Epigenetic mechanisms underlying arsenic-induced toxicity

Accepted Manuscript Epigenetic mechanisms underlying arsenic-induced toxicity Cassandra J. Meakin, Elizabeth M. Martin, Rebecca C. Fry PII:

S2468-2020(17)30049-9

DOI:

10.1016/j.cotox.2017.06.003

Reference:

COTOX 53

To appear in:

Current Opinion in Toxicology

Received Date: 25 April 2017 Revised Date:

30 May 2017

Accepted Date: 1 June 2017

Please cite this article as: C.J. Meakin, E.M. Martin, R.C. Fry, Epigenetic mechanisms underlying arsenic-induced toxicity, Current Opinion in Toxicology (2017), doi: 10.1016/j.cotox.2017.06.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Epigenetic mechanisms underlying arsenic-induced toxicity

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Cassandra J. Meakin1, Elizabeth M. Martin1 and Rebecca C. Fry1*

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Public Health, University of North Carolina, Chapel Hill, North Carolina, USA

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Rebecca C. Fry

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Department of Environmental Sciences and Engineering, Gillings School of Global

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Department of Environmental Sciences and Engineering, Gillings School of Global

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Public Health, 135 Dauer Drive, CB 7431, University of North Carolina, Chapel Hill,

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North Carolina, USA. Phone: (919) 843-6864; email: [email protected]

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Running title: Arsenic-associated modification of the epigenome

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Key Words: Arsenic exposure, CpG DNA methylation, histone modification, miRNA

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expression

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Acknowledgements: This research was supported by grants from the National Institute

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of Environmental Health Sciences (T32ES007018, R01ES0019315, P42ES005948), and

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the B.B. Parker Fellowship. We would like to acknowledge Caroline Reed for her

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assistance with the figure.

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Disclosure of potential conflict of interests: The authors claim no competing financial

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interests.

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Abstract: Millions of individuals world-wide are exposed to potentially harmful levels of inorganic arsenic that exceed the World Health Organization (WHO) recommended limit

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of 10 µg/L. To date, studies on arsenic-associated epigenetic modification suggest

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numerous mechanisms by which inorganic arsenic impacts DNA methylation, miRNA

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expression, and modified histones. For example, evidence supports that inorganic arsenic

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modifies the enzymatic activity of DNA methyltransferases, histone deacetylase (HDAC)

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and histone acetyltranferase (HAT). Both in vitro and in vivo evidence support that

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inorganic arsenic acts as an epigenetic modifier of genes involved in critical cellular

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functions such as cellular growth and immune response. Future research should focus on

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scientific gaps related to the functional consequences of the epigenetic marks, the impact

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of sexual dimorphism of epigenetic marks, the cross-talk among epigenetic modifiers,

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and the temporal stability of epigenetic marks.

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Introduction In spite of the World Health Organization’s (WHO) recommended limit of 10 µg/L, there are more than 100 million exposed to harmful levels of inorganic arsenic in

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drinking water [1]. For example, arsenic has been documented to be present in drinking

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water at levels exceeding 1,000 µg/L in Bangladesh [2,3], 800 µg/L in the United States

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[4], and 800 µg/L in Mexico [5]. Given its toxicity and high environmental prevalence,

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inorganic arsenic is ranked as the highest priority agent by the Agency for Toxic

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Substances and Disease Registry (ATSDR) [6]. Arsenic exposure continues to represent

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a major public health threat for populations worldwide.

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The major route of exposure to inorganic arsenic for the general population is through contaminated drinking water impacted largely by geologic sources [1]. In terms

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of associated health effects, chronic inorganic arsenic exposure has been linked to

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cancers of numerous organs including the lung, liver, bladder and kidney [7]. Arsenic

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exposure is also associated with non-cancer endpoints such as skin lesions, diabetes

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mellitus, and increased blood pressure in adults [1,8]. Of concern, prenatal and early life

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exposures to arsenic are associated with the increased risk of low birth weight,

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susceptibility to infection, preterm birth, and later life cancers [1,9].

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Decades of arsenic-focused toxicology studies have aimed at elucidating the

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specific biological mechanisms underlying disease. Among the many proposed cellular

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mechanisms that underlie arsenic-associated disease are altered DNA repair capacity,

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altered signal transduction, induction of reactive oxygen species (ROS) and epigenetic

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modifications [10]. The epigenome is composed of biological and chemical modifications

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that do not modify the base sequence of DNA, but nevertheless can alter gene expression

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and activity. Epigenetic modifications serve as a regulatory mechanism controlling

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mRNA (gene) expression through chromatin and DNA accessibility [11]. The

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epigenome serves as a mediator between environmental exposures, the inherited genome,

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and disease development [12].

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In the past decade, there has been an expansion in the literature that supports the importance of the epigenome in maintaining cellular homeostasis and the implication for

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disease development within the context of arsenic exposure [13]. Specifically,

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perturbations of the epigenome, including altered DNA methylation, histone

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modification, and altered miRNA expression, have been associated with arsenic

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exposure. Importantly, various arsenic-associated diseases apparent early in life such as

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adverse birth outcomes as well as later-life diseases such as diabetes and cancer may have

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epigenetic underpinnings [13].

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Of relevance to both arsenic-associated disease and to arsenic-associated changes to the epigenome, is the complex mechanism by which arsenic is metabolized. Once

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inorganic arsenic enters the body, it is metabolized from iAs to monomethylated arsenic

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(MMA) by the enzyme arsenic (+3 oxidation state) methyltransferase (As3mt) [14].

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A3MT functions by utilizing the substrate, S-Adenosyl methionine (SAM), as a methyl

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donor [15]. Finally, MMAs are further metabolized to form dimethylated arsenic (DMA)

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by As3mt, which can also be excreted from the body in addition to monomethylated

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forms of arsenic. Because metabolism leads to the elimination of arsenic, this process has

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been regarded as a detoxification mechanism with more efficient metabolizers of arsenic

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exhibiting higher percentages (60-80%) of DMAs in urine compared to MMAs (10-20%)

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and iAs (10-20%) [16]. In support of this, higher percentages of MMAs in the urine of

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exposed individuals have been linked to lower birth weight in infants, cancer, and

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cardiovascular disease [17,18]. The metabolism of arsenic has also been tied to

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differential DNA methylation in humans [19].

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This review details studies that investigate the role of the epigenome as a

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contributor to arsenic-induced toxicity and a mediator of adverse health outcomes. The

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complex mechanisms underlying arsenic-associated changes to the epigenome and their

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role in arsenic-induced toxicity are described. Similarities among studies from both

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human populations and in vitro studies are detailed and current gaps in the literature are

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described.

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Arsenic-induced changes in DNA methylation

Of the various forms of epigenetic modifications, DNA methylation is the most

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thoroughly investigated. In cytosines proximal to guanines or CpG (cytosine-phosphate-

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guanine) dinucleotides, methyl groups can be transferred from SAM to cytosines to form

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5-methyl cytosine (5-MeC). These processes are facilitated by the DNMT enzyme family

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[20]. In general, CpG methylation has been described as a transcriptional silencing

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mechanism, as it is postulated that the addition of methyl groups impedes transcription

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factor binding to DNA to initiate transcription. More specifically, the transcription

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blockade is thought to be the result of the recruitment of methyl-CpG-binding domain

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proteins to the binding site or through direct methylation of the binding site [21].

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To date, there are three major mechanisms by which inorganic arsenic is proposed

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to impact DNA methylation. First, arsenic can alter the activity and expression of DNMT,

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the enzyme that facilitates DNA methylation. Generally, DNMT expression and activity

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are reduced in the presence of arsenic [22]. Second, the availability of SAM has been

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shown to be reduced in the presence of arsenic [23]. S-adenosine methionine (SAM) is

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one of the main substrates that DNMT uses as a methyl donor to execute CpG

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methylation and a major cofactor required for arsenic metabolism [24]. Third, the

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alterations of gene-specific DNA methylation patterning in response to environmental

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contaminants, may be mediated through transcription factor binding as described by the

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transcription factor occupancy theory [25]. This theory states that targeted DNA binding

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by transcription factors alters the access of DNMT to the targeted DNA sequence, thus,

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affecting gene-specific DNA methylation patterning [25]. This collective evidence

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suggests that arsenic directly influences the process of DNA methylation by altering the

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availability and activity of enzymes that facilitate CpG methylation processes.

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At present, extensive literature has demonstrated the effects of arsenic on DNA

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methylation patterns through the use of in vitro techniques that span the assessment of

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various cell lines. The broad array of cell lines assessed include lung cells (A549), colon

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cancer cells (Caco-2), kidney cells (UOK), liver cells (HepG2), urinary bladder cells

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(UROtsa), normal human urothelial cell line (SV-HUC-1), and embryonic kidney cells

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(HEK-293), among others [7]. The in vitro experimentation has identified various target

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genes that display altered DNA methylation following exposure to arsenic. For example,

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exposure of A549 and Caco-2 cells to 0-10 µM sodium arsenite for seven days, resulted

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in both global DNA methylation changes as well as changes in the methylation of tumor

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promoter 53 (TP53) [26,27]. Of importance to human health, TP53 functions to control

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tumor proliferation, DNA repair, and cell death [27]. Subsequent dysregulation of this

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gene by epigenetic modifications has been implicated in cancer development, thus

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targeted and global methylation of this gene is a potential mechanism for disease

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development [27]. Additional studies have found targeted hypermethylation and reduced

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expression of death-associated protein kinase (DAPK) to be observed following exposure

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of SV-HUC-1, a non-cancer cell line, to 2-10 µM of sodium arsenite for 48 hours [28].

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Reduced expression of DAPK has been implicated in the development of various types of

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cancer due to DAPK’s function as a tumor suppressor and mediator of cell death, which is

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integral for healthy cell growth and proliferation [29]. It should be noted that due to the

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complexities of culturing primary cells, there are few studies that have used primary cell

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lines to investigate the mechanisms of arsenic-associated DNA methylation. To date this

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lack of assessment of primary cells represents a substantial gap in the literature.

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Nevertheless, cancerous cell lines are able to provide researchers with substantial data

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regarding epigenetic modifications in relation to arsenic exposure.

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In human populations, chronic arsenic exposure is associated with both altered global and gene-specific DNA methylation patterns [19,30-35]. For obvious reasons, the

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majority of these studies have been carried out using DNA isolated from peripheral blood

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leukocytes and not target tissues. As an example, Chanda et al. identified

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hypermethylation of TP53 in chronically exposed adult populations with and without

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cancer [36]. The exposure in the population ranged from <50 to >500 µg/L iAs for over 6

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months. Interestingly, these results overlap with those observed in the A549 and Caco-2

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cell lines. These data further validate the potential for DNA methylation to be implicated

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in the development of certain cancers, as key genes involved in apoptosis and cellular

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proliferation are demonstrated to be dysregulated following exposure to arsenic in human

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populations and in in vitro studies [26,27].

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Additional overlaps between DNA methylation patterns observed in cell culture and human populations are seen in results identified by Chen et al. [37]. In this human

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population study, researchers isolated cells from urothelial tumors, which were induced

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by arsenic exposure, and identified targeted hypermethylation of DAPK. This gene also

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displayed altered DNA methylation in the non-cancer cell line, SV-HUC-1, exposed to

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arsenic [28,37]. Taken together, these data highlight overlap of genes that display

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arsenic-associated DNA methylation in both cell culture and human populations. The

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results support the value of in vitro studies to examine potential mechanisms underlying

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adverse outcomes seen in human populations exposed to arsenic.

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With respect to prenatal exposure in human populations, arsenic has been

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associated with both gene-specific hyper- and hypomethylation [38-44]. A recent study in

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a prenatally-exposed population in New Hampshire examined CpG methylation in the

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placenta. The exposure levels in the population ranged up to 67.5 µg/L arsenic in

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drinking water [45]. Arsenic levels in the placenta were observed to be significantly

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correlated with gene-specific CpG methylation in the target tissue of the placenta [44].

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Specifically, LYR motif-containing protein 2 (LYRM2) displayed the most CpG

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significant methylation in the promoter region of the gene [44]. While the specific role

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that this gene plays in the placenta is unknown, it has been postulated that these findings

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demonstrate arsenic’s ability to alter gene function as expression was reduced in samples

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with the highest levels of methylation [44]. In a separate prenatally exposed population in

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Mexico where exposure ranged up to 236.0 µg As/L the investigators identified lower

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birth weight in infants in relation to arsenic exposure [17]. The DNA methylation of an

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imprinted gene known to play a role infant birth weight was subsequently identified,

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namely potassium voltage-gated channel subfamily Q member 1 (KCNQ1) [38]. This

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study was also unique is its assessment of the relationship between altered DNA

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methylation and functional consequences in gene expression as KCNQ1 expression

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decreased as methylation increased. Importantly, KCNQ1 also is involved in the

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development of diabetes, a disease associated with arsenic exposure. In addition to

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altered DNA methylation of genes involved in birth weight, prenatal arsenic exposure has

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been associated with altered expression of genes involved in inflammatory pathways such

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as tumor necrosis factor (TNF) regulated pathways, nuclear factor κ-light-chain-enhancer

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of activated B cells (NF- κB), transforming growth factor β (TGF-β), Interleukin-10 (IL-

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10) signaling, tumor protein 53 (p53) signaling, and the glucocorticoid receptor signaling

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pathway (Figure 1) [46]. These data highlight the broad epigenomic effects of arsenic

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exposure impacting numerous biological pathways.

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Interestingly, trends for sex-specific DNA methylation patterns in response to arsenic exposure have been observed [47,48]. In a prenatally-exposed Bangladeshi

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population where arsenic ranged up to 661 µg/L As in drinking water, global CpG

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methylation was demonstrated to be decreased in females and increased in males [47].

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Recently, a study by Niedzwiecki et al. investigated the effects of arsenic on global DNA

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methylation in a Bangladeshi adult population population exposed up to 300 µg/L As in

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drinking water. Results indicated that there was no association between arsenic exposure

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and global methylation in females. In contrast, in males arsenic was positively associated

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with global methylation patterns [48]. A limitation of these studies was a lack of the

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assessment of gene expression. Thus, future research should focus on how methylation

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patterns influence gene expression and whether sex-specific methylation patterns could

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potentially underlie sexual dimorphism of disease in relation to arsenic exposure [47]. Due to limitations in sample collection from human populations, many studies do

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not have access to RNA for functional validation of the impact of DNA methylation. For

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those studies have been able to incorporate these critical biological endpoints, the

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importance of genome location of the methyl marks in relation to functional changes in

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gene expression has been noted. Specifically, CpG methylation in the promoter region of

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DNA has been correlated with gene silencing, while methylation of the gene body has

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been associated with gene activation [38]. Studies should aim to examine both DNA

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methylation and gene expression from cells to confirm the functional role of the

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epigenetic modification. Taken together, arsenic exposure is associated with global and

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gene-specific hypo- and hypermethylation. The role of these alterations as they related to

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potential adverse health outcomes mediated through gene silencing, activation, or by

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altering biological pathways remains under investigation.

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Arsenic-induced changes in histone modifications Another potential mediator of mRNA expression and an environmentally-

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responsive epigenetic mechanism is histone modification. The histones are proteins

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which serve as a structure around which DNA can supercoil. This wrapping of DNA

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results in a condensed structure is termed the nucleosome and comprises chromatin [49].

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Histone modifications can impact chromatin structure and allow for the necessary regions

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of DNA to be accessible for their respective DNA binding elements [49]. The acetylation

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of histones has been correlated with increased gene and protein expression due to a

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weakening of DNA-protein contact [49]. Conversely, histone deacetylation has been

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correlated with decreased gene and protein expression due to increased DNA-protein

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contact [49]. In contrast, histone methylation has been shown to have a variable effect on

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transcriptional activity due to the specific location and quantity of the methyl groups on

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the histone tails [49]. Histone modifications are mediated by the enzyme families: histone

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acetyltranferases (HAT), histone deacetylases (HDAC), histone methyltranferases,

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histone demethylases, serine and threonine kinases, and phosphatases [49].

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The mechanistic basis by which arsenicals impact histones is centered on the

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ability of arsenic to affect the enzymes that execute the modification of histones.

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Specifically, arsenic has been shown to interact with histone methyltransferases, HAT,

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HDAC, and kinases [50]. In vitro studies have demonstrated that arsenic can directly

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interact with the enzymes HDAC and HAT [51,52]. The alterations in histone acetyl

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groups from these interactions are dependent upon cell type, duration, and dose of

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exposure [53,54]. It has been shown that arsenic can inhibit the activity of HDAC, which

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results in an increase in global histone acetylation [51]. In contrast, it has also been

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demonstrated that direct binding of arsenic to HAT enzymes results in decreased

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acetylation of histone tails in human embryonic kidney (HeLa) cells [52]. Additionally,

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kinases have been shown to phosphorylate histones in response to arsenic exposure

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depending on the dose and duration of arsenic exposure [55], and histone

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methyltransferase mRNA and protein expression have been demonstrated to increase in

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response to arsenic exposure [56].

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With respect to arsenic exposure in vitro, various histone modifications have been

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observed. The majority of studies that investigated the relationship between iAs and post-

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translational histone modifications (PTHMs) were conducted in vitro utilizing human

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diploid fibroblast cells (WI-38), HeLa adenocarcinoma cell line and normal human

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fibroblasts hybrid (CGL-2), human malignant melanoma epithelial-like cell line

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(RPMI7951), human urothelium non-tumorigenic (UROtsa), human liver carcinoma

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(Hep-G2), human lung carcinoma (A549), and keratinocytes (HaCaT) cell lines [7]. In

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relation to non-cancer cell lines, data from non-tumorigenic UROtsa cell lines exposed to

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1 µM iAs3 for over 30 days demonstrated an increase in H3K4Me2 and an increase in

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H3K9Me2, which are associated with transcriptional activation and repression,

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respectively [57,58]. A separate study utilized Hep-G2 cell lines treated with 7.5 µM of

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sodium arsenite for 24 hours demonstrated an increase in H3K9ac, which is associated

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with transcriptional activation [51]. These results demonstrate arsenic’s potential to

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induce transcriptionally-repressive or activating post-translational histone modifications

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that may subsequently affect gene expression and disease development. These differences

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also could be a result of prior epigenetic modifications being present in cancerous cell

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lines that non-tumorigenic cell lines may not possess. To address this issue, future in vitro

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studies should utilize primary or non-tumorigenic cell lines in conjunction with cancerous

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cell lines to alleviate potential confounding or misinterpretation of in vitro results.

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A few human population-based studies have investigated histone modifications in

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response to arsenic [53,54,59]. A recent study in an exposed population in China

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investigated specific arsenic-responsive histone modifications from coal burning and

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found altered global levels of H3K18ac and H3K36me3 in peripheral blood leukocytes

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[59]. These histone modifications were postulated to be involved in mediating the

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transcription of oxidative stress response genes following oxidative stress from exposure

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to arsenic [59]. Additionally, a study in a Bangladeshi population investigated the

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impacts of arsenic exposure via drinking water ranging up to 500 µg/L As on histone

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modifications in the serum of exposed individuals [54]. Exposure to arsenic was

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associated with increases in H3K9Me2 and decreases in H3K9Ac, which are implicated

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in transcriptional repression and gene silencing and can impact tumor suppression and

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cell proliferation [54]. Jensen et al. examined UROtsa cells exposed to arsenic and

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demonstrated increases in H3K9Me2 following exposure to arsenic [57]. In a separate

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study, in a cohort in Italy exposed to arsenic via inhalation where the mean concentration

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was 0.10 µg/m3 As, there was an increase in H3K4Me2 which is associated with

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transcriptional activation and gene expression [53]. These results are in line with those

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observed in UROtsa cell lines as increases in H3K4Me2 were observed following

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treatment with arsenic [57]. These studies are of particular interest to the field of

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epigenetics as they demonstrate how route of exposure can differentially affect PTHMs

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and potential health outcomes. In these cases, inhalation exposure resulted in histone

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modifications that increased transcriptional activity, while exposures via drinking water

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resulted in transcriptionally repressive PTHMs. Future research is needed to elucidate

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how the route of exposure influences subsequent epigenetic marks.

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Several studies have also shown the potential for cross-talk between several

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epigenetic modifications such as histone modifications and DNA methylation [38,60,61].

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It has been suggested that DNA methylation may guide histone modifications following

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transcription and, conversely, histone modifications may influence DNA methylation

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through interactions between DNA methyltransferases and histones [61]. Additionally,

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differential methylation of the enzymes that modify histones have been observed [38].

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Future research should further investigate these relationships as epigenetic modifications

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remain largely complex and likely do not act alone.

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As was observed for DNA methylation, trends for sex-specific patterns of histone modifications following exposure to arsenic have been observed. In female and male

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adults exposed to arsenic via drinking water ranging up to 500 µg/L As, variable histone

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methylation and acetylation patterns at differing locations on their respective histone tails

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were observed [54]. Specifically, H3K27me3 was positively associated with water

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arsenic levels in females and negatively associated in males. The opposite trend was

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observed with respect to H3K27ac where females displayed a negative association

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between H3K27ac and arsenic water levels and males displayed a positive association

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[54]. These data are significant as these observed associations could be an underlying

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mechanism for sex-specific patterns of disease [54]. Collectively, human population and

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in vitro data demonstrate that arsenic exposure influences histone modifications

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associated with both gene activation and repression.

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Arsenic-induced changes in miRNA expression

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microRNAs (miRNAs) represent a third type of epigenetic regulator. miRNAs are

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small, non-coding RNA sequences about 21-33 nucleotides in length that are known

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regulators of gene expression through complimentary binding of the miRNA to the 3’ or

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5’ regions of mRNA. In some cases miRNAs have been linked to transcriptional

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activation, but more often miRNAs are implicated in gene silencing activity through the

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targeting of specific transcripts for degradation or through a transcription blockage [62].

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As with mRNAs, miRNAs are transcribed within the nucleus. However, after

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transcription inside the nucleus, miRNAs form hairpin structures and exit the nucleus as

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pre-miRNA. The pre-miRNA is then processed further into mature miRNA and

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incorporated into RISC complexes [62].

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The mechanisms underlying arsenic-induced miRNA expression changes are not well characterized, however, it is well established that transcription factors represent

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critical regulators in the control of miRNA expression [63]. The study of the interplay

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between miRNAs and transcription factor regulation represents a current area of

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investigation, as this could represent a potential therapeutic method to combat miRNA-

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mediated diseases [63]. One potential mechanism by which arsenic may modify miRNA

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expression is through the induction of environmentally-responsive transcription factors

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[64]. In support of this, a recent meta-analysis has demonstrated that that there is a

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enrichment for common transcription factor binding site in the promoter region

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sequences of the arsenic-responsive miRNAs [64]. Future research is needed to validate

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the role of arsenic-responsive transcription factors as they influence miRNAs.

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A majority of studies that investigate arsenic-induced miRNA expression changes have been conducted in vitro and spanned the use of human lymphocyte (TK-6), human

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bladder carcinoma (T24), and human liver carcinoma (Hep-G2) cell lines [7]. HepG-2

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cells treated with 4 µM arsenic trioxide for 24 hours displayed upregulation of miR-24,

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miR-29a, miR-30a, and miR-210 [65]. These findings are particularly pertinent to the

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field of epigenetically-mediated disease as miR-29a demonstrated a therapeutic effect in

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liver carcinoma cells through the induction apoptosis and inhibition of cellular growth

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and proliferation, making this miR a potential target for hepatocellular carcinoma therapy

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[65].

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At present, there are only a few human population-based studies that have examined the relationships between altered miRNA expression in response to arsenic

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exposure and the potential for these alterations to result in disease development [7]. For

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example, in a study that investigated the health impacts from prenatal exposure ranging

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up to 236.0 μg As/L in drinking water in Mexico, researchers found a set of differently

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expressed miRNAs and mRNAs that were implicated in innate and adaptive immune

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response [53]. Analyses of these differentially expressed miRNAs revealed enrichment in

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pathways associated with inflammatory disease and response, cancer, diabetes, and

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respiratory disease, among others. mRNA analysis demonstrated several mRNAs to also

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be implicated in immune response [66]. Subsequent expression levels of arsenic-

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associated mRNAs and miRNAs were evaluated and miRNA expression levels were

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found to be negatively associated with mRNA levels, which is important, which indicates

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miRNAs may be responsible for reducing expression or silencing of important genes

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involved in immune response [66]. Given that prenatal exposure to arsenic is associated

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with an increased incidence of infant infections [45], arsenic responsive miRNAs related

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to innate and adaptive immune signaling could be a potential mechanism underlying this

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susceptibility. Future research is needed to examine miRNAs in response to arsenic

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exposure in human populations. Additionally, the potential for cross-talk between

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miRNAs, methylation, and histone modifications needs to be examined.

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In vitro studies have identified arsenic exposure has a variable effect on miRNA expression profiles depending on specific cell type, duration, and dose of exposure [7].

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Given the few studies of miRNAs expression in humans, it is not surprising that there is

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little overlap between observed miRNA expression in human populations exposed to

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arsenic and cell lines. Nevertheless, several studies have confirmed altered miRNA

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expression to be implicated in disease development and immune response, which makes

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miRNAs a possible mediator for arsenic-induced disease development [66,67].

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Summary/Future research needs

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This review provides a concise view of the current literature summarizing known arsenic-associated epigenetic modifications and their potential implications in the

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development of disease. Current literature on the mechanisms underlying arsenic-

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associated changes to the epigenome are detailed. In addition, overlaps between data

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derived from in vitro and human population studies are described. From a mechanistic standpoint, the collective literature supports that arsenic

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dysregulates the epigenome in four primary ways. First, it alters the availability of the

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substrate S-adenosine methionine (SAM) [23]. Second, it modifies transcription factor

383

binding and subsequent availability of DNA to DNA methyltransferase (DNMT) [25].

384

Third, arsenic exposure directly alters the activity of enzymes that modify histones or

385

methylate DNA, such as histone deacetylase (HDAC), histone acetyltransferase (HAT),

386

and DNMT, respectively [13]. Fourth, arsenic may induce activation of environmentally-

387

responsive transcription factors that modify access to DNMT and impact gene-specific

388

methylation [25] or increase the expression of arsenic-responsive miRNAs [64]. These

389

epigenetic modifications are important as they are associated with disease intermediates

390

including cytokines and inflammatory-related proteins, innate and adaptive immune

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response proteins, and are also associated with adverse health outcomes including

392

diabetes development, cancer, and fetal growth [13,38,66]. Still, more research is needed

393

to validate the precise mechanisms by which arsenic induces epigenetic modifications.

394

Comparisons of data derived from in vitro and human population-based studies

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demonstrate overlaps in the epigenetic patterns observed. For example, human exposure

396

and in vitro studies both demonstrated a common gene to be hypermethylated in response

397

to arsenic [26,27,36]. Additionally, histone modifications involved in both transcriptional

398

repression and activation displayed overlaps between studies using non-tumorigenic cell

399

lines and blood-derived assessments in human populations [53,54,57]. Given that there

400

are very few human studies that have investigated miRNA changes in response to arsenic,

401

overlaps between the data derived from cell culture and human populations are sparse.

402

Increasing the number of human population-based studies that assess miRNA expression

403

would provide more information on how miRNAs are altered by arsenic.

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There are several major gaps in the current research on arsenic-associated

405

epigenetic modifications. Related to cell types, many of the in vitro studies thus far have

406

been conducted using cancer cell lines. While overlaps have been observed with specific

407

gene targets, the utilization of cancerous cell lines to quantify epigenetic changes may

408

confound data these cells may possess epigenetic marks prior to exposure that would not

409

be present in normal/primary cells. To address this, future research should focus on the

410

utilization of primary cell lines to model arsenic exposure in human populations to

411

control for potential confounding that may arise from the use of non-normal cell lines.

412

Another limitation is that many human exposure studies that investigate DNA

413

methylation patterns in response to arsenic do not have access to RNA to validate

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expression levels in relation to methylation. To further validate the functional

415

implications of the epigenetic modifications in relation to arsenic, future studies should

416

incorporate mRNA validation into results. These data can be used to substantiate the links

417

between DNA methylation in response to arsenic, gene expression, and the dysregulation

418

of biological pathways that can lead the development of disease. Additionally, many

419

human exposure studies only provide exposure and epigenetic data from one collection

420

time point instead of over time. While costs and experimental feasibility must be

421

considered, designing human exposure studies that examine exposures and the

422

epigenome over time could provide more insight on the stability of epigenetic marks.

423

Finally, there is some evidence for the potential for cross-talk between various epigenetic

424

mechanisms and that those epigenetic mechanisms may be present in a sex-dependent

425

manner [38,48]. Integration of a variety of epigenetic data sets could elucidate the

426

potential cross-talk that is occurring among epigenetic mechanisms and potentially

427

identify sex-specific trends in epigenetic marks following exposure to arsenic.

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In conclusion, there is strong evidence that arsenic exposure is associated with

429

epigenetic changes including DNA methylation alterations, histone tail modifications,

430

and altered expression of miRNAs. Moreover, increasing evidence supports these

431

epigenetic alterations are associated with both cancer and non-cancer health outcomes in

432

human populations. Future research should aim to address the gaps detailed in this review

433

pertaining to arsenic’s effects on the epigenome.

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identifying sites of 5-methylcytosine alterations that predict functional

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changes in gene expression in newborn cord blood and subsequent birth

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outcomes. Toxicol Sci 2015, 143:97-106. Using site-specific DNA methylation

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analyses, the investigators identified that CpG methylation in the promoter region

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of the DNA is most predictive of gene expression levels and functional outcomes.

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This finding demonstrates that the locaiton of CpG methylation plays a significant

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role in gene and protein expression and functional outcomes as a reuslt of

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enivornmental toxicant exposure.

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exposure to be significantly correlated with CpG methylaiton in placental tissue.

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which could be an underlying mechanism for arsneic-induced birth outcomes.

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the associations between global %5-hmC in blood DNA and arsenic exposure

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Highlights: Millions of individuals around the world are exposed to potentially harmful levels

recommended limit of 10 ppb.

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of inorganic arsenic that exceed the World Health Organization (WHO)

Arsenic exposure has been associated with altered DNA methylation patterning, disrupted histone modifications, and altered miRNA expression in vitro and in

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exposed human populations.

Arsenic potentially impacts DNA methylation patterning by altering the activity

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of DNA methyltransferase (DNMT), modifying the availability of S-adenosine methionine (SAM), and modifying transcription factor occupancy. Arsenic exposure is associated with the modification of histone tails through its action on histone deacetylase (HDAC) and histone acetyltranferase (HAT)

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activity.

The genes that are dysregulated via epigenetic mechanisms include those involved in inflammatory pathways, innate and adaptive immune response pathways,

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diabetes-related pathways, and imprint gene-controlled growth pathways.