Angela M. Devlin

Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses

Background: In animal models, variations in early maternal care are associated with differences in hypothalamic-pituitary-adrenal (HPA) stress response in the offspring, mediated via changes in the epigenetic regulation of glucocorticoid receptor (GR) gene (Nr3c1) expression. Objective: To study this in humans, relationships between prenatal exposure to maternal mood and the methylation status of a CpG-rich region in the promoter and exon 1F of the human GR gene (NR3C1) in newborns and HPA stress reactivity at age 3 months were examined. Methods: The methylation status of a CpG-rich region of the NR3C1 gene, including exon 1F, in genomic DNA from cord blood mononuclear cells was quantified by bisulfite pyrosequencing in infants of depressed mothers treated with a serotonin reuptake inhibitor antidepressant (SRI) (n=33), infants of depressed non treated mothers (n=13) and infants of non depressed/non treated mothers (n=36). To study the functional implications of the newborn methylation status of NR3C1 in newborns, HPA function was assessed at 3 months using salivary cortisol obtained before and following a non noxious stressor and at a late afternoon basal time. Results: Prenatal exposure to increased third trimester maternal depressed/anxious mood was associated with increased methylation of NR3C1 at a predicted NGFI-A binding site. Increased NR3C1 methylation at this site was also associated with increased salivary cortisol stress responses at 3 months, controlling for prenatal SRI exposure, postnatal age, and pre and postnatal maternal mood. Conclusions: Methylation status of the human NR3C1 gene in newborns is sensitive to prenatal maternal mood and may offer a potential epigenetic process that links antenatal maternal mood and altered HPA stress reactivity during infancy.

Metabolic Interactions of Alcohol and Folate

The goals and objectives of these studies, conducted over the past 30 y, were to determine: a) how chronic alcoholism leads to folate deficiency and b) how folate deficiency contributes to the pathogenesis of alcoholic liver disease (ALD). The intestinal absorption of folic acid was decreased in binge drinking alcoholics and, prospectively, in volunteers fed alcohol with low folate diets. Monkeys fed alcohol for 2 y developed decreased hepatic folate stores, folic acid malabsorption and decreased hepatic uptake but increased urinary excretion of labeled folic acid. Micropigs fed alcohol for 1 y developed features of ALD in association with decreased translation and activity of intestinal reduced folate carrier. Another study in ethanol-fed micropigs demonstrated abnormal hepatic methionine and DNA nucleotide imbalance and increased hepatocellular apoptosis. When alcohol feeding was combined with folate deficiency, micropigs developed typical histological features of ALD in 14 wk, together with elevated plasma homocysteine levels, reduced liver S-adenosylmethionine and glutathione and increased markers for DNA and lipid oxidation. In summary, chronic alcohol exposure impairs folate absorption by inhibiting expression of the reduced folate carrier and decreasing the hepatic uptake and renal conservation of circulating folate. At the same time, folate deficiency accelerates alcohol-induced changes in hepatic methionine metabolism while promoting enhanced oxidative liver injury and the histopathology of ALD.

Prenatal Exposure to Maternal Depressed Mood and the MTHFR C677T Variant Affect SLC6A4 Methylation in Infants at Birth

Background Prenatal and early postnatal exposure to maternal depression may “program” childhood behavior via epigenetic processes such as DNA methylation. Methylenetetrahydro-folate reductase (MTHFR) is an important enzyme in the generation of methyl groups for DNA methylation. The common MTHFR C677T variant is associated with depression in men and non-pregnant women, and with global changes in DNA methylation. This study investigated the effect of maternal MTHFR C677T genotype on antenatal maternal mood, and their impact on the gene-specific methylation in pregnant women and their newborn infants. The methylation status of SLC6A4, which encodes the transmembrane serotonin transporter, and BDNF, which encodes brain derived neurotrophic factor, were assessed because of their potential role in behaviour. Methods/Principal Findings Depressed mood was assessed by the Edinburgh Postnatal Depression Scale (EPDS) and the Hamilton Rating Scale for Depression (HAM-D) in women (n = 82, all taking folate) during the 2nd and 3rd trimesters of pregnancy. The methylation status of SLC6A4 and BDNF were assessed in 3rd trimester maternal peripheral leukocytes and in umbilical cord leukocytes collected from their infants at birth. Women with the MTHFR 677TT genotype had greater 2nd trimester depressed mood (p<0.05). Increased 2nd trimester maternal depressed mood (EPDS scores) was associated with decreased maternal and infant SLC6A4 promoter methylation (p<0.05), but had no effect on BDNF promoter methylation. Conclusions These findings show that the MTHFR C677T variant is associated with greater depressed mood during pregnancy. We further showed that prenatal exposure to maternal depressed mood affects gene-specific DNA methylation patterns. These findings support the concept that alterations in epigenetic processes may contribute to developmental programming of behaviour by maternal depression.

Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2′-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.

Childhood Obesity and Cardiovascular Dysfunction

Obesity-related cardiovascular disease in children is becoming more prevalent in conjunction with the rise in childhood obesity. Children with obesity are predisposed to an increased risk of cardiovascular morbidity and mortality in adulthood. Importantly, research in children with obesity over the last decade has demonstrated that children may exhibit early signs of cardiovascular dysfunction as a result of their excess adiposity, often independent of other obesity-related comorbidities such as dyslipidemia and insulin resistance. The clinical evidence is accumulating to suggest that the cardiovascular damage, once observed only in adults, is also occurring in obese children. The objective of this review is to provide a synopsis of the current research on cardiovascular abnormalities in children with obesity and highlight the importance and need for early detection and prevention programs to mitigate this potentially serious health problem.

Evaluation of genetic variants in the reduced folate carrier and in glutamate carboxypeptidase II for spina bifida risk

Genetic variants in folate metabolism have been reported to increase risk for neural tube defects (NTD). The first such sequence change was the 677C-->T substitution in methylenetetrahydrofolate reductase (MTHFR), but additional sequence changes have been identified in enzymes or transporters for folates. Two recently identified variants are the 1561C-->T (H475Y) mutation in glutamate carboxypeptidase II (GCPII) and the 80A-->G (H27R) change in the reduced folate carrier RFC-1. We examined a group of mothers of spina bifida offspring, and a group of control women, for the above polymorphisms to assess their impact on NTD risk as well as on homocysteine and nutrient (RBC folate, serum folate, and serum cobalamin) levels. The GCPII variant (in the heterozygous state) did not influence NTD risk or metabolite levels; homozygous mutant (YY) women were not observed in our study group. The homozygous mutant (RR) genotype for the RFC-1 gene was not associated with a significant difference in NTD risk (OR=1.39, 95% CI=0.55-3.54), but there was a borderline significant (p=0.065) decrease in RBC folate levels, compared with the HH genotype. However, the combination of the RR genotype for RFC-1 and low RBC folate was associated with a significant 4.6-fold increase in NTD risk (OR=4.6, 95% CI=1.47-14.37). Since this small study is the first to demonstrate increased risk for women with the RFC-1 variant for having a child with a NTD, additional larger studies are required to confirm this change as another potential genetic modifier for spina bifida risk.

Folate deficiency, methionine metabolism, and alcoholic liver disease ☆

Methionine metabolism is regulated by folate, and both folate deficiency and abnormal hepatic methionine metabolism are recognized features of alcoholic liver disease (ALD). Previously, histological features of ALD were induced in castrated male micropigs fed diets containing ethanol at 40% of kilocalories for 12 months, whereas in male micropigs fed the same diets for 12 months abnormal methionine metabolism and hepatocellular apoptosis developed. Folate deficiency may promote the development of ALD by accentuating abnormal methionine metabolism. Intact male micropigs received eucaloric diets that were folate sufficient, folate deficient, or each containing 40% of kilocalories as ethanol for 14 weeks. Folate deficiency alone reduced hepatic folates by one half, and ethanol feeding alone reduced methionine synthase, S-adenosylmethionine (SAM), and glutathione (GSH) levels and elevated plasma malondialdehyde (MDA) levels. The combined regimen elevated plasma homocysteine, hepatic S-adenosylhomocysteine (SAH), urinary 8-hydroxy-2-deoxyguanosine (oxy(8)dG), an index of DNA oxidation, and serum aspartate aminotransferase (AST) levels. Terminal hepatic histopathologic characteristics included typical features of steatonecrosis and focal inflammation in pigs fed the combined diet, with no changes in the other groups. Hepatic SAM levels correlated with those of GSH, whereas urinary oxy(8)dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH. The results demonstrate the linkage of abnormal methionine metabolism to products of DNA and lipid oxidation and to liver injury. The finding of steatonecrosis and focal inflammation only in the combined diet group supports the suggestion that folate deficiency promotes and folate sufficiency protects against the early onset of methionine cycle-mediated ALD.

Hypermethylation of Fads2 and altered hepatic fatty acid and phospholipid metabolism in mice with hyperhomocysteinemia.

Alterations in lipid metabolism may play a role in the vascular pathology associated with hyperhomocysteinemia (HHcy). Homocysteine is linked to lipid metabolism through the methionine cycle and the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine (PE) methyltransferase, which is responsible for the synthesis of 20–40% of liver PC. The goal of the present study was to determine if the reduced methylation capacity in HHcy is associated with alterations in liver phospholipid and fatty acid metabolism. Mice heterozygous for disruption of cystathionine β-synthase (Cbs+/-) fed a diet to induce HHcy (HH diet) had higher (p < 0.001) plasma total homocysteine (30.8 ± 4.4 μm, mean ± S.E.) than C57BL/6 mice (Cbs+/+) fed the HH diet (7.0 ± 1.1 μm) or Cbs+/+ mice fed a control diet (2.3 ± 0.3 μm). Mild and moderate HHcy was accompanied by lower adenosylmethionine/adenosylhomocysteine ratios (p < 0.05), higher PE (p < 0.05) and PE/PC ratios (p < 0.01), lower PE methyltransferase activity (p < 0.001), and higher linoleic acid (p < 0.05) and lower arachidonic acid (p < 0.05) in PE. Mice with moderate HHcy also had higher linoleic acid and α-linolenic acid (p < 0.05) and lower arachidonic acid and docosahexaenoic acid (p < 0.05) in liver PC. The first step in the desaturation and elongation of linoleic acid and linolenic acid to arachidonic acid and docosahexaenoic acid, respectively, is catalyzed by Δ(6)-desaturase (encoded by Fads2). We found hypermethylation of the Fads2 promoter (p < 0.01), lower Fads2 mRNA (p < 0.05), and lower Δ(6)-desaturase activity (p < 0.001) in liver from mice with HHcy. These findings suggest that methylation silencing of liver Fads2 expression and changes in liver fatty acids may contribute to the pathology of HHcy.

Cerebral Vascular Dysfunction in Methionine Synthase–Deficient Mice

Background—Methionine synthase (MS) catalyzes the folate-dependent remethylation of homocysteine to methionine. We tested the hypothesis that deficiency of MS impairs endothelial function in mice heterozygous for disruption of the Mtr gene, which encodes MS. Methods and Results—Plasma total homocysteine was similar in wild-type (Mtr+/+) and heterozygous (Mtr+/−) mice fed a control diet (4.5±0.3 and 5.3±0.4 &mgr;mol/L, respectively) and mildly elevated in Mtr+/+ and Mtr+/− mice fed a low-folate (LF) diet (7.5±0.7 and 9.6±1.2 &mgr;mol/L, respectively; P<0.001 versus control diet). Dilatation of cerebral arterioles to the endothelium-dependent dilator, acetylcholine (10 &mgr;mol/L) was blunted in Mtr+/− mice compared with Mtr+/+ mice fed the control diet (21±4 versus 32±4%; P<0.05). Both Mtr+/+ and Mtr+/− mice exhibited impaired dilatation of cerebral arterioles to acetylcholine when they were fed the LF diet (12±2 and 14±2%, respectively; P<0.01 versus Mtr+/+ mice fed the control diet). Elevated levels of superoxide and hydrogen peroxide were detected by confocal microscopy in cerebral arterioles of Mtr+/− mice fed the control diet and in both Mtr+/+ and Mtr+/− mice fed the LF diet. Conclusions—These findings demonstrate that defective homocysteine remethylation caused by deficiency of either MS or folate produces oxidative stress and endothelial dysfunction in the cerebral microcirculation of mice.

Ethnic-specific differences in vitamin D status is associated with adiposity.

Background Low circulating 25 hydroxyvitamin D [25(OH)D] concentrations are common in obesity (BMI ≥30 kg/m2) and a negative relationship with body fat distribution has recently been reported. Ethnic-specific differences in body fat distribution have been described with South Asians are reported to have greater visceral adipose tissue (VAT), which could influence circulating 25(OH)D concentrations. The objective of this study is to investigate the relationship between plasma 25(OH)D, adiposity, and body fat distribution in Europeans and South Asians. Methods/Principal Findings 187 Europeans and 192 South Asians were assessed for demographics, anthropometrics, and plasma 25(OH)D concentrations. Subcutaneous adipose tissue (SAT) and VAT were quantified by CT scan, and percent body fat by DEXA. Data were assessed by general linear models. South Asians had lower (P<0.001) plasma 25(OH)D concentrations and higher VAT (P = 0.04) than Europeans. Plasma 25(OH)D concentrations were negatively (P<0.05) associated with BMI, waist circumference, percent body fat, total adipose tissue, VAT, and SAT in unadjusted models and negatively (P<0.05) associated with VAT, SAT, and percent body fat after adjusting for BMI, ethnicity, age, and season of blood collection in males and females. When percent body fat, VAT, and SAT were included in the same model, only VAT remained negatively (P<0.05) associated with plasma 25(OH)D concentrations. Ethnicity remained significant in all models (P<0.001). Conclusion Compared to other adipose tissue compartments, VAT may have a distinct role in determining plasma 25(OH)D concentrations, which may account for the lower levels in South Asians.