Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Epigenetics in chemical-induced genotoxic
carcinogenesis
Grace A. Chappell and Julia E. Rager
Abstract
The relationship between genotoxicity and cancer has been
extensively demonstrated and is well accepted; however, the
role of epigenetic regulators that are involved in genotoxic
events and carcinogenic processes is less understood.
Epigenetic mechanisms are known to play important roles in
carcinogenesis, and connections between epigenetics and
DNA damage and repair have been reported. This mini review
discusses some of the known relationships between epige-
netic mechanisms and DNA damage, focusing on DNA
methylation, histone modification, and microRNAs. Examples
include: (i) preferential binding of reactive chemicals or me-
tabolites to methylated cytosines at mutational “hotspots” that
lead to carcinogenesis, (ii) histone modifications that affect
chromatin structure, resulting in altered accessibility of DNA to
damaging agents and/or DNA repair machinery and/or
recruitment of DNA repair machinery to sites of damage, and
(iii) changes in abundance of microRNAs that regulate the
expression of genes involved in DNA damage response. Still,
there remains a need for further research on these topics, as
key findings on epigenetic alterations will advance the under-
standing of chemical-induced cancer, including cases that
involve genotoxicity. A better understanding of the role of the
epigenome in chemical carcinogenesis will contribute to im-
provements in human health hazard assessments and
screening efforts for potential carcinogens.
Addresses
ToxStrategies, Inc., Austin, TX, USA
Corresponding author: Chappell, Grace A. (gchappell@toxstrategies.
com)
Current Opinion in Toxicology 2017, 6:10–17
This review comes from a themed issue on Genomic Toxicology:
Epigenetics
Available online 15 June 2017
For a complete overview see the Issue and the Editorial
http://dx.doi.org/10.1016/j.cotox.2017.06.007
2468-2020/© 2017 Elsevier B.V. All rights reserved.
Keywords
Epigenetics, Epigenomics, Genotoxicity, Cancer, microRNAs, DNA
methylation, Histone modifications, DNA damage.
1. Introduction
Epigenetic alterations represent molecular modifica-
tions to genetic material that do not involve changes to
the nucleotide sequence. Evidence of the involvement
Current Opinion in Toxicology 2017, 6:10–17
Toxicology
of epigenetic alterations in carcinogenesis, including
that which is chemically-induced, is continually
increasing in abundance, strength and reliability [1].
Parallel to the increased inclusion of epigenetic end-
points in toxicology studies, there are ongoing discus-
sions on how such data can be integrated into chemical
evaluations. For example, the integration of epigenetics
data into toxicological assessments has been the focus of
recent working group meetings (e.g., ToxicoEpigenetics
meeting organized by the United States Environmental
Protection Agency (USEPA) and the Society of Toxi-
cology in 20161; Epigenetics and Environmental Origins
of Cancer meeting organized by the International
Agency for Research on Cancer in 20162; Epigenetics
and Risk Assessment workshop organized by the USEPA
in 20153). The induction of epigenetic alterations has
also been included as one of the recently proposed ten
key characteristics of carcinogens [2].
Genotoxicity is another of the proposed ten key char-
acteristics of carcinogens [2], and has long been recog-
nized to play an important role in chemical
carcinogenesis [3]. Genotoxicity is defined as the po-
tential of a chemical to damage DNA, which can result
in heritable mutations through cell divisions [4]. If not
properly repaired, such mutations may ultimately lead to
carcinogenesis via activation of oncogenes and/or inac-
tivation of tumor suppressors [3,4]. However, not all
genotoxic agents are mutagenic, as some damage can be
repaired by normal cell processes, including those
involving epigenetic mechanisms as discussed below.
Additionally, DNA damage associated with chemical
exposures may act as an initiating event in carcinogen-
esis, or it may occur within the sequelae of molecular
initiating events. Determining the step at which DNA
damage occurs in chemical-induced carcinogenesis re-
mains a critical issue in understanding the mode of
action of hazardous chemicals, a key issue contributing
to chemical safety assessments [5].
While specific aberrations to the epigenetic “code”
represent potential non-genotoxic mechanisms of
carcinogenesis, such alterations may occur concur-
rently with genotoxic events, as there is evidence that
certain epigenetic mechanisms are related to DNA
damage and repair [6e13]. Broadly, existent epige-
nomic features (i.e. the entirety of DNA and histone
1
http://www.toxicology.org/events/shm/cct/Toxicoepigenetics.asp.
2
http://iarc-conference2016.com/ege-meeting.
3
https://cfpub.epa.gov/ncea/risk/recordisplay.cfm?deid=308271.
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 Epigenetics and genotoxicity Chappell and Rager 11

modifications that determine the chromatin state) may
influence DNA damage; chemical-induced epigenetic
alterations may occur in tandem with DNA damage;
DNA damage may cause epigenetic changes; and some
epigenetic mechanisms may cooperate in DNA
damage repair. We present herein a brief discussion of
several evident relationships between chemical-
induced DNA damage and epigenetic alterations, and
highlight examples of chemicals for which epigenetic
alterations, DNA damage, and carcinogenesis have
been demonstrated.
2. The relationship between epigenetic
alterations and DNA damage
Although somewhat limited, there is evidence that all
major types of epigenetic and epigenomic alterations
(DNA methylation, histone modifications, chromatin
remodeling, and expression of non-coding RNAs) have
relationships to DNA damage (Fig. 1). An overview of
these relationships, focusing on DNA methylation, his-
tone modifications, and microRNAs, follows.
2.1. DNA methylation and DNA damage
DNA methylation refers to the addition of methyl
groups from the universal donor S-adenosyl-L-methio-
nine to DNA cytosine residues. The patterning and
maintenance of DNA methylation influences many
important normal cellular processes (e.g. embryonic
development, transcription, genomic imprinting, and
chromatin structure), while aberrant DNA methylation
patterns at both the gene and global level have been
identified in many types of cancer [14].
There is an apparent relationship between DNA
methylation/demethylation and DNA damage/repair
systems [15] (Fig. 2AeC). For example, 5-
methylcytosine (5mC) and 5-hydroxymethylcytosine
(5hmC) patterns are proposed to influence the likeli-
hood of DNA damage caused by genotoxic agents. 5mCs
represent the most abundant type of DNA methylation
and are regulated by DNA methyltransferases
(DNMTs), enzymes that catalyze de novo methylation
during development and propagate methylation

Fig. 1

Overview of representative relationships between epigenetic mechanisms in DNA damage response. This schematic provides a brief overview of several
ways in which epigenetic alterations are involved in the cellular response to DNA damage, some of which are inter-related, and depicts the relationships
between these epigenomic features within the cell. (A) Acetylation at specific lysine residues represents a histone modification that is altered by DNA
damage and is also commonly directly (and indirectly) involved in DNA damage response. (B) Covalent binding of some chemicals to DNA exhibits site-
specific preference for methylated cytosines. Changes to DNA methylation may result in response to DNA damage, which can affect transcription of
genes related to DNA damage repair and/or cancer. (C) Alterations in the expression level and nuclear transport of miRNAs as a result of DNA damage
can regulate genes involved in DNA repair and/or cell cycle. Double-ended arrows emphasize that these alterations are, in many cases, not permanent
(although in some exposure scenarios they can become static and/or permanent). Abbreviations: DNMTs: DNA methyltransferases; HATs: histone
acetyltransferases; miRNA: microRNA.

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12 Genomic Toxicology: Epigenetics
Fig. 2
Example relationships between DNA methylation/demethylation systems and DNA damage/repair systems. (A) DNA methylation can influence DNA
damage susceptibility through changes in chromatin structure. (B) DNA damage can influence DNA methylation profiles through changes in chromatin
structure, which then impacts the accessibility for maintenance methylation and DNA repair machinery. (C) DNA methylation can alter the expression of
genes involved in DNA damage repair, which can then influence DNA damage repair capacity. Note that in these instances, directionality can be reversed,
or modified altogether, as epigenetic profiles can be dynamic.
patterns during cellular replication [16]. 5hmCs are
catalyzed by enzymes of the ten-eleven translocation
(TET) family and can exist as intermediates during
demethylation processes, and are potentially associated
with carcinogenesis [16].
Cytosine methylation influences local chromatin struc-
ture (i.e. the degree of compaction or openness of DNA
packaging) [15], affecting DNA accessibility to tran-
scription factors and covalent binding of DNA-reactive
compounds (Fig. 2A). Conversely, DNA adducts may in-
fluence local chromatin structure, which can impact the
accessibility of DNA methylation maintenance machin-
ery and/or DNA repair machinery (Fig. 2B) [15]. Both of
these mechanisms can then affect methylation profiles, as
methylation maintenance machinery can add methyl
groups to cytosines, and DNA repair systems can actively
remove and replace methylated cytosines with un-
methylated cytosines [15,16]. Further, DNA methyl-
ation status can influence DNA repair via the silencing of
tumor suppressor genes involved in DNA damage
response through hypermethylation of CpG dinucleotide-
dominant regions (CpG “islands”) that are often present
within gene promoters and are typically predominantly
unmethylated (active) in a normal cellular state (Fig. 2C)
[6]. Some DNA repair proteins can also specifically bind
to methylated CpG sites and increase DNA damage repair
efficiency. For example, methyl CpG-binding proteins
MBD4 and CTCF bind to methylated CpG sites and are
involved in mismatch repair, and nucleotide excision
repair [6] and DNA repair [1,17], respectively. These
mechanisms demonstrate important links between DNA
damage response and DNA methylation.
Current Opinion in Toxicology 2017, 6:10–17
2.1.1. Example toxicant: benzo[a]pyrene
Benzo[a]pyrene (BaP) represents a well-studied geno-
toxic carcinogen, causing cancer of the skin and lung in
humans and multiple sites in animal models [18], that
has also been shown to alter DNA methylation. BaP
exposure can cause DNA lesions through metabolism to
the diol-epoxide, benzo[a]pyrene-7,8-diol-9,10-epoxide
(BPDE), which reacts predominantly at the N2-posi-
tion of guanine, resulting in BPDE-N2-dG adducts [18].
Studies evaluating BaP responses have shown a direct
impact of methylation on adduct formation. For example,
methylated CpG sites have been identified in vitro as
preferential binding targets for BaP metabolites within
specific codons in the p53 tumor suppressor gene body, at
locations that are commonly mutated in smoking-
associated lung cancers [13,15], as well as at codon 14
of the K-ras proto-oncogene, which is also frequently
mutated in various human cancers [15]. The occurrence
of 5mC at these mutation sites has also been correlated
with high levels of BPDE adducts [19], further sub-
stantiating the relationship between exposure, methyl-
ation status, DNA damage, and mutagenesis.
Relatedly, BaP exposure-induced DNA damage has been
shown to impact DNA methylation. BPDE-N2-dG ad-
ducts have been shown to cause local relaxation of the
chromatin structure, which can increase access for DNA
repair systems, and such DNA repair mechanisms can, in
turn, induce DNA demethylation (reviewed in Ref.
[15]). BPDE-N2-dG adducts have also been shown to
reduce the binding of the prokaryotic DNA methyl-
transferase M.HhaI [20]. While evidence of these
mechanisms in humans is limited, one study reported an
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association between increased BPDE adducts and
changes in global and gene-specific promoter DNA
methylation in circulating lymphocytes from coke-oven
workers [7]. These findings demonstrate the potential
of genotoxic carcinogens to impact DNA methylation/
demethylation levels, which have been shown to func-
tion in tandem with DNA damage/repair systems.
2.2. Histone modifications and DNA damage
Modifications to histones, proteins around which DNA is
coiled, occur post-transcriptionally, and include the
addition/removal of: phosphoryl, methyl or acetyl groups;
ubiquitin; or small ubiquitin-like modifier proteins on
specific amino acid residues of histone tails. Although
dynamic histone modifications are essential to normal
cellular processes, aberrant histone modifications have
been related to carcinogenesis [1,14]. Of pertinence to
genotoxic chemical carcinogenesis, histone modifications
represent essential events in DNA damage response and
repair [8,11,12] (Fig. 3AeC), as they can influence the
chromatin landscape, which determines the accessibility
of DNA to repair machinery (Fig. 3A) and, potentially, to
further DNA damaging events [15]. Specific histone
modifications co-localize with DNA damage and serve as
binding sites for repair proteins (Fig. 3B). An established
example is the phosphorylation of the histone 2 variant
H2AX at sites of DNA breakage, which is involved in the
recruitment of DNA repair proteins and is required for
homologous recombination [21]. Additionally, acetyla-
tion of lysines 9 and 14 of histone 3 has been shown to be
essential for efficient nucleotide excision repair in yeast
[11], and co-localization of acetylated lysine 56 on his-
tone 3 with DNA double strand breaks in mammalian
cells in vitro suggests a role in DNA damage repair [8].
The level of these histone acetylation marks, among
Fig. 3
Example relationships between histone modifications and DNA damage/repair systems. (A) DNA damage can affect histone modifications that influence
local chromatin structure, which, in turn, influences accessibility to DNA repair enzymes. (B) Examples of histone modifications that recruit DNA repair
machinery to sites of DNA breakage. (C) Histone methylation status can influence DNA methylation in a canonical fashion, typically involving DNMTs,
ultimately influencing expression of genes involved in DNA damage repair (among other processes), which can lead to increased prevalence of
mutations.
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Epigenetics and genotoxicity Chappell and Rager 13
others, are dynamically altered following DNA damage
(Fig. 3A) [21]. Further, histone modifications (particu-
larly methylation of specific lysine residues) can influ-
ence cytosine methylation through complex (and not
fully characterized) relationships involving binding do-
mains and transcription factors related to DNA methyl-
transferases (Fig. 3C) [22].
2.2.1. Example toxicant: 1,3-butadiene
1,3-Butadiene is metabolized to DNA- and protein-
reactive intermediates that bind with DNA and cause
damage, including DNAeDNA cross-links [23]. Addi-
tionally, epigenetic alterations have been observed in
various tissues of mice exposed via inhalation to this
toxicant, with varying patterns of both DNA adducts and
epigenetic alterations between target and non-target
tissues of tumorigenesis [23]. For example, a loss of
histone marks that are essential for DNA repair were
significantly decreased in the lung of exposed mice,
which was also the tissue with the highest level of DNA
adducts. Further, an increase in histone marks that
confer increased transcriptional activation and genomic
instability (e.g. acetylation of lysine 27 of histone 3) was
observed in the liver, which is a target tissue of
1,3-butadiene-induced tumorigenesis in the mouse. In
contrast, histone modifications that confer transcrip-
tional inactivation (e.g. trimethylation of lysines 9 and
27 on histone 3) were observed in the kidney, a tissue
that is not a target of butadiene-induced tumorigenesis
in the mouse [23]. This finding is important because
DNA adducts are observed in both target (liver and
lung) and non-target (kidney) tissues of tumorigenesis
in butadiene-exposed mice, indicating that the epige-
netic marks may be specific to the carcinogenic process
under the conditions evaluated.
Current Opinion in Toxicology 2017, 6:10–17
14 Genomic Toxicology: Epigenetics
2.3. Differential microRNA expression and DNA
damage
MicroRNAs (miRNAs) are short (17e24 nucleotides),
noncoding RNAs that post-transcriptionally regulate
gene expression through direct RNA binding, recruit-
ment of chromatin modifying enzymes to target genes,
and/or the recruitment of proteins to form ribonucleo-
protein complexes [24]. miRNAs are known to regu-
late a variety of molecular and cellular processes that
occur in both normal and pathologic states, including
cell differentiation, proliferation, and migration, and the
initiation/progression of disease [24].
There is a wealth of data demonstrating aberrant
miRNA expression in various types of cancer [25], and
disruptions in miRNA expression have been associated
with exposure to various genotoxic agents [26].
Although their exact role is unclear, there is evidence
that specific miRNAs are involved in DNA damage
response. For example, induction of processing and nu-
clear export of miRNAs has been demonstrated in
response to DNA damage [10], and cell-cycle arrest has
been shown to be induced by miR-34, which shows
increased expression in response to DNA damage [9].
While the mechanisms linking miRNA expression
modulation with DNA damage response are still being
elucidated, recent data show that the modulation of
DNA-damage responsive miRNAs can occur in a manner
that is dependent upon ATM serine/threonine kinase, a
primary sensor of DNA damage [9,10].
2.3.1. Example toxicant: formaldehyde
Formaldehyde represents an example of a carcinogen
that impacts miRNA expression [27,28] and causes DNA
damage [29]. The mode of action underlying tumors of
the airway has been postulated to involve increased cell
proliferation resulting from cell damage, while both
genotoxicity and cytotoxicity have been considered to
play a role in nasal tumors [30]. The evaluation of DNA
adducts associated with formaldehyde exposure requires
the delineation between baseline levels of DNA adducts
formed by endogenously-produced formaldehyde and
that formed by exogenous sources of formaldehyde. This
delineation can be achieved using isotopically labeled
formaldehyde and mass spectrometry methods [29].
These methods have been employed in parallel with
genome-wide miRNA expression analyses, generating
data to enable the identification of potential relation-
ships between DNA damage and miRNAs.
Exogenously produced adducts have been evaluated
alongside miRNA expression profiles in the nasal
epithelium of nonhuman primates exposed to 0, 2, and
6 ppm formaldehyde for two days [27,29]. Using these
published data, the relationship between exposure-
altered miRNA expression and exogenous N2-HOMe-
dG levels was assessed (Appendix A). Two miRNAs
were significantly associated with exogenous adduct
Current Opinion in Toxicology 2017, 6:10–17
levels: miR-152 and miR-142-3p (Fig. 4A). Transcrip-
tional targets of these two miRNAs were predicted using
experimentally and computationally-derived in-
teractions, and showed significant enrichment for the
functional category ‘DNA replication, recombination,
and repair’ (Fig. 4B), among others (Appendix A).
Although these miRNAs have not been confirmed to
interact directly with formaldehyde-induced adducts,
and the potential delineation between adduct presence
and general cytotoxicity/tissue damage requires exami-
nation, these data clearly demonstrate that formalde-
hyde can significantly alter the expression of miRNAs,
including miRNAs that regulate transcriptional targets
involved in DNA damage response signaling.
3. Plasticity, temporality, and specificity of
epigenetic features in genotoxic
carcinogenesis
Plasticity is a principal feature of epigenetic alterations,
and is an important aspect to consider for causal infer-
ence. While identification of static vs. transient epige-
netic marks may provide insight into their influence on
disease progression (as discussed recently in Refs.
[31,32]), non-permanent changes to the epigenome
may still influence DNA damage response and carcino-
genesis, and should not be undervalued as events
involved in disease progression.
Epigenetic changes have been demonstrated as early
events in carcinogenesis [1], in some cases preceding
DNA damage [9,15]. Further, some epigenetic marks are
observed in surrogate tissues, indicating their sensitivity
to environmental exposure [26,28]. Together, these
qualities position epigenetic endpoints as candidates for
efficacious biomarkers of exposure and disease, a topic
discussed in detail in another article in this special issue.
It has also been demonstrated that epigenetic profiles
differ between target and non-target tissues of tumori-
genesis [28], while DNA adduct measures did not
delineate sites of tumorigenesis in specific cases [23],
exemplifying the potential utility of epigenetic markers
in cancer evaluations.
Some epigenetic alterations are relatively non-specific,
such as global DNA hypomethylation, commonly
found across many cancer types and etiologies. In other
instances, epigenetic alterations are more specific,
including increased potency of DNA binding at meth-
ylated CpG sites only at certain codons [13], gene-
specific hyper and/or hypomethylation [3,7], and vari-
able histone alterations related to DNA damage
response between species [1] and exposure scenarios
(e.g., [8] vs [11]). The identification of epigenetic al-
terations that are chemical-, cancer type-, species- and/
or tissue-specific is essential to enhance our under-
standing of the role of specific epigenetic events and
features in defined cancer pathways.
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 Epigenetics and genotoxicity Chappell and Rager 15

Fig. 4

Formaldehyde-altered miRNAs and their relationships to DNA damage. (A) Expression levels of formaldehyde-altered miRNAs, miR-142-3p and miR-
152, are associated with exogenous DNA adduct levels in nasal epithelial tissue of nonhuman primates. The plotted miRNAs show expression alterations
significantly associated with formaldehyde exposure (p < 0.05 for 0 vs. 2 ppm FA and 0 vs. 6 ppm FA) and with the levels of exogenous N2-HOMe-dG
mono-adducts (p < 0.05 for adduct levels vs. expression levels). (B) Transcriptional targets of miR-142-3p and miR-152 are related to DNA replication,
recombination, and repair signaling. Interactions are shown with either solid lines, representing experimentally validated interactions, or dashed lines,
representing computationally predicted interactions. Abbreviations: BH, Benjamini-Hochberg; FA, formaldehyde.

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16 Genomic Toxicology: Epigenetics
In vivo studies that include multiple time-points and/or
duration of exposure, as well as evaluations at various
stages of tumorigenesis, are essential to better under-
stand the plasticity and temporality of epigenetic
events. Further, the measurement of functional end-
points such as DNA damage and the expression and
activity of genes and enzymes that are involved in DNA
repair in parallel with epigenetics endpoints will enable
a better understanding of the role of epigenetic events
in DNA damage and repair. The continuing increase in
application of various “-omics” methods, including
transcriptomics and genome-wide methylation studies,
should contribute to a rise in the presentation of such
datasets.
4. Summary and conclusions
Epigenetic mechanisms are important factors in carci-
nogenesis related to DNA-damaging agents. We have
highlighted examples of the roles of DNA methylation,
histone modifications, and miRNA expression in response
to DNA damage-inducing chemicals, underscoring that
alterations to the epigenome can potentially influence
carcinogenic potential. Although we present several ex-
amples, there is currently a general paucity in the avail-
able data regarding epigenetic mechanisms among
carcinogens for which a genotoxic mechanism is consid-
ered a potential factor in the carcinogenic potential [26].
Finally, evidence of association vs. direct involvement of
epigenetic alterations and DNA damage represents an
area requiring further elucidation. The demonstration of
direct relationships (protective or deleterious) between
the epigenome and DNA damage in chemically-altered
cells presents an opportunity to advance the under-
standing of underlying mechanisms of chemical carcino-
genesis; specifically, to better understand the relationship
between genotoxicity, a time-tested feature of many
cancer-causing chemicals, and epigenetic alterations, a
relatively new, but important, molecular modulator of
carcinogenesis. Such findings will enhance the applica-
tion of epigenetics data in hazard and risk assessments.
Acknowledgments
No external funding was received for this work.
Appendix A. Supplementary data
Supplementary data related to this article can be found
at http://dx.doi.org/10.1016/j.cotox.2017.06.007.
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