Arch Toxicol DOI 10.1007/s00204-014-1426-0

REVIEW ARTICLE

DNA methylation alterations in response to prenatal exposure of maternal cigarette smoking: A persistent epigenetic impact on health from maternal lifestyle? Christina H. Nielsen · Agnete Larsen · Anders L. Nielsen 

Received: 25 September 2014 / Accepted: 25 November 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Despite increased awareness, maternal cigarette smoking during pregnancy continues to be a common habit causing risk for numerous documented negative health consequences in the exposed children. It has been proposed that epigenetic mechanisms constitute the link between prenatal exposure to maternal cigarette smoking (PEMCS) and the diverse pathologies arising in later life. We here review the current literature, focusing on DNA methylation. Alterations in the global DNA methylation patterns were observed after exposure to maternal smoking during pregnancy in placenta, cord blood and buccal epithelium tissue. Further, a number of specific genes exemplified by CYP1A1, AhRR, FOXP3, TSLP, IGF2, AXL, PTPRO, C11orf52, FRMD4A and BDNF are shown to have altered DNA methylation patterns in at least one of these tissue types due to PEMCS. Investigations showing persistence and indications of trans-generational inheritance of DNA methylation alterations induced by smoking exposure are also described. Further, smoking-induced epigenetic manifestations can be both tissue-dependent and gender-specific which show the importance of addressing the relevant sex, tissue and cell types in the future studies linking specific epigenetic alterations to disease development. Moreover, the effect of paternal cigarette smoking and second-hand smoke exposure is documented and accordingly not to be neglected in future investigations and data evaluations. We also outline possible directions for the future research to address how DNA methylation alterations induced by maternal lifestyle, exemplified by smoking, have direct

C. H. Nielsen · A. Larsen · A. L. Nielsen (*)  Department of Biomedicine, Bartholin Building, Aarhus University, 8000 Aarhus C, Denmark e-mail: [email protected]

consequences for fetal development and later in life health and behavior of the child. Keywords  Epigenetics · Environment · Developmental origins of health and disease (DOHaD) · DNA methylation · Smoking · Trans-generational effects

Introduction The developing fetus has potential to adapt to the intrauterine environment to minimize the adverse effects of harmful environmental exposures and to optimize growth to the given conditions. Such environmental adaptations can be beneficial in the womb, but they also represent a risk to the long-term health for the child. In this line, the widely applicable developmental origins of health and disease (DOHaD) hypothesis states that the intrauterine environment has capability to “program” the fetus by inducing subtle changes in organ functions which can pre-dispose to disease in adulthood (Barker 2007; Ganu et al. 2012; Wadhwa et al. 2009). The DOHaD hypothesis is often exemplified by a fetus in a low-nutrient environment, which as a consequence optimizes its metabolism in “anticipation” of a calorie-restricted postnatal life which will then conflict with a high-nutrient postnatal diet. The DOHaD hypothesis thereby can be used to explain the higher rates of obesity and diabetes in children born small for gestational age. In recent years, mounting evidence has been obtained to suggest that epigenetic mechanisms such as DNA methylation, histone modifications, and microRNA (miRNA) gene regulation have key functions in the DOHaD hypothesis (Wadhwa et al. 2009). In relation to the DOHaD hypothesis, it is important to note that an epigenetic response to an environmental exposure in utero will not always be detrimental but

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may represent a protective adaptation with beneficial outcome for the child in later life, potentially benefiting future generations as well. In order to unambiguously show a role of epigenetics in the DOHaD hypothesis, an association between a specific intrauterine environment and a specific epigenetic alteration has to be shown. This has been difficult to prove given the plethora of putative confounding factors of genetic, environmental, temporal and stochastic origin. Given the long latency of the development of many complex human diseases proposed to implicate the DOHaD hypothesis, and the challenge proposed by the lack of suitable longitudinal samples with high numbers of biospecimens, a proved linking of epigenetics with DOHaD remains to be firmly established (Ganu et al. 2012). Although an increasing number of association studies link epigenetics, e.g., DNA methylation to disease, only few reproducible data have emerged linking specific epigenetic alterations to specific environmental exposures, emphasizing that there is still an important piece missing in the DOHaD hypothesis (Swanson et al. 2009). In recent years, data have emerged linking the epidemiologically well-described hazardous environmental exposure “maternal smoking during pregnancy” with genome-wide and gene-specific epigenetic changes, in particular alterations in DNA methylation. Maternal cigarette smoking during pregnancy represents an environmental exposure on the fetus with potential lifelong consequences for the health status, and thus, it can be implemented in the DOHaD hypothesis (Swanson et al. 2009). The evidence that maternal smoking affects epigenetics in the fetus highlights the importance of studying the association between smoking exposure of the fetus, epigenetic alterations, and later in life increased disease risk. The World Health Organization has reported that in the Western world approximately 25 % of women above the age of 15 are smoking cigarettes (Ng and Zelikoff 2007). Despite increased awareness and legal regulations for well over three decades, in the order of 50 % of these continue to smoke during their entire pregnancy (Doherty et al. 2009). Moreover, in the developing countries, considerable increase in smoking status among females is observed. Cigarette smoke contains more than 4,000 different chemicals (Sen et al. 2007). At least 60 of these are classified as carcinogens, while other components have adverse effects on the cardiovascular, respiratory, reproductive, and nervous systems (Ng and Zelikoff 2007). In humans, several components of cigarette smoke can pass the placental barrier, including nicotine, pesticides, polycyclic aromatic hydrocarbons (PAHs), and heavy metals (Knopik et al. 2012). Notably, fetal nicotine concentrations can be as much as 15 % higher than maternal concentrations in consequence of maternal smoking during pregnancy. Epidemiological studies have documented a strong link between prenatal exposure to maternal cigarette

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smoking (PEMCS) and a variety of unwanted pregnancy outcomes (Knopik et al. 2012). Among these, perinatal complications such as preterm delivery, intrauterine growth restrictions and low birth weight are strongly associated with PEMCS (Ng and Zelikoff 2007). In addition, multiple developmental impairments (Knopik et al. 2012), several respiratory symptoms (Duijts 2012), neuropsychiatric diseases, e.g., attention deficit hyperactivity disorder (ADHD; Morris et al. 2011), various metabolic diseases (Doherty et al. 2009) and cancers (Doherty et al. 2009) are associated with PEMCS. It has been a long-term challenge for the scientific community to understand the mechanisms by which PEMCS causes these unwanted health outcomes, and in this line, it has been proposed that cigarette smoke induces stable alterations in the gene expression program in placental and fetal cells. Epigenetics is the field of science focusing on the regulation of the cellular gene expression program in communication with the cellular environment, and it is likely that epigenetics, at least in part, can explain the mechanisms linking PEMCS to diverse disease pathologies. Focusing on DNA methylation, this review describes relevant epigenetic mechanisms and the methods currently used to investigate epigenetic changes as a consequence of maternal smoking. Furthermore, we review the literature dealing with the influence of PEMCS on the offspring’s epigenome.

Concepts of epigenetics In the 1940s, Conrad Waddington introduced the term “epigenetics” in modern biology with the meaning “beyond the genetics” (Lim and Brunet 2013). Epigenetics is briefly defined as the study of stable yet reversible changes in gene expression that are not caused by changes in the sequence of DNA. The main modes of epigenetic gene regulation include histone modifications, DNA methylation and hydroxyl methylation, chromatin remodeling, noncoding RNA-mediated gene regulation including miRNA-mediated regulation, and protein prions (Maccani and Marsit 2009). Histone modifications and DNA methylation both have capabilities to modulate the chromatin structure which depends on the exact combination of DNA and corresponding histone proteins winding the DNA, and they do so through posttranslational chemical changes (Maccani and Marsit 2009). Open-structured chromatin regions are termed euchromatin and are in general transcriptionally competent, whereas heterochromatin is defined as locked chromatin structures usually transcriptionally incompetent (Jorde et al. 2009). Through such chromatin structural changes epigenetics can affect the transcriptional activity of a given DNA independent of the underlying sequence (Szyf and Bick 2013). Methionine/homocysteine

Arch Toxicol

metabolism influences trans-methylation reactions including DNA methylation, whereas DNA methylation is the action of enzymatic-mediated addition of a methyl group to cytosine nucleotides most often present in the CpG dinucleotide composition. The transfer of methyl groups is catalyzed by DNA methyltransferases, and these enzymes transfer one methyl group from the methyl donor S-adenosylmethionine onto the 5′ position of the cytosine ring (Szyf and Bick 2013). DNA methylation is a reversible process either passively or actively mediated. Genome-wide, most CpG dinucleotides are methylated but at promoter and enhancer regulatory elements a highly complex methylation picture emerges. Transcriptional regulatory ciselements composed of a high percentage of C and G-rich regions are called CpG islands and are often identified in gene promoter and/or enhancer regions (Maccani and Marsit 2009). In a common scenario, when a CpG island in the promoter region of a gene is highly methylated, i.e., “hyper-methylated”, the gene in question will be effectively silenced. On the contrary, when a given CpG island is mostly un-methylated, i.e., “hypo-methylated”, the gene will be prone to expression (Szyf and Bick 2013). The DNA methylation itself does not prompt this transcriptional repression. This is rather a result of the binding of various trans-factors, with inherent specificity for recognition of methylated cytosine within their DNA binding domain, to the methylated stretches of the DNA (Maccani and Marsit 2009). This again can result in heterochromatin formation and transcriptional repression. Notably, DNA methylation can also have regulatory importance outside the context of CpG islands, and again the general, however not consistent, functional consequence is a correlation between a higher general level of DNA methylation in specific regulatory regions and concordant gene silencing. For many genes, DNA methylation patterns are cell and tissue specific and can be highly dynamic both in a normal differentiation and developmental program and in response to environmental exposures (Szyf et al. 2008). Methylation patterns of both germ line and somatic cell lineages are established during the early period of embryonic development (Maccani and Marsit 2009). After fertilization, DNA methylation in the zygote’s genome is largely erased during the cleavage phase of development. This is followed by a de novo methylation in between the implantation and gastrulation phases to reestablish DNA methylation patterns (Knopik et al. 2012). This DNA methylation programming is not restricted to the embryo and is also present in extra-embryonic tissue, but the general DNA methylation level is higher in somatic compared to extra-embryonic lineages (Jaenisch 1997). Appropriate removal and resetting of methylation patterns are indispensable, which makes this period a critical window during which the environment can influence on the epigenetic pattern of the offspring (Maccani and Marsit

2009). This gene–environment interaction through DNA methylation can be implemented in the DOHaD hypothesis, which attempt to explain the influence of the in utero environment on the development of the offspring (Knopik et al. 2012).

Detecting alterations in DNA methylation induced by maternal smoking The studies concerning altered DNA methylation patterns as a consequence of maternal smoking which are reviewed in this paper have used a range of different methodologies. To help evaluate the derived results and comparability between studies, we here shortly describe the techniques used to quantitatively and qualitatively estimate DNA methylation levels. The early studies in the field focused on the overall level of CpG methylation without addressing specific genes and CpG sites. Immunologically based techniques such as ELISA were normally used for this purpose—either alone or in combination with methylationdependent enzymatic digestions of the genomic DNA. Alternatively, some studies used labeling of genomic DNA using radioactive marked methyl donors with subsequent quantitative measurement of the DNA-labeling capacity per genome unit, reflecting the potential number of un-methylated acceptor CpG sites available. These low-resolution methods have been used to address whether a genome is hypo- or hyper-methylated in response to maternal smoking. Other analyses directly address particular genomic CpG sites genome-wide or in selected gene sets. Such studies provide more precise information about the level of DNA methylation at a given genomic position, and directly usable used to confirm and deny suggested associations between a given exposure and an altered level of DNA methylation in a specific gene or region. Methylationdependent restriction enzyme digestions of the genomic DNA followed by Southern blotting or PCR can be used to detect methylation at a given position given that the sequence overlaps a recognition site for a relevant restriction enzyme. However, the most used methods for CpGspecific DNA methylation analyses include an initial treatment with sodium bisulfite. This treatment converts un-methylated cytosines into uracils, but maintains methylated cytosines as cytosines, hence making it possible to detect methylation at CpG sites by subsequent recognition of unmodified/modified cytosines (Tollefsbol 2011). The detection of cytosine conversion is then carried out by various methods including methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) or DNA sequencing techniques such as high-throughput pyrosequencing (Wang et al. 2013). Comparison of results from untreated to bisulfite-treated DNA will thus give

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quantitative data concerning DNA methylation status. High-throughput next-generation sequencing of bisulfitetreated DNA with subsequent alignment with results from untreated DNA or a reference genome is without a doubt the most comprehensive method currently available for determining DNA methylation patterns. However, due to high cost and technical difficulties, also at the level of bioinformatics, this procedure is still not state of art and no such studies have to our knowledge addressed genomewide DNA methylation alterations in relation to maternal smoking. As an alternative, several array-based methods have been used to address DNA methylation genome wide. The disadvantage with this methodology is that only a predetermined subset of CpGs representing CpG islands and promoter regions can be examined. The Illumina Infinium Human Methylation (IIHM) Bead Chip arrays enable sitespecific distinction of methylation of 27,000 and 450,000 CpG sites, respectively, across the human genome. After bisulfite treatment, the DNA sample is amplified and enzymatically fragmented before the application to array bead chip (Weisenberger et al. 2008). Annealing occurs during hybridization at oligomers linked to a methylated or an un-methylated bead. Afterward, single base extension of fluorescently labeled dideoxynucleotides allows for quantification of signal intensity (Weisenberger et al. 2008). DNA methylation values, termed beta values, are recorded for each locus in each sample and reported as a number between zero and one. Theoretically, a beta value of zero represents an un-methylated site, while a beta value of one indicates a completely methylated CpG (Weisenberger et al. 2008). However, a beta value is a ratio without the ability to ever reach exactly zero or one and identified beta value differences affecting beta values close to zero and one shall be very carefully evaluated due to an inherited uncertainty in this range. The Illumina Golden Gate panel is in principle similar to the IIHM arrays, but instead it analyzes only a subgroup of 1,505 CpG loci predetermined to be of particular functional interest in DNA methylation analyses. A confounder effect of the widely used techniques for DNA methylation in studies of PEMCS is that the bisulfite treatment not distinguish between DNA methylation and DNA hydroxymethylation with the latter representing an intermediate in the active mediated de-methylation process by the ten–eleven translocation methylcytosine dioxygenase TET family of proteins (Kinney and Pradhan 2013). Whereas DNA methylation normally is considered a mark for transcriptional repression if present at promoter regions, this is not the case for DNA hydroxymethylation (Kinney and Pradhan 2013). Thus, cautions should be taken for functional interpretation of PEMCS-induced DNA methylation changes using the current state of art techniques. Finally, methylated DNA immunoprecipitation (MeDIP) techniques have been developed but yet not implemented in large-scale

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PEMCS studies. MeDIP can be used at single gene level or genome wide through next-generation sequencing and is based on immunoprecipitation of specifically methylated DNA, which is pre-fragmented, and MeDIP can be used for quantification of DNA methylation over genomic regions without addressing methylation of the specific CpG sites. One advantage of MeDIP is that the technique can distinguish DNA methylation from DNA hydroxymethylation with the latter detectable by use of specific antibodies. Thus, this immunoprecipitation-based technique can be important in future analyses to separate PEMCS-induced DNA methylation from DNA hydroxymethylation.

DNA methylation changes in response to maternal smoking: an exposure affecting multiple cell types So far, human studies linking altered DNA methylation patterns to PEMCS have been focusing on placenta, umbilical cord blood or buccal cells from life–born children. However, it could also be highly relevant to study more unavailable tissues such as skeletal muscles, liver and the CNS (Suter and Aagaard 2012). Some material can be collected from abortions but ethical considerations and the limitation in sample availability importantly asks for further development of animal models and in vitro cell culture models mimicking fetal cigarette smoke exposure. Epigenetic profiles are usually considered to be very cell type and tissue-specific complicating comparisons of epigenetic studies of one tissue with studies using other types of tissue (Suter and Aagaard 2012). It has been argued that placental changes in DNA methylation will reflect changes in fetal tissues such as the brain even if the epigenetic picture revealed is not exhaustive (Lee and Ding 2012). Given that epidemiological data have pointed toward maternal smoking influencing the neuronal function in the progeny, e.g., studies have found an association between PEMCS and an increased risk of developing ADHD (Morris et al. 2011), examination of DNA methylation patterns directly in a CNS cellular background will be highly interesting. In one of the few comparative studies addressing PEMCSmediated epigenetic alterations in different tissues, Novakovic et al. compared PEMCS-induced DNA methylation changes in placental tissue, umbilical cord blood and buccal cells (Novakovic et al. 2013). PEMCS was associated with a 10 % lower level of methylation in the AhRR gene in cord blood mononuclear cells, while the level of methylation in buccal epithelium and placenta was unaffected by smoking in the same pregnancies (Novakovic et al. 2013). This study clearly indicates that in utero exposure to cigarette smoke causes different epigenetic manifestations in different tissues. The subsequent description of the literature concerning PEMCS-mediated DNA methylation

Arch Toxicol

alterations will hence be describing the findings according to tissue type, i.e., looking separately at placenta, cord blood and buccal cells. Moreover, since DNA methylations are known to be highly dynamic over the life span (Lillycrop et al. 2014), age should be considered as a very serious potential confounder in data interpretations, making analyses of different age groups difficult to compare (Suter and Aagaard 2012). We have thus focused the initial section of the review on DNA methylation analyses from fetal tissue and children at the age of six or younger as we envisage that epigenetic differences present in early life are more likely to be directly associated with the examined exposure to maternal smoking and to a lesser degree represent confounding exposures from the childhood environment and secondary epigenetic effects. Examples of results from older individuals are presented later in the paper. DNA methylation changes detected in placenta The placenta is a fetal-maternal endocrine organ responsible for maintaining and regulating pregnancy stages (Lee and Ding 2012). Throughout the in utero development of the fetus, the placenta is crucial to health, growth and survival of the fetus. The placenta not only nurtures the fetus by regulating the exchange of respiratory gases and nutrients, it also protects it from an attack by the maternal immune system and forms the blood–placental barrier in order to protect the fetus from harmful xenobiotic exposures such as maternal cigarette smoking (Maccani and Marsit 2009). The placenta can be considered an important and rather accessible record of in utero exposures and pathology. One comprehensive study using placental tissue specimens found 623 genes to be differently expressed between smokers and non-smokers (see Table  1; Suter et al. 2011). In addition, 1,024 placental CpGs were differently methylated, but of these only 38 CpG sites differed more than 10 % in methylation status also pointing at the relative modest quantitative effect expectable for DNA methylation changes in environmental exposure analyses. Among these, five CpG sites were replicated by sequencing and were corresponding to the genes PURA, GTF2H2, GCA, GPR135, and HKR1. Four hundred and thirty-eight genes showed a significant correlation between expression and CpG methylation status. Furthermore, functional pathway analysis of these genes revealed an affiliation to oxidative phosphorylation signature pathways, which is consistent with current studies demonstrating significantly increased presence of markers of oxidative damage in placental tissue among smokers (Suter et al. 2011). A significant association between PEMCS and fetal growth attenuation was observed and after correction for potential confounders such as gender, six differently methylated CpG sites were attributed to

maternal smoking-mediated birth weight reduction (Suter et al. 2011). Wilhelm-Benartzi et al. also found a correlation between maternal smoking status, placental DNA methylation levels and birth weight percentile (Table 1; Wilhelm-Benartzi et al. 2012). In this study, methylation levels of long interspersed nuclear element-1 (LINE-1) and AluYb8 repetitive elements were determined, and further a genome-wide DNA methylation analysis was applied to some of the samples. LINE and Alu repeat elements together constitute nearly one-third of the human genome, thereby acting as surrogate markers for the global DNA methylation (Wilhelm-Benartzi et al. 2012). The methylation level of AluYb8 was significantly higher among infants exposed to cigarette smoke, indicating that PEMCS induces global DNA hyper-methylation. Moreover, an increasing AluYb8 methylation level was associated with a higher degree of methylation at CpG loci located in polycomb group targets, which represents strict developmental and cell differentiation regulated genes, making methylation changes in AluYb8 a marker of altered methylation levels in these important loci (Wilhelm-Benartzi et al. 2012). Another study on both placental and cord blood samples did not find any association between Line-1 methylation and PEMCS (Table 1; Michels et al. 2011). The placental expression level of cytochrome P450-1A1 aryl hydrocarbon hydroxylase (CYP1A1), a gene known to be important for nicotine metabolism, was 4.4-fold upregulated in smokers compared to non-smokers. Consistent with the expression data, hypo-methylation of crucial CYP1A1 promoter regions was detected among smokers (Table 1) with the CYP1A1 promoter region I exhibiting a very significantly decreased level of methylation in smokers compared to non-smokers (Suter et al. 2010). Interestingly, this promoter proximal region includes a well-characterized xenobiotic response element (XRE)-mediating expression of CYP1A1 and an inverse correlation between the percentage of methylation of this region and CYP1A1 expression was described (Suter et al. 2010). PAH compounds together with nitrosamines comprise likely hazardous species in cigarette smoke. Cytochrome P450 aryl hydrocarbon hydroxylases catalyzes the conversion of PAHs into reactive hydrophilic intermediates that have the potential to form harmful DNA adducts. The binding of PAH compounds to their intracellular aryl hydrocarbon receptor (AhR) results in a nuclear AHR translocation and subsequent generation of an AhR/ARNT transcription factor complex which through epigenetic regulation can induce CYP1A1 expression. The induced CYP1A1 levels drive conversion of PAH into hydrophilic intermediates which by phase II enzymes, such as the glutathione S-transferase (GST) family, via conjugation are excreted as polar electrophiles in a detoxification process or in an alternative pathway produce DNA adducts. The pathways

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346 Cord blood samples were analyzed by bisulfite conversion and qMSP

418 Samples were pyrosequenced

527 Samples applied to IIHM 27 Bead Chip and data replication using IIHM 450 K bead chip data from whole blood. Joubert et al. (2012)

Hinz et al. (2012)

Murphy et al. (2012)

Breton et al. (2014)

AluYb8 element hypo-methylation indicating a lower level of global methylation. AXL and PTPRO genes both hyper-methylated

FOXP3 gene hyper-methylated. A low number of Tregs was significantly linked to the development of AD IGF2 gene hyper-methylated in males. The methylation level was significantly linked to gene expression Increased DNA methylation of FRMD4A and C11orf52 genes

Global DNA methylation showed an inverse dose–response relationship to serum cotinine levels Methylation status of 26 CpGs was significantly associated with PEMCS. AhRR and GFI1 genes hypo-methylated. CYP1A1 and MYO1G genes hyper-methylated AhRR gene hypo-methylated TSLP gene hypo-methylated

622 Genes were differently expressed, and 1,024 CpG sites were differently methylated. Among smokers, 438 genes showed a significant correlation between expression and methylation AluYb8 element hyper-methylation among exposed individuals. AluYb8 methylation related to methylation at CpG loci located in polycomb group targets No association was found between LINE-1 methylation and PEMCS CYP1A1 gene hypo-methylated

Results

AXL promotes anti-apoptosis, mitogenesis and cell survival. PTPRO has been linked to differentiation of neurons during gestation

FRMD4A encodes a Arf6 activating scaffolding protein. FRMD4A is hyper-methylated by BaP and associated with nicotine dependence. C11orf52 overlaps the heat shock 27-kDa protein HSPB2 gene

See above TSLP skew the immune system toward Th2 dominance, and the TSLP protein has been shown to be required in atopic dermatitis FOXP3 is essential for Treg induction and stability. Hyper-methylation of the gene causes a decrease in the number of Tregs Deregulation of IGF2 expression has been linked to overgrowth disorders and cancer

AhRR is a negative regulator of AhR, which binds xenobiotics and induces expression of CYP1A1. Novakovic et al. (2013)



CYP1A1 metabolically activates PAH compounds into reactive oxygen intermediates

Several of the 438 differentially methylated genes are affiliated to the oxidative phosphorylation signature pathways. It has been shown that markers of oxidative damage are increased among smokers The methylation level of AluYb8 is a surrogate for the global DNA methylation. Polycomb group targets are important for tissue development and differentiation –

Gene function

To avoid confounders, studies included in this table are only included if investigating DNA methylation effects of PEMCS in children beneath age of 6 years

Breton et al. (2009)

348 Samples were pyrosequenced. 272 samples were applied to Illumina Golden gate panel followed by pyrosequencing

Bisulfite treatment and MSP of 46 samples 14 + 150 Samples were applied to IIHM 27 K bead chip. Validated by MDFS

Novakovic et al. (2013) Wang et al. (2013)

Buccal cells

1,062 Samples were applied to IIHM 450 K bead chip

Joubert et al. (2012)

Cord blood Guerrero-Preston et al. (2010)

ELISA for global DNA methylation on 30 samples

319 Placental and cord blood samples were pyrosequenced qMSP and bisulfite sequencing of 34 samples

Michels et al. (2011)

Suter et al. (2010)

380 Placental samples were pyrosequenced. 184 of these were further applied to an IIHM 27 K Bead Chip

36 Matched samples were applied to gene expression arrays and IIHM 27 Bead Chips

Methods

Wilhelm-Benartzi et al. (2012)

Placenta Suter et al. (2011)

Study

Table 1  Summary of DNA methylation studies in placental, cord blood and buccal epithelium cells

Arch Toxicol

Arch Toxicol maternal smoking

CYP1A1

PAH AhR

PAH extra-cellular

chap chap

AhR

PAH PAH

DNA methylation changes detected in umbilical cord blood

chap

chap chap chap

PAH

CYP1A1 mRNA

cytoplasm nucleus PAH AhR

AhR

ARNT

ARNT

DNMT DRF

PEMCS DNA-demethylation placenta

M

M

XRE

CYP1A1-gene

TET BP AhR

ARNT

DDRF

XRE

CYP1A1-gene

nucleus extra-cellular cytoplasm

CYP1A1 mRNA CYP1A1

DNA adducts

GST

detoxification

CYP1A1

PAH BPDE

CYP1A1

CYP1A1

CYP1A1

Fig. 1  Model of placenta PEMCS DNA methylation alterations of the CYP1A1 gene integrated with the metabolic pathways of PAHs. Arrows symbolize the event flow. Cigarette smoke included chemicals such as PAHs bind to the cytosolic located and inactive transcription factor AhR. This results in dissociation of AhR and an associated chaperone protein complex which results in a nuclear translocation of AhR. In the nucleus, AHR is dimerizing with ARNT and the resulting AhR/ARNT complex binds the XRE in the CYP1A1 gene promoter with following increased CYP1A1 expression. The enhanced cytoplasmic CYP1A1 levels metabolize PAHs to epoxides such as B[a]-7,8-dihydrodiol-9,10-epoxide (BPDE) which can be detoxified through GSTs or produce DNA adducts. It can be hypothesized that PEMCS associated XRE demethylation is mechanistic mediated through a shift in DNA methylase (DNMT) and DNA demethylase (TET) enzymatic activities through TET interactions the AhR/ARNT transcription factor complex either directly or through bridging proteins (BP). Existence of independent initial functioning DNA demethylase recruitment factors (DDRF) thereby stimulating AhR/ARNT binding by epigenetic modulating the corresponding XRE in a process also including removal of DNA methylase recruitment factors (DRF) is plausible and testable. The metabolic section of the figure was inspired from Kishi et al. (2008)

are summarized in Fig. 1. In line with the observations of PEMCS-induced placental DNA methylation alterations, Suter et al. speculated that smoking-mediated hypo-methylation of the XRE in the CYP1A1 promoter may serve as an epigenetic signature for a transcriptional memory of “high” CYP1A1 expression due to an in utero exposure of maternal smoking, and that this could predispose for a lifelong generation of reactive and carcinogenic DNA adducts (Suter et al. 2010).

A significantly lower methylation level has been observed in cord blood from newborn babies prenatally exposed to maternal cigarette smoke compared to non-exposed babies (Table  1; Guerrero-Preston et al. 2010). The study compared newborn babies with high, low and very low cotinine levels in cord blood serum and found that PEMCS caused a global DNA hypo-methylation (Guerrero-Preston et al. 2010). Cotinine is an alkaloid found in tobacco and is also a metabolite of nicotine. The study was based on an earlier study, which confirmed the link between active smoking and high levels of cotinine in cord serum (Ng and Zelikoff 2007). The global DNA hypo-methylation in cord blood is contradictory to the finding in placental tissue, which is in line with other observations that epigenetic measurements not necessarily can be transmitted from one tissue type to another. However, in one study, the investigation was based on pyrosequencing of a surrogate marker, while the other performed using an ELISA-based method, making study comparison not straightforward. Epigenomewide association studies (EWAS), which represents the epigenetic pendant to the classical genome-wide association studies (GWAS), harbors the potential to identify DNA methylation changes as a response to a specific type of exposure in utero and to associate with a specific disease outcome. Few EWAS data are published concerning maternal cigarette smoking but the largest EWAS to date includes screening of 1,062 cord blood samples using the IIHM 450 K array (Joubert et al. 2012). In this study, a statistically significant association between maternal plasma cotinine and DNA methylation status at 26 CpGs representing four genes was observed (Table 1; Joubert et al. 2012). Among the 26 CpGs, eight CpGs representing hypomethylation were detected in the growth factor independent one transcription repressor (GFI1) gene; four CpGs representing hypo-methylation in the AhRR gene; four CpGs representing hyper-methylation in the CYP1A gene, and four CpGs representing hyper-methylation in the myosin 1G (MYO1G) gene. Increasing cotinine levels were associated with a significant decrease in the methylation level of AhRR in three out of four CpGs and in all CpGs of the GFI1 region. In contrast, the CYP1A1 and MYO1G CpGs all had a higher methylation level with increasing levels of cotinine (Joubert et al. 2012). AhRR is a negative regulator of AHR function which mediates induction of the CYP1A1 expression as mentioned above (Novakovic et al. 2013). See Fig. 2 for a summary. Similarly, Novakovic et al. found that PEMCS induced a 10 % lower methylation level in a specific CpG (cg05575921 on the IIHM 450 K array) in the AhRR gene (Table 1; Novakovic et al. 2013). Another genome-wide scan detected DNA methylation differences in three genes, thymic stromal lymphopoietin (TSLP),

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maternal smoking

M

M

M

M

M

AhRR-gene

XRE

XRE

AhRR-gene

AhRR

AhRR-gene

AhRR

M

XRE

AhR

AhR

persistent PEMCS induced DNA-demethylation in cord blood

DDRF

M

ARNT

BP

AhRR-gene

TET

ARNT

AhRR

AhR activation

M

M

M

maternal smoking

M

XRE

CYP1A1-gene

AhR activation

DNA adducts BPDE

PAH

CYP1A1

AhRR

detoxification

TET BP

M

XRE

M

M

PAH

CYP1A1

M

CYP1A1-gene

M

XRE

Fig. 2  Model linking PEMCS-induced DNA methylation alterations in the AhRR and CYP1A1 genes in cord blood with the PAH metabolic pathway. Arrows symbolize the event flow. PEMCS results in opposite DNA methylation alterations in the AhRR and CYP1A1 genes with hypo- and hyper-methylation, respectively. AhRR gene DNA demethylation is observed throughout the gene body, and it can be hypothesized that this is a result of either a passive demethylation event following cell divisions or an experimental testable active process in where DDRFs or the intron one located XREs mediates increased recruitment of DNA demethylase activity. The concordant increased cellular levels of the AhRR transcription repressor results in titration of the available amounts of ARNT through AhRR/ARNT dimerization with resulting decreased amounts of dimeric AhR/ARNT transcription factors (Hahn et al. 2009). For the AhR/ARNT-activated CYP1A1 gene, the shift toward

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M

BPDE

AhR

M

ARNT

AhRR

DRF

persistent PEMCS induced DNA-methylation in cord blood

detoxification

AhRR

DNA adducts

BP

CYP1A1-gene

CYP1A1-gene

DNMT

M

ARNT

AhRR

AhR

ARNT

DDRF

XRE

M

M

CYP1A1-gene

AhRR/ARNT binding exposes the CFYP1A1 gene promoter toward a shift in recruitment of demethylase activity in an abnormal ratio to DNA methylase activity and subsequent epigenetic repression of the CYP1A1 gene. We note that HDAC recruitment to the CYP1A1 gene is experimentally shown and that HDAC and DNMT recruitment can be interconnected (Hahn et al. 2009). On top of this, PEMCS potentially can direct epigenetic silencing of the CYP1A1 gene through AhRR-independent pathways to recruit DNA methylase activity to the promoter by DRFs. The resulting final increased CYP1A1 gene methylation and accordingly decreased level of cellular available CYP1A1 proteins will result in a longitudinal decreased capacity to cope with PAH chemical detoxification. Note the observed cord blood PEMCS effect on CYP1A1 gene methylation is inversely correlated to placenta

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glutathione S-transferase theta 1 (GSTT1) and cytochrome b5 reductase 3 (CYB5R3) among newborns prenatally exposed to maternal cigarette smoke compared to nonexposed (Table 1; Wang et al. 2013). However, only a smoking-induced decrease of the DNA methylation level in the TSLP gene displayed significance by replication. This DNA methylation alteration was observed in a CpG island in the 5′-region of the TSLP gene, and an inverse correlation was observed between the degree of TSLP DNA methylation and both cotinine levels and TSLP expression. Development of atopic dermatitis (AD) is in epidemiological studies shown to be associated with maternal smoking status during pregnancy, and interestingly, the TSLP 5′-CpG island methylation level was significantly lower in AD children than in children without the disease (Wang et al. 2013). The TSLP protein can activate dendrite cells that promote differentiation of naïve T cells to Th2 cells and thereby functional skew the immune system. Moreover, TSLP may activate Langerhans cells in the epidermis and induce production of pro-inflammatory cytokines and thereby also potentially be involved in AD. Thus, a smoking-induced DNA hypo-methylation of the TSLP gene might be in direct association with the AD risk. Another gene found to be associated with maternal smoking and AD is the forkhead/winged-helix family of transcriptional regulators member FOXP3 (Table 1; Hinz et al. 2012). PEMCS was observed to cause a decrease in the number of cord blood regulatory T lymphocytes (Tregs) concordant with a FOXP3 gene hyper-methylation in the treg-specific demethylated region (TSDR). Hypo-methylation of the TSDR is required for correct expression of the FOXP3 transcription factor and Treg functionality (Hinz et al. 2012). Very interestingly, in line with these observations, we note that also AhR is also involved in immune responses (Hao and Whitelaw 2013). Activation of AhR can lead to immunosuppression in a mechanism which potentially skew the Th1/Th2 balance toward Th1 dominance and thereby a boosted Treg cell differentiation (Hao and Whitelaw 2013). In addition, AhR activation can induce Th17 cell polarization and the severity of autoimmune diseases (Hao and Whitelaw 2013). How activation of the AhR pathway by PEMCS influences the immune system in utero and after birth remains to be clearly elucidated. Finally, the DNA methylation level of the gene expression regulating differentially methylated region (DMR) of the Insulin-like Growth Factor 2 (IGF2) gene has also been associated with PEMCS, and with exposure induced IGF2 DNA methylation differences only significant in males (see also below; Murphy et al. 2012). The study showed that infants born to smokers displayed higher DMR methylation levels and a lower degree of IGF2 expression than those born to never smokers or those born to mothers quitting smoking during the pregnancy (Murphy et al. 2012). Thus, like described

for the AhRR gene an early maternal response during pregnancy by quitting smoking seems to eliminate acquirement of at least some PEMCS-induced IGF2 DNA methylation alterations in the progeny. It was estimated that for every 1 % decrease in DMR methylation an approximately twofold increase in expression was obtained (Murphy et al. 2012). The IGF2 gene DMR DNA methylation and expression analysis are consistent with several other studies showing that the DNA methylation status of the DMR is functionally sensitive to environmental exposures (Suter et al. 2013). The IGF2 gene is an imprinted gene in which DMR DNA methylation is involved silencing the maternal allele resulting in only the paternal allele to be expressed. Correct IGF2 gene regulation is an essential determinant for correct fetal growth and IGF2 deregulation is associated with human diseases such as obesity, metabolic disorder, imprinting disorders and cancer (OMIM 147470). DNA methylation changes detected in buccal cells In buccal cells, measurements on the repetitive element AluYb8 showed a significantly lower level of DNA methylation among children prenatally exposed to maternal cigarette smoking compared to non-exposed children (Table 1; Breton et al. 2009). For LINE1, no significant change in DNA methylation was observed between exposed and nonexposed children (Breton et al. 2009). The AluYb8 result is contradictory to the findings in placental tissue, which used the same detection method, but similar to the results in cord blood serum, which used another methodology; again suggesting that PEMCS causes different epigenetic manifestations dependent on tissue type. In the same study, the AXL and PTPRO genes were identified to have PEMCS-specific DNA methylation alterations using first a genome-wide analysis based on microarrays followed by replication by pyrosequencing. The AXL and PTPRO genes showed significantly higher methylation status, 0.37 and 0.34, respectively, in smoking exposed children (Breton et al. 2009). The examined CpG site in the AXL gene was located in the promoter region at position −223 and is part of the core promoter region described including a Sp1/Sp3 transcription factor binding site. The gene expression of AXL has been correlated with DNA methylation levels in this Sp1/Sp3 transcription factor binding site supporting a functional role of the DNA methylation status. The AXL protein is a receptor tyrosine kinase which promotes antiapoptosis, mitogenesis, cell invasion, and cell survival. The differentially methylated site in the PTRPO gene is located in position −371 in the promoter region, and again PTPRO gene expression has been linked with DNA methylation levels (Breton et al. 2009). The PTPRO protein is a protein tyrosine phosphatase receptor. Importantly, in relation to disease phenotypes, e.g., ADHD associated with PEMCS,

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the PTPRO protein is linked to differentiation and axonogenesis of central and peripheral nervous system neurons during gestation (Breton et al. 2009).

Persistence and heredity of smoking‑induced epigenetic changes? It is not clear whether the alterations in DNA methylation described in response to maternal smoking are adaptive changes proving to be beneficial later in life, or whether the changes are merely functionally neutral biological biomarkers, or perhaps even detrimental to the health of the affected children later on. Moreover, it is still not known if epigenetic changes induced by PEMCS are transient or persistent. In other words, does an alteration in DNA methylation persist throughout life, or will the methylation pattern return to the baseline existing in non-exposed individuals? These are interesting questions, which have not yet been fully answered, maybe because of the comprehensive nature of such investigations. Optimally, these kinds of studies would include an assessment of the DNA methylation of a specific gene in a specific type of tissue at the fetal stage followed by frequent assessments of the methylation level in the same gene, the same type of tissue, and in the same individuals throughout life. The DNA methylation and de-methylation machineries remain active and dynamic throughout life, supporting the hypothesis that DNA methylation patterns might change later in life (Szyf et al. 2008). Persistence of cigarette smoke induced epigenetic changes Only a couple of studies have specifically addressed whether maternal smoking-induced DNA methylation alterations can be persistent throughout childhood. Novacovic et al. found that a significant difference in mean methylation of the AhRR gene between children prenatally exposed to cigarette smoke and non-exposed individuals had remained throughout the first 18 months of their lives (see Table 2; Novakovic et al. 2013). This suggests that PEMCS causes persistent alterations in the methylation status of the AhRR gene. From this study, an additional important notion is that even if the progeny was not exposed to household smoking in the postnatal environment persistent differences in AhRR DNA methylation were still present, hence supporting the fact that such differences are due to smoking exposure in utero and not solely induced by continued second-hand exposure to smoking after birth. A disadvantage of this study of persistence is that the analyses included only a few types of tissue sampled from a small study population of just a few individuals (Novakovic et al. 2013). To date, the association between tobacco smoking exposure and DNA

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methylation status within the AhRR gene remains the most convincing example of the relationship between a specific defined environmental exposure and persistent differences in DNA methylation (Novakovic et al. 2013). In this line, a study among adult smokers and nonsmokers showed that the AhRR gene DNA methylation status in blood of previous smokers approaches the levels of never smokers within the first years of quitting smoking, but it never reaches normal levels and remains 3–4 % lower (Zeilinger et al. 2013). AhR binds to numerous xenobiotics including nicotine and caffeine, and AHR induces the expression of CYP1A1 and other genes involved in the removal of toxic chemicals (Figs. 1, 2; Hahn et al. 2009). AhR also participates in other cellular pathways including cell cycle control mechanisms. AhRR-mediated repression of AHR requires binding of AhRR to the protein AhR nuclear translocator (ARNT), a dimerization partner of AhR for mediating gene activation (Hahn et al. 2009). Thus, it can be envisaged that increased AhRR expression, obtained through hypo-methylation of specific CpGs in the gene as a consequence of maternal smoking, could result in an attenuation of the cellular response toward smoking thus affecting PAH detoxification throughout life. In the study by Joubert et al., it was shown that maternal smoking-induced divergent DNA methylation alterations of the AhRR and CYP1A1 genes, hypo- and hyper-methylation, respectively, indicating that a concordant increase in AhRR expression and decrease in CYP1A1 expression can be involved in the cellular response toward smoke exposure (Joubert et al. 2012). It has not yet been directly shown that an increased AhRR expression results in decreased CYP1A1 expression. Notably, the DNA hypomethylations observed in the AhRR gene resides in CpGs within the gene body where the regulatory consequence of DNA methylation remains unclear and the functional consequences for the particular modifications are indeed not yet solved. But the association between hypo-methylation of such CpGs and increased AhRR expression observed in blood samples from adult smokers supports a functional role of these CpGs in the AhRR gene body. In Fig. 2, we present a model for the PEMCS epigenetic changes in the AhRR and CYP1A1 genes and the interconnection. The contrast between umbilical cord blood samples showing an increased CYP1A1 methylation in response to PEMCS and the findings by Suter et al. (2010) who reported a decrease in CYP1A1 methylation in placental tissue underlines the complexity involved in epigenetics. A hypothesis could be that different tissues cope with environmental exposures differently where the placenta mediates an immediate defense response by accumulating and detoxifying of PEMCS-related exposures including PAH chemicals as part of the blood-placenta barrier. The fetus, on the other hand, seems, in accordance with the DOHaD hypothesis, to display an adaptive response to the toxic environment in utero

PEMCS was significantly associated with decreased Sat2 methylation Peripheral blood cells from women at the age of 38–48 years old were bisulfite sequenced Flom et al. (2011)

Replication of IIHM 450 K bead chip data from whole blood (mean age 9 years) in cord blood-derived IIHM 450 K bead data

Prenatally exposed individuals showed a fourfold increased level of DNA methylation in the BDNF-6 promoter compared to the non-exposed A modest increase in DNA methylation of FRMD4A and C11orf52 Toledo-Rodriguez et al. (2010) Breton et al. (2014)

Bisulfite pyrosequencing of cord blood cells in six newborn twin pairs. A novel bisulfite pyrosequencing after 18 months, but this time in peripheral blood mononuclear cells Blood cells from 78 adolescents were bisulfite sequenced Novakovic et al. (2013)

A significant difference in mean methylation of AHRR had remained between exposed and non-exposed children

Methods Study

Table 2  Summary of studies directing the persistence of PEMCS-induced DNA methylation alterations

Results

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all though it remains to be experimentally tested whether PEMCS mediates hyper-methylation of CYP1A1 in the fetus or this is a secondary effect arising after birth. Moreover, the exact molecular mechanisms linking PEMCS with DNA methylation alterations need resolution. In a study of adolescents, individuals in utero exposed to smoking had a fourfold higher level of DNA methylation in the promoter six of the alternative spliced brain-derived neurotropic factor (BDNF) gene. This could indicate that PEMCS might lead to a long-term functional down-regulation of this BDNF isoform (Table 2; Toledo-Rodriguez et al. 2010). In addition, an inverse association between PEMCS and Sat2 methylation was observed in middle-aged women, even after adjustment for environmental tobacco exposure during childhood and adult smoking status (Table 2; Flom et al. 2011). A disadvantage of this type of studies is that the DNA methylation level was only measured at a single point relatively late in life. Hence, the observed differences in DNA methylation levels could just as well have emerged after birth due to other environmental exposures or as secondary effects to other epigenetic alterations occurring in utero. In this line, we note that decreased methylation of the IGF2 gene has been observed 60 years after exposure in adults exposed in utero to the Dutch famine during World War II, indicating that at least some in utero environmental exposures have the ability to cause persisting DNA methylation alterations (Suter et al. 2013). A very recent study further supports persistence of PEMCS-induced DNA methylation alterations. Breton et al. presented DNA methylation data using the IlHM27 K array and whole blood from 527 individuals, ages 5–12, from a population of asthmatic patients (Breton et al. 2014). PEMCS is known to be associated with an increased risk of developing asthma (Duijts 2012). Nineteen loci were identified to have PEMCS associated DNA methylation patterns, and two of these loci were replicated in additional sample sets including a collection of umbilical cord blood samples. The two loci, FRMD4A and C11orf52, both had modest (2 %) increase in methylation by PEMCS. The replicative identification of a similar set of PEMCS-induced DNA methylation alterations in umbilical cord blood and in children ages 5–12 supports that at least some of these DNA methylation alterations can be persistent throughout childhood (Breton et al. 2014). In years to come, it will be interesting to monitor the persistence of PEMCS-induced DNA methylation alterations in a much more longitudinal perspective and for a higher number of genes. Heredity of cigarette smoke induced epigenetic changes Persistence of epigenetic changes throughout the entire body and during life is not a prerequisite to heredity if, e.g., gametic cells have epigenetic alterations at the time of

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conception, for other factors need to be considered in transgenerational considerations of epigenetic inheritance. A pregnant woman (F0 generation) not only carries her fetal offspring (F1) within, but also the primordial germ cells of the third generation (F2), which are lying within the F1 fetus (Curley et al. 2011). Thus, an environmental exposure such as PEMCS not only directly affects the epigenome of the pregnant woman herself and her fetal offspring, but also the epigenome of her grandchild (F2). To demonstrate that any PEMCS-induced epigenetic change is inherited transgenerationally, the change must be identified in the first non-exposed generation, which would be the F3 generation (Curley et al. 2011) As described earlier, the methylation in the zygote’s genome is almost completely reestablished during embryogenesis (Knopik et al. 2012). Nevertheless, transgenerational inheritance has been observed in several species and for an increasing number of traits (Lim and Brunet 2013). In humans, some classes of genes have shown the capacity to maintain an altered methylation across generations despite of the reprogramming (Curley et al. 2011). However, to our knowledge, no human studies have identified epigenetic changes in fourth generation caused by PEMCS. Recently, Joubert et al. addressed trans-generational inheritance of smoking-induced DNA methylation alterations taking basis in previously presented cord blood DNA methylation data (Joubert et al. 2012, 2014). They questioned the presence of association between pre-conceptional cessation of maternal or paternal smoking and offspring DNA methylation and if there is any association between grandmothers smoking during pregnancy and grandchild DNA methylation status independent of maternal smoking status. The presented findings did not support epigenetic trans-generational inheritance within the 26 CpG sites examined and which were predetermined to have most significant altered DNA methylation status as a consequence of smoking exposure (Joubert et al. 2012, 2014). Furthermore, it was examined whether throughout pregnancy sustained PEMCS is required for offspring DNA methylation alterations with the latter supporting the in utero effect (Joubert et al. 2012, 2014). The results showed that a sustained exposure through at least 18 weeks in pregnancy was identified to be required for DNA methylation alterations to be significantly present at time of birth. In this line, Novakovic et al. identified no evidence that smoking during first trimester followed by cessation is sufficient to induce DNA methylation differences that are measurable at time of birth (Novakovic et al. 2013). These results are in contrast to the general assumption that early pregnancy represents the most sensitive period for environmental induced epigenetic changes. In conclusion, at present, the published results support that smoking-induced differential DNA methylation patterns will reflect and require a sustained in utero exposure rather than reflect a trans-generational epigenetic inheritance of pre-determined DNA methylation patterns.

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Additional considerations and studies directing the association between PEMCS and DNA methylation alterations A possible confounder in studies addressing the association between maternal smoking during pregnancy and epigenetic alterations in the child is the reliability of selfreported smoking status during pregnancy. Most, if not all, pregnant women are well aware of the fact that smoking may be harmful to the child hence understating or denying their smoking habits. Determination of plasma cotinine concentrations, a biomarker for tobacco smoke, has been used as an additional direct measure for the maternal smoking status (Joubert et al. 2012). Using this method, Shipton and co-workers found that one in five smoking women fail to report their smoking habits when asked, and later studies support these findings (Dietz et al. 2011; Shipton et al. 2009). Since mothers smoking during pregnancy usually continue after birth, another confounder is the continued exposure from household smoking throughout childhood. Similarly, exposure of the fetus of a non-smoking woman through household smoking also represents a potential confounder when assessing the consequences of PEMCS. Second-hand smoking has some of the same health consequences as direct smoking exposure and it may thus be difficult to separate the consequences of first- and secondhand maternal smoking (Knopik et al. 2012). In one of the few studies addressing this, Guerrero-Preston et al. investigated the global DNA methylation level in umbilical cord blood, ranging the global DNA methylation level according to the maternal smoking status during pregnancy as non-smoker, second-hand smoker or active smoker. It was observed that, compared to newborns with non-smoking mothers living in a non-smoking environment, newborns exposed to second-hand smoking from the household during pregnancy displayed a significantly lower level of global DNA methylation, all though an even lower DNA methylation level was observed in the infants exposed to maternal cigarette smoking in utero (Guerrero-Preston et al. 2010). This indicates that maternal smoking and passive smoking exposure from the household to some extend may have similar impact on the progeny DNA epigenome. Thus, second-hand smoke exposure during pregnancy as well as exposure from persisting household smoking during childhood should be accounted in future epigenetic analyses (Suter and Aagaard 2012). This review focuses on the epigenetic effects of maternal cigarette smoking during pregnancy, but the paternal smoking status might also influence the epigenome of the child in paths besides second-hand exposure described above. Epidemiological studies have shown that paternal exposure to PAHs is strongly related to childhood leukemia and

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greater BMI of the offspring (Soubry et al. 2014). In addition, cigarette smoke has been shown to induce changes in gene expression of human spermatozoa, indicating a possible epigenetic mechanism for the consequences of paternal cigarette smoking (Soubry et al. 2014). While the mother through her smoking habit possibly affect the offspring epigenetically both before and during pregnancy, the paternal smoking status only impacts on the offspring before conception, at least ignoring the possible effects mediated by second-hand smoke exposure. An increasing number of animal studies confirm that paternal pre-conceptional insults affect the epigenome of the offspring and several epigenetic windows of susceptibility to environmental effects have been suggested (Soubry et al. 2014). The first window of susceptibility is during the embryonic development, where the primordial germ cells undergo genome-wide epigenetic erasure and reprogramming of DNA methylation patterns. Other windows are the paternal pre-puberty and spermatogenesis (Soubry et al. 2014). Of particular interest for this review are epidemiologic studies showing that paternal initiation of smoking in pre-puberty is associated with increased risk of metabolic diseases for grandsons (Pembrey et al. 2006). Gender specificity is observed here, since the effect for granddaughters is non-significant. If and how these consequences of heredity of paternal smoking are mediated by specific alterations in DNA methylation still awaits clarification. Thus, even though only a sparsely number of studies have investigated the influence of paternal cigarette smoking on the human offspring, the available data indicates epigenetic mechanisms to participate in mediating the health risk consequences in progeny of paternal cigarette smoking. Related to the gender-specific effect of exposure to smoking exposure described above, Murphy et al. found a gender-specific increase in the methylation level of the transcriptional regulatory region of the IGF2 gene (Murphy et al. 2012). When stratified by gender of the infant, only males exposed to maternal cigarette smoking in utero showed a significantly different methylation level when compared to non-exposed infants, whereas no differences were found in females (Murphy et al. 2012). The opposite was observed in a study investigating the DNA methylation level of the AXL gene promoter (Breton et al. 2011). In this study, buccal cells from 173 children at the age of 11 were analyzed by MSP and pyrosequencing. The children exposed to maternal smoking during pregnancy showed a 2.3 % increase in DNA methylation, but when stratified by gender no effect was seen in the males, while the females showed a 3.4 % increase (Breton et al. 2011). No gender specificity was observed in the DNA methylation of AhRR between infants exposed to maternal smoking and infants of non-smoking mothers (Novakovic et al. 2013). The results indicate that for some genes, progeny

gender act as a modifier toward the status of DNA methylation difference induced by PEMCS, and this should be an included variable in future PEMCS DNA methylation studies. The AXL gene, like the IGF2 gene, is imprinted and the expression is maternal transmitted and dependent on DNA methylation status of the gene (Breton et al. 2011). It is an open question whether imprinted genes are particular prone to environmental exposures because the particular cellular functions of this type of genes are driving force for DNA methylation alterations and/or because only a single allele is active. In the latter scenario, any environmentalinduced DNA methylation change will have greater impact on gene expression (Breton et al. 2011). Further investigations will be needed to elucidate the biological mechanisms linking gene imprinting with environmental exposures like PEMCS and disease associations identified by epidemiologic studies.

Conclusion remark Many publications from the recent years have taken basis in the DOHaD hypothesis and carefully examined the proposed link between PEMCS, epigenetic changes and diverse pathological conditions. In placenta, umbilical cord blood, whole blood and buccal epithelium tissue, PEMCS has been shown to cause differences in the global DNA methylation pattern (Breton et al. 2009; GuerreroPreston et al. 2010; Wilhelm-Benartzi et al. 2012). However, in placental tissue, PEMCS was shown to result in hyper-methylation, while hypo-methylation was observed in the other tissues, suggesting a different manifestation of PEMCS dependent on tissue type. Furthermore, genespecific significant alterations in DNA methylation patterns were observed for CYP1A1, AhRR, FOXP3, TSLP, IGF2, AXL, PTPRO, C11orf52, FRMD4A, and BDNF genes in at least one of these tissues among individuals exposed to maternal cigarette smoke when compared to non-exposed (Breton et al. 2009; Hinz et al. 2012; Joubert et al. 2012; Murphy et al. 2012; Novakovic et al. 2013; Suter et al. 2010; Wang et al. 2013). Low birth weight and atopic dermatitis are among the health outcomes related to both PEMCS and identified alterations in DNA methylation patterns (Hinz et al. 2012; Suter et al. 2011; Wang et al. 2013; Wilhelm-Benartzi et al. 2012). Interestingly, two of the presented placental studies indicated that PEMCS induces increased levels of reactive oxygen species, making this a potential focus for future studies (Suter et al 2010, 2011). Development of in vitro cellular model systems for efficient measuring the functional effect of PEMCS-induced DNA methylation alterations is urgently needed. For this could be used relevant fetal-derived primary cell lines exposed to nicotine/cotinine and benzo[a]pyrene (BaP),

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a common polycyclic aromatic hydrocarbon presents in cigarette smoke, followed by DNA methylation and gene expression analyses as well as ectopic-induced epigenetic modulation by targeting epigenetic modifiers, i.e., through the Cas9/CRISPR-system. Few reports have addressed the persistence of PEMCS-induced DNA methylation changes as well as the question of trans-generational heritability. Such analyses will need a future substantiation. Together with the functional characterization of identified DNA methylation alterations, this could be the initial important step to clearly correlate specific DNA methylation changes to the increased risk and pathology of specific diseases associated with PEMCS and subsequently developing of diagnostic epigenetic biomarkers and eventual novel treatment protocols. Some studies have showed gender specificity for PEMCS-induced DNA methylation alterations, suggesting future studies to consider the distribution of gender within the study population (Breton et al. 2011; Murphy et al. 2012). Other potential confounders are second-hand smoke exposure and paternal smoking habitude in pre-conception timing. Current estimates suggest that more than 126 million non-smoking adults are exposed to second-hand smoke, and pregnant women are no exception (Knopik et al. 2012). Second-hand exposure to cigarette smoke has been found to cause the same epigenetic effects in the newborn babies as PEMCS, though to a lesser extent, thus indicating that second-hand smoke exposure should be considered as a serious confounder (Guerrero-Preston et al. 2010; Suter and Aagaard 2012). In this review, we have highlighted the focus the scientific community has placed on investigations concerning the influence that maternal smoking during pregnancy has on the DNA methylation in progeny. Whereas epigenetic data for miRNA expression and concordant downstream gene regulation now also are emerging (Herberth et al. 2014; Maccani et al. 2010), investigations of histone modifications and additional epigenetic pathways are only sparsely described. It will be important to substantiate such epigenetic analyses to have a more comprehensive picture of the association between maternal cigarette smoking during pregnancy, specific epigenetic alterations acquired by the progeny, and the subsequently increased risks for specific diseases. Acknowledgments  This work was supported by The Lundbeck Foundation, Dines Hansens Legat, and Læge Sofus Carl Emil Friis og hustru Olga Doris Friis Legat.

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DNA methylation alterations in response to prenatal exposure of maternal cigarette smoking: A persistent epigenetic impact on health from maternal lifestyle?

Despite increased awareness, maternal cigarette smoking during pregnancy continues to be a common habit causing risk for numerous documented negative ...
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