pathological tissue vs normal mucosa (P=0.08), mainly due to a much higher SAH concentration (P<0.005). Colonic folate concentration was significantly diminished in malignant tissue (P<0.0001). Plasma homocysteine concentration was within the normal to high range, and folate and vitamin B12 plasma concentrations were within the low to normal range as compared with normative values. Our results contribute to the hypothesis that altered DNA methylation and methyl metabolism is associated with colorectal neoplasia.

Polyamines and DNA methylation in childhood leukaemia

Both polyamine metabolism and DNA methylation play an important role in normal and malignant growth. Specific enzyme inhibitors or drugs that interfere with these metabolic pathways have proven to be potential anticancer agents. Since DNA methylation and polyamine metabolism depend on a common substrate, i.e. S-adenosylmethionine, interaction between both pathways can be expected. Little is known about the relationship between these pathways but studies are available indicating that polyamines and DNA methylation are directly or indirectly interconnected, metabolically as well as physiologically with respect to the regulation of cell growth, differentiation and cancer development. These considerations give rise to the possibility that, by targeting both pathways, a more profound and effective inhibitory effect on the growth of malignant cells can be achieved. In previous studies we showed that 6-MP (6-mercaptopurine) as well as MTX (methotrexate), well-known drugs in the treatment of acute lymphoblastic leukaemia, inhibit DNA methylation and induce apoptosis in malignant blood cells. Our recent results show that combined treatment with 6-MP, MTX and drugs interfering with polyamine metabolism has additive/synergistic effects on the growth, cell viability and/or apoptotic death of leukaemic cells. Such a combination therapy could have great clinical value for patients in which therapy using inhibitors of thiopurines/purine metabolism has failed.