Long-Term Effects of Chromatin Remodeling and DNA Damage in Stem Cells Induced by Environmental and Dietary Agents

Abstract

The presence of histones acts as a barrier to protein access; thus chromatin remodeling must occur for essential processes such as transcription and replication. In conjunction with histone modifications, DNA methylation plays critical roles in gene silencing through chromatin remodeling. Chromatin remodeling is also interconnected with the DNA damage response, maintenance of stem cell properties, and cell differentiation programs. Chromatin modifications have increasingly been shown to produce long-lasting alterations in chromatin structure and transcription. Recent studies have shown environmental exposures in utero have the potential to alter normal developmental signaling networks, physiologic responses, and disease susceptibility later in life during a process known as developmental reprogramming. In this review we discuss the long-term impact of exposure to environmental compounds, the chromatin modifications that they induce, and the differentiation and developmental programs of multiple stem and progenitor cell types altered by exposure. The main focus is to highlight agents present in the human lifestyle that have the potential to promote epigenetic changes that impact developmental programs of specific cell types, may promote tumorigenesis through altering epigenetic marks, and may be transgenerational, for example, those able to be transmitted through multiple cell divisions.

I. INTRODUCTION

A. Chromatin Remodeling and Epigenetics

Epigenetics is the study of heritable changes in gene expression without a change in the DNA sequence. Nucleosomes are composed of 147 base pairs of DNA wrapped around core histone proteins H2A, H2B, H3, and H4.^1–3 Histone H1 acts to link histones together and to the nuclear scaffold. H3 and H4 termini extend from the nucleosome and can be modified chemically by acetylation, methylation, ubiquitination, phosphorylation, sumoylation, citrullination, and ADP-ribosylation.³ Modifications promote either open or closed chromatin,³ which in turn influences multiple cellular processes such as transcription and replication.^4–6 Chromatin remodeling is also interconnected with the DNA damage response, maintenance of stem cell properties, and cell differentiation programs. Patterns of histone modification are maintained after replication and thus inherited through multiple cellular generations. DNA methylation predominantly involves the covalent addition of a methyl group (CH3) to cytosine in the context of CpG in DNA, creating a significant epigenetic marker of transcriptional inactivity. Patterns of DNA methylation are generated during development involving de novo methylation and demethylation mediated by DNA methyltransferases (DNMTs). DNMT3 regulates de novo methylation during development, and DNMT1 maintains DNA methylation patterns following replication. Global genome methylation patterns are highly specific depending on developmental stage and type of tissue.

This review focuses on commonly used environmental and dietary compounds previously known to have or suspected of having carcinogenic or mutagenic properties but recently identified as having the potential to disrupt chromatin remodeling and epigenetic regulation in stem cells, induce long-term changes in developmental programs, and promote tumorigenesis. Furthermore, we highlight recent reports that have shown that in utero exposure to these compounds can also promote epigenetic modifications that in turn induce gene expression changes that persist throughout life.^7,8

B. The Connection Between Epigenetic Alterations and DNA Double-Strand Breaks

The DNA double-strand break response (DDR) is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell cycle checkpoints, and multiple enzymatic activities to repair broken ends of DNA. DNA double-strand breaks (DSBs) have the highest potential to promote illegitimate repair mechanisms and the accumulation of mutations and are considered the critical primary lesions in the formation of chromosomal rearrangements associated with disease and tumorigenesis. Recent advances in the understanding of the interplay between chromatin remodeling, epigenetics, and the DDR have been reviewed.^9–11 New emerging evidence extends earlier findings with the potentially pathological repercussions of restoring chromatin structure, resulting in a DSB-induced epigenetic memory of damage.

Chromatin dynamics and changes in chromatin architecture that occur for repair of DSBs include nucleosome eviction from DSBs, relaxation of heterochromatin structure, and localized chromatin destabilization at DSBs.¹² After DNA damage, chromatin structure is altered by adenosine triphosphate–dependent chromatin remodeling, the incorporation of histone variants into nucleosomes, and modifications to covalent histones. These histone modifications include phosphorylation of H1; acetylation of H2A and phosphorylation and ubiquitination of H2AX; acetylation and methylation of H3; and phosphorylation and acetylation of H4.³ Among the different histone modifications, phosphorylation of all 4 histones as well as the variant H2AX plays a primary role in DNA damage response by facilitating the access of repair proteins to DNA breaks.^13,14 Phosphorylation of H2AX (γH2AX) spreads over large chromatin domains away from a DSB. This chromatin marking and large-scale chromatin reorganization recruits repair factors, recombination proteins, and chromatin remodeling complexes involved in DNA repair pathways.¹⁵

Heterochromatin is the tightly compacted DNA structure that acts as a barrier to DNA repair processes. As a result, heterochromatic (HC) DSBs are generally repaired more slowly than euchromatic DSBs,¹⁶ and heterochromatin and euchromatin use distinct remodeling complexes and pathways for DSB repair.^17–21 DSB repair may be stalled within HC regions if a series of dynamic and localized changes fail to occur.^18,22,23 The ataxia telangiectasia mutated protein and DDR mediator proteins overcome constraints posed by heterochromatin superstructure to promote repair through the modulation of 2 HC factors: KAP-1 corepressor and HP1 chromodomain protein.¹⁹ Activation of the ataxia telangiectasia mutation signaling pathway and the subsequent phosphorylation of KAP-1 trigger HC modifications required for DSB repair.²⁴ In addition, studies have shown that histone acetyltransferase complexes act with the adenosine triphosphate–dependent switch/sucrose nonfermentable and remodel structure of chromatin (RSC)-containing chromatin remodeling complexes to facilitate DNA repair.²⁵

Polycomb group (PcG) proteins, which have well-established roles in gene regulation, were recently found to accumulate on chromatin surrounding DNA damage.²⁶ PcG proteins form complexes involved in the epigenetic regulation of gene expression. PcG repressive complexes catalyze posttranslational modifications critical to their gene silencing function, including histone H3K27 di- and trimethylation (H3K27me2, H3K27me3) and histone H2A ubiquitination.^27–29 PcG and polycomb recessive complex (PRC) components found to respond to DNA damage include BMI-1, MEL-18, EZH2 methyltransferase, EZH1, EED, and SUZ12,³⁰ suggesting that DNA methylation modifications occur as part of the DDR. Recruitment of PcG protein BMI1 promotes mono-ubiquitination of H2A and DNA DSB repair.³¹

Signal transduction pathways in DDR communicate with chromatin remodeling factors through protein-protein interactions. The chromatin remodeling protein scaffold matrix attachment region 1 (SMAR1) binds other SMAR1 elements along with histone deacetylase (HDAC) 1 and p53 to form a repressor complex to downregulate transcription.^32,33 The chromatin remodeling factor Tip49 recruits Rad51, the homolog of bacterial RecA and major homologous recombination repair protein, to DNA damage sites.³⁴ In cells depleted of Tip49, Rad51 recruitment can be restored by addition of an HDAC inhibitor, underscoring the interplay of epigenetic markers and DDR. In addition to DNA repair, the p53 signaling pathway is associated with chromatin changes that mainly involve the histone acetyltransferase Tip60 to modulate the fate of a cell between cell cycle arrest and apoptosis. ³⁵ Numerous chromatin remodeling factors involved in DNA methylation and demethylation also play a role in DDR. In the thymus, genotoxic stress decreases global DNA methylation by a reduction in DNMT1, DNMT3a, DNMT3b, and methyl-binding proteins MeCP2 and MBD2.³⁶ That these alterations are carried into the offspring of exposed individuals led the authors to suggest profound epigenetic dysregulation, which in turn could lead to genome destabilization and possibly serve as a precursor for transgenerational carcinogenesis.³⁶

C. Stem Cells and Induced Pluripotent Stem Cells

Stem cells are the focus of study in many laboratories because of their unique properties of self-renewal and pluripotency that allow for both maintenance of the stem cell pool as well as generation of various cell types through differentiation. Induced pluripotent stem cells (iPSCs) have allowed for the study of the developmental processes of multiple tissue and organ systems.^37–40 Stem cell models have been used in drug discovery and toxicology screens and the development of biomarker panels. ^41–43

Because stem cells give rise to all of the mature cells in an organism, they have become a promising source for potential replacement therapies in the treatment of diseases,⁴⁴ although both genetic integrity and a “correct” epigenome will be important factors in both their experimental and therapeutic usefulness. Extensive literature exists analyzing transcriptional and proteomic profiles as well as the chromatin modification profiles of stem cells, embryonic stem (ES) cells, iPSCs, and differentiated cell types to create signatures of “stemness.” ^45–61

The genomic instability of human ES cells and iPSCs in vitro has been reported. Common abnormalities of ES cells are gains of chromosomes 12, 17, and 20.^62–67 Chromosome 12 contains genes implicated in cell survival, such as STELLAR, GDF3, and NANOG, thus likely giving cells a proliferative advantage. It is interesting to note that trisomy of chromosome 12 is one of the most commonly described mosaicisms in amniocentesis⁶⁸ and is characteristic of germ cell tumors in almost all cases.⁶⁹ A gain of chromosome 17 is similarly associated with tumorigenesis, specifically breast cancer,⁷⁰ and amplifications of chromosome 20 are observed in a wide variety of tumors.^71,72 The gain of chromosome X in stem cells cultured in vitro⁷⁰ is associated with trisomy 17 as well, although it is unclear whether this provides a proliferative or survival advantage; a loss of chromosome X also has been reported in vitro.⁶⁴ The fact that these chromosomal abnormalities are common in both cultured stem cells and in multiple in vivo settings suggests that stem cells may be particularly susceptible to specific alterations. iPSCs have shown copy number variations, an abnormal karyotype, and point mutations.⁴² In addition, they have DNA methylation and histone modification defects and altered X chromosome inactivation.⁴² Some studies have indicated that iPSCs may “remember” their previous somatic cell fate as stored within the epigenome,^49,72,74 reducing their experimental and therapeutic potential.

Transcriptional regulation and gene expression patterns occur in the context of chromatin. In general, stem cells have epigenetic signatures that are characteristic of an active chromatin state; for example, chromatin is generally decondensed. Chromatin is reorganized during differentiation programs, and chromatin immunoprecipitation (ChIP) sequencing approaches have demonstrated that chromatin structure at promoters and regulatory regions correlates with active, repressive, or poised “bivalent” transcriptional states, which in turn correlate with the state of cell differentiation.^75–78

Stem cell–specific structures are regulated not only by epigenetic markers but also by higher-order chromatin structures, which are not discussed here (reviewed in Ref. 78). Global levels of DNA methylation in stem cells are similar to levels in somatic cells; however, 25% of methylation sites in stem cells are in non-CpG sites, indicating a unique methylation program.⁷⁹ Multiple studies demonstrate that histone acetylation is central to the maintenance of pluripotency. H3K9 acetylation is higher overall in human stem cells and necessary to maintain an undifferentiated state, whereas H3K9 deacetylation occurs with differentiation.⁸⁰ HDAC inhibitors such as butyrate and valproic acid increase overall levels of H3K9ac⁸⁰ as well as the expression of pluripotency genes such as Nanog, Sox, and Oct4⁸¹ and promote stem cell and iPSC survival.^82,83 Other histone modifications associated with transcriptionally active chromatin also have been shown to be increased in stem cells, including H3K14 acetylation, H3K4 trimethylation (H3K4me3), and H3K36 di- and trimethylation (H3K36me2, H3K36me3).^84,85 Levels of trimethylated H3K27 (H3K27me3) remain unchanged during differentiation but preferentially localize at the nuclear periphery in stem cells, and this localization decreases during differentiation.⁸⁶ It is interesting to note that both H3K27me2 and H3K27me3 markers are mediated by the PRC2 and present at polycomb target genes known to be suppressed in stem cells.^87,88 Taken together, these observations suggest that polycomb target genes are located at the nuclear periphery in stem cells and that this localization may serve as an additional epigenetically regulated repressive mechanism.⁷⁹ Whole-genome ChIP sequencing approaches have shown that both H3K4me3 (a marker of active promoters) and H3K27me3 (a marker of silenced promoters) colocalize in ES cells for 16–22% of genes, most of which are required for developmental regulation, creating a “bivalent” marker hypothesized to poise such genes for future expression while maintaining repression in the stem cell state.^{75,77,78,89,90}

Thus, stem cells have specific epigenetic markers important for maintaining stem cell pluripotency and self-renewal that, when disrupted, could lead to dysregulated differentiation or tumorigenesis. One well-defined example of this is in the initiation of acute myeloid leukemia with rearrangements of the mixed lineage leukemia (MLL) gene. MLL is a homologue of Drosophila melanogaster trithorax and is a positive regulator of Hox gene expression by H3K4 methylation. Hox gene expression is also negatively regulated by H3K27 methylation by polycomb group proteins, thus conferring a delicate balance of epigenetic markers. Disruption of these opposing epigenetic regulatory factors through MLL chromosomal translocation leads to hyperactivation of Hox genes and, ultimately, to leukemogenesis.⁹¹ The mechanisms by which stem cells might transform into cancer stem cells remain widely unknown; however, repeated exposure to agents that damage DNA or disrupt epigenetic gene regulation may cause stem cells to become more similar to cancer stem cells and eventually initiate disease. In support of this, repeated exposure of cultured stem cells to toxic stress and metals has been shown to promote differentiation at the expense of an accumulating stem cell pool, induce abnormal cell signaling and global proteomic alterations analogous to those observed in transformed cells, acquire multiple tumor cell characteristics, and lead to an enrichment of cancer stem cells.^51,92–94

II. ENVIRONMENTAL TOXINS

A. Aldehydes and Alcohols

Carbonyl compounds are stable intermediates of photochemical oxidation of most hydrocarbons and are the precursors to free radicals and ozone; thus environmental exposure can be pervasive. Higher levels of reactive aldehydes such as acetylaldehyde and formaldehyde have been measured in ambient air samples of urban communities and are linked to toxicity, mutagenicity, and carcinogenicity^95–99 (Fig. 1). Exposure to ozone during exercise results in ozonation of lipids to produce aldehydes in fluid in the epithelial lining of the airway in humans.¹⁰⁰ Reactive aldehydes and acetaldehyde are also by-products of endogenous cellular metabolism and have been found to have genotoxic effects. Bone marrow failure in Fanconi anemia may result in part from aldehyde-mediated genotoxicity in the hematopoietic stem and progenitor cell pool. In support of this, mouse hematopoietic stem and progenitor cells are more susceptible to acetaldehyde toxicity compared with mature blood precursors.¹⁰¹ Hematopoietic stem cells from Aldh2^−/− Fancd2^−/− mice that are deficient in the Fanconi anemia pathway–mediated DNA repair and in endogenous acetaldehyde detoxification undergo a more than 600-fold reduction in numbers, display a predisposition to leukemia, and require Aldh2 for protection against acetaldehyde toxicity. ¹⁰¹ Another endogenous source of acetaldehyde is as the first product from the breakdown of alcohol in cells. It has been previously proposed that acetaldehyde generated from alcohol metabolism reacts in cells to generate DNA lesions that form interstrand crosslinks (ICLs).¹⁰² Since the Fanconi anemia– and breast cancer–associated DNA damage response network plays a crucial role in protecting cells against ICLs, Marietta et al.¹⁰³ tested the proposed role of acetaldehyde in generating ICLs. They exposed human lymphoblastoid cells from normal individuals, a patient with xeroderma pigmentosum complementation group A, a patient with Fanconi anemia G, and a patient with Fanconi anemia A to acetaldehyde and studied the activation of the Fanconi anemia– and breast cancer–associated network. Their study reported that acetylaldehyde in a dose range of 0.1–1 mM stimulates FANCD2 monoubiquitination, BRCA1 phosphorylation at Ser1524, and γH2AX at Ser139 in a dose-dependent manner. These results demonstrate interplay between multiple DDR networks and may also support differential tissue specificity of alcohol-related carcinogenesis.¹⁰³ The data also support findings of association between alcohol intake and increased breast cancer risk. Chronic exposure to ethanol induces DNA damage and an induction in the levels of the Fanconi anemia D2 (FANCD2) protein in both human neural precursor SH-SY5Y cells in culture and in the midbrain of C57BL/6J mice in vivo.¹⁰⁴ FANCD2 response induced by alcohol thus plays a role in DDR in post-mitotic neurons and neural precursor cells.

FIG. 1.

Environmental toxins. The chemical structure and biologic consequences of aldehydes and alcohols as well as benzene and its metabolites are shown.

Alcohols and aldehydes are linked with altered histone H3K9 acetylation (H3K9ac) and altered cellular differentiation in bone marrow stem cells, cardiac progenitor cells, and hepatocytes.^105–110 A genome-wide reduction in H3K9ac typically occurs during human ES cell differentiation, and HDAC activity is required for ES cell differentiation. ^80,111 In cardiac progenitor cells, low levels of ethanol, acetaldehyde, and acetate promoted a more than 2-fold increase in histone H3K9ac without affecting the proliferation of cells consistent with the maintenance of a progenitor state.¹¹² High concentrations sufficient to produce a 30% reduction in cell viability also increased H3K9ac by more than 5-fold.¹¹² In addition, high concentrations significantly elevated the expression of GATA4 and Mef2c genes related to heart development, resulting in their impaired differentiation.¹¹² Consistent with these findings, the deregulation of genes that play a role in heart development has been proposed to be one of the mechanisms for the occurrence of congenital heart disease due to alcohol exposure during pregnancy.¹¹³

Occupational and environmental exposures to formaldehyde are prevalent. Formaldehyde is produced on a large scale in the manufacture of resins, particleboard, plywood, leather goods, paper, and pharmaceuticals. Formaldehyde is known to have genotoxic and mutagenic potential. It has been demonstrated that formaldehyde induces genotoxicity by causing DNA-protein crosslinks.¹¹⁴ In addition, lysine residues in the N-terminal tail and the globular fold domain of histone H4 have been identified in in vitro studies as binding sites for formaldehyde at concentrations from 5 to 100 mM, suggesting another mechanism of formaldehyde affecting epigenetic regulation.¹¹⁵

B. Benzene and Its Metabolites

Benzene is a ubiquitous pollutant and one of the top production chemicals in the United States (Fig. 1). It is used in the manufacturing industry and is a combustion product of cigarette smoke. Benzene is carcinogenic and causes primarily hematopoietic cancers in humans.^116,117 It has been reported that it acts through its metabolites, especially 1,4-benzoquinone (1,4-BQ), as a strong topoisomerase II (topoII) poison causing DNA DSBs.¹¹⁸ 1,4-BQ (25 μM) in vitro stimulates 8-fold DNA cleavage by topoII at sites close to defined chromosome breakpoints in leukemia. Benzene metabolites 1,4-BQ (1–10 μM) or 1,4-hydroquinone (10–100 μM) cause DNA damage and fragmentation in cultured HL60 cells though the generation of hydrogen peroxide oxidative stress, leading to apoptosis.¹¹⁹ Benzene and its metabolites, including benzoquinone, also influence the downstream DNA repair of DSBs. As little as 1 μM benzoquinone was sufficient to increase homologous recombination repair by 2.7-fold in a Chinese hamster ovary cell line containing a neomycin gene direct repeat recombination substrate.¹²⁰ Global genomic hypomethylation is a common event in cancer and frequently observed in hematopoietic malignancies, including leukemia. Gasoline station attendants are exposed to benzene fumes, and this exposure has been thought to lead to higher rates of lymphatic and hematopoietic cancers.¹²¹ In support of this, one study showed a 1.6% decrease in global DNA methylation in these workers, suggesting an epigenetic mechanism of benzene action and cancer.¹²²

Studies have extended earlier cell culture studies to in vivo mouse models, showing alterations in epigenetic markers and developmental reprogramming. Neonatal exposure to 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene resulted in the activation of constitutive androstane receptor; a permanent increase of H3K4 mono-, di-, and trimethylation (H3K4me, H3K4me2, H3K4me3); and a decrease of H3K9 trimethylation (H3K9me3) within the Cyp2B10 locus.¹²³ These epigenetic changes were maintained in mice throughout life and resulted in a permanent change in drug metabolism in the liver.¹²³ Taken together, the in vitro and in vivo studies provide further support for the interplay between the DDR, DNA repair, and long-term chromatin remodeling.

C. Trace Metals

Trace amounts of metallic compounds are pervasive in the environment (Fig. 2). They are present in air, water, and food, and occupational exposure to them may occur through industrial production and waste disposal. Several studies have determined that trace metals cross the placenta,^124–129 and the presence of cadmium, copper, chromium, nickel, lead, and zinc in placentas correlates with the response of the biomarker metallothionein and delta-amniolevulinic acid dehydratase, and lipid peroxidation.¹³⁰ Consistent with environmental exposure to these elements, both levels of metals and biomarker responses were statistically significantly related to maternal dietary habits, consumption of canned food and bottled mineral water, as well as smoking.¹³¹ Animal models similarly have shown that sodium arsenite in drinking water administered to pregnant C57BL6/J mice resulted in dose-dependent accumulation in newborn pups.¹³¹ Trace metals elicit pleiotropic biochemical and physiological effects such as mimicry of binding in protein active sites; oxidative changes in lipid, proteins, or DNA; impaired transfer of nutrients to the fetus; low birth weight; and developmental delay. The variation of chemical properties and the reactive toxicities of each indicate that a uniform mechanism of action for all toxic metals is unlikely.

FIG. 2.

Environmental toxins: trace metals. The biologic consequences of exposure to trace metals are shown.

Recent reports have demonstrated that trace quantities of metals directly promote overall histone production, specific epigenetic modifications, and heritable changes in gene expression.¹³² Most important, these changes have been shown to occur in stem cells, potentially being transgenerational. Dimethylation of H3 leads to gene silencing,¹³³ and exposure to multiple metals has been linked to this phenomenon. Zinc can modulate overall histone gene expression and possibly mediate effects on chromatin regulation.¹³⁴ Treatment of human mononuclear THP-1 cells with 50 μmol/L zinc sulfate for 40 hours decreased H2B transcription by 1.58-fold. Zinc deprivation by treatment with 2.5 μmol/L of the membrane permeable zinc chelator TPEN conversely led to a 4.38-fold increase in H2B transcription. Exposure to cadmium, chromium, mercury, and nickel leads to global changes in DNA methylation and histone modifications.¹³² Acute in vitro exposure of mouse ES cells to arsenic, cadmium, mercury, and nickel led to a more than 50% decrease in H3K27 monomethylation (H3K27me), suggesting global induction of transcriptional repression.¹³⁵ Nickel ion exposure at 250 μM or higher increased global H3K9me and H3K9me2 by 2- to 3-fold in a time-dependent manner in cell lines of different lineages, including murine ES cells and embryonic fibroblast cells and human lung carcinoma, osteosarcoma, and embryonic kidney cells.¹³⁶ Furthermore, nickel ions induced gpt transgene silencing and exhibited inhibition of H3K9 demethylation, which led to, or permitted, the observed increase in H3K9me2.¹³⁶ Analysis of peripheral blood mononuclear cells of humans with occupational exposure to nickel suggests that chronic exposure leads to further epigenetic changes in vivo: these samples showed an increase in H3K9me3.¹³⁷ It is interesting to note that epigenetic alterations by metal exposure may be sex-specific: peripheral blood mononuclear cell analysis of women exposed to arsenic in drinking water demonstrated an increase in H3K9me2 and a decrease in H3K9ac, both markers of repression, but led to opposite changes in similarly exposed men.¹³⁷

Low concentrations of trace metals are also sufficient to induce multiple cellular effects. Prolonged in vitro exposure of mouse ES cells to low concentrations (<IC₅₀) of arsenic, cadmium, copper, lead, lithium, mercury, and nickel led to decreased cell proliferation; altered expression of cell differentiation markers Oct-4 and egfr; altered expression of DNA repair genes Rad-18, Top-3a, and Ogg-1; and overall decreased total production of histone protein.¹³⁵

As a downstream result of transcriptional silencing by alterations in epigenetic markers, exposure leads to defects in cellular differentiation pathways. The arsenic derivative arsenite suppresses the expression of cellular differentiation markers to inhibit signaling pathways, maintain proliferative ability, and suppress the differentiation of keratinocyte progenitor cells, as well as transform human prostate epithelial progenitor cells into a cancer stem-cell phenotype.^138–141 In one study, SCC9 human squamous carcinoma cells that exhibited a keratinocyte progenitor cell phenotype were stably transfected with constructs containing the proximal human involucrin promoter, either wild-type or mutated at both AP1 sites, were examined for their transcriptional activity using luciferase reporter activities with and without treatment with arsenate. It is notable that effects were detectable with a nontoxic concentration within the range of environmental exposures (2 μM sodium arsenate or sodium arsenite). As one marker of inhibition of differentiation, arsenite resulted in a significant reduction of c-Fos transcription factor and of acetylated H3 at the proximal and distal AP1 response elements of the involucrin gene promoter and of coactivator p300 at the proximal element of the involucrin gene promoter, as shown by chIP studies. Treatment with arsenite led to the dramatic suppression of the transcriptional activity of the involucrin gene to 2% of the level observed in the absence of any treatment.

Some research has extended cell culture studies to examine the long-term effects in vivo. Exposure of C57Bl6/J mice to 100 μg/L arsenic in drinking water from 1 week before conception until birth resulted in offspring with global H3K9 hypoacetylation, changes in functional annotation with highly significant representation of Krüppel-associated box transcription factors in brain samples, and long-term memory impairment compared to unexposed controls.¹⁴² Timed-pregnant Long–Evans hooded rats exposed to 200 ppm lead acetate in deionized drinking water during pregnancy delivered offspring with age-related neuropathological characteristics analogous to those seen in Alzheimer’s disease. These characteristics were accompanied by changes in the methylation patterns of key Alzheimer’s disease genes.¹⁴³ Continued exposure to lead during the postnatal period resulted in a transient increase in β-amyloid precursor protein (APP). Messenger RNA expression during the first month after birth followed by a return to basal levels by 1 year, but a surprising subsequent delay of overexpression at 20 months after exposure to lead had ceased. These data suggest that environmental influences occurring during brain development predetermined methylation patterns, gene expression, and regulation of APP later in life, potentially altering the course of amyloidogenesis. These studies support the fetal basis of adult disease hypothesis, which states that many adult diseases have a fetal origin.^144–148 Injury or environmental influences occurring at critical periods of organ development in the fetus at early stages of cell differentiation could lead to alterations in gene expression or gene imprinting, which can result in “programmatic” changes in gene expression and functional deficits evident later in life.

Epidemiological studies have well documented metals as human carcinogens associated with skin, lung, liver, and bladder cancers; however, the underlying mechanisms have not been clear. Cancer incidence increases with chronic exposure to metals such as arsenic, cadmium, chromium, and nickel.^149–152 Studies associate arsenic exposure to multiple cancer types in human subjects and gene-specific DNA hypermethylation.^153–157 This direct link between arsenic, tumorigenesis, and hypermethylation was further documented by low-dose (0.5 μM) exposure to arsenic trioxide that led to a transformation of BALB/c 3T3 cells and a dramatic increase in the tumor growth of these cells in a xenograft mouse model.¹⁵⁸ Furthermore, these cells exhibited activated polycomb group proteins BMI1 and SUZ12, increased H3K27me3, and suppression of p16 and p19 that could be rescued by small hairpin RNA to either BMI1 or SUZ12.¹⁵⁸

III. DIETARY EXPOSURES AND SUPPLEMENTS

A. Bisphenol A and Other Estrogens

Bisphenol A (BPA) is a hormonally active environmental xenoestrogen widely used in the production of polycarbonate plastics and resins, including some dental composites (Fig. 3). Human exposure to BPA comes mainly from daily diet; it leaches from food and drink packaging, water pipes, and dental sealants.¹⁵⁹ BPA can cross the placenta and has been detected in urine, amniotic fluid, maternal and fetal plasma, placental tissue, and the breast milk of lactating mothers.^159–162 Estrogens are both natural hormones produced in the body and widely used in hormone supplement therapy. 17β-Estradiol is an endogenous estrogen. Genistein is a soy phytoestrogen present in foods, particularly soybeans, and infant soy formulas^163,164 (Fig. 3). Genistein and other estrogen derivatives are also available at health food stores as dietary and menopausal supplements.¹⁶⁵ The validated and widely used ES cell test for toxicity^166,167 demonstrated that exposure to BPA or genistein significantly altered genome-wide methylation patterns and decreased ES cell-to-cardiomyocyte differentiation. ^168,169 Furthermore, the combination of BPA and genistein had a synergistic effect at lower concentrations, similar to those observed in human blood or sera.¹⁶⁸

FIG. 3.

Estrogenic compounds: dietary exposures and supplements. The chemical structure and biologic consequences of xenoestrogens as well as phytoestrogens and bioflavonoids are shown. Of note is the similar phenolic ring structure of phytoestrogen and bioflavonoid compounds to etoposide, a potent inhibitor of topoII.

The epigenetically toxic effects of environmental chemicals like BPA and phthalates include DNA methylation, histone modification, and changes in the level of microRNA expression.⁷ Some of these effects have been found to be transgenerational. Numerous studies show that exposure to xenoestrogens can developmentally reprogram multiple organ systems. Differences in the ability of xenoestrogens to induce developmental reprogramming are likely driven by intrinsic differences in their binding to specific estrogen receptor subtypes. In the female reproductive tract, exposure is associated with alterations in morphology, hormonal response, and gene expression and promotes diverse outcomes such as obesity and cancer later in life.^170–172 BPA is an endocrine disruptor, causing an adverse effect on mammalian reproduction because of the impaired development of germ cells. BPA has been reported to play a role in modulating germ cell differentiation, retinoic acid signaling, and the expression of germ cell marker genes in mouse ES cells.¹⁷³ After administration of 50 μM BPA, upregulation of meiotic entry gene Stra8 (20-fold), upregulation of ovarian markers Foxl2 and Wnt4 (15- to 20-fold), and suppression of testicular markers Sox9 and Fgf9 were detected, showing that, in addition to germ cell differentiation, BPA also affects testicular and ovarian development. BPA dosing in pregnant C57BL/6J mice from embryonic day 8.5 to day 13.5 accelerated neurogenesis in the developing neurocortex and decreased the number of detectable neural stem/progenitor cells.¹⁷⁴ Animal studies also have reported that postnatal exposure to BPA accelerates neurogenesis and causes neuronal migration defects that impair neurocortex development in embryos.¹⁷⁵ BPA modulates adipogenic differentiation of cultured human primary adult stem cells¹⁷⁶ and suppresses adipogenic differentiation of mouse mesenchymal stem cells.¹⁷⁷

Physiologically relevant doses of BPA or estradiol have been reported to increase susceptibility to adult-onset prostate precancerous lesions and hormonal carcinogenesis. This imprinting involves epigenetic changes such as permanent alterations in the DNA methylation patterns of multiple cell signaling genes.¹⁷⁸ Developmental exposure to estradiol and BPA leads to an increase in the susceptibility to prostate carcinogenesis with aging through epigenetic regulation.¹⁷⁸ In normal prostates, gradual methylation occurs within the specific genomic cluster containing the gene for phosphodiesterase type 4 variant 4 (PDE4D4), which is an enzyme responsible for the breakdown of cyclic adenosine monophosphate. This methylation is associated with decreased expression. In contrast, exposure of neonatal Sprague-Dawley rats to BPA (10 μg/kg) or 17β-estradiol 3-benzoate (2500 μg/kg or 0.1 μg/kg) resulted in early and prolonged hypomethylation at this site and continued, elevating PDE4D4 gene expression throughout life, consistent with observed hypomethylation of this gene in prostate cancer cells. Several genes showed changes in methylation in response to neonatal estrogen treatments, many of which are permanent.

Estrogens also have been linked to the generation of DNA DSBs or inhibition of their repair. Exposure of primary gingival fibroblasts to dental adhesives containing BPA derivatives produced increased numbers of DNA breaks, marked damaged chromosomes with gH2AX, altered cell cycle profiles, and produced slower kinetics of repair. ^179–181 Sensitivity to BPA derivatives may be global because exposure of keratinocytes, skin fibroblasts, intestinal cells (line LS174T), and hepatoma cells (line HepG2) all mark damaged DNA by gH2AX.^182,183

B. Bioflavonoids as Topoisomerase II Inhibitors

Bioflavonoids comprise a diverse group of polyphenolic compounds divided into 3 main groups: flavones, flavonols, and isoflavones (Fig. 3). The most common sources of these bioflavonoids are fruits, vegetables, soy, tea, coffee, and wine.¹⁸⁴ Genistein is both an estrogen derivative (discussed earlier), available at health food stores as a dietary and menopausal supplement,¹⁶⁵ and a soy phytoestrogen present in foods, particularly soybeans, and infant soy formulas.^163,164 Its effects in cells are thus likely pleiotropic, acting through both mechanisms. Because of their antioxidant capacity, flavonoids are used for their presumed health benefits, such as protection against cardiovascular diseases, cancer, and inflammation. Flavonoid supplements are available worldwide over the counter in pharmacies and drugstores.

However, accumulating evidence indicates that dietary flavonoids are potent inhibitors of topoII (topoII α and topoII β in mammalian cells^185,186). DNA topoisomerases are essential cellular enzymes that cause topological changes in the DNA for processes such as replication and transcription. Inhibitors of topoII block the relegation of the transient DSBs made by topoII, leading to cell death at high doses and potentially leading to illegitimate repair, genome instability, and chromosomal abnormalities in surviving cells. The chemotherapeutic agents etoposide, doxorubicin, daunorubicin, and mitoxantrone^185,187 are well-characterized topoII inhibitors that are typically used as comparison controls during the testing of other potential inhibitors. Etoposide is composed of a polycyclic ring system (rings A–D), a glycosidic moiety at the C4 position, and a pendant ring (E ring) at the C1 position.^188–190 The binding of etoposide to topoII is driven by interactions with the A ring and the B ring,¹⁸⁹ while the E ring is important for etoposide function.¹⁹⁰ Several studies have reported that etoposide promotes MLL rearrangements in mouse ES cells,¹⁹¹ primitive hematopoietic stem cells, and in human fetal hematopoietic stem cells.^192–195 Other anticancer agents, including teniposide, anthracyclines, and dactinomycin, also are associated with MLL rearrangements due to topoII inhibition and enhanced DNA cleavage, leading to defective DNA repair and chromosomal translocations.¹⁹⁶

Multiple bioflavonoid compounds are also polyphenolic ring structures and thus may biochemically and mechanistically act similarly to etoposide.¹⁸⁶ Etoposide, genistein, and quercetin contain pendant rings that feature a 4′-OH group that is essential for drug action.^186,197,198 The 5′-OH group of genistein plays an important role in mediating binding to topoII, and the 4′-OH plays a significant role in function.¹⁸⁶ TopoII inhibition by bioflavonoids was investigated in an in vitro plasmid DNA cleavage assay using purified recombinant wild-type human topoIIα and IIβ; it showed that these compounds were active against topoIIβ.¹⁸⁶ Genistein (50 μM) was shown to be the most effective of the bioflavonoids tested and stimulated enzyme-mediated DNA cleavage ~10-fold,¹⁸⁶ whereas 100 μM genistein efficiently induced topoII-DNA cleavage complexes in both cultured mouse myeloid progenitor cells (32Dc13) and Top2β knockout mouse embryonic fibroblasts, and it was suggested that these complexes are processed by proteasome, which led to chromosome rearrangements.¹⁹⁹ Cultured human lymphocytes treated with 50 μM genistein display chromosome abnormalities in metaphase karyotypic analyses.²⁰⁰ DSBs with the MLL gene breakpoint cluster region were induced by bioflavonoid exposure both in primary human progenitor hematopoietic cells from healthy newborns and adults as well as in hematopoietic progenitor cell lines (BV173 and K562).²⁰¹ Quercetin, genistein, and kaempferol induced DSBs in primary human hematopoietic CD34+ stem cell–enriched cells (at doses of 25 and 50 μM).²⁰² Besides chromosomal translocations, monosomy or trisomy of MLL also was reported in cells exposed to quercetin.²⁰²

It is important to note that synthetic flavonoids are able to cross the placenta in the rat and are found in all fetal tissues (17% of the initial dose), including the fetal brain.²⁰³ Maternal and fetal distributions of the synthetic radioactively labeled bioflavonoid EMD-49209 were detectable 1–24 hours after intravenous injection into pregnant Wistar rats. Transplacental exposure to high but biological amounts of the flavonoids genistein and quercetin in Atm-ΔSRI mutant mice with an impaired capacity for DSB repair led to the detection (using inverse polymerase chain reaction) of a 2-fold higher number of MLL rearrangements compared with their wild-type siblings.¹⁸⁴ Parallel in vitro studies with bone marrow cells exposed to genistein (50 μM) or quercetin (50 μM) showed 2.1–5.0 rearrangements/80 ng genomic DNA (1 per 13,000 cells) for quercetin or genistein compared with 0.2 translocations/80 ng genomic DNA for wild-type cells. Thus, the risk of these rearrangements due to in utero exposure to these bioflavonoids increases in the presence of compromised DNA repair, although in that study MLL rearrangements were detectable in all samples regardless of diet or mutational status.¹⁸⁴

The epigenetic and transgenerational effects of these dietary compounds were addressed in a study that showed that exposure of progeny to genistein through maternal diet during pregnancy can have long-lasting effects on the progeny.²⁰⁴ Mice (129/SvJ:C57BL/6J background) ~8 weeks old were given genistein (270 mg/kg of feed) from conception until birth. Genistein induced epigenetic changes and altered the color of the coat of agouti mice. The progeny of these mice had a significant downregulation of genes involved in hematopoiesis of bone marrow cells, increased erythropoiesis, and a permanent signature hypermethylation of repetitive elements in hematopoietic lineages. Thus prenatal exposure to genistein affected the epigenetic signature of hematopoietic cells and caused long-lasting alterations in gene expression.

Exposure to the flavonoid quercetin during pregnancy can result in long-term changes in iron homeostasis during adulthood.²⁰⁵ Quercetin is a strong iron chelator and has the ability to cross the placenta and accumulate in the fetus. In this study, female mice (129/SvJ:C57BL/6J background) were given quercetin (302 mg/kg feed) from 3 days before conception until the end of gestation. Mice prenatally exposed to quercetin had upregulated iron-associated cytokine expression and significantly increased iron storage in the liver (~94 ng/mg for quercetin exposure vs. ~62 ng/mg for control). Quercetin exposure was associated with hypermethylation of repetitive elements, and these epigenetic modifications could cause these long-term changes in cytokine gene expression. All of these changes led to a shift toward a higher expression of cytokines associated with inflammation in the liver of adult mice that were prenatally exposed to quercetin.

Quercetin also has been shown to affect the xenobiotic metabolism of chemical carcinogens in mice that were prenatally exposed to this compound. ²⁰⁶ Mice (129/SvJ:C57BL/6J background) were given quercetin (1 mmol or 302 mg/kg of feed) from 3 days before conception until the end of gestation. Mice exposed to quercetin showed altered biotransformation of the environmental pollutant benzo[a]pyrene. This occurred because of the altered gene expression of metabolic enzymes such as Cyp1a1, Cyb1b1, Nqo1, and Ugt1a6, which persisted into adulthood in a tissue- and sex-dependent manner. These long-lasting changes were associated with epigenetic alterations since prenatal quercetin exposure led to hypomethylation of repetitive elements SINEB1. These persistent alterations in the metabolic enzymes of adult mice may affect cancer risk because of environmental chemical carcinogens.

IV. CONCLUDING REMARKS

The exposure to environmental and dietary agents discussed above is widespread today. Because of the beneficial effects assumed to be associated with the use of bioflavonoids, their use as dietary supplements is increasingly popular and widespread. However, a growing body of evidence is emerging regarding the long-term implications and adverse effects of using these compounds in an unrestricted manner. Multiple environmental toxins and dietary agents have the potential to cause long-term epigenetic changes, leading to dysregulation of multiple cellular functions and developmental pathways. Epigenetic modulation, cell differentiation, gene expression, signal transduction, and illegitimate DNA repair all are associated with human diseases and cancer.²⁰⁷ It is important to note that epigenetic alterations following exposure will continue to affect cellular development long after exposure has ceased. In view of the variety of adverse effects these agents have in exposed individuals, it is important to raise public awareness, set guidelines, and regulate the use and market availability of such compounds to reduce the risk of disease. Since the role of in utero exposures in causing long-term transgenerational effects has been demonstrated to be critical, it is important to address the susceptibility of different stages of cell differentiation to the deleterious molecular changes induced by these agents. However, a system to directly and rapidly examine the role of a large number of different compounds, both individually and in combination, in inducing the genetic/epigenetic changes discussed above is lacking, and thus testing has been limited to small isolated studies. Even the inherent genomic instability of existing stem cell lines or the memory of previous epigenetic codes of iPSCs suggest that they may not always mimic in vivo consequences. The development of model systems for cells at different stages of differentiation with different susceptibilities to epigenetic alterations will provide useful insights into the comparative risk to human populations and how the timing or stage of development may affect in vivo consequences to individuals.