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Epigenetics investigates heritable changes in gene expression occurring without changes in DNA sequence. Several epigenetic mechanisms, including DNA methylation, histone modifications, and microRNA (miRNA) expression, can change genome function under exogenous influence. Here, we review current evidence indicating that epigenetic alterations mediate toxicity from environmental chemicals.
In-vitro, animal, and human investigations have identified several classes of environmental chemicals that modify epigenetic marks, including metals (cadmium, arsenic, nickel, chromium, methylmercury), peroxisome proliferators (trichloroethylene, dichloroacetic acid, trichloroacetic acid), air pollutants (particulate matter, black carbon, benzene), and endocrine-disrupting/reproductive toxicants (diethylstilbestrol, bisphenol A, persistent organic pollutants, dioxin). Most studies conducted so far have been centered on DNA methylation, whereas only a few investigations have studied environmental chemicals in relation to histone modications and miRNA.
For several exposures, it has been proved that chemicals can alter epigenetic marks and that the same or similar epigenetic alterations can be found in patients with the disease of concern or in diseased tissues. Future prospective investigations are needed to determine whether exposed subjects develop epigenetic alterations over time and, in turn, which such alterations increase the risk of disease. Also, further research is needed to determine whether environmental epigenetic changes are transmitted transgenerationally.
Identifying the effects of environmental exposures on human health is a major objective of life sciences and biomedical research. In environmental health, the recognition that exposures could produce DNA mutations represented a major landmark for risk assessment and prevention. Consequently, chemical agents have been categorized according to their capability to alter the DNA sequence. Such information has been fundamental to determine environmental risks and shape current regulatory efforts for exposure reduction. Recent evidence suggests that the molecular influence of the environment may extend well beyond the interaction with the DNA sequence.[3,4] Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. Epigenetic mechanisms are flexible genomic parameters that can change genome function under exogenous influence. Here, we review current evidence indicating that epigenetic mechanisms can mediate the toxicity of environmental chemicals.
The current field of epigenetics includes a number of mechanisms, including DNA methylation, histone modification, and microRNAs.[6,7] (Fig 1) DNA methylation is a covalent modification, heritable by somatic cells after cell division. 5-methyl-cytosine (5MeC) represents 2-5% of all cytosines in mammalian genomes and is found primarily on CpG dinucleotides. DNA methylation is involved in regulating many cellular processes, including chromatin structure and remodeling, X-chromosome inactivation, genomic imprinting, chromosome stability, and gene transcription.[9,10] Generally, gene promoter hypermethylation is associated with decreased expression of the gene. However, more than 90% of all genomic 5-methylcytosines are not directly related to gene function, as they lie on CpG dinucleotides located in transposable repetitive elements, also referred to as transposons. Alu and LINE-1 (Long Interspersed Nuclear Element-1) are the most common and well-characterized transposable sequences and measurements of Alu and LINE-1 methylation have been used to estimate global genomic DNA methylation content. Global hypomethylation, as well as hypomethylation of transposable repetitive elements, have been associated with reduced chromosomal stability and altered genome function.[13,14]
Histones are globular proteins that undergo posttranslational modifications that alter their interaction with the DNA and other nuclear proteins. H3 and H4 histones have long tails protruding from the nucleosome, which can be covalently modified by acetylation, methylation, ubiquitination, phosphorylation, sumoylation, citrullination, and ADP-ribosylation, and thus influence chromatin structure and gene expression (Fig 2).
microRNAs (miRNA) are single-stranded RNAs of ≈21–23 nucleotides in length that are transcribed from DNA but not translated into proteins (non-coding RNAs); Mature miRNAs are partially complementary to one or more messenger RNA (mRNA) molecules. miRNA main function is to down-regulate gene expression by interfering with mRNA functions (Fig 3).[16,17]
Some environmental factors have been linked to aberrant changes in epigenetic pathways both in experimental and epidemiological studies. In addition, epigenetic mechanisms may mediate specific mechanisms of toxicity and responses to certain chemicals. Whereas mechanisms of action of some of these agents are understood, for others the mode of action remains to be completely elucidated. Because these epigenetic changes are small, potentially cumulative, and they may develop over time, it may be difficult to establish the cause-effect relationships among environmental factors, epigenetic changes and diseases. Below, we review the effects of individual environmental toxicants on epigenetic mechanisms, as derived from in-vitro, animal, or human studies, which we summarized in Table 1 (effects on DNA methylation), Table 2 (effects on histones modifications), and Table 3 (effects on miRNA).
Several studies have established an association between DNA methylation and environmental metals, including nickel, cadmium, lead, and particularly arsenic.[19-21] Metal-induced oxidative stress may represent a unifying process to account for these findings across different metals. Metals are known to increase production of reactive oxygen species (ROS) in a catalytic fashion via redox cycling.[23,24] Oxidative DNA damage can interfere with the ability of methyltransferases to interact with DNA, thus resulting in a generalized altered methylation of cytosine residues at CpG sites.
Cadmium is an established carcinogen that has very low mutagenicity. Many possible mechanisms of cadmium carcinogenesis have been suggested and, among them, induction of ROS and alteration of DNA methylation seem to play a predominant biological role. Takiguchi et al. showed that cadmium reduces genome methylation, inhibiting DNA methyltransferases in a noncompetitive manner. This finding is suggestive of interference in enzyme-DNA interaction, possibly through an interaction of cadmium with the methyltransferase DNA binding domain. Cadmium can also inhibit DNA methylation in proto-oncogenes inducing oncogene expression and resulting in cell proliferation.[28,29]
Arsenic is an established carcinogen that lacks carcinogenicity in animal models. Inorganic arsenic is enzymatically methylated for detoxication, using up S-adenosyl-methionine (SAM) in the process. The observation that DNA methyltransferases also require SAM as their methyl donor suggested a role for DNA methylation in arsenic carcinogenesis and other arsenic-related effects.  In rat-liver epithelial cell lines treated with chronic low arsenic doses, Zhao et al. showed malignant transformation associated with depressed SAM levels, global DNA hypomethylation, and decreased DNA methyltrasferase activity. Following this findings, several studies have shown that arsenic is associated with gene-specific hypermethylation [31-34], as well as global DNA hypomethylation [35-37]. An unexpected finding was recently reported in vivo, as a global dose-dependent hypermethylation of blood DNA was observed in Bangladeshi adults with chronic arsenic exposure. This effect was modified by folate, suggesting that arsenic-induced increases in DNA methylation were dependent from methyl availability. The same group, however, reported that lower blood DNA methylation was a risk factor for arsenic-induced skin lesions in a related Bangladeshi population.
In a human study from India, significant DNA hypermethylation of p53 and p16 promoter regions was observed in blood DNA of subjects exposed to toxic level of arsenic compared to controls. In this study, hypermethylation showed a dose-response relationship with arsenic measured in drinking water.
Arsenic toxicity has been recently related to changes in miRNA expression. Marsit et al. showed alterations in miRNA profiles of human lymphoblastoid cells grown under sodium arsenite treatment. Interestingly, Arsenic altered expression of specific miRNAs that were involved in one-carbon metabolism.
The mechanisms underlying nickel health-related effects, including carcinogenicity and cardiorespiratory disease, are still largely unknown. It has been proposed that nickel may replace magnesium in DNA interactions, enhance chromatin condensation, and trigger de novo DNA methylation. In chinese hamster G12 cells transfected with E.Coli gtp gene, Lee et al. demonstrated nickel-induced hypermethylation leading to the inactivation of the expression of the transfected gene.
Several studies have shown that nickel affects histone modifications. Exposure to soluble NiCl2 has been shown to reduce histone acetylation, increase demethylation of H3K9, and increase monoubiquitination of H2A and H2B in-vitro. Broday et al. studied nickel effects, at nontoxic levels, on yeast and mammalian cells and found a decrease in histone H4 acetylation, affecting only lysine 12 in mammalian cells and all of the four H4 lysines in yeasts.
Nickel ion exposure has been shown to increase global H3K9 mono- and dimethylation, both of which have been associated with increased DNA methylation and long-term gene silencing. Nickel ions also interfere with the removal of histone methylation in vivo and directly decrease the activity of a Fe(II)-2-oxoglutarate-dependent histone H2K9 demethylase in nuclear extract in vitro. In human lung cells exposed to soluble nickel compounds, three major changes in histone modifications have been observed: i) loss of acetylation of H2A, H2B, H3 and H4; ii) increased H3K9 dimethylation; iii) increased ubiquitinylation of H2A and H2B.[41-46]
It has been proposed that the binding of Ni2+ is able to promote a secondary structure with organized side-chain orientation on the N-terminal tail of histone H4. Acetylation of lysine 12 and 16 in yeast exposed to nickel was more robustly affected than lysine 5 and 8. Nickel binding to histidine 18 in histone H4 may be accountable for this effect, acting as an anchoring binding site for metal ions.
Kondo et al. investigated p16 methylation using a methylation-specific PCR method in 30 lung cancer cases associated with chromate exposure and 38 non-chromate lung cancers. A variety of genetic changes in lung cancers from chromate-exposed subjects is known, but the epigenetic effects of chromium are still poorly understood. Kondo et al showed that chromate exposure influenced p16 hypermethylation measured in lung cancer tissues, compared to tissues from non-chromate lung cancer. Chromium has been shown to reduce in-vitro H3 phosphorilation and trimethylation, as well as various acetylation marks in H3 and H4.
Methylmercury is an environmental contaminant and a potential neurotoxic agent that may be present at high levels in sea-food. Perinatal exposure to methylmercury causes persistent changes in learning and motivational behavior in mice. Developmental exposure to low levels of methylmercury induces epigenetic suppression of BDNF (Brain Derived Neurotrophic Factor) gene expression in the hippocampus and predisposes mice to depression.
Trichloroethylene (TCE), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) are environmental contaminants that are peroxisome proliferators and carcinogenic in mouse liver. Decreased methylation in the promoter regions of the c-jun and c-myc genes and increased levels of their mRNAs and proteins were found in livers of mice exposed to TCE, DCA, and TCA. Methionine supplement prevented both the decreased methylation and the increased levels of the mRNAs and proteins of the two protooncogenes. This work supported the hypothesis that these carcinogens may act by depleting the availability of SAM, whereas methionine would prevent DNA hypomethylation by maintaining adequate SAM levels.
Exposure to air pollution, particularly to particulate matter (PM), has been associated with increased morbidity and mortality from cardiorespiratory disease, as well as with lung cancer risk.[52-56] In a human study, we recently investigated the effects of particulate matter (PM) exposure on global (estimated through Alu and LINE-1 repeated elements) and gene-specific methylation in workers of a steel plant with well-characterized exposure to PM with aerodynamic diameters <10 μm (PM10). iNOS (inducible Nitric Oxide Synthase) promoter methylation was significantly lower in post-exposure blood samples compared to baseline. Long-term exposure to PM10 was negatively associated with methylation in both Alu and LINE-1. In a recent investigation, we showed that exposure to black carbon (BC), a marker of traffic particles, was also associated with decreased DNA methylation in LINE-1, measured in 1,097 blood DNA samples from the Normative Aging Study (NAS), a repeated measure investigation of elderly men in the Boston area. As global DNA hypomethylation has been found in patients with cancer  or cardiovascular disease, such changes may reproduce epigenetic processes related to disease development and represent mechanisms by which particulate air pollution affects human health.
In an animal study, Yauk et al. showed that sperm DNA of mice exposed to steel plant air was hypermethylated compared to control animals and this change persisted following removal from the environmental exposure. This finding calls for further research to determine whether air pollutants produce DNA methylation changes that are transmitted transgenerationally.
In a recent investigation, we investigated whether DNA methylation changes are induced by low-benzene exposure in peripheral blood DNA of gasoline station attendants and traffic police officers. High-level exposure to benzene has been associated with increased risk of acute myelogenous leukemia (AML),  which is characterized by aberrant global hypomethylation and gene-specific hypermethylation/hypomethylation. In our study, airborne benzene exposure was associated with a significant reduction in global methylation measured in LINE-1 and Alu. Airborne benzene was also associated with hypermethylation in p15 and hypomethylation of the MAGE-1 cancer-antigen gene. This findings show that low-level benzene exposure may induce altered DNA methylation reproducing the aberrant epigenetic patterns found in malignant cells.
RDX is a common environmental pollutant resulting from military and civil activities that has been associated with neurotoxicity, immunotoxicity and increased risk of cancer. Zhang and Pan evaluated the role of RDX in modifying miRNA expression in mouse liver and brain, as measured by miRNA expression microarrays. Several miRNAs were found to be differentially expressed in exposed mice, with specific miRNA expression profiles in gene pathways related to cancer, toxicant-metabolizing enzymes, and neurotoxicity.
Developing organisms are extremely sensitive to perturbation by endocrine-disrupting chemicals with ormone-like activity. In mammal germ cells and preimplantation embryos, DNA methylation undergoes two distinct cycles of demethylation/remethylation in which methylation patterns are reprogrammed genome wide and cells with a broad developmental potential are generated. Available evidence from animal models indicate that exposure to xenobiotics during critical periods of mammalian development may induce persistent and heritable changes of epigenetic states.
Diethylstilbestrol (DES) is a developmental stage-specific non-genotoxic carcinogen that was used in the past to prevent miscarriages in pregnant women.  In mice models, pre- and neonatal DES exposure causes a wide range of gene expression changes. Exogenous estrogen treatment results in persistent expression of certain genes, including lactoferrin, epidermal growth factor, and protooncogenes such as c-fos, c-jun and c-myc.[67-70] Xie et al. have provided data supporting an indirect relationship between exogenous estrogen and methylation changes, showing that estrogen inhibited catechol-O-methyltransferase (COMT) gene transcription. COMT is an ubiquitous enzyme catalyzing the transfer of the methyl groups from SAM to one of the hydroxyl groups of catechols in presence of Mg2+. Inhibition of this enzyme results in inhibition of the methylation process.
BPA is a chemical with estrogenic properties that is present in many commonly used products including food and beverage containers, baby bottles, and dental composites. Dolinoy et al  used a rat exposure model to show that maternal exposure to BPA modified methylation of the metastable loci Avy and CapbIAP. Interestingly, this effect on DNA methylation and the associated change in coat color of the exposed animals were prevented by maternal dietary supplementation with a source of methyl group such as folic acid or with the phytoestrogen genistein. 
Rusiecki et al. evaluated the relationship between plasma POP concentrations and blood global DNA methylation, estimated in Alu repeated elements, in 70 Greenlandic Inuit, a population presenting some of the highest reported levels of POPs worldwide. In this work, a significant inverse linear relationships was found for DDT, DDE, β-BHC, oxychlordane, α-chlordane, mirex, several PCBs, and sum of all POPs. 
Dioxin toxicity is mediated by the Aryl-hydrocarbon Receptor (AhR) (reviewed in [74-76]) and appears to require altered transcription of target genes. Moffat et al. hypothesized that miRNAs might be responsible for this mRNA downregulation in dioxin/AhR-related pathways but, using two different miRNA array platforms and Real time PCR, they just found a few changes of small entity in miRNA levels, thus indicating a limited role of miRNA in dioxin toxicity.
In animal studies, several chemicals including alloxan, cyclophosphamide, orthoaminoasotoluol,, benzopyrene,, diethylstilbestrol (DES), and vinclozolin have been reported to induce transgenerational phenotypic effects. Transgenerational transmission of chemically-induced epigenetic changes have been suggested as a potential mechanisms for these effects. Anway et al. showed that gestational exposure of female rats to the endocrine disruptor vinclozolin at the time of gonadal sex determination caused a variety of abnormalities in the offspring that were then transmitted down the male line for at least three generations. The high incidence of the defects (approximately 90% of all males in all generations) and the absence of abnormalities when passed down the female line suggested gametic epigenetic inheritance. In this study, altered DNA methylation in two candidate genes was seen in sperm from vinclozolin-exposed males, and these abnormal methylation patterns were inherited. These results indicate that exposure of germ cells, possibly at a specific developmental stage, is necessary to produce heritable epigenetic changes. In addition, epigenetic mechanisms may underlie the effects of in utero and early life exposures on adult health, as in-utero/early-life exposures to epigenetically-active chemicals may produce health effects later in life even independently of environmental risk factors in adults. As reported in the sections above, most of the studies on epigenetic effects of environmental chemicals have shown changes in DNA methylation, histone modifications or microRNA in somatic cells of adult individuals. Whether epigenetic changes observed in somatic cells are correlated with germline epigenetic changes is uncertain. Environmentally-induced epigenetic somatic alterations may be sufficient to cause anomalies in biological functions, but these changes are not heritable per se and may not be associated to any transgenerational risk.
In the last few years, several investigations have examined the relation between exposure to environmental chemicals and epigenetics, and identified several toxicants that modify epigenetic marks. Most of the studies conducted so far have been centered on DNA methylation, whereas only a few recent investigations have studied the effects of environmental chemicals on histone modications and miRNA. Epigenetics holds substantial potential for developing biological markers to predict which exposures would put exposed subjects at risk and which individuals will be more susceptible to develop disease. In human studies, this will require the use of laboratory methods with enhanced precision and sensitivity, so that epigenetic changes can be detected as early as possible and well ahead of disease diagnosis. It is worth noting that several human studies have investigated the effects of environmental exposures on tissues, such as blood, that are easy to obtain, but that, as effects of environmental chemicals may be tissue or even cell specific, do not necessarily represent epigenetic patterns in the target tissues. For several exposures, however, it has been proved that chemicals can alter epigenetic marks and that the same or similar epigenetic alterations can be found in patients with the disease of concern or in diseased tissues. At the current stage, the missing link is to determine whether environmentally-induced epigenetic alterations are part of the causative pathways that leads to the disease development. Future prospective investigations are needed to determine whether exposed subjects develop epigenetic alterations over time and, in turn, whether such alterations increase the risk of disease.