![A comparison of time points for erasure and reprogramming of the DNA methylome prior to fertilization and during embryogenesis in mammals and fish. The methylome is erased and reprogrammed in primordial germ cells at approximately the time of birth or hatching. Imprinting follows in mammals, but has not been established in fish. Following fertilization (F1) the methylome is again erased and reprogrammed between the zygote and blastocyst stage in male and female mammals, but the paternal methylome appears to be maintained in fish at this stage. Adapted from Head, 2014 [23].](https://www.researchgate.net/profile/Susanne-Brander/publication/320116105/figure/fig2/AS:556283155542016@1509639647410/A-comparison-of-time-points-for-erasure-and-reprogramming-of-the-DNA-methylome-prior-to_Q320.jpg)
![Recent research [26] suggests that epigenetic change often precedes and may even trigger or increase the likelihood for mutations resulting in altered DNA sequence. Thus epigenetic variation is the mechanism by which organisms can adapt to rapid environmental perturbations, such as pollution, but these modifications on the surface of DNA or histones may in turn increase the probability that a genomic region might mutate, facilitating adaptation over the long term. This suggests that the Lamarckian basis for inheritance facilitates rapid evolution while providing the stimulus for mutations that enable long-term Darwinian adaptation. Images sourced from Wikimedia.](https://www.researchgate.net/profile/Susanne-Brander/publication/320116105/figure/fig5/AS:556283155103747@1509639647574/Recent-research-26-suggests-that-epigenetic-change-often-precedes-and-may-even-trigger_Q320.jpg)
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The Role of Epigenomics in Aquatic Toxicology
Susanne M. Brander,
a,
* Adam D. Biales,
b
and Richard E. Connon
c
a
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, USA
b
National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio
c
Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California,
USA
Abstract—Over the past decade, the field of molecular biology
has rapidly incorporated epigenetic studies to evaluate organism–
environment interactions thatcan result in chronic effects. Such responses
arise from early lifestage stress, the utilization of genetic information over
an individual’s lifetime, and transgenerational inheritance. Knowledge of
epigenetic mechanisms provides the potential for a comprehensive
evaluation of multigenerational and heritable effects from environmental
stressors, such as contaminants. Focused studies have provided a greater
understanding of how many responses to environmental stressors are
driven by epigenetic modifiers. We discuss the promise of epigenetics and
suggest future research directions within the field of aquatic toxicology,
with a particular focus on the potential for identifying key heritable marks
with consequentialimpacts at the organism and populationlevels. Environ
Toxicol Chem 2017;36:2565–2573. #2017 SETAC
Keywords—Epigenetics; Aquatic toxicology; Contaminants; Ecotoxico-
genomics; Stressors
Introduction
The pivotal role played by the epigenome in orchestrating the
expression of genes that drive cellular differentiation and the
response to environmental change (e.g., temperature, nutri-
tion, pollution) is now undisputed. The epigenome is a suite of
mitotically and/or meiotically heritable changes in gene
function that occur via mechanisms other than direct changes
in deoxyribonuclease (DNA) sequence. Epigenetic regulation
of gene function is associated with a wide variety of
mechanisms that both positively or negatively impact gene
transcription or the production of protein products. Some of
the more well-known mechanisms include DNA methylation
and modification of histones through the methylation or
acetylation of lysine residues. Functional groups added to
specific regulatory regions of DNA (either upstream of genes
or bound to the tails of histones that provide DNA’s
framework) dictate which regions of coding DNA are
accessible to transcriptional machinery. Epigenetics involves
the reduction or prevention of transcription via methylation of
cytosine nucleobases in regulatory regions, as well as either
decreased or increased transcription via methylation or
acetylation of lysine in histone tails, respectively. The
processes of histone modification and DNA methylation are
directed by, and transcripts can be deactivated prior to,
translation by short-stranded noncoding ribonucleic acids
(RNAs) [1].
We now understand that these 3 major epigenetic mecha-
nisms, DNA methylation, histone modification, and non-
coding RNAs (ncRNAs), are shared across most taxa, and
that the response to environmental stressors, including
aquatic pollutants, is influenced by epigenetic modifiers [2].
Thus there is great interest in the study of epigenetics in the
many model and nonmodel organisms used for experimen-
tation in the field of aquatic toxicology. In many cases
organisms with rapid developmental life stages greatly
facilitate investigations into somatic epigenetic change as
well as the potential for epigenetic transgenerational
inheritance.
Much of the current thinking about epigenetics and its role
in adaptation and disease manifestation is derived from
In This Issue:
ET&C FOCUS
Focus articles are part of a regular series intended to sharpen understanding of current and
emerging topics of interest to the scientific community.
* Address correspondence to branders@oregonstate.edu
Published online in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/etc.3930
#2017 SETAC
Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017 2565
research with mammals and plants. Technical advances
have greatly expanded our ability to interrogate the
epigenome in new species, with greater resolution and on
an unprecedented scale (epigenome wide). Moreover, much
has been learned through the combination of epigenome-
wide studies and whole transcriptome studies [3]. Recent
research has challenged the most fundamental understand-
ing of the relationship between epigenetic mechanisms and
phenotypic plasticity and has forced researchers to consider
the degree to which results can be extrapolated across
taxa [4–6]. Despite these unknowns, it is clear that
epigenetics is a major engine driving adaptation, which
has implications in many areas of basic and applied
ecological research. In the present study we build on a
previous Focus publication [7], discussing new examples of
epigenetic research specifically within the field of aquatic
toxicology, along with suggested directions for new
research. We also give an overview of emerging questions
regarding recently recognized links between epigenetic
change and classical Darwinian mechanisms of inheritance.
Definitions
Adverse outcome pathway
(AOP)
A structured representation of biological events leading to adverse effects that are relevant
to risk assessment.
Bivalent chromatin Chromatin that is intermediate between heterochromatin and euchromatin.
Blastocyst A mammalian blastula in which some differentiation of cells has occurred, the inner cell mass
of the blastocyst will become an embryo.
CpG island Regulatory regions of DNA where cytosine (C) nucleobases occur adjacent to a guanine (G)
in the linear sequence of bases (sharing a phosphodiester bond –p) at a high frequency,
often in a promoter region where transcription factors bind.
DNA methylation Methyl groups added by DNA methyltransferases to cytosine nucleobases, which typically
cause a decrease in the expression of upstream genes.
Endocrine-disrupting
compound
A chemical capable of imitating, inhibiting, or interfering with the action or production of
endogenous hormones.
Epigenetic transgenerational
inheritance
The transmission of epigenetic tags or states via the germline without direct exposure of the
affected generation.
Epigenetic trap A maladaptive epigenetic response caused when evolutionarily novel factors (e.g.,
pollutants, other temporary stressors) disrupt epigenetic machinery.
Epigenome A suite of mitotically or meiotically heritable changes in gene function that cannot be
explained by changes in DNA sequence.
Epimutation An aberrant chromatin state leading to altered gene expression patterns that occurs in the
absence of a change in DNA sequence.
Euchromatin Chromatin that is loosely held together resulting in DNA being accessible by transcription
factors and RNA polymerase.
Heterochromatin Chromatin that is tightly held together resulting in DNA being inaccessible by transcription
factors and RNA polymerase.
Histone acetylation Acetyl groups added by histone acetyltransferases to lysine residues present in histone tails,
generally resulting in open or euchromatin and increased expression of associated genes.
Histone variant Histones that differ slightly in amino acid sequence from common varieties and play a role in
the remodeling of chromatin (e.g., H3.3).
Methyl-binding domain
protein
Proteins that bind methylated DNA and recruit multiprotein complexes resulting in histone
modification and remodeling, activating or repressing gene activity.
Methylome The set of nucleic acid methylation modifications in an organism’s genome or in a particular
cell.
ncRNA Noncoding RNAs that bind to and thus interfere with the translation of mRNA transcripts
into proteins or direct chromatin remodeling.
Nucleosome A structural unit of a eukaryotic chromosome that consists of a length of DNA coiled around
a core of histones.
Parthenogenesis A form of asexual reproduction in an embryo develops from an unfertilized egg, resulting in
a genetically identical copy of the parent.
Primordial germ cells Cells in an embryo that develop into stem cells that generate reproductive gametes (sperm
and eggs) at reproductive maturation.
RNA polymerase II A protein–protein complex that catalyzes the transcription of DNA to generate pre-mRNA
and noncoding RNA.
Somatic epigenetic
transmission
The transmission of epigenetic tags or states during mitosis, within an organism’s lifetime.
Zygote A diploid cell resulting from the fusion of two haploid gametes; a fertilized egg (ovum).
2566 Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017
Epigenetic Mechanisms
Epigenetics includes both transgenerational (i.e., germline
transmission of information) and nontransgenerational (i.e.,
somatic, mitotic stability) factors. Epigenetic information can
be maintained after cell division, can spread between cells, and
can even pass via the germline from one generation to the next.
Because of transgenerational factors, DNA methylation is the
most broadly assessed mechanism in the study of multigenera-
tional impacts resulting from environment–organism interactions.
Nontransgenerational epigenetics can be used to evaluate disease
progression during the lifetime of an individual, and to associate
events from early life stages that chronically affect growth,
development, and reproduction [8]. However, so-called non-
transgenerational, somatic cell effects can become transgenera-
tional, most noticeably in parthenogenetic individuals (e.g., some
cladoceran species), because this form of asexual reproduction
results in direct germline transmission of epigenetic information.
By far the best-studied type of epigenetic mark is DNA
methylation. Most commonly this involves the addition of a
methyl group to the pyrimidine ring of a cytosine base,
giving rise to a 5-methylcytosine (5-mC). Methylation of
cytosines, often those contained within a (cytosine-
phosphodiester-guanine [CpG]) island upstream of a gene
promoter, usually results in decreased expression of the
affected gene because 5-mCs prevent or inhibit access by
transcription factors [9]. However, as the number of
genome-wide association studies and now epigenome-
wide association studies continue to grow, it is becoming
evident that, at least on the genome scale, the relationship
between gene expression and methylation levels is not as
clear as once believed.
Although DNA methylation can repress gene transcription by
blocking the binding of the transcriptional machinery to
DNA, the primary mechanism for modulating gene activity is
through the modification of chromatin structure (Figure 1).
Chromatin, the framework around which DNA is wrapped,
exists in 2 distinct formations: euchromatin or heterochro-
matin. Loosely wound euchromatin is accessible by RNA
polymerase II and transcription factors that can range from
general coactivators or corepressors to specific nuclear
receptor dimers or monomers (i.e., bound estrogen or
glucocorticoid receptors). Heterochromatin, around which
DNA is tightly assimilated, generally cannot be easily
accessed by the multitude of proteins that regulate gene
expression. Evidence is mounting that ncRNAs, DNA
methylation, and histone modification act in concert to direct
chromatin structure and the expression of associated
genes [1].
Histone acetylation and methylation, a highly conserved
process, involves the addition of one of these functional
groups to a lysine (K) on the tail of a specific histone [7]. It is
widely established that lysine 9 on histone H3 (H3K9) is one
FIGURE 1: Chromatin exists as euchromatin or heterochromatin, with an intermediate bivalent state. Loosely wound euchromatin is accessible by
RNA polymerase II and transcription factors. The genes contained within heterochromatin generally cannot be expressed. DNA methyltransferases,
histone deacetylases, and methyltransferases produce a heterochromatic state, whereas histone acetyltransferases and ten-eleven translocation,
along with some types of DNA repair, contribute to a euchromatic state. Evidence suggests that noncoding RNAs, DNA methylation, and histone
acetylation and histone methylation act together to influence chromatin structure and the expression of associated genes.
Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017 2567
of the most common sites for methylation (which can be
heritable), whereas H3K27 is frequently acetylated. Methyl-
ation or acetylation at one of these sites induces a
heterochromatic or euchromatic state, respectively. In
contrast, methylation of H3K4 results in increased association
with active promoter elements. Thus, in terms of histone
modifications, the relationship between chromatin structure
and acetylation or methylation status may be even less clear-
cut than 5-mC tags on DNA. In fact, recent research has
confirmed the existence of so-called bivalent chromatin,
which is intermediate between euchromatin and heterochro-
matin [1]. Histone variants, which differ slightly in amino
acid sequence from their commonly present siblings, also
appear to play a large and highly conserved role in the
remodeling of chromatin. For example, the variant H3.3 is
present in actively transcribed chromatin, whereas mac-
roH2A.1 and the isoform H2A.2 can have either positive or
negative effects on transcription depending on the gene of
interest. These variants appear to be conserved across
phylogenetic groups [2,10].
While known to also be an important component of the
epigenetic machinery, the role of ncRNAs is currently the
least well-understood mechanism. These ncRNAs govern 2
different types of interrelated activities, on the one hand
interfering with already transcribed messenger RNA by
binding to it and preventing translation (microRNAs
[miRNAs]), and on the other directing the remodeling of
chromatin to silence specific genes [11]. These changes
appear to be capable of persisting through many rounds of
mitosis. Studies that have used injectable ncRNAs have found
that the miRNA possessed by sperm can reduce maternal
transcripts in early zygotes, reprogramming the phenotype for
stress response [12].
Modifications of histones and DNA are initiated, removed,
and maintained through the action of a number of proteins
and protein complexes. Methylation status of genes across
the genome is established by the de novo DNA methyl-
transferases DNMT3a and b, which methylate previously
unmethylated DNA, and maintained by DNMT1, which
copies pre-existing methylation during replication [13].
Mechanisms of demethylation are less well understood and
were, until recently, considered a passive process, whereby
methylation was lost because of reduced DNMT1 activity.
However, mechanisms of active demethylation have been
identified. One such mechanisms relies on the ten-eleven
translocation family of proteins, which, through a series of
reactions converts 5-mC to 5-hydroxymethylcytosine and
eventually to 5-formylcytosine and 5-carboxylcytosine,
which are subsequently converted to unmethylated cytosine
through thymine–DNA glycosylase activity [14]. Alterna-
tive demethylation pathways involve DNA repair pathways
and associated proteins (base excision repair glycosylases
and Gadd45) [15]. Histone methylation is accomplished
through the action of the histone lysine methyltransferases,
such as the SET-domain protein methyltransferase
family [16].
Acetylation is another histone modification process associ-
ated with epigenetic regulation, where the presence of acetyl
groups signifies active genes. Acetylation is accomplished
through the actions of a diverse group of histone acety-
transferases that usually occur in large multi-subunit
complexes, where the constituents of the complex target the
histone acetytransferase activity [17]. Acetylation of histones
is reversible, and transcriptional levels of acetylated genes are
modulated through the addition and removal of acetyl groups
through an interplay of histone acetytransferases and histone
deacetylases. The DNA methylation and histone modification
are linked through proteins containing methyl-binding
domains. The methyl-binding domain proteins bind methyl-
ated DNA and recruit multiprotein complexes resulting in
histone modification and remodeling. The functional signifi-
cance of this is that methyl-binding domain proteins are able
to either activate or repress gene activity [18].
Early Life Exposures
It has been established, at least in vertebrates, that organisms
begin life with an epigenetic blueprint. Shortly after fertilization,
however, prior epigenetic marks are erased, and the genome
undergoes de novo methylation. This occurs again at
approximately the time of birth or hatching in primordial
germ cells, which will eventually mature and develop into
gametes that may pass this information to the next generation.
However, some of the similarities between what is established in
mammals and what has been observed in the organisms (i.e.,
fish) used to study the effects of water-borne pollutants may end
there (Figure 2). Much of what is known regarding epigenetics in
aquatic organisms results from the study of zebrafish (Danio
rerio). For example, in zebrafish it appears that genomic
imprinting does not occur as it does in mammals [19].
Furthermore, CpG island number is lower compared with
mammalian genomes, whereas the methylation levels of these
regions is comparatively higher in zebrafish [19].
The developmental origins of health and disease is a widely
accepted paradigm that can be applied across taxa. This
expression refers to early life exposures, during the sensitive
periods of embryogenesis or the young juvenile period, that
result in adverse or diseased adult phenotypes (i.e., metabolic
disorders, infertility) as a result of somatic epigenetic
transmission. Environmental chemicals are hypothesized to
interfere with the process of methylation in primordial germ
cells and/or to cause the addition of de novo methyl tags. A
recent study in zebrafish measured global methylation and
site-specific addition or removal of 5-mCs following exposure
to several classes of compounds (endocrine disruptors, heavy
metals, pharmaceuticals) from 0 to 72 d post fertilization. It
was demonstrated that while all exposures resulted in
morphological deformities, only one compound (5-azacyti-
dine), a known inhibitor of DNA methylation, caused
significant changes in global methylation. However, several
endocrine-disrupting compounds ([EDCs], bisphenol A
[BPA], diethystilbestrol, and 17a-ethinylestradiol [EE2])
2568 Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017
caused locus-specific alterations in methylation activity,
particularly at the vasa gene, which is important for germ
cell development [20]. Other studies [19] have shown changes
in global methylation levels in zebrafish following exposure
to environmental toxicants, specifically for genes involved in
early development, metabolism, reproduction, and oncogen-
esis. Nonmodel species such as the self-fertilizing mangrove
killifish (Kryptolebias marmoratus; Figure 3), and the inland
silverside (Menidia beryllina), a euryhaline group spawner,
are also sensitive to early life exposure, which results in
negative phenotypic outcomes in adults or F1 offspring
exposed as gametes [21,22]. As mentioned above, fish in
general have a higher percentage of global DNA methylation
in comparison with other vertebrates, and in addition it
appears that the paternal methylome plays a stronger role than
that of the mother during embryogenesis (Figure 2) [23]. Thus
continued study of the epigenome in zebrafish and other
ecotoxicologically relevant fish species is of great impor-
tance, because it is evident there are vast differences between
higher and lower vertebrates.
Two of the best studied invertebrates with regard to
epigenetics are Daphnia species (invertebrate cladoceran)
and the Pacific oyster (Crassostrea gigas). Although
invertebrate methylation levels are variable compared with
the more highly methylated genomes of vertebrates [9], there
are many similarities between aquatic invertebrates and fish.
Similar to fish, the embryonic stage of daphnid species
appears to be most sensitive to the alteration of epigenetic tags
via the influence of environmental cues, such as temperature
changes or pollutant exposure. Typically, progeny are
genetically identical, because under normal conditions
offspring are produced via parthenogenesis, which makes
cladocerans an ideal organism for the study of epigenetically
influenced phenotypes [24]. It has already been established
that Daphnia magna possesses the same DNA
FIGURE 2: A comparison of time points for erasure and reprogramming of the DNA methylome prior to fertilization and during embryogenesis in
mammals and fish. The methylome is erased and reprogrammed in primordial germ cells at approximately the time of birth or hatching. Imprinting
follows in mammals, but has not been established in fish. Following fertilization (F1) the methylome is again erasedand reprogrammed between the
zygote and blastocyst stage in male and female mammals, but the paternal methylome appears to be maintained in fish at this stage. Adapted from
Head, 2014 [23].
FIGURE 3: Nonmodel species such as the self-fertilizing mangrove
killifish (Kryptolebias marmoratus) are being used to investigate
epigenetic mechanisms. Image sourced from F. Silvestre, Laboratory
of Evolutionary and Adaptive Physiology.
Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017 2569
methytransferases as humans and fish and that DNA
methylation occurs. Global methylation levels are altered
by exposure to chemicals such as 5-azacytidine (also in
zebrafish), vinclozolin, genistein, and zinc. Furthermore, it
has been demonstrated that histones H3 and H4 are both
methylated and acetylated, also similar with respect to
vertebrates [2].
In oysters, specifically C. gigas, the methylome has been
evaluated in many tissue types and developmental stages [9].
Thus it is known that DNA methylation occurs and is
correlated with gene expression [25], and that (as with other
organisms) the sensitivity of early embryonic stages should be
emphasized in future studies. Investigations into the nature
and timing of epigenetic reprogramming in the early life of
invertebrates and comparisons with what is known in fish and
mammals in the context of response to environmental
stressors are greatly needed.
Transgenerational Effects
Epigenetic transgenerational inheritance is described as
transmission of epigenetic tags via the germline, without
direct exposure of the affected generation [26]. In mammals it
is established that EDCs such as phthalates and diethylstilbes-
trol (now banned) cause increased DNA methylation, and that
these epigenetic tags lead directly to adverse reproductive
health outcomes. However, the large body of work on
transgenerational epigenetic effects is based on the placental
model of development, which allows for the exposure of both
the F1 (fetus) and F2 (fetal germ cells) generations during
mammalian gestation. In the majority of nonmammalian
models, such as those used in aquatic toxicology research,
transgenerational effects manifest in the F2 generation of F0
exposed individuals, because it is only possible for the F1
generation to be directly exposed (as unfertilized eggs or
preejaculated sperm). However, in organisms that host the
development of their young, such as live bearing fish or
cladocerans (i.e., D. magna), true transgenerational effects
cannot be observed until the F3 generation (Figure 4), because
even the F2 generation can be directly exposed while the germ
cells or offspring, respectively, develop inside the mother [24].
Although carrying studies out to the F3 generation and beyond
can pose a challenge, typical test species used in aquatic
toxicology are short-lived, with relatively brief generation
times, making such experiments more feasible.
Transgenerational studies have been performed in relatively
few species, but existing studies suggest that early life
exposures (discussed in the section Early life exposures) can
result in altered phenotypes for several subsequent gener-
ations. In medaka fish (Oryzias latipes), exposure of embryos
to BPA or EE2 for the first 7 d following fertilization resulted in
reduced fertilization rates in the F2 and F3 adults and hence
reduced survival rates in the F3 and F4 embryos, but no effects
were observed in F0 or F1. Notably, analytical chemistry
deducedthateachF0embryoabsorbedamerepg/mg(partper
trillion) concentration of contaminant on average [27].
Parental early life exposure to benzo[a]pyrene in zebrafish
resulted in deformities that continued through to the F2 and
F3 generations [28]. What is missing from these studies is
the mechanism by which these phenotypic abnormalities
were transferred to generations not directly exposed to the
respective toxicants. Furthermore, in fish, it appears that the
paternal exposure history may be more important than that
of the mother, because in zebrafish paternal methylation
marks are maintained while maternal methylation is
erased [23]. Studies that compare transgenerational effects
between maternal and paternal exposures across aquatic test
species are greatly needed.
In invertebrates such as Daphnia species, it has been posited
that intersex in offspring produced during environmental
challenges (altered temperature or other stressors) results
from transgenerational inheritance of epigenetic modifica-
tions, but this has yet to be proved experimentally [24]. As
mentioned previously, what makes this model unique is that
offspring are usually genetically identical, and thus any
phenotypic differences between siblings produced via
parthenogenesis would be the result of epigenetic modifica-
tions. Although data are currently lacking, evidence that
daphnids undergo transgenerational epigenetic inheritance
exists. For example, daphnids do produce intersex offspring
under certain environmental conditions. Mothers that are
removed from the conditions that triggered intersex progeny
continue to produce them for some time. Other daphnid
phenotypes such as the formation of helmets and neckteeth,
which are induced by predator cues (e.g., kairomones) and
some synthetic chemicals, also appear to be inherited across
FIGURE 4: Cladoceran species (e.g., Ceriodaphnia dubia,Daphnia
magna) hold great promise as model organisms for epigenetic research.
Progeny are typically genetically identical, because offspring
are normally produced via parthenogenesis, and thus F2 generation
germ cells can be directly exposed during F1generation developmentin
the brood pouch. Environmentally induced phenotypes have already
been documented, as has DNA methylation.
2570 Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017
generations rather than after direct exposure [24]. Future
studies should evaluate the presence of inducible phenotypes
out to at least the F3 and F4 generations, which is highly
feasible in an organism with a generation time ranging from a
few days to a few weeks. It is notable that plants, which are by
far the most extensively studied group of living organisms
when it comes to epigenetics, can pass phenotypically
inherited traits for hundreds of generations [26]. We have
likely only begun to scratch the surface of the potential for
comprehending such transmission in relatively understudied
aquatic organisms.
Rapid Evolution
Transgenerational inheritance of epigenetic marks appears to
facilitate rapid evolution via increasing phenotypic plasticity.
However, most studies still consider epigenetically triggered
phenotypic change as being separate from classical Darwinian
inheritance. Recent research suggests that epigenetic change
often precedes and may even trigger or increase the likelihood
for mutations resulting in altered DNA sequence. Thus
epigenetic variation is the mechanism by which organisms
can adapt to rapid environmental perturbations, such as
pollution, but these modifications on the surface of DNA or
histones can in turn increase the probability that a genomic
region might mutate [29]. This has been demonstrated with
transposons, the activity of which may be suppressed by a
high degree of DNA methylation but mobilized via other
epigenetic states. Furthermore, the frequency of both trans-
locations and inversions can be influenced by all 3 previously
described main epigenetic mechanisms. In fact, it has been
noted by evolutionary biologists that there is a mismatch
between rates of mutation at the genotypic and phenotypic
levels, and that epigenetic change appears to be the missing
puzzle piece between the two [26]. Such changes may be
beneficial to the species at hand if the parental environment
matches that of the offspring or grand-offspring and beyond,
but considering the often pulse-like or seasonal nature of
pollutant introduction in the wild, this may not hold true for
the study of long-term transgenerational effects induced via
toxicant exposure and could result in a so-called epigenetic
trap [29]. Furthermore, existing studies on the long-term
adaptation of aquatic species to pollutants or exposure to other
stressors show that trade-offs or evolutionary constraints may
be inherent in the process of evolving to tolerate toxi-
cants [30], but this may be dependent on the species, the
length of exposure, and the type of stressor.
The evolutionary implications of this hypothesis are colossal,
considering that the underpinnings of Darwinian inheritance
(changes in DNA sequence) relied on a rejection of Lamarck’s
hypothesis, which was that traits could be inherited from one’s
environment. In essence this implies that the Lamarckian basis
for inheritance may facilitate rapid evolution while providing
the stimulus for mutations that enable long-term adaptation[26]
(Figure 5). Put simply, the two processes appear to be highly
integrated with and potentially reliant on each other, and thus
should be studied together when possible, particularly in the
context of exposure to environmental stressors.
Analytical Approaches
Standard molecular biology and biochemical techniques, and
more recently high-throughput sequencing approaches, can be
used to determine epigenetic modifications of DNA, histones,
and ncRNAs. Powerful approaches such as the low-cost
reduced representation bisulfite sequencing and chromatin
immunoprecipitation assays that use antibodies for specific
histone modifications are commonly used for investigating
DNA methylation and histone modification, respectively [31].
Because promoter regions are relatively free of nucleosomes,
there are now a number of genome-wide approaches that
allow the identification of these regions, as well as techniques
that detect protein-mediated DNA interaction sites and DNA
binding proteins [32]. These techniques, as with most high-
throughput molecular approaches, are developing rapidly
alongside bioinformatics approaches that integrate different
analytical platforms, such as the analysis of DNA methylation
data alongside gene expression [31]. We recommend the
recent publications by Yong et al. [31], and Kagohara
et al. [32], which provide thorough reviews of these
techniques, as well as their utility for transgenerational and
nontransgenerational studies [31,32].
Epigenetics in an
Ecotoxicological Framework
It has become clear that epigenetic mechanisms allow
organisms to adapt to local environments and environmental
cues. Epigenetic markers have been shown to be altered
following chemical exposure. There is a well-established
linkage among epimutations, dysregulation of epigenetic
enzymes (e.g., DNMTs), and the manifestation of disease
states [33]. All these findings suggest that epigenetics is
obviously relevant to our understanding of the impacts of
pollutant release into environmental media, an area that
remains relatively poorly studied. Epigenetics may be useful
for understanding and predicting differences in susceptibility
within and among populations. For example, hypermethyla-
tion of CYP enzymes may result in increased sensitivity to
chemical exposure. This has been observed in the human
pharmacological arena, where it has clear clinical rele-
vance [34]. The ubiquity of epigenetic modification and
disease manifestation strongly suggests that they have clear
relevance to the development of adverse outcome path-
ways [35]. For example, epigenetic changes have been found
in all cancer types studied and are often found in early stages
of tumorigenesis [34]. As more detailed information on
epigenetic mechanisms comes to light across taxa, it is
possible that DNA methylation, histone modification, or
ncRNAs could eventually be considered causal molecular
initiating events. To this end, epigenetics, used in combina-
tion with other omics modalities and traditional endpoints
Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017 2571
focused on different levels of the biological hierarchy, may be
useful in unraveling causes of ecological impairment in
complex exposure scenarios, particularly those involving
multiple stressors. Different endpoints may provide informa-
tion on different temporal scales, which may be useful in
understanding when the critical exposure occurred. Moreover,
differentially methylated genes may provide insights into
impacted biological pathways, providing clues as to additional
potential drivers of adverse outcomes. The role of epigenetics
in understanding the dynamics of ecosystem function and
response to stressors remains poorly characterized. It is clear,
however, that epigenetics provides the foundation for local and
short-term adaptation to environmental change and that
epigenetic dysregulation is a key player in the development
of adverse outcomes. The breadth of applications for
epigenetics within ecotoxicology and associated environmen-
tal regulation are just now being considered, and it is up to the
aquatic research community to fill in the many existing gaps in
knowledge for model and nonmodel species alike.
Acknowledgment
Funding from the Environmental Protection Agency (EPA STAR # 835799,
to S.M. Brander and R.E. Connon) made this work possible.
Disclaimer
The views expressed in this Focus article are those of the authors and do not
necessarily represent the views or policies of the US Environmental
Protection Agency.
Questions for future
research
1. How direct is the connection between epigenetic
tags in the promoter region of a gene (DNA
methylation) and reduced gene expression?
2. Is transgenerational inheritance and the potential
for rapid evolution driven by pollutant exposure
caused by integrated changes between the epige-
nome and DNA mutations?
3. Does transgenerational inheritance consistently
persist beyond the F2 or F3 generation (depend-
ing on reproductive mode) in aquatic toxicology
model and nonmodel species?
4. How do multiple stressors (temperature, acidification,
hypoxia) influence epigenetic and hence genetic
change, in the context of pollutant exposure under
global climate change?
5. How quickly can aquatic organisms adapt to pol-
lutant or stressor exposure? Are there trade-offs or
evolutionary constraints?
6. What are the implications of parental exposure of
the father, versus the mother? In fish, for example,
is paternal exposure history of more importance
across species?
FIGURE 5: Recent research [26] suggests that epigenetic change often precedes and may even trigger or increase the likelihood for mutations
resulting in altered DNA sequence. Thus epigenetic variation is the mechanism by which organisms can adapt to rapid environmental perturbations,
such as pollution, but these modifications on the surface of DNA or histones may in turn increase the probability that a genomic region might
mutate, facilitating adaptation over the long term. This suggests that the Lamarckian basis for inheritance facilitates rapid evolution while
providing the stimulus for mutations that enable long-term Darwinian adaptation. Images sourced from Wikimedia.
2572 Environmental Toxicology and Chemistry, Vol. 36, No. 10, October, 2017
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