Postnatal stability,tissue, and time specific effects of AHRR methylation change in response to maternal smoking in pregnancy

The intrauterine environment has the potential to “program” the developing fetus in a way that can be potentially deleterious to later health. While in utero environmental/stochastic factors are known to influence DNA methylation profile at birth, it has been difficult to assign specific examples of epigenetic variation to specific environmental exposures. Recently, several studies have linked exposure to smoking with DNA methylation change in the aryl hydrocarbon receptor repressor (AHRR) gene in blood. This includes hypomethylation of AHRR in neonatal blood in response to maternal smoking in pregnancy. The role of AHRR as a negative regulator of pathways involved in pleiotropic responses to environmental contaminants raises the possibility that smoking-induced hypomethylation is an adaptive response to an adverse in utero environmental exposure. However, the tissue specificity of the response to maternal smoking, and the stability of the methylation changes early in life remain to be determined. In this study we analyzed AHRR methylation in three cell types—cord blood mononuclear cells (CBMCs), buccal epithelium, and placenta tissue—from newborn twins of mothers who smoked throughout pregnancy and matched controls. Further, we explored the postnatal stability of this change at 18 months. Our results confirm the previous association between maternal smoking and AHRR methylation in neonatal blood. In addition, this study expands the region of AHRR methylation altered in response to maternal smoking during pregnancy and reveals the tissue-specific nature of epigenetic responses to environmental exposures in utero. Further, the evidence for postnatal stability of smoking-induced epigenetic change supports a role for epigenetics as a mediator of long-term effects of specific in utero exposures in humans. Longitudinal analysis of further specific exposures in larger cohorts is required to examine the extent of this phenomenon in humans.     

Locus-specific DNA methylation in the placenta is associated with levels of pro-inflammatory proteins in cord blood and they are both independently affected by maternal smoking during pregnancy

We investigated the impact of maternal smoking during pregnancy on placental DNA methylation and how this may mediate the association between maternal smoking and pro-inflammatory proteins in cord blood. The study population consisted of 27 individuals exposed to maternal smoking throughout pregnancy, 32 individuals exposed during a proportion of the pregnancy, and 61 unexposed individuals. Methylation of 11 regions within 6 genes in placenta tissue was assessed by pyrosequencing. Levels of 7 pro-inflammatory proteins in cord blood were assessed by electrochemiluminescence. Differential methylation was observed in the CYP1A1 promoter and AHRR gene body regions between women who smoked throughout pregnancy and non-smokers on the fetal-side of the placenta and in the GFI1 promoter between women who quit smoking while pregnant and non-smokers on the maternal-side of the placenta. Maternal smoking resulted in elevated levels of IL-8 protein in cord blood, which was not mediated by DNA methylation of our candidate regions at either the maternal or the fetal side of the placenta. Placental DNA methylation was associated with levels of inflammatory proteins in cord blood. Our observations suggest that maternal smoking during pregnancy affects both placental DNA methylation and the neonate's immune response.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10^?7 and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

Increased risk of cancer mortality by smoking-induced aryl hydrocarbon receptor repressor DNA hypomethylation in Japanese population: A long-term cohort study

BackgroundSmoking is well known to be a major risk factor for cancer, and to decrease the levels of aryl hydrocarbon receptor repressor (AHRR) DNA methylation. AHRR is a key regulator for AHR signaling, which is involved in chemical metabolism and cancer development. Therefore, smoking-induced AHRR DNA hypomethylation may be associated with cancer development. However, it has not been reported that association between AHHR DNA methylation and cancer mortality in Asian population. Hence, we examined whether AHRR DNA methylation levels were associated with cancer mortality in a Japanese population.MethodsThis study was conducted with 812 participants (aged 38–80 years) who received a health check-up in 1990, and did not have a clinical histories. We followed up the participants until the end of 2019 (median: 27.8 years), and 100 participants died from cancer. The AHRR DNA methylation levels in peripheral blood mononuclear cells (PBMCs) were measured by the pyrosequencing method. We calculated the hazard ratio (HR) and 95% confidence interval (CI) for cancer mortality according to the baseline levels of AHRR DNA methylation.ResultsWe found that AHRR DNA hypomethylation was associated with a higher risk of all cancer mortality, especially smoking related cancers and lung cancer. (all cancer: HR, 1.28, 95% CI, 1.09–1.51; smoking-related cancers: HR, 1.35, 95% CI, 1.12–1.62; lung cancer: HR, 1.68, 95% CI, 1.24–2.26).ConclusionsSmoking-induced AHRR DNA hypomethylation in PBMCs was associated with the risk of cancer mortality in Japanese population; therefore, hypomethylation of AHRR may be a useful biomarker of cancer mortality risk.     

Global DNA hypomethylation is associated with in utero exposure to cotinine and perfluorinated alkyl compounds

Environmental exposures in utero may alter the epigenome, thus impacting chromosomal stability and gene expression. We hypothesized that in utero exposures to maternal smoking and perfluoroalkyl compounds (PFCs) are associated with global DNA hypomethylation in umbilical cord serum. Our objective was to determine if global DNA methylation could be used as a biomarker of in utero exposures to maternal smoking and PFCs. Using an ELISA-based method, global DNA methylation was quantified in umbilical cord serum from 30 newborns with high (>10 ng/ml, mean 123.8 ng/ml), low (range 1–10 ng/ml, mean 1.6 ng/ml) and very low (<1 ng/ml, mean 0.06 ng/ml) cord serum cotinine levels. Y chromosome analysis was performed to rule out maternal DNA cross-contamination. Cord serum global DNA methylation showed an inverse dose response to serum cotinine levels (p < 0.001). Global DNA methylation levels in cord blood were the lowest among newborns with smoking mothers (mean = 15.04%; 95% CI, 8.4, 21.7) when compared to babies of mothers who were second-hand smokers (21.1%; 95% CI, 16.6, 25.5) and non-smokers (mean = 29.2%; 95% CI, 20.1, 38.1). Global DNA methylation was inversely correlated with serum PFOA (r = -0.35, p = 0.06) but not PFOS levels. Serum Y chromosome analyses did not detect maternal DNA cross-contamination. This study supports the use of global DNA methylation status as a biomarker of in utero exposure to cigarette smoke and PFCs.Key words: epigenomics, umbilical cord serum, hypomethylation, cigarette smoke, perfluorooctane sulfonate, perfluorooctanoate, global DNA methylation     

Association of maternal and nutrient supply line factors with DNA methylation at the imprinted IGF2/H19 locus in multiple tissues of newborn twins

Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p < 0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.     

Global DNA hypomethylation is associated with in utero exposure to cotinine and perfluorinated alkyl compounds

Environmental exposures in-utero may alter the epigenome, thus impacting chromosomal stability and gene expression. We hypothesized that in utero exposures to maternal smoking and perfluoroalkyl compounds (PFCs) are associated with global DNA hypomethylation in umbilical cord serum. Our objective was to determine if global DNA methylation could be used as a biomarker of in utero exposures to maternal smoking and PFCs. Using an ELISA-based method, global DNA methylation was quantified in umbilical cord serum from 30 newborns with high (>10 ng/ml, mean 123.8 ng/ml), low (range 1-10 ng/ml, mean 1.6 ng/ml) and very low (&lt;1 ng/ml, mean 0.06 ng/ml) cord serum cotinine levels. Y chromosome analysis was performed to rule out maternal DNA cross-contamination. Cord serum global DNA methylation showed an inverse dose response to serum cotinine levels (p&lt;0.001). Global DNA methylation levels in cord blood were the lowest among newborns with smoking mothers (mean=15.04%; 95% CI, 8.4, 21.7) when compared to babies of mothers who were second-hand smokers (21.1%; 95% CI, 16.6, 25.5) and non-smokers (mean=29.2%; 95% CI, 20.1, 38.1). Global DNA methylation was inversely correlated with serum PFOA (r= -0.72, p &lt;0.01) but not PFOS levels. Serum Y chromosome analyses did not detect maternal DNA cross-contamination. This study supports the use of global DNA methylation status as a biomarker of in utero exposure to cigarette smoke and PFCs.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10⁻⁷ and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

Maternal cigarette smoking during pregnancy and offspring DNA methylation in midlife

Maternal smoking in pregnancy (MSP) has been associated with DNA methylation in specific CpG sites (CpGs) in infants and children. We investigated whether MSP, independent of own personal active smoking, was associated with midlife DNA methylation in CpGs that were previously identified in studies of MSP-DNA methylation in children. We used data on MSP collected from pregnant mothers of 89 adult women born in 1959–1964 and measured DNA methylation in blood (granulocytes) collected in 2001–2007 (mean age: 43 years). Seventeen CpGs were differentially methylated by MSP, with multiple CpGs mapping to CYP1A1, MYO1G, AHRR, and GFI1. These associations were consistent in direction with prior studies (e.g., MSP associated with more and less methylation in AHRR and CYP1A1, respectively) and, with the exception of AHRR CpGs, were not substantially altered by adjustment for active smoking. These preliminary results confirm prior prospective reports that MSP influences the offspring DNA methylation, and extends the timeframe to midlife, and suggest that these effects may persist into adulthood, independently of active smoking.     

Heritability and role for the environment in DNA methylation in AXL receptor tyrosine kinase

DNA methylation in AXL, a receptor tyrosine kinase relevant in cancer and immune function, is reportedly highly heritable. We present evidence to suggest that heritability of DNA methylation in AXL is variable, dependent on population characteristics and cell type studied. Moreover, environmental exposures in utero, particularly exposure to maternal smoking, contributes to variation in DNA methylation of select CpG loci that can affect calculations of heritability. Children exposed to maternal smoking in utero had a 2.3% increase (95 % CI 0.3, 4.2) in DNA methylation in AXL, which was magnified in girls as compared to boys. These results present compelling evidence that environmental exposure to tobacco smoke during pregnancy may alter DNA methylation levels in subtle but potentially important ways, and that these changes are persistent years after birth.     

Maternal smoking during pregnancy and cord blood DNA methylation: new insight on sex differences and effect modification by maternal folate levels

Maternal smoking during pregnancy may affect newborn DNA methylation (DNAm). However, little is known about how these associations vary by a newborn’s sex and/or maternal nutrition. To fill in this research gap, we investigated epigenome-wide DNAm associations with maternal smoking during pregnancy in African American mother-newborn pairs. DNAm profiling in cord (n = 379) and maternal blood (n = 300) were performed using the Illumina HumanMethylation450 BeadChip array. We identified 12 CpG sites whose DNAm levels in cord blood were associated with maternal smoking, at a false discovery rate <5%. The identified associations in the GFI1 gene were more pronounced in male newborns than in females (P = 0.002 for maternal smoking × sex interaction at cg18146737). We further observed that maternal smoking and folate level may interactively affect cord blood DNAm level at cg05575921 in the AHRR gene (P = 5.0 × 10^?4 for interaction): compared to newborns unexposed to maternal smoking and with a high maternal folate level (>19.2 nmol/L), the DNAm level was about 0.03 lower (P = 3.6 × 10^?4) in exposed newborns with a high maternal folate level, but was 0.08 lower (P = 1.2 × 10^?8) in exposed newborns with a low maternal folate level. Our data suggest that adequate maternal folate levels may partly counteract the impact of maternal smoking on DNAm. These findings may open new avenues of inquiry regarding sex differences in response to environmental insults and novel strategies to mitigate their intergenerational health effects through optimization of maternal nutrition.     

DNA methylation in neonates born to women receiving psychiatric care

Prenatal exposure both to maternal psychiatric illness and psychiatric medication has been linked with adverse child outcomes that affect physiological, emotional and psychiatric development. Studies suggest that epigenetic mechanisms, such as DNA methylation, may facilitate these effects. In this report, we explore the association between maternal psychiatric illness and treatment during pregnancy and neonatal DNA methylation patterns in a prospectively-characterized clinical cohort of 201 dyads. Associations between the percent of umbilical cord blood DNA methylated at 27,578 CpG sites and maternal psychiatric diagnosis, symptoms and antidepressant use were evaluated by fitting a separate linear mixed effects model for each CpG site. There were no significant changes in neonatal DNA methylation attributable to maternal psychiatric diagnosis or depressive symptoms during pregnancy. Exposure to an antidepressant medication was associated with differential methylation of CpG sites in TNFRSF21 and CHRNA2 (false discovery rate < 0.05), but the average difference in methylation for both CpG sites was less than 3% between each group. The results were not specific to type of antidepressant or duration of the exposure. This study suggests that there are no large effects of maternal psychiatric illness, depressive symptoms or prenatal exposure to antidepressants on neonatal DNA methylation. Delineation of the influence of maternal psychiatric illness and pharmacological exposures on the developing fetuses has critical implications for clinical care during pregnancy.     

Aberrant 5’-CpG Methylation of Cord Blood TNFα Associated with Maternal Exposure to Polybrominated Diphenyl Ethers

Growing evidence suggests that maternal exposures to endocrine disrupting chemicals during pregnancy may lead to poor pregnancy outcomes and increased fetal susceptibility to adult diseases. Polybrominated diphenyl ethers (PBDEs), which are ubiquitously used flame-retardants, could leach into the environment; and become persistent organic pollutants via bioaccumulation. In the United States, blood PBDE levels in adults range from 30–100 ng/g- lipid but the alarming health concern revolves around children who have reported blood PBDE levels 3 to 9-fold higher than adults. PBDEs disrupt endocrine, immune, reproductive and nervous systems. However, the mechanism underlying its adverse health effect is not fully understood. Epigenetics is a possible biological mechanism underlying maternal exposure-child health outcomes by regulating gene expression without changes in the DNA sequence. We sought to examine the relationship between maternal exposure to environmental PBDEs and promoter methylation of a proinflammatory gene, tumor necrosis factor alpha (TNFα). We measured the maternal blood PBDE levels and cord blood TNFα promoter methylation levels on 46 paired samples of maternal and cord blood from the Boston Birth Cohort (BBC). We showed that decreased cord blood TNFα methylation associated with high maternal PBDE47 exposure. CpG site-specific methylation showed significantly hypomethylation in the girl whose mother has a high blood PBDE47 level. Consistently, decreased TNFα methylation associated with an increase in TNFα protein level in cord blood. In conclusion, our finding provided evidence that in utero exposure to PBDEs may epigenetically reprogram the offspring’s immunological response through promoter methylation of a proinflammatory gene.     

Adaptations of placental and cord blood ABCA1 DNA methylation profile to maternal metabolic status

In utero environmental perturbations have been associated with epigenetic changes in the offspring and a lifelong susceptibility to cardiovascular diseases (CVD). DNA methylation at the ATP-binding cassette transporter A1 (ABCA1) gene was previously associated with CVD, but whether these epigenetic marks respond to changes in the maternal environment is unknown. This study was undertaken to assess the associations between the maternal metabolic profile and ABCA1 DNA methylation levels in placenta and cord blood. Placenta and cord blood samples were obtained at delivery from 100 women including 26 with impaired glucose tolerance (IGT) diagnosed following a 75 g-oral glucose tolerance test (OGTT) between week 24 and 28 of gestation. ABCA1 DNA methylation and mRNA levels were measured using bisulfite pyrosequencing and quantitative real-time PCR, respectively. We report that ABCA1 DNA methylation levels on the maternal side of the placenta are correlated with maternal high density lipoprotein cholesterol (HDL-C) levels (r < –0.21; P < 0.04) and glucose levels 2 h post-OGTT (r = 0.25; P = 0.02). On the fetal side of the placenta, ABCA1 DNA methylation levels are associated with cord blood triglyceride levels (r = –0.28; P = 0.01). ABCA1 DNA methylation variability on both sides of the placenta are also associated with ABCA1 mRNA levels (r < –0.35; P = 0.05). As opposed to placenta, cord blood DNA methylation levels are negatively correlated with maternal glucose 2 h post-OGTT (r = –0.26; P = 0.02). In conclusion, the epivariations observed in placenta and cord blood likely contribute to an optimal materno–fetal cholesterol transfer. These in utero epigenetics adaptations may also potentially trigger the long-term susceptibility of the newborn to dyslipidemia and CVD.     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating Insulin-like Growth Factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p=0.03, and 4.9%, p=0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p=0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.     

DNA methylation in neonates born to women receiving psychiatric care

Prenatal exposure both to maternal psychiatric illness and psychiatric medication has been linked with adverse child outcomes that affect physiological, emotional and psychiatric development. Studies suggest that epigenetic mechanisms, such as DNA methylation, may facilitate these effects. In this report, we explore the association between maternal psychiatric illness and treatment during pregnancy and neonatal DNA methylation patterns in a prospectively-characterized clinical cohort of 201 dyads. Associations between the percent of umbilical cord blood DNA methylated at 27,578 CpG sites and maternal psychiatric diagnosis, symptoms and antidepressant use were evaluated by fitting a separate linear mixed effects model for each CpG site. There were no significant changes in neonatal DNA methylation attributable to maternal psychiatric diagnosis or depressive symptoms during pregnancy. Exposure to an antidepressant medication was associated with differential methylation of CpG sites in TNFRSF21 and CHRNA2 (false discovery rate < 0.05), but the average difference in methylation for both CpG sites was less than 3% between each group. The results were not specific to type of antidepressant or duration of the exposure. This study suggests that there are no large effects of maternal psychiatric illness, depressive symptoms or prenatal exposure to antidepressants on neonatal DNA methylation. Delineation of the influence of maternal psychiatric illness and pharmacological exposures on the developing fetuses has critical implications for clinical care during pregnancy.     

Potential risk of asthma associated with in utero exposure to xenobiotics

The incidence of asthma, a complex disease and significant public health problem, has been increasing over the last 30 years for unknown reasons. Changes in environmental exposures or lifestyle may be involved. In some cases asthma may originate in utero or in early life. Associations have been found between in utero exposures to several xenobiotics and increased risk of asthma. There is convincing evidence that maternal smoking and/or in utero and perinatal exposure to environmental tobacco smoke are associated with increased risk of asthma. Similar effects have been demonstrated in animal models of allergic asthma. Evidence also suggests that in utero and/or early‐life exposures to various ambient air pollutants may increase the risk of asthma although supporting animal data are very limited. A few studies have suggested that in utero exposure to acetaminophen is associated with increased risk of asthma; however, animal data are lacking. Various vitamin deficiencies and supplements during pregnancy have been studied. In general, it appears that vitamins A, C, and E have protective effects and vitamins D and B may, in some instances, increase the risk, but the data are not conclusive. Some studies related to in utero exposures to polychlorinated biphenyls and bisphenol A and asthma risk are also reported. The underlying mechanisms for an association between xenobiotic exposures and asthma remain a matter of speculation. Genetic predisposition and epigenetic changes have been explored. The developing immune, respiratory, and nervous systems are potential targets. Oxidative stress and modulation of inflammation are thought to be involved. Birth Defects Research (Part C) 99:1–13, 2013. © 2013 Wiley Periodicals, Inc.