Environ Sci Pollut Res (2015) 22:16262–16276
DOI 10.1007/s11356-014-3466-7
DANIO RERIO AS A MODEL IN AQUATIC TOXICOLOGY AND SEDIMENT RESEARCH
Zebrafish as a model to study the role of DNA methylation
in environmental toxicology
Jorke H. Kamstra & Peter Aleström & Jan M. Kooter &
Juliette Legler
Received: 28 May 2014 / Accepted: 14 August 2014 / Published online: 31 August 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Environmental epigenetics is a rapidly growing
field which studies the effects of environmental factors such
as nutrition, stress, and exposure to compounds on epigenetic
gene regulation. Recent studies have shown that exposure to
toxicants in vertebrates is associated with changes in DNA
methylation, a major epigenetic mechanism affecting gene
transcription. Zebra fish, a well-known model in toxicology
and developmental biology, are emerging as a model species
in environmental epigenetics despite their evolutionary dis-
tance to rodents and humans. In this review, recent insights in
DNA methylation during zebra fish development are
discussed and compared to mammalian models in order to
evaluate zebra fish as a model to study the role of DNA
methylation in environmental toxicology. Differences exist
in DNA methylation reprogramming during early develop-
ment, whereas in later developmental stages, tissue distribu-
tion of both 5-methylcytosine and 5-hydroxymethylcytosine
seems more conserved between species, as well as basic DNA
(de)methylation mechanisms. All DNA methyl transferases
Responsible editor: Philippe Garrigues
J. H. Kamstra (*) : J. Legler
Institute for Environmental Studies, VU University Amsterdam,
1081 HV Amsterdam, The Netherlands
e-mail: jorke.kamstra@vu.nl
J. Legler
e-mail: juliette.legler@vu.nl
P. Aleström
Faculty of Veterinary Medicine and Biosciences, Dept. of Basic
Science and Aquatic Medicine, Norwegian University of Life
Sciences, 0033 Oslo, Norway
e-mail: peter.alestrom@nmbu.no
J. M. Kooter
Department of Molecular Cell Biology, Section Genetics, VU
University Amsterdam, 1081 HV Amsterdam, The Netherlands
e-mail: j.m.kooter@vu.nl
identified so far in mammals are present in zebra fish, as well
as a number of major demethylation pathways. However,
zebra fish appear to lack some methylation pathways present
in mammals, such as parental imprinting. Several studies
report effects on DNA methylation in zebra fish following
exposure to environmental contaminants, such as arsenic,
benzo[a]pyrene, and tris(1,3-dichloro-2-propyl)phosphate.
Though more research is needed to examine heritable effects
of contaminant exposure on DNA methylation, recent data
suggests the usefulness of the zebra fish as a model in envi-
ronmental epigenetics.
Keywords Environmental epigenetics . Zebra fish .
5-Methylcytosine . 5-Hydroxymethylcytosine .
Environmental toxicology . Development . Transgenerational
effects
Introduction
As shown by a multitude of studies over the past decades,
epigenetic modifications alter the expression of underlying
genes and are critically important in the development and
homeostasis during life (Bird 2007). Epigenetic marks include
covalent DNA base (cytosine methylation), histone modifica-
tions (e.g., lysine and arginine methylation, lysine acetylation
and ubiquitination, serine phosphorylation), noncoding
RNAs, and chromatin structure organization of the DNA itself
and are embodied in the field of epigenetics (Bird 2007).
Importantly, DNA and histone modifications can be inherited
during cell division and to some extent over generations
(Probst et al. 2009). Epigenetic modifications are known
to be highly dynamic and are affected by external stimuli,
such as changes in internal and external environment,
nutrition, and exposure to compounds. The effects of these
stimuli on epigenetic gene regulation are described as
Environ Sci Pollut Res (2015) 22:16262–16276
environmental epigenetics (Jirtle and Skinner 2007). These
changes may lead to a variety of either beneficial or adverse
phenotypical outcomes, as well as disease-related effects, and
in some cases even over generations (Fukata and Mori 2004;
Bollati and Baccarelli 2010; Baylin and Jones 2011; Wang
et al. 2012). Transgenerational effects have been linked to
aberrant DNA methylation patterns, emphasizing the impor-
tance of this epigenetic mark (Skinner 2013; Vandegehuchte
and Janssen 2014).
So far, research in DNA methylation has focused pre-
dominantly on mice and human, especially in the fields of
medicine and environmental epigenetics (Skinner 2013).
Many studies report aberrant DNA methylation levels and
patterns in virtually all cancers (Gokul and Khosla 2013).
Due to drawbacks in using rodent models for epigenetic
research such as economical and ethical considerations,
DNA methylation studies in other species, including zebra
fish, are emerging rapidly in the field of epigenetics
(Williams et al. 2014). Despite the evolutionary distance
to mammals, zebra fish share 70 % of their genes with
human and have proven to be a suitable model organism in
many studies, including epigenetics. The transparent na-
ture of the early embryo, its well-characterized develop-
mental stages, its rapid development, and the ease of cul-
ture make this species an extremely useful experimental
model (Strähle et al. 2012).
During zebra fish embryogenesis, several developmental
stages can be distinguished (Kimmel et al. 1995), and dur-
ing development, changes in the epigenetic landscape are
highly dynamic (Mhanni and McGowan 2004; MacKay
et al. 2007; Lindeman et al. 2010b; Andersen et al. 2012a,
b; Jiang et al. 2013; Potok et al. 2013), leading to tightly
controlled gene expression (Aanes et al. 2011). During the
zygotic stage (0.25 h postfertilization (hpf)), maternal
mRNAs are present and determine the fate of cells during
the first hours of embryonic development. Many maternal
mRNAs encode for chromatin remodeling factors during
this stage, and no apparent zygotic transcription is observed
(Aanes et al. 2011). This period of embryonic cell divisions
in the absence of de novo transcription is relatively long, ten
cell cycles, or ∼3 h, after which zygotic genome activation
(ZGA) takes place during the midblastula transition (MBT,
3.3 hpf, 1,000 cells) (Aanes et al. 2011). The majority of
maternal mRNAs are degraded by the aid of zygotic
miRNAs (miR-430) during this stage (Schier 2007). At
gastrula, when the germline is formed (50 % epiboly,
5.3 hpf), genes involved in cell cycle, cell regulation, and
early development are expressed (Mathavan et al. 2005;
Aanes et al. 2011), and the epigenetic landscape will be-
come more complex due to the formation of differentiated
cells (Andersen et al. 2012a). During segmentation and later
stages, genes involved in organogenesis are expressed
(Mathavan et al. 2005).
16263
The advantages of using zebra fish in epigenetic research
are numerous. For instance, due to techniques like zinc finger
nucleases and the more recently developed transcription
activator-like effector nucleases (TALENs) (Cermak et al.
2011) and clustered, regularly interspaced, short palindromic
repeats (CRISPR/Cas9) (Hwang et al. 2013; Wang et al.
2013), the design and generation of mutants of proteins in-
volved in epigenetic processes have become possible to
achieve for many laboratories. Moreover, since many genes
involved in epigenetic processes in the early embryo are
maternally transferred, prolonged survival is possible in these
mutants during the first stages of development. Furthermore,
if needed mRNAs from these maternal genes can be silenced
by antisense morpholinos (MOs) (Landsverk et al. 2010;
Pikulkaew et al. 2011). Recent research in zebra fish DNA
methylation has focused mainly on processes during early
zebra fish development and the basic enzyme systems in-
volved in DNA methylation (e.g., methyl transferases, de-
methylation mechanisms, methylcytosine-binding proteins).
Since DNA methylation patterns during embryonic develop-
ment as well as the (de) methylation pathways in mammals are
extensively studied (Law and Jacobsen 2010; Smallwood and
Kelsey 2012; Wu and Zhang 2014), comparisons with recent
findings in the zebra fish are timely. The purpose of this
review is to describe and compare features of DNA methyla-
tion in the zebra fish with those of mammals in order to
evaluate their applicability as a model to study the effects of
environmental toxicants in humans.
DNA methylation
DNA methylation, a covalent modification of a methyl group
on the 5 position of cytosine (mC), is an epigenetic mark
involved in gene regulation and genome maintenance. In
vertebrates, DNA methylation is predominantly located at a
cytosine-guanine dinucleotide (CpG). Non-CpG methylation
has also been found, for example, embryonic stem cells, but
the levels are much lower, and the significance of this mark is
as yet unknown (Lister et al. 2009). Although silencing of
genes is an important feature of promoter methylation, as
shown by the inactive X chromosomes and transposons
and imprinted and tissue-specific genes, recent studies
indicate that DNA methylation is more dynamic and
plays a more complex role in gene regulation (reviewed
by Jones 2012).
Until recently, distinct differences in CpG islands (CGIs)
occurrence between mammals and cold blooded vertebrates
were observed. A CGI is commonly defined as a DNA region
of more than 200 bp long with a relatively high guanosine and
cytosine (GC) content in a CpG dense region. In mammals,
CGIs are often found at transcription start sites (TSSs) of
genes (>70 %) and remain mostly unmethylated and are
16264
important for gene regulation (Deaton and Bird 2011). CGIs
are identified using various algorithms; however, a definite
definition of a CGI has not yet been reported (Gardiner-
Garden and Frommer 1987; Takai and Jones 2002; Wu et al.
2010). In cold blooded vertebrates, these algorithms generally
calculate a much lower CGI occupancy at TSSs compared to
mammals, indicating an evolutionary difference between
mammals and cold blooded vertebrates in CGI occurrence
and perhaps in function (Long et al. 2013). For example, using
the algorithm of Gardiner-Garden and Frommer (1987) within
the UCSC genome browser, Potok and colleagues (2013)
found a total amount of 12,683 CGIs in zebra fish, a lower
number than in mouse and human (16,026 and 28,691, re-
spectively) (Table 2). Zebra fish CGIs were located at TSSs,
repeat sequences, and gene bodies of metabolic and develop-
mental genes, but only 16 % actually spanned TSSs compared
to >50 % in mouse and humans (Potok et al. 2013). Therefore,
these algorithms need to be adapted to the specific genomic
features of zebra fish. For example, Andersen and coworkers
(2012b) used the Takai and Jones algorithm with modified
parameters for observed to expected (o/e) ratio of CpG sites,
i.e., the observed number of CpG sites versus theoretical
number of CpG sites in a given sequence. By determining
the percent GC and CpG content at regions 1 kb upstream of
the transcription start sites of zebra fish genes, they
could divide the promoters analyzed into 65 % high
and 35 % low CpG promoters, suggesting that this
approach is more conserved between vertebrate taxa
and comparable to the >70 % CGIs found in mouse
and human promoters using the Gardiner-Garden and
Frommer algorithm.
Another abundant modification of cytosine is 5-
hydroxymethylcytosine (hmC). In mammals, this cytosine
modification has been observed in the early embryo inner cell
mass and embryonic stem cells, and high levels have been
found in brain neurons and bone marrow (Pfeifer et al. 2013;
Wu a n d Z h a n g 2 0 1 4 ) . T h e e x a c t f u n c t i o n s o f
hydroxymethylation are not known up to this date, but one
is an intermediate step during DNA demethylation, together
with the less abundant 5-formylcytosine (fC) and 5-
carboxylcytosine (cC) (Wu and Zhang 2010). Since many
tissues have a higher hmC content than expected, it might
serve as an epigenetic mark during stem cell and neuronal
development (Pfeifer et al. 2013). hmC is enriched in genic
regions and is present in gene bodies of transcribed genes,
indicating its specific functional role (Nestor et al. 2012).
Also, studies hint to the existence of specific hmC interacting
proteins, which are known to be involved in epigenetic pro-
cesses (Yildirim et al. 2011; Mellén et al. 2012; Wu and Zhang
2014). Despite the increasing knowledge about DNA
(hydroxy)methylation, still many aspects of these epigenetic
marks remain unanswered (Vastenhouw and Schier 2012;
Jones 2012; Wu and Zhang 2014).
Environ Sci Pollut Res (2015) 22:16262–16276
DNA methyltransferases in zebra fish
DNA methyltransferases (DNMTs) methylate cytosines at the
C5 position with S-adenosyl methionine (SAM) as a methyl
donor (Hermann et al. 2004). The DNMT family consists of
different members which can be roughly divided into mainte-
nance and de novo methyltransferases. In mammals, five
different isoforms of DNMTs are known: DNMT1,
DNMT2, DNMT3a, DNMT3b, and DNMT3L (Table 1).
DNMT1 is involved in maintenance methylation, whereas
DNMT3a and DNMT3b are involved in de novo methylation.
DNMT3L lacks the catalytic subunit and has a supporting role
in de novo methylation (Hermann et al. 2004). DNMT2,
which harbors only the catalytic domain of a DNA methyl-
transferase, is thought to be involved in RNA cytosine meth-
ylation (Smith et al. 2011). Zebra fish have eight different
dnmt genes, of which six are most likely de novo Dnmts as
they are most similar to the mammalian DNMT3 members. It
appears that zebra fish lack a Dnmt3L protein, since it has not
been located in the zebra fish genome up to this date (Smith
et al. 2011) (Table 1). All zebra fish Dnmts are maternally
transferred to the zygote, and mRNA levels decline during the
first stages of development and increase again after ZGA
(Martin et al. 1999; Smith et al. 2011). Gene expression of
the Dnmt3b orthologs (dnmt3, dnmt4, and dnmt7) increases
up to 6 hpf and gradually declines again towards undetectable
levels at 72 hpf, whereas expression of DNMT3a orthologs
(dnmt6 and dnmt8) steadily increases after 6 hpf. All DNMT3
orthologs are also detectable in muscle and brain tissue, but
are consistently higher in brain (Smith et al. 2011).
DNMTs are evolutionarily conserved proteins, as shown
by the overall sequence similarity of DNMTs from different
species (Mhanni et al. 2001) and the observation that ectopic
expression of the human DNMT1 and DNMT3b in zebra fish
can take over the function of the zebra fish orthologs (Rai et al.
2006, 2010). Additional evidence of evolutionary conserva-
tion is found in the interactions of DNMTs with other proteins.
In mice, UHRF1 (DNMT1-recruiting proteins, ubiquitin-like,
Table 1 DNMTs isoforms in mice and zebra fish
Mice Zebra fish
Maintenance methylation DNMT1 Dnmt1
RNA methylation DNMT2 Dnmt2
De novo methylation DNMT3a Dnmt6
Dnmt8
De novo methylation DNMT3b Dnmt3
Dnmt4
Dnmt5
Dnmt7
Cofactor DNMT3L N/A
From Shimoda et al. (2005) and Law and Jacobsen (2010)
Environ Sci Pollut Res (2015) 22:16262–16276
containing PHD and RING finger domains) and proliferating
cell nuclear antigen (PCNA) recruit DNMT1 to
hemimethylated CpG sites after DNA replication (Law and
Jacobsen 2010). Similarly, in zebra fish, interaction of Dnmt1
with Uhrf1 has been observed (Tittle et al. 2011). Both Uhrf1
and Dnmt1 mutants show similar phenotypical defects in
zebra fish lens development along with a lower global mC
content, indicating that Uhrf1 is essential for maintenance
methylation, though a direct interaction between Uhrf1 and
Dnmt1 was not reported (Tittle et al. 2011). Further evidence
of Uhrf1 conservation was found in a genome-wide methyla-
tion study where both zebra fish and mice Uhrf1 mutants
showed similar demethylation patterns (Feng et al. 2010).
Interaction of Dnmt1 with H3K9 methyltransferase suppres-
sor of variegation 3-9 homologue 1 (Suv39h1) and euchro-
matic histone-lysine N-methyltransferase 2 (Ehmt2 or G9a)
has also been observed in zebra fish (Rai et al. 2006, 2010).
Interestingly, similar interactions between DNMTs and these
proteins have also been found in mammalian in vitro and
in vivo studies (Rai et al. 2006).
In zebra fish, Dnmt3 to Dnmt8 are the main contributors to
de novo methylation, similar to DNMT3A and DNMT3B in
mammals (Table 1). In mammals, a DNMT3A/DNMT3L
tetramer is thought to be involved in methylating imprinted
genes (Yokomine et al. 2006; Law and Jacobsen 2010). Since
no DNMT3L ortholog seems to be present in zebra fish, it is
unlikely that zebra fish harbor a similar imprinting mechanism
(Wu et al. 2011). The fact that parthogenesis and gynogenesis
result in the normal development of zebra fish demonstrates a
fundamental difference between fish and mammals in this
mechanism of epigenetic control of development (Corley-
Smith et al. 1996). In mice, the DNMT3A/DNMT3L tetramer
is also targeted towards transposable elements by Miwi2
proteins loaded with piRNAs, in order to silence transposable
elements through CpG methylation (Law and Jacobsen 2010).
This piRNA pathway has also been demonstrated in zebra fish
(Houwing et al. 2007; Wei et al. 2012), but no interaction with
Dnmts or DNA methylation has been reported yet, presum-
ably due to the nonexistence of the DNMT3L ortholog. The
reason why zebra fish contains more de novo Dnmts than
mammals is currently not clear; possibly, these enzymes har-
bor tissue or promoter- or gene-specific functions. For exam-
ple, tissue-specific expression has been observed for Dnmt4,
Dnmt6, and Dnmt8 in zebra fish embryos (Takayama et al.
2014). Also, it has been demonstrated that of all five de novo
Dnmt homologs, only Dnmt7 morpholinos showed effects on
promoter methylation of no tail, a gene essential for notochord
and tail development, whereas no effects on global methyla-
tion levels were observed, indicating locus-specific recruit-
ment of this Dnmt (Yamakoshi and Shimoda 2003).
Interactions of DNMT3B with several histone methyltrans-
ferases (G9a, SUV39h1, enhancer of zeste homologue 2
(EHZ2), SET domain bifurcated 1 (SETDB1)) have been
16265
reported in mammals (Law and Jacobsen 2010). The first
evidence of similar interactions in zebra fish was obtained
with Dnmt3, an ortholog of DNMT3B, which was shown to
interact with zebra fish G9a (Rai et al. 2010). G9a
morpholinos exhibited similar deficiencies in brain develop-
ment as with Dnmt3 knockouts. Interestingly, phenotypes
could be rescued by overexpression of both zebra fish and
human G9a, thereby supporting the evolutionary conservation
of this pathway (Rai et al. 2010). In the same study, it was
shown that both Dnmt1 and Dnmt3 are essential for retinal
development, with Dnmt3 being vital for early stages of
differentiation and Dnmt1 for terminal differentiation (Rai
et al. 2010). Recently, expression of all de novo Dnmts in
the developing eye of zebra fish has been observed, with
specific functions of different Dnmts in different regions.
However, there is an overlap in expression of the various
Dnmts (Seritrakul and Gross 2014).
In summary, these data show that similar interactions be-
tween DNA methyltransferases and histone methyltransfer-
ases occur between species, indicating strong conservation of
these epigenetic pathways. However, some interactions ob-
served in mice, such as Suv39h1-DNMT3b, have not been
observed in zebra fish up to this date.
Demethylation pathways in zebra fish
In mammals, demethylation of cytosines occurs both passive-
ly and actively during development and life. Passive demeth-
ylation occurs during DNA replication and cell division when
hemimethylated DNA is not methylated by the maintenance
DNMT1 (Wu and Zhang 2010). Passive demethylation ap-
pears to be the mechanism for the global erasure in both
primordial germ cells (PGCs) and in the zygote, where the
paternal methylome is first oxidized by ten-eleven transloca-
tion (TET) enzymes (Smallwood and Kelsey 2012; Wu and
Zhang 2014). Active demethylation generally follows two
pathways in mammals. One involves deamination of methyl-
ated cytosines by activation-induced cytosine deaminases
(AIDs) or apolipoprotein B mRNA-editing enzyme
(APOBEC), creating a GT mismatch which is removed via
base excision repair (BER) using thymine DNA glycolase
(TDG) and methyl-binding domain protein 4 (MBD4) (Law
and Jacobsen 2010; Wu and Zhang 2014). A second pathway
involves the formation of a hmC intermediate by TET en-
zymes, followed by two possible routes: (1) deamination and
BER via the previously mentioned pathway or (2) further
oxidation of hmC to cC, which is removed by a yet to be
discovered carboxylase or via BER (Pastor et al. 2013; Wu
and Zhang 2014). In zebra fish, both enzyme families are
present. Aid/Apobec deamination has been more thoroughly
studied, and evidence of Tet involvement in zebra fish de-
methylation mechanisms is emerging.
16266
Demethylation via deamination by Aid/Apobec
Until recently, the role of the AID/Apobec-mediated mC
demethylation pathway in zebra fish was controversial.
Although mismatch repair of GT by TDG and BER has been
shown in several studies, human recombinant AID and
APOBEC deaminate cytosines poorly and have an even lower
activity towards mC (Pastor et al. 2013). However, purified
zebra fish Aid has a high deamination activity on both C and
mC, suggesting that demethylation via Aid/Apobec is a plau-
sible pathway in zebra fish (Abdouni et al. 2013). The func-
tionality of this cytosine modification mechanism is supported
by experiments whereby the demethylation of methylated
DNA fragments introduced in embryos was monitored (Rai
et al. 2008). Gradually, up to 13 hpf, the DNA fragments were
demethylated and remethylated at 28 hpf. At 13 hpf, mRNA
levels of aid, apobec2a, and apobec2b were increased.
Repression of aid, apobec2a, and apobec2b expression via
MOs showed reduction in demethylation, but only when all
three genes were repressed (Rai et al. 2008). Interestingly,
overexpression of Aid/Apobec did not increase demethyla-
tion. Only when hMBD4 was coinjected, demethylation was
observed of both inserted methylated DNA fragments and the
genomic DNA itself. Catalytically inactive (for thymine
deglycosylase) hMBD4 coinjected with Aid revealed GT
mismatches, suggesting active deamination of methylated
CpG sites (Rai et al. 2008). Although AID is able to deaminate
both C and mC, the involvement of a methyl domain-binding
protein in cooperation with deaminases could provide the
specificity towards mC.
Involvement of TET enzymes in DNA demethylation
Oxidation of mC to hmC by TET enzymes and subsequent
removal of the modified base is a second route towards
demethylation. In mice, the hmC intermediate has been found
in the zygote on the paternal genome prior to erasure of the
methylation marks (Ruzov et al. 2011); however, subsequent
demethylation is believed to occur passively (Wu and Zhang
2014). Furthermore, hmC has been found in several tissues,
including brain, embryonic stem cells, and PGCs (Globisch
et al. 2010; Nestor et al. 2012). Surprisingly and in contrast to
mammals, no detectable levels of hmC are present in embryos
up to the tenth somite stage (14 hpf) (Almeida et al. 2012).
These observations indicate that demethylation of the paternal
genome does not involve hmC. However, low levels of hmC
are detectable at the 64-cell stage with a more sensitive hmC
pull-down method at specific differentially methylated regions
(DMRs) (e.g., hoxc and dnmt3) (Jiang et al. 2013; Potok et al.
2013). However, loci with hmC were rare and it is unlikely
that this mark is an intermediate in the global erasure of
methylation, as observed in mammals. Analysis of hmC at
Environ Sci Pollut Res (2015) 22:16262–16276
the zygotic stage, which to our knowledge has not yet been
performed, could give additional proof that there is no
reprogramming via hmC. At later embryonic stages, hmC
has been found with immunostaining in the tail section
(Jiang et al. 2013) and skeletal muscle, retina, and brain
regions (Almeida et al. 2012), a similar tissue distribution as
observed in mammals (Globisch et al. 2010; Nestor et al.
2012), however, no data is available on the quantitative mea-
sures in various tissues of zebra fish. Where present, hmC is
accompanied by mRNA increases of all three tet genes
(Almeida et al. 2012; Ge et al. 2014). In zebra fish, a specific
role of Tet2 has been discovered in erythropoiesis, accompa-
nied by lower promoter methylation of genes involved in
erythropoiesis (Ge et al. 2014). These are the first indications
of similar demethylation mechanisms at later developmental
stages in mammals and zebra fish.
Methylation during different developmental stages
of zebra fish
The studies presented in this review use many techniques for
either fundamental research on DNA methylation or effects on
DNA methylation following exposure to compounds. A vari-
ety of analyses can be used for specific, global, and genome-
wide DNA methylation analysis, to assess differences in DNA
methylation during development or following exposures to
environmental toxicants. Table 3 presents an overview of some
of the major techniques applied in the studies described below.
The overall guanosine and cytosine (GC) content in the
zebra fish genome is slightly lower compared to mammals
(36.8 versus 42.1, respectively), whereas the o/e is higher
(0.53 in zebra fish versus 0.17 and 0.20 in mouse and human,
respectively) (Table 2). Methylation levels of CpGs in zebra
fish are generally higher (60–90 % for human/mouse and 70–
95 % for zebra fish) (Table 2) (Potok et al. 2013), with the
highest levels observed in sperm (91–95 %), whereas oocytes
exhibit lower levels (75–80 %) (Jiang et al. 2013; Potok et al.
2013). Oocytes of mice also have a lower methylation level
than sperm (approx. 40 versus 82 %, respectively), although
the overall methylation levels are generally lower in both germ
cell types compared to zebra fish (Kobayashi et al. 2012;
Smith et al. 2012).
During the development of the zebra fish embryo, methyl-
ation of gene promoters is highly dynamic and varies between
gametes and developmental stage (Fig. 1). In both zebra fish
sperm and oocytes, many promoter regions of genes involved
in early development and metabolism are hypomethylated and
remain hypomethylated throughout early development up to
the sphere stage (Potok et al. 2013). Hypermethylated promot-
er regions in oocytes and sperm are generally found in genes
important for later developmental stages, such as neurogenesis.
Interestingly, promoter regions of germ-specific genes (piwi,
Environ Sci Pollut Res (2015) 22:16262–16276 16267

Table 2 Genomic features of human, mice, and zebra fish analysis, since a reduction of methylation by half was ob-
Human Mice Zebra fish served at every cell replication, whereas de novo methylation
of oocyte-specific hypomethylated loci has been observed in
Number of chromosomes 23 20 25 zebra fish with a SNP in the maternal DNA (Jiang et al. 2013).
Total genome (bp) 3.1×109a 2.7×109a 1.4×109a These data suggest that passive demethylation and de novo
Protein coding genes 20,770b 23,139b 26,241b methylation occur simultaneously and that the maternal
CpG sites 2.82×107a 2.13×107a 2.53×107a methylome is reprogrammed to that of the paternal genome.
%CG 42.5a 42.2a 36.8a Though the global erasure of methylation prior to de novo
CGI 28,691a 16,026a 12,683a methylation observed in mammals during early embryogene-
%mdC in CpG context 60–90 %c 60–90 %c 70–95a sis (Smallwood and Kelsey 2012) has not been observed in
%mC of total C ∼1–2 %d ∼3–4 %e ∼8–9 %f zebra fish, there are some features that resemble mammals. In
o/e CpG 0.20a 0.17a 0.53a mice, Smith and coworkers (2012) observed that, despite the
Imprinting Yes Yes No global erasure of DNA, oocyte-specific hypermethylated
(compared to sperm) sites were hypomethylated at E7.5 and
a
Potok et al. (2013) oocyte-specific hypomethylated sites were hypermethylated
b
Ensembl (2013) at E7.5. In other words, the methylome of oocyte-specific
c
Tucker (2001) DMRs is reprogrammed to that of sperm, in both mice and
d
Li and Liu (2011) zebra fish. Furthermore, in both species, the number of
e
Globisch et al. (2010) hypermethylated DMRs in sperm was higher than in oocytes.
f
Rai et al. (2008) Also, the methylome of sperm resembles that of somatic
tissue, and regardless of global erasure of DNA methylation
dnmt6, and vasa) are hypomethylated in sperm and during embryogenesis, the methylome of oocytes is
hypermethylated in oocytes, even though these genes are reprogrammed towards the paternal (or somatic) state, which
known to be maternally transferred (Potok et al. 2013). The would imply that this reprogramming mechanism is rather
developmental genes that are only hypomethylated in sperm conserved between species.
include all the 48 hox genes (Wu et al. 2011; Jiang et al. 2013; As the zebra fish embryo passes through the different
Potok et al. 2013). In oocytes, it appears that in zebra fish, but stages during development, cells differentiate and migrate to
not in mice, the hox gene promoters are hypermethylated specific regions, and organogenesis starts. At this point of
(Potok et al. 2013). Since many genes involved in early devel- development, the epigenetic complexity of the embryo in-
opment were already hypomethylated in sperm, the creases and the embryo cannot be seen as one homogenous
methylome of zebra fish sperm can already be regarded as cell mass, and therefore, specific tissues, rather than whole
programmed for embryonic development. embryos should be compared for methylation status. A num-
During the one- to two-cell stage, a hypomethylated state ber of promoter regions of genes have been shown to be
of the zebra fish genome has been observed in one study, with differentially methylated during myogenesis (Potok et al.
lower methylation levels compared to sperm and oocytes and 2013). For example, promoters of genes encoding for tran-
increasing methylation levels up to 4 hpf (Mhanni and scription factors are hypermethylated at the sphere stage (plu-
McGowan 2004) (Fig. 1). The hypomethylated state at the ripotent state) compared to adult skeletal muscle tissue (ter-
one- to two-cell stage, however, has not been confirmed in minally differentiated), along with genes involved in metabo-
later studies, and no additional information is available on the lism and germ line maintenance (hox, vasa, piwil1, and dazl)
actual methylation status during the formation of the zygote. (Potok et al. 2013). When comparing DMRs (∼3,000) of
Two recent studies by Potok et al. (2013) and Jiang et al. sphere and muscle tissue, ∼2,000 became hypermethylated
(2013) examined DNA methylation at later stages (mixture of in muscle, predominantly in the gene bodies, while ∼1,000
2–16- and 16-cell stage, respectively), using whole-genome became unmethylated (Potok et al. 2013). The function of
shotgun bisulfite sequencing and confirmed the increase in gene body methylation is basically unknown; it occurs mainly
methylation up to the sphere stage. in introns and possibly acts on enhancers (Potok et al. 2013)
More specific analysis of methylation during early devel- and/or is a mechanism for silencing of repeat elements and is
opment has revealed some striking features. For example, it also positively correlated with gene expression (Jones 2012).
has been observed that oocyte-specific hypomethylated re- Additionally, the entire hoxc gene cluster is hypomethylated in
gions are de novo methylated to a level that is similar to the sperm, whereas most of the genes in this cluster are
hypermethylated state of sperm, whereas hypermethylated hypermethylated in muscle tissue (Wu et al. 2011). During
regions in the oocyte are passively demethylated (Potok myogenesis, some muscle-specific genes are hypomethylated
et al. 2013). Also, passive demethylation at oocyte-specific (musk, dystrophin) whereas other genes involved in muscle
hypermethylated loci has been shown by bisulfite sequencing development (myocyte enhancer factors, e.g., pbx1, myod1)
 16268

Table 3 Methods used to assess DNA methylation in zebra fish

Method Principle Advantage Disadvantage Study

Global analysis
Methylation-sensitive Methylation-sensitive restriction enzymes Fast, cost effective Not quantitative, no information on Martin et al. (1999), Macleod et al. (1999),
restriction digestion (HPAII/MSPI), combined with Southern specific loci Mhanni and McGowan (2004)
blots
Liquid chromatography- Global analysis of fully digested DNA, Quantitative, fast, combined with mass No information on specific loci Rai et al. (2008), Globisch et al. (2010),
based methods quantitative analysis of single nucleosides spectrometry scan for multiple Ge et al. (2014), Olsvik et al. (2014)
cytosine modifications
Immunostaining Use mCpG- or hmCpG-specific antibodies, Tissue-specific distribution in whole Not quantitative, no information on Li et al. (2009), Ceccaldi et al. (2011),
use with immunoblots or whole embryo body specific loci Almeida et al. (2012)
staining
Genome-wide analysis
Whole-genome bisulfite Whole-genome analysis of Complete methylome analyzed Expensive, no discrimination Jiang et al. (2013), Potok et al. (2013)
sequencing (WGBS) bisulfite-converted DNA between mC and hmC
Reduced representation Representation of high CG content loci with Input amount DNA low, relatively Analysis of only 1 % of the genome, Gu et al. (2011), Smith et al. (2012)
bisulfite sequencing methylation-insensitive restriction cheap covering only 7 % promoters in
(RRBS) digestions (MspI) zebra fish
Methyl DNA Immunoprecipitation of mC followed by Only mC is analyzed, also hmC Bias in cross-reactivity antibodies, Andersen et al. (2012b), Olsvik et al. (2014)
immunoprecipitation microarrays or sequencing immunoprecipitation no single CpG resolution with
(MeDIP) arrays
Specific analysis
Bisulfite sequencing Sanger sequencing, pyrosequencing on Quantitative information on specific PCR bias Strömqvist et al. (2010), Fang et al. (2013),
bisulfite-converted DNA loci, at single-CpG sites, multiplex Hernández et al. (2013), Corrales et al.
sequencing (2014a)
Methylation-sensitive High -resolution melting on Quantitative information on specific No information of single CpG sites Kamstra et al. (2014)
high-resolution melting bisulfite-converted DNA loci, high throughput, correction for
(MS-HRM) PCR bias
Environ Sci Pollut Res (2015) 22:16262–16276
Environ Sci Pollut Res (2015) 22:16262–16276
Fig. 1 Dynamic methylation during early zebra fish development. a Red
indicates maternal; blue paternal; and green, diploid embryonic genome.
Maternal genome is reprogrammed during early development, with pas-
sive demethylation starting at zygote and de novo methylation from two-
cell stage. The paternal genome is unaffected. No data on PGC program-
ming is available. The gray box indicates a sensitive window for inter-
ference by Dnmt inhibitor exposure. b–d Examples of methylation
were already hypomethylated at early developmental stages
(Potok et al. 2013). The hypomethylated status of these genes
suggests a permissive state for transcription of the promoter in
terminal differentiation.
When comparing MBT embryos with ZF4 fibroblasts, a
cell line derived from 24-hpf zebra fish embryos, representing
a more developed tissue, gene promoters with more than one
CGI are hypermethylated in ZF4 cells. Genes with multiple
CGIs often encode transcription factors (such as hox genes),
and hypermethylation in ZF4 cells suggests a long-term si-
lenced state of these genes (Andersen et al. 2012b).
Pluripotency control genes (pou5f1, sox3, klf4, otx1b, and
vasa) are hypermethylated in ZF4 cells when compared to
MBT embryos, in correspondence with their repressed state in
the somatic cell type line (Lindeman et al. 2010b). After MBT,
many developmental gene promoters are hypomethylated and
have bivalent histone marks, making them poised for tran-
scription (Andersen et al. 2012b). It is possible that after
differentiation, these developmental genes are subsequently
permanently silenced by H3K9me3 and H3K27me3, which is
reinforced and stabilized by de novo promoter methylation
(Jones 2012) (see below).
16269
dynamics a specific loci. b Methylation levels of many developmental
genes (e.g., vasa, piwil1, dazl, dnmt3) decrease up to the sphere stage and
increase at later developmental stages; however, c some developmental
genes are already hypomethylated. d Muscle-specific genes, musk and
dystrophyn, are hypermethylated in sperm and hypomethylated in oocytes
(not shown), and methylation increases during development and de-
creases in specific tissues at later stages
Histone modifications associated with DNA methylation
during development
Modifications of histones alter transcriptional activity of chro-
matin and may influence DNA methylation. Among the many
known histone modifications, H3K4me3 is associated with
gene expression, while H3K9me3 and H3K27me3 are asso-
ciated with gene repression. H3K9me3 and H3K27me3 are
often accompanied by DNA methylation and silencing of
specific loci (Ruthenburg et al. 2007; Lindeman et al. 2011).
The bivalent H3K4me3 and H3K27me3 mark is often found
at developmental genes poised for transcriptional
(in)activation during embryonic development and in embry-
onic stem cells (Ruthenburg et al. 2007; Vastenhouw and
Schier 2012).
In zebra fish development, before, during, and after ZGA,
hypomethylated DNA regions contain H3K4me3-marked nu-
cleosomes, though no information was reported about the
actual transcriptional activity of hypomethylated promoters
with H3K4me3 marks (Andersen et al. 2012b). However,
Potok et al. (2013) did report an association between
hypomethylated promoters, H3K4me3 marks and
16270
gene expression. At ZGA and later, an increase in H3K9me3
and H3K27me3 was observed, due to a more epigenetic
complex state of determined and differentiated cells in later
development (Lindeman et al. 2010a). At sphere stage (MBT),
a number of developmental genes were marked with a com-
bination of H3K4me3 and H3K27me3 and low promoter
methylation, correlated with low expression levels
(Andersen et al. 2012a; Potok et al. 2013). The relation
between histone marks and DNA methylation argues that
histone modification analysis should be taken into consider-
ation together with DNA methylation analysis when assessing
toxicant-induced effects on the epigenome.
DNA methylation in environmental toxicology
Interference with enzyme systems
Altered global DNA methylation levels are often associated
with compounds that interfere with methyltransferases or its
substrate SAM. Among compounds that have been thorough-
ly investigated for their hypomethylating effects and are often
used as model compounds in studies that assess DNA meth-
ylation are the DNMT inhibitors 5-aza-cytidine (5-AC) or 5-
aza-deoxycytidine (5-AdC). These cytosine analogs have the
potency to inhibit DNMTs as they incorporate in DNA and
covalently bind DNMTs, eventually leading to loss of DNMT
activity. As a demonstration of this inhibition, levels of both
methylated and hydroxymethylated DNA are decreased by 5-
AC exposure in a concentration-dependent manner (Fig. 2)
(Kamstra et al. 2014, in preparation). In an earlier study with
5-AC exposed to zebra fish, severe phenotypical changes and
lower overall methylation levels were also observed (Martin
et al. 1999), where the timing of exposure to 5-AC appears to
be critical. The most pronounced morphological effects were
found after exposure to 75 μM 5-AC in embryos exposed
from 1 to 24 hpf. Shorter windows of exposure from 1 to 4 hpf
appeared to be critical and resulted in similar results, whereas
exposure from 6 to 24 hpf showed almost no effects on
morphology (Martin et al. 1999). This sensitive early stage
window corresponds with the methylation dynamics during
early development and indicates that this time window must
be included in the studies with compounds that are susceptible
to interfere with Dnmt enzymes (Fig. 1).
Several environmental pollutants have been found to alter
global DNA methylation levels in humans and other model
organisms. For example, changes in global methylation have
been observed in an in vitro mouse adipocyte differentiation
model following exposure to the organotin tributyltin (TBT)
and the plasticizer bisphenol A (BPA) (Bastos Sales et al.
2013). One of the most studied compounds showing effects
on global DNA methylation are arsenic (As) compounds.
Genome-wide hypomethylation has been found after sodium
Environ Sci Pollut Res (2015) 22:16262–16276
Fig. 2 De(hydroxy)methylation in 48-hpf embryos following continuous
exposure of 5-AC from 4–16-cell stage. a Control versus b 5-AC
(75 μM)-exposed zebra fish, showing short tail phenotype and heart
edema. c mC levels decline with increasing concentrations of 5-AC. A
similar trend is observed with hmC. Significance was determined with
one-way ANOVA, following a Bonferroni’s multiple comparisons test
(P<0.05) using GraphPad Prism 5.04. Methylation analysis was carried
out only in embryos showing no phenotypical effects
arsenite exposure in rat liver cells (Zhao et al. 1997) as well as
in livers from mice exposed to sodium arsenite via drinking
water for 48 weeks (Chen et al. 2004). The hypomethylating
effect of As in liver could be explained by its methylated
metabolites, as metabolism involves SAM-dependent arsenic
methyltransferases, thereby depleting the methyl pool for
DNMTs (Hamdi et al. 2012). In zebra fish exposed to As, an
aberrant DNA methylation pattern has been observed in trunk
and tail, with hypomethylation after 24 hpf and hypermethy-
lation after 48 hpf compared to controls (Li et al. 2009).
However, exposure levels of As in these experiments were
very high, and it is not clear if these effects were nonspecific
due to toxicity and apoptosis which was found in the same
regions (Li et al. 2009).
Changes in global methylation levels have also been ob-
served in zebra fish embryos exposed to the polycyclic aro-
matic hydrocarbon benzo[a]pyrene (B[a]P) (Fang et al. 2013;
Corrales et al. 2014a) and the organophosphorus flame retar-
dant tris(1,3-dichloro-2-propyl)phosphate (TDCPP) (McGee
et al. 2012) (Table 4). With B[a]P, a known mutagen through
its formation of DNA adducts, decreased methylation levels
could be due to adduct formation which preferentially takes
place on a (hemi)methylated CpG consensus, subsequently
leading to GC to TA conversion (Weisenberger and Romano
1999). Therefore, these effects, albeit compound specific, are
likely due to chemical modifications of DNA by B[a]P and
not necessarily by inhibition of DNMTs. In the study with
Table 4 Overview of studies examining global DNA methylation and specific gene promoter methylation in zebra fish embryos following exposure to several compounds

Compound Concentration (M) Stage Exposure time Effect Assay Study

Benzo[a]pyrene 1.0E−7 1 hpf 96 h Global hypomethylation ELISA Fang et al. (2013)

Environ Sci Pollut Res (2015) 22:16262–16276

Specific: vasaa
Benzo[a]pyrene 2.0E−7 Mother/embryo 3.3 and 96 h Global hypomethylation ELISA- based bisulfite Corrales et al. (2014a, b)
Specific: c-fos, cyp1b1, dazl, gstp1, mlh1, nqo1, multiplex deep
pten, p53, sox2, sox3a sequencing
Sodium arsenite 2.0E−3 1 hpf 24–48 h Global hypomethylation (24 hpf), global Immunostaining Li et al. (2009)
hypermethylation (48 hpf)
Tris(1,3-dichloro-2-propyl) phosphate 3.0E−6 1 hpf 2h Global hypomethylation Methylation-sensitive McGee et al. (2012)
restriction enzymes
5-Azacytidine 7.5E−5 1 hpf 24 h Global hypomethylation (48 hpf) Methylation-sensitive Martin et al. (1999)
restriction enzymes
5-Azacytidine 10b Adult female 32 days Global hypomethylation (F0), many differentially HPLC-UV MeDIP array Olsvik et al. (2014)
methylated genes in F0 and F1, but few in F2
(one unkown and adra2aa)
Flavone derivates 2.5E−5 1 hpf 24 h Global hypomethylation Immunostaining Ceccaldi et al. (2011)
17α ethinylestradiol 3.4E−10 5–7 monthsa 14 days Vitellogenin I hypomethylation Bisulfite sequencing Strömqvist et al. (2010)
2,3,7,8-Tetrachlorodibenzo-p-dioxin 0.02b Adult female 47 days Few differentially methylated genes in F0, but few MeDIP array Olsvik et al. (2014)
in F1 (F2 not analyzed)
Methyl mercury 10b Adult female 47 days Many differentially methylated genes in F0 and F1, MeDIP array Olsvik et al. (2014)
but one in F2 (rRNA)
a
In liver—functions of genes: vasa and dazl, germ cell development; c-fos, proto-oncogene; cyp1b1, steroid metabolism; gstp1, glutathione metabolism; nqo1, reduction of quinones; mlh1, mismatch
repair; pten, tumor suppressor; p53, tumor suppressor, apoptosis; sox2 and sox3, development, adra2a, adrenoceptor alpha 2A
b
Via feed (milligrams per kilogram)
16271
16272
TDCPP, methylation was measured at a very early time point
(2 hpf), and at this early stage, exposure to the chemical could
influence cell proliferation, leading to developmental delay
and causing different methylation levels relative to controls. It
would be important to know if methylation changes caused by
exposure during early development persist to later stages.
Given the limited number of studies on toxicant effects on
DNMTs, further research is clearly needed. To address this
issue, Ceccaldi and colleagues (2011) have developed a
screening assay in 96-well plates coated with double-
stranded DNA labelled with a fluorophore at one end and a
biotin on the other end. When DNMT3A/3L complex is added
in combination with SAM, DNA will become methylated.
With methylation-sensitive restriction enzymes, these methyl-
ated pieces of DNA are protected from cleavage and retain
their fluorescence after washing. Upon addition of an inhibitor
the DNA remains unmethylated, will be cleaved and the
fluorescent signal is lost (Ceccaldi et al. 2011). With this
method, over 100 flavone and flavanone derivates and, more
recently, over 1,000 small organic compounds could be effi-
ciently screened, some of them are more potent DNMT inhib-
itors than 5-AC (Ceccaldi et al. 2011, 2013). Moreover, zebra
fish embryos exposed to two potent DNMT-inhibiting deri-
vates of flavones identified by this screening assay showed
similar phenotypical changes as with 5-AC exposure
(Ceccaldi et al. 2011). In future research, similar in vitro
assays could be developed for enzymes such as zebrafish
Apobec and Tet as well as for histone methyltransferases, of
which an assay for histone methyltransferase G9a has already
been developed (Ceccaldi et al. 2013).
Compound-specific effects on DNA methylation at specific
loci
As global methylation analyses do not reveal specific gene
targets altered by toxicant exposure, research on specific
promoter methylation of relevant genes in different species
is increasing (Vandegehuchte and Janssen 2014). Examples of
endocrine-disrupting chemicals (EDCs) that are able to differ-
entially methylate specific gene promoters in species other
than zebra fish are diethylstilbestrol (DES) (Li et al. 2003),
bisphenol A (Dolinoy et al. 2007), TCDD (Wu et al. 2004),
the phytoestrogen genistein (Fukata and Mori 2004), TBT
(Kirchner et al. 2010), and the organophosphate pesticide
diazinon (Zhang et al. 2012). Furthermore, specific
hypomethylating effects on the Pparγ2 promoter have been
observed following exposures of polybrominated biphenyl
ether-47 (BDE-47) in an in vitro adipocyte differentiation
model (Kamstra et al. 2014). These compounds could alter
methylation via endocrine pathways; however, it is not ruled
out that these compounds could also act via interfering with
the methylation pathways, as is suggested with As. Therefore,
it is not known whether these changes in methylation are
Environ Sci Pollut Res (2015) 22:16262–16276
caused by compounds directly or as a consequence of their
(upstream) toxicological mode of action.
There is as of yet little known about promoter-specific
methylation in zebra fish. In 3.3- and 96-hpf zebra fish ex-
posed to B[a]P, differentially methylated promoters were
found in genes that are involved in early development and
metabolism and in proto-oncogenes (Table 4) (Fang et al.
2013; Corrales et al. 2014a). In this study, however, the
changes in promoter methylation were modest and require
conformation. In adult zebra fish, demethylation of the vitel-
logenin (vtg) gene promoter was observed in livers following
exposure to 0.34 nM 17α ethinylestradiol (EE2) for 14 days
(Table 4) (Strömqvist et al. 2010). These results might indicate
an effect of estrogenic compounds on the methylation status of
the vtg promoter, and since EE2 has shown to increase vtg
mRNA at even lower concentrations, this promoter demeth-
ylation could have a positive effect on the gene expression of
vtg (Strömqvist et al. 2010).
Since DNA methylation can be inherited, many studies on
mammalian models focus on the transgenerational effects of
environmental stressors on the methylome (Skinner 2013;
Vandegehuchte and Janssen 2014). In zebra fish,
transgenerational studies are emerging (Corrales et al.
2014b; Baker et al. 2014); however, limited data is available
about transgenerational effects of toxicants on DNA methyl-
ation. A recent study assessed the transgenerational effects of
5-AC, TCDD, and MeHg in zebra fish (Olsvik et al. 2014).
Adult female zebra fish were exposed via feed to the com-
pounds (Table 4), and offspring from two generations were
assessed at 3 dpf for methylation changes. Limited effects on
promoter methylation in the second generation were observed
with all compounds, indicating a lack of transgenerational
effects of these toxicants in zebra fish (Table 4). However, it
should be noted that only females were exposed, which ex-
cludes possible effects on male spermatogenesis. Also, the
exposure window did not include DNA methylation
reprogramming during embryonic and PGC development
and therefore could be a less sensitive time window for
transgenerational effects. Furthermore, in F1 and F2 genera-
tions, whole embryos were assessed for methylation changes
which could dilute tissue-specific effects on the methylome.
Clearly, with more improved study designs, more research on
transgenerational effects in the zebra fish is warranted.
Conclusions and outlook
The genome of zebra fish is heavily methylated, like that of
other vertebrates, and it is therefore not surprising that they
have maintenance and several de novo DNMTs and methyl
CpG-binding (MBD) proteins (Bogdanović and Veenstra
2009). In addition to mC, hmC has also been found as well
Environ Sci Pollut Res (2015) 22:16262–16276
as TET enzymes responsible for the oxidation of mC into
hmC. Furthermore, demethylation in zebra fish involves de-
amination by Aid and Apobec enzymes, followed by BER.
Thus, zebra fish share the basic methylation and demethyla-
tion protein machinery with mammals, implying that zebra
fish could be used as a model system or reporter for the
identification of compounds in the environment that affect
DNA methylation. Despite the similarities in enzymes and
other proteins involved DNA methylation, there are differ-
ences in the ways DNA methylation is applied to control, for
example, development in zebra fish and mammals. The most
striking difference is that in contrast to most mammals, only
the maternal genome of the zygote is reprogrammed, whereas
the paternal genome is unchanged. Consequently, it appears
that no mediated hmC reprogramming takes place in the
paternal genome.
Recent studies emphasize the complexity of DNA methyl-
ation and the use of it to control gene expression, including
tissue- and life stage-specific variation and the interactions
with environmental factors. Zebra fish are a promising model
system in environmental toxicology to study epigenetic ef-
fects, since a number of studies show changes in mC levels
and mC patterns after exposure to toxicants. Regarding the
screening of epigenetic toxicity of EDCs, it has been proposed
to adapt the newly developed zebra fish embryo toxicity test
(OECD guideline 236) to include measurements of global and
gene-specific methylation changes (Greally and Jacobs 2013).
Some considerations need to be taken into account in
further application of zebra fish as a model for DNA methyl-
ation in environmental toxicology. First, differentially meth-
ylated DNA regions following exposure to environmental
contaminants are hard to predict and therefore genome-wide
analyses (e.g., MeDIP arrays, WGBS) are recommended as
the first screening step for effects on DNA methylation after
exposure. This may reveal specific sequences that can be used
as epigenetic biomarkers, which later can be analyzed with
more specific DNA methylation analyses in high-throughput
screens, like MS-HRM or pyrosequencing. This would allow
the screening of multiple compounds at different stages of
zebra fish development and exposure windows. Second, it is a
challenge to assess chemical-mediated methylation effects
during the dynamic demethylation and remethylation events
before ZGA onset. This is an extremely important stage to
include in toxicity testing, but care needs to be taken to ensure
that early exposures do not inhibit cell proliferation of devel-
oping zebra fish, resulting in differences in methylation dy-
namics between controlled and exposed groups, rather than
chemical-specific effects on methylation. Third, no informa-
tion is available on the role of DNA methylation in parent of
origin methylation marks and PGC development in zebra fish.
Comparative studies should be conducted to map differences
in methylation dynamics of PGC development between mam-
mals and zebra fish. Such studies could provide pivotal
16273
information regarding predicted transgenerational effects be-
tween species. Transgenerational effects in zebra fish remain
to be studied in greater detail, similar to rodent studies, in
order to assess whether heritable effects of DNA methylation
exist across species. The strength of the zebra fish is that
genetic tools are available to understand the mechanisms
underpinning DNA methylation changes. Functional studies,
like gene inactivation or overexpression, as well as protein
analysis, can be performed to further elucidate the role of
proteins involved in methylation pathways and their interac-
tion with toxicants. Such studies can provide additional infor-
mation improving the applicability of zebra fish as a suitable
model for understanding the role of epigenetics in toxicology.
Acknowledgments JK’s work on global and genome-wide methylation
in zebra fish was performed at NMBU Vetbio, Department BasAm in
Oslo. JK was supported by the Netherlands Organization of Scientific
Research (NWO), project number ASPASIA/864.09.005, and partly by
the Research Council of Norway through its Centre of Excellence funding
scheme, project number 223268/F50, Centre for Environmental Radio-
activity (CERAD) 2013-2022 (JK: Bis-seq, zebra fish exposures and lab
consumables). JL was supported by NWO, project number
VIDI/864.09.005.
References
Aanes H, Winata CL, Lin CH et al (2011) Zebrafish mRNA sequencing
deciphers novelties in transcriptome dynamics during maternal to
zygotic transition. Genome Res 21:1328–1338. doi:10.1101/gr.
116012.110
Abdouni H, King JJ, Suliman M et al (2013) Zebrafish AID is capable of
deaminating methylated deoxycytidines. Nucleic Acids Res 41:
5457–5468. doi:10.1093/nar/gkt212
Almeida RD, Loose M, Sottile V et al (2012) 5-Hydroxymethyl-cytosine
enrichment of non-committed cells is not a universal feature of
vertebrate development. Epigenetics 7:383–389. doi:10.4161/epi.
19375
Andersen IS, Ostrup O, Lindeman LC et al (2012a) Epigenetic complex-
ity during the zebrafish mid-blastula transition. Biochem Biophys
Res Commun 417:1139–1144. doi:10.1016/j.bbrc.2011.12.077
Andersen IS, Reiner AH, Aanes H et al (2012b) Developmental features
of DNA methylation during activation of the embryonic zebrafish
genome. Genome Biol 13:R65. doi:10.1186/gb-2012-13-7-r65
Baker TR, Peterson RE, Heideman W (2014) Using zebrafish as a model
system for studying the transgenerational effects of dioxin. Toxicol
Sci 138:403–411. doi:10.1093/toxsci/kfu006
Bastos Sales L, Kamstra JH, Cenijn PH et al (2013) Effects of endocrine
disrupting chemicals on in vitro global DNA methylation and adi-
pocyte differentiation. Toxicol Vitr 27:1634–1643. doi:10.1016/j.
tiv.2013.04.005
Baylin SB, Jones PA (2011) A decade of exploring the cancer epige-
nome—biological and translational implications. Nat Rev Cancer
11:726–734. doi:10.1038/nrc3130
Bird A (2007) Perceptions of epigenetics. Nature 447:396–398. doi:10.
1038/nature05913
Bogdanović O, Veenstra GJC (2009) DNA methylation and methyl-CpG
binding proteins: developmental requirements and function.
Chromosoma 118:549–565. doi:10.1007/s00412-009-0221-9
Bollati V, Baccarelli A (2010) Environmental epigenetics. Hered (Edinb)
105:105–112. doi:10.1038/hdy.2010.2
16274
Ceccaldi A, Rajavelu A, Champion C et al (2011) C5-DNA methyltrans-
ferase inhibitors: from screening to effects on zebrafish embryo
development. Chembiochem 12:1337–1345. doi:10.1002/cbic.
201100130
Ceccaldi A, Rajavelu A, Ragozin S et al (2013) Identification of novel
inhibitors of DNA methylation by screening of a chemical library.
ACS Chem Biol 8:543–548. doi:10.1021/cb300565z
Cermak T, Doyle EL, Christian M et al (2011) Efficient design and
assembly of custom TALEN and other TAL effector-based con-
structs for DNA targeting. Nucleic Acids Res 39:e82. doi:10.1093/
nar/gkr218
Chen H, Li S, Liu J et al (2004) Chronic inorganic arsenic exposure
induces hepatic global and individual gene hypomethylation: impli-
cations for arsenic hepatocarcinogenesis. Carcinogenesis 25:1779–
1786. doi:10.1093/carcin/bgh161
Corley-Smith GE, Lim CJ, Brandhorst BP (1996) Production of andro-
genetic zebrafish (Danio rerio). Genetics 142:1265–1276
Corrales J, Fang X, Thornton C et al (2014a) Effects on specific promoter
DNA methylation in zebrafish embryos and larvae following
benzo[a]pyrene exposure. Comp Biochem Physiol C Toxicol
Pharmacol 163:37–46. doi:10.1016/j.cbpc.2014.02.005
Corrales J, Thornton C, White M, Willett KL (2014b) Multigenerational
effects of benzo[a]pyrene exposure on survival and developmental
deformities in zebrafish larvae. Aquat Toxicol 148:16–26. doi:10.
1016/j.aquatox.2013.12.028
Deaton AM, Bird A (2011) CpG islands and the regulation of transcrip-
tion. Genes Dev 25:1010–1022. doi:10.1101/gad.2037511
Dolinoy DC, Huang D, Jirtle RL (2007) Maternal nutrient supplementa-
tion counteracts bisphenol A-induced DNA hypomethylation in
early development. Proc Natl Acad Sci U S A 104:13056–13061.
doi:10.1073/pnas.0703739104
Ensembl (2013) Mouse, zebrafish and human assembly and gene anno-
tation. http://www.ensembl.org/. Accessed 28 Aug 2013
Fang X, Thornton C, Scheffler BE, Willett KL (2013) Benzo[a]pyrene
decreases global and gene specific DNA methylation during
zebrafish development. Environ Toxicol Pharmacol 36:40–50. doi:
10.1016/j.etap.2013.02.014
Feng S, Cokus SJ, Zhang X et al (2010) Conservation and divergence of
methylation patterning in plants and animals. Proc Natl Acad Sci U
S A 107:8689–8694. doi:10.1073/pnas.1002720107
Fukata H, Mori C (2004) Epigenetic alteration by the chemical sub-
stances, food and environmental factors. Reprod Med Biol 3:115–
121
Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate ge-
nomes. J Mol Biol 196:261–282
Ge L, Zhang R-P, Wan F et al (2014) TET2 plays an essential role in
erythropoiesis by regulating lineage-specific genes via DNA oxida-
tive demethylation in a zebrafish model. Mol Cell Biol 34:989–
1002. doi:10.1128/MCB.01061-13
Globisch D, Münzel M, Müller M et al (2010) Tissue distribution of 5-
hydroxymethylcytosine and search for active demethylation inter-
mediates. PLoS One 5:e15367. doi:10.1371/journal.pone.0015367
Gokul G, Khosla S (2013) DNA methylation and cancer. In: Kundu TK
(ed) Epigenetics: development and disease. Springer, Dordrecht, pp
597–625
Greally JM, Jacobs MN (2013) In vitro and in vivo testing methods of
epigenomic endpoints for evaluating endocrine disruptors. ALTEX
30:445–471
Gu H, Smith ZD, Bock C et al (2011) Preparation of reduced
representation bisulfite sequencing libraries for genome-scale
DNA methylation profiling. Nat Protoc 6:468–481. doi:10.
1038/nprot.2010.190
Hamdi M, Yoshinaga M, Packianathan C et al (2012) Identification of an
S-adenosylmethionine (SAM) dependent arsenic methyltransferase
in Danio rerio. Toxicol Appl Pharmacol 262:185–193. doi:10.1016/
j.taap.2012.04.035
Environ Sci Pollut Res (2015) 22:16262–16276
Hermann A, Gowher H, Jeltsch A (2004) Biochemistry and biology of
mammalian DNA methyltransferases. Cell Mol Life Sci 61:2571–
2587. doi:10.1007/s00018-004-4201-1
Hernández HG, Tse MY, Pang SC et al (2013) Optimizing methodologies
for PCR-based DNA methylation analysis. Biotechniques 55:181–
197. doi:10.2144/000114087
Houwing S, Kamminga LM, Berezikov E et al (2007) A role for Piwi and
piRNAs in germ cell maintenance and transposon silencing in
zebrafish. Cell 129:69–82. doi:10.1016/j.cell.2007.03.026
Hwang WY, Fu Y, Reyon D et al (2013) Efficient genome editing in
zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229.
doi:10.1038/nbt.2501
Jiang L, Zhang J, Wang J-J et al (2013) Sperm, but not oocyte, DNA
methylome is inherited by zebrafish early embryos. Cell 153:773–
784. doi:10.1016/j.cell.2013.04.041
Jirtle RL, Skinner MK (2007) Environmental epigenomics and disease
susceptibility. Nat Rev Genet 8:253–262. doi:10.1038/nrg2045
Jones PA (2012) Functions of DNA methylation: islands, start sites, gene
bodies and beyond. Nat Rev Genet 13:484–492. doi:10.1038/
nrg3230
Kamstra JH, Hruba E, Blumberg B et al (2014) Transcriptional and
epigenetic mechanisms underlying enhanced in vitro adipocyte dif-
ferentiation by the brominated flame retardant BDE-47. Environ Sci
Technol. doi:10.1021/es405524b
Kimmel CB, Ballard WW, Kimmel SR et al (1995) Stages of embryonic
development of the zebrafish. Dev Dyn 203:253–310. doi:10.1002/
aja.1002030302
Kirchner S, Kieu T, Chow C et al (2010) Prenatal exposure to the
environmental obesogen tributyltin predisposes multipotent stem
cells to become adipocytes. Mol Endocrinol 24:526–539. doi:10.
1210/me.2009-0261
Kobayashi H, Sakurai T, Imai M et al (2012) Contribution of intragenic
DNA methylation in mouse gametic DNA methylomes to establish
oocyte-specific heritable marks. PLoS Genet 8:e1002440. doi:10.
1371/journal.pgen.1002440
Landsverk ML, Weiser DC, Hannibal MC, Kimelman D (2010)
Alternative splicing of sept9a and sept9b in zebrafish produces
multiple mRNA transcripts expressed throughout development.
PLoS One 5:e10712. doi:10.1371/journal.pone.0010712
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying
DNA methylation patterns in plants and animals. Nat Rev Genet 11:
204–220. doi:10.1038/nrg2719
Li W, Liu M (2011) Distribution of 5-hydroxymethylcytosine in different
human tissues. J Nucleic Acids 2011:870726. doi:10.4061/2011/
870726
Li S, Hansman R, Newbold R et al (2003) Neonatal diethylstilbestrol
exposure induces persistent elevation of c-fos expression and hypo-
methylation in its exon-4 in mouse uterus. Mol Carcinog 38:78–84.
doi:10.1002/mc.10147
Li D, Lu C, Wang J et al (2009) Developmental mechanisms of arsenite
toxicity in zebrafish (Danio rerio) embryos. Aquat Toxicol 91:229–
237. doi:10.1016/j.aquatox.2008.11.007
Lindeman LC, Reiner AH, Mathavan S et al (2010a) Tiling histone H3
lysine 4 and 27 methylation in zebrafish using high-density micro-
arrays. PLoS One 5:e15651. doi:10.1371/journal.pone.0015651
Lindeman LC, Winata CL, Aanes H et al (2010b) Chromatin states of
developmentally-regulated genes revealed by DNA and histone
methylation patterns in zebrafish embryos. Int J Dev Biol 54:803–
813. doi:10.1387/ijdb.103081ll
Lindeman LC, Andersen IS, Reiner AH et al (2011) Prepatterning of
developmental gene expression by modified histones before zygotic
genome activation. Dev Cell 21:993–1004. doi:10.1016/j.devcel.
2011.10.008
Lister R, Pelizzola M, Dowen RH et al (2009) Human DNA methylomes
at base resolution show widespread epigenomic differences. Nature
462:315–322. doi:10.1038/nature08514
Environ Sci Pollut Res (2015) 22:16262–16276
Long HK, Sims D, Heger A et al (2013) Epigenetic conservation at gene
regulatory elements revealed by non-methylated DNA profiling in
seven vertebrates. Elife 2:e00348. doi:10.7554/eLife.00348
MacKay AB, Mhanni AA, McGowan RA, Krone PH (2007)
Immunological detection of changes in genomic DNA methylation
during early zebrafish development. Genome 50:778–785. doi:10.
1139/g07-055
Macleod D, Clark VH, Bird A (1999) Absence of genome-wide changes
in DNA methylation during development of the zebrafish. Nat
Genet 23:139–140. doi:10.1038/13767
Martin CC, Laforest L, Akimenko MA, Ekker M (1999) A role for DNA
methylation in gastrulation and somite patterning. Dev Biol 206:
189–205. doi:10.1006/dbio.1998.9105
Mathavan S, Lee SGP, Mak A et al (2005) Transcriptome analysis of
zebrafish embryogenesis using microarrays. PLoS Genet 1:260–
276. doi:10.1371/journal.pgen.0010029
McGee SP, Cooper EM, Stapleton HM, Volz DC (2012) Early zebrafish
embryogenesis is susceptible to developmental TDCPP exposure.
Environ Health Perspect 120:1585–1591. doi:10.1289/ehp.1205316
Mellén M, Ayata P, Dewell S et al (2012) MeCP2 binds to 5hmC enriched
within active genes and accessible chromatin in the nervous system.
Cell 151:1417–1430. doi:10.1016/j.cell.2012.11.022
Mhanni AA, McGowan RA (2004) Global changes in genomic methyl-
ation levels during early development of the zebrafish embryo. Dev
Genes Evol 214:412–417. doi:10.1007/s00427-004-0418-0
Mhanni AA, Yoder JA, Dubesky C, McGowan RA (2001) Cloning and
sequence analysis of a zebrafish cDNA encoding DNA (cytosine-5)-
methyltransferase-1. Genesis 30:213–219. doi:10.1002/gene.1067
Nestor CE, Ottaviano R, Reddington J et al (2012) Tissue type is a major
modifier of the 5-hydroxymethylcytosine content of human genes.
Genome Res 22:467–477. doi:10.1101/gr.126417.111
Olsvik PA, Williams TD, Tung H-S et al (2014) Impacts of TCDD and
MeHg on DNA methylation in zebrafish (Danio rerio) across two
generations. Comp Biochem Physiol C Toxicol Pharmacol 165C:
17–27. doi:10.1016/j.cbpc.2014.05.004
Pastor WA, Aravind L, Rao A (2013) TETonic shift: biological roles of
TET proteins in DNA demethylation and transcription. Nat Rev Mol
Cell Biol 14:341–356. doi:10.1038/nrm3589
Pfeifer GP, Kadam S, Jin S-G (2013) 5-Hydroxymethylcytosine and its
potential roles in development and cancer. Epigenetics Chromatin 6:
10. doi:10.1186/1756-8935-6-10
Pikulkaew S, Benato F, Celeghin A et al (2011) The knockdown of
maternal glucocorticoid receptor mRNA alters embryo development
in zebrafish. Dev Dyn 240:874–889. doi:10.1002/dvdy.22586
Potok ME, Nix DA, Parnell TJ, Cairns BR (2013) Reprogramming the
maternal zebrafish genome after fertilization to match the paternal
methylation pattern. Cell 153:759–772. doi:10.1016/j.cell.2013.04.030
Probst AV, Dunleavy E, Almouzni G (2009) Epigenetic inheritance
during the cell cycle. Nat Rev Mol Cell Biol 10:192–206. doi:10.
1038/nrm2640
Rai K, Nadauld LD, Chidester S et al (2006) Zebra fish Dnmt1 and
Suv39h1 regulate organ-specific terminal differentiation during de-
velopment. Mol Cell Biol 26:7077–7085. doi:10.1128/MCB.00312-
06
Rai K, Huggins IJ, James SR et al (2008) DNA demethylation in
zebrafish involves the coupling of a deaminase, a glycosylase, and
gadd45. Cell 135:1201–1212. doi:10.1016/j.cell.2008.11.042
Rai K, Jafri IF, Chidester S et al (2010) Dnmt3 and G9a cooperate for
tissue-specific development in zebrafish. J Biol Chem 285:4110–
4121. doi:10.1074/jbc.M109.073676
Ruthenburg AJ, Li H, Patel DJ, Allis CD (2007) Multivalent engagement
of chromatin modifications by linked binding modules. Nat Rev
Mol Cell Biol 8:983–994. doi:10.1038/nrm2298
Ruzov A, Tsenkina Y, Serio A et al (2011) Lineage-specific distribution
of high levels of genomic 5-hydroxymethylcytosine in mammalian
development. Cell Res 21:1332–1342. doi:10.1038/cr.2011.113
16275
Schier AF (2007) The maternal-zygotic transition: death and birth of
RNAs. Science 316:406–407. doi:10.1126/science.1140693
Seritrakul P, Gross JM (2014) Expression of the de novo DNA methyl-
transferases (dnmt3 - dnmt8) during zebrafish lens development.
Dev Dyn 243:350–356. doi:10.1002/dvdy.24077
Shimoda N, Yamakoshi K, Miyake A, Takeda H (2005) Identification of a
gene required for de novo DNA methylation of the zebrafish no tail
gene. Dev Dyn 233:1509–1516. doi:10.1002/dvdy.20455
Skinner MK (2013) Environmental epigenetics and transgenerational
epigenetic inheritance. In: Jirtle RL, Tyson FL (eds)
Environmental epigenomics in health disease. Springer, Berlin, pp
245–256
Smallwood SA, Kelsey G (2012) De novo DNA methylation: a germ cell
perspective. Trends Genet 28:33–42. doi:10.1016/j.tig.2011.09.004
Smith THL, Collins TM, McGowan RA (2011) Expression of the dnmt3
genes in zebrafish development: similarity to Dnmt3a and Dnmt3b.
Dev Genes Evol 220:347–353. doi:10.1007/s00427-010-0347-z
Smith ZD, Chan MM, Mikkelsen TS et al (2012) A unique regulatory
phase of DNA methylation in the early mammalian embryo. Nature
484:339–344. doi:10.1038/nature10960
Strähle U, Scholz S, Geisler R et al (2012) Zebrafish embryos as an
alternative to animal experiments—a commentary on the definition
of the onset of protected life stages in animal welfare regulations.
Reprod Toxicol 33:128–132. doi:10.1016/j.reprotox.2011.06.121
Strömqvist M, Tooke N, Brunström B (2010) DNA methylation levels in
the 5′ flanking region of the vitellogenin I gene in liver and brain of
adult zebrafish (Danio rerio)—sex and tissue differences and effects
of 17alpha-ethinylestradiol exposure. Aquat Toxicol 98:275–281.
doi:10.1016/j.aquatox.2010.02.023
Takai D, Jones PA (2002) Comprehensive analysis of CpG islands in
human chromosomes 21 and 22. Proc Natl Acad Sci U S A 99:
3740–3745. doi:10.1073/pnas.052410099
Takayama K, Shimoda N, Takanaga S et al (2014) Expression patterns of
dnmt3aa, dnmt3ab, and dnmt4 during development and fin regen-
eration in zebrafish. Gene Expr Patterns 14:105–110. doi:10.1016/j.
gep.2014.01.005
Tittle RK, Sze R, Ng A et al (2011) Uhrf1 and Dnmt1 are required for
development and maintenance of the zebrafish lens. Dev Biol 350:
50–63. doi:10.1016/j.ydbio.2010.11.009
Tucker KL (2001) Methylated cytosine and the brain: a new base for
neuroscience. Neuron 30:649–652
Vandegehuchte MB, Janssen CR (2014) Epigenetics in an ecotoxicolog-
ical context. Mutat Res Genet Toxicol Environ Mutagen 764–765:
36–45. doi:10.1016/j.mrgentox.2013.08.008
Vastenhouw NL, Schier AF (2012) Bivalent histone modifications in
early embryogenesis. Curr Opin Cell Biol 24:374–386. doi:10.
1016/j.ceb.2012.03.009
Wang J, Wu Z, Li D et al (2012) Nutrition, epigenetics, and metabolic
syndrome. Antioxid Redox Signal 17:282–301. doi:10.1089/ars.
2011.4381
Wang H, Yang H, Shivalila CS et al (2013) One-step generation of mice
carrying mutations in multiple genes by CRISPR/Cas-mediated
genome engineering. Cell 153:910–918. doi:10.1016/j.cell.2013.
04.025
Wei C, Salichos L, Wittgrove CM et al (2012) Transcriptome-wide
analysis of small RNA expression in early zebrafish development.
RNA 18:915–929. doi:10.1261/rna.029090.111
Weisenberger DJ, Romano LJ (1999) Cytosine methylation in a CpG
sequence leads to enhanced reactivity with benzo[a]pyrene diol
epoxide that correlates with a conformational change. J Biol Chem
274:23948–23955
Williams TD, Mirbahai L, Chipman JK (2014) The toxicological applica-
tion of transcriptomics and epigenomics in zebrafish and other tele-
osts. Brief Funct Genomics 13:157–171. doi:10.1093/bfgp/elt053
Wu SC, Zhang Y (2010) Active DNA demethylation: many roads lead to
Rome. Nat Rev Mol Cell Biol 11:607–620. doi:10.1038/nrm2950
16276
Wu H, Zhang Y (2014) Reversing DNA methylation: mechanisms,
genomics, and biological functions. Cell 156:45–68. doi:10.1016/j.
cell.2013.12.019
Wu Q, Ohsako S, Ishimura R et al (2004) Exposure of mouse preimplan-
tation embryos to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) al-
ters the methylation status of imprinted genes H19 and Igf2. Biol
Reprod 70:1790–1797. doi:10.1095/biolreprod.103.025387
Wu H, Caffo B, Jaffee HA et al (2010) Redefining CpG islands using
hidden Markov models. Biostatistics 11:499–514. doi:10.1093/
biostatistics/kxq005
Wu S-F, Zhang H, Hammoud SS et al (2011) DNA methylation profiling
in zebrafish. Methods Cell Biol 104:327–339. doi:10.1016/B978-0-
12-374814-0.00018-5
Yamakoshi K, Shimoda N (2003) De novo DNA methylation at the CpG
island of the zebrafish no tail gene. Genesis 37:195–202. doi:10.
1002/gene.10245
Environ Sci Pollut Res (2015) 22:16262–16276
Yildirim O, Li R, Hung J-H et al (2011) Mbd3/NURD complex
regulates expression of 5-hydroxymethylcytosine marked
genes in embryonic stem cells. Cell 147:1498–1510. doi:10.
1016/j.cell.2011.11.054
Yokomine T, Hata K, Tsudzuki M, Sasaki H (2006) Evolution of the
vertebrate DNMT3 gene family: a possible link between existence
of DNMT3L and genomic imprinting. Cytogenet Genome Res 113:
75–80. doi:10.1159/000090817
Zhang X, Wallace AD, Du P et al (2012) Genome-wide study of DNA
methylation alterations in response to diazinon exposure in vitro.
Environ Toxicol Pharmacol 34:959–968. doi:10.1016/j.etap.2012.
07.012
Zhao CQ, Young MR, Diwan BA et al (1997) Association of arsenic-
induced malignant transformation with DNA hypomethylation and
aberrant gene expression. Proc Natl Acad Sci U S A 94:10907–
10912