Toxicology and Applied Pharmacology 396 (2020) 115002

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Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/taap

Exposure to low doses of inorganic arsenic induces transgenerational T

changes on behavioral and epigenetic markers in zebrafish (Danio rerio)
Selma Vallesa, Jorge Hernández-Sáncheza, Víctor René Dippa, Darién Huerta-Gonzáleza,
⁎
Tatiana N. Olivares-Bañuelosb, Jose González-Fragaa, Ulises Bardullasa,
a
Facultad de Ciencias, Universidad Autónoma de Baja California (UABC), Carretera Tijuana-Ensenada 3917, Playitas, 22860 Ensenada, B.C., Mexico
b
Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, Ensenada, Baja California, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: The ability of environmental pollutants to alter the epigenome with resultant development of behavioral al-
Neurodevelopment genes terations has received more attention in recent years. These alterations can be transmitted and affect later
Behavioral toxicology generations that have not been directly in contact with the contaminant. Arsenic (As) is a neurotoxicant and
Epigenetics potent epigenetic disruptor that is widespread in the environment; however, the precise potential of As to
Transgenerational
produce transgenerational effects is unknown. Our study focused on the possible transgenerational effects on
Zebrafish
behavior by ancestral exposure to doses relevant to the environment of As, and the epigenetic mechanisms that
could be involved. Embryos of F0 (ancestral generation) were directly exposed to 50 or 500 ppb of As for
150 days. F0 adults were raised to produce the F1 generation (intergeneration) and subsequently the F2 gen-
eration (transgeneration). We evaluated motor and cognitive behavior, neurodevelopment-related genes, and
epigenetic markers on the F0 and F2 generation. As proposed in our hypothesis, ancestral arsenic exposure
altered motor activity through the development and increased anxiety-like behaviors which were transmitted to
the F2 generation. Additionally, we found a reduction in brain-derived neurotrophic factor expression between
the F0 and F2 generation, and an increase in methylation on histone H3K4me3 in the nervous system.

1. Introduction
Arsenic is recognized as being one of the chemicals that causes
major concern for global public health due to its dispersion in the en-
vironment, particularly in groundwater (Shankar et al., 2014). For 2.5
billion people, groundwater is their only source of water, and at least
20% of the world's population does not have access to a safe source of
water for consumption (UNESCO, 2012). At present, an estimated 200
million people are at risk of consuming water contaminated with As
above the allowed limit of 10 μg/L; these people live in countries such
as México, Taiwan, and Bangladesh (Del Razo et al., 1993; Kinniburgh
and Kosmus, 2002).
The ability of As to produce neurotoxicity has been widely described
in human and animal models (Rodriguez et al., 2003; Tyler and Allan,
2014). The mechanism of As-induced neurotoxicity is closely related to
the enhancement of reactive oxygen species (Chattopadhyay et al.,
2010) and oxidative damage in lipids and proteins in the brain (Garcia-
Chavez et al., 2006). Together, these factors can lead to behavioral
disorders; for example, exposure to low doses of As can alter cognitive
function, producing a significant decrease in the IQ in humans
(Carpenter et al., 1998; Calderon et al., 2001), and impulsivity and
alteration in the attention status (Rodriguez-Barranco et al., 2017). The
effect of As on behavior has been extensively studied, but mainly on
rodents (Rodriguez et al., 2003).
Several studies have shown that epigenetic change by exposure to
chemical pollutants is associated with the development of neurological
and neurodegenerative diseases (Landgrave-Gomez et al., 2015), and As
is a potent inducer of epigenetic modifications, including DNA methy-
lation (Zhang et al., 2014), changes in miRNA abundance, and post-
translational modifications in histone posttranslational modifications
(HPTMs) (Bailey and Fry, 2014). However, it has been challenging to
interpret the functional meaning of these modifications. Studies in an-
imals and humans have associated As exposure in utero with an in-
creased risk of developing chronic degenerative diseases as an adult
(Smith et al., 2012; Farzan et al., 2013). In rodents, gestational ex-
posure induced changes in the methylation pattern in specific regions of

Abbreviations: As, arsenic; HPTMs, histone posttranslational modifications; HPF, hours post-fertilization; DPF, days post-fertilization; NI, navigation index; H3K9ac,
histone 3 lysine 9 acetylation; H3K4me3, histone 3 lysine 9 trimethylation.
⁎
Corresponding author at: Universidad Autónoma de Baja California, Facultad de Ciencias, Ensenada, B.C., Mexico.
E-mail address: bardullas@uabc.edu.mx (U. Bardullas).

https://doi.org/10.1016/j.taap.2020.115002
Received 10 December 2019; Received in revised form 26 March 2020; Accepted 7 April 2020
Available online 08 April 2020
0041-008X/ © 2020 Elsevier Inc. All rights reserved.
S. Valles, et al.
Fig. 1. Timeline and experimental design of the transgenerational study and the markers evaluated in the three generations. The F0 generation was continuously
exposed to As, while the generation F1 (intergeneration) exposed only as a germ cell and F2 without any exposure to As represents transgenerational generation.
the brain (the hippocampus and cerebral cortex) on the reelin gene
(RELN) and protein phosphatase 1 (PP1), associated with neuronal
plasticity, causing alterations in learning functions and adult rodent
memory (Martinez et al., 2011). Likewise, C57Bl6/J mice exposed to
low doses of As during their embryonic development presented a global
hypoacetylation in the histone 3 lysine 9 segment, which was associated
in the adult stage with the disruption of spatial and episodic memory
(Cronican et al., 2013).
In recent years, evidence has been generated on a growing number
of substances, mainly on endocrine disruptors that are capable of in-
ducing adverse effects across generations, including alterations in re-
productive and endocrine physiology (Xin et al., 2015. However, few
studies with metals (Carvan 3rd et al., 2017; Xu et al., 2015), polycyclic
aromatic hydrocarbons (Knecht et al., 2017; Vignet et al., 2015) poly-
chlorinated biphenyls and polybrominated diphenyl ethers (Alfonso
et al., 2019) have addressed behavioral alterations produced by trans-
generational exposure. Although arsenic is one of the most studied
substances, and it is a well-described neurotoxin, its potential as an
inducer of transgenerational effects on behavior has never been ad-
dressed.
The zebrafish, as a model, has a high potential for transgenerational
studies on chemical exposure because its external embryonic develop-
ment means that the F2 generation is the first that will not have been
exposed to the xenobiotic (Baker et al., 2014). Although compared with
mammals, fish have a higher level of global methylation 7%–8% of all
cytosines. The main epigenetic mechanisms and the DNA methylation
pattern is similar between zebrafish embryos and mammals (Williams
et al., 2014). In zebrafish, methylation patterns can be copied to the
daughter's DNA after mitosis and transmitted to her progeny through
sperm (Jiang et al., 2013). This organism also has a metabolism that has
comparable characteristics with other vertebrates, such as the presence
of arsenic enzyme 3-methyltransferase and a profile of As species si-
milar to that detected in mammalian tissues, including those of humans
(Hamdi et al., 2012).
We previously reported in the parental generation (F0) that chronic
exposure to 500 ppb As, alters motor function and induces behaviors
associated with anxiety from the embryo to the adult stage, and ex-
posure to 50 or 500 ppb As affects associative learning and sensor-
imotor response during adult phase (Dipp et al., 2018). In this study, we
present evidence that ancestral exposure to doses relevant to the en-
vironment of As induces behavioral alterations in the zebrafish model
that are passed on to other generations without exposure; we also ex-
plore some epigenetic markers that could be involved in the inheritance
of these alterations.
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Toxicology and Applied Pharmacology 396 (2020) 115002
2. Material and methods
2.1. Parental chronic exposure (F0) and obtaining the F1 and F2 generation
Ninety zebrafish, previously acclimatized to laboratory conditions
for one month, were crossed in a 3 female: 2 male ratio. At 4 h post-
fertilization (hpf), the embryos were randomly mixed and distributed in
60 mm petri dishes (50 embryos per dish) containing a nominal con-
centration of sodium arsenite (0, 50, or 500 ppb) in E2 medium without
methylene blue.
We built a flow-through system in order to expose the organisms for
an extended period of time while minimizing the stress of handling. The
system delivers fresh solutions (0, 50, or 500 ppb As, as required) with
the use of peristaltic pumps in 5/L glass tanks and four repetitions per
treatment. The pH of the reservoirs was maintained at 7.0 to minimize
the oxidation of arsenite to arsenate. The temperature of the water was
maintained at 26.5 °C ± 1.5 °C, and we monitored the water quality
parameters weekly. Early larvae, from five to 9 days post-fertilization
(dpf), were grown, at a density of 100 larvae per liter and in a poly-
culture system with arsenic solutions in a static condition (Best et al.,
2010). From nine dpf, the flow of As solution started gradually, with a
total replacement of arsenic solution in the treatment tanks every 48 h.
At 30 dpf, the fish from the same treatments were randomly pooled
together at a density of 10 fish per liter and were fed with brine shrimp
nauplii twice a day. We monitored the As concentration in the experi-
mental tanks, artemia nauplii cultures, and the reverse osmosis water,
colorimetrically, with an arsenic low range test kit (Hach, US) once a
week. In the following generations, we used a new continuous flow
system, including glass tanks, to avoid any contamination.
To produce the F1 and F2 generation, 50 females and 36 males of
each exposure were randomly crossed in the RO water for 3 consecutive
days; an appropriate number of embryos were obtained ( ± 1200 em-
bryos). The embryos grew without As in the same feeding and density
conditions and water quality parameters as the F0. The experimental
design and timeline for the present study are presented in Fig. 1.
2.2. Embryotoxicity
Embryos of 4 hpf were placed individually, during blastulation, in
96-well plates in two replicates, and filled with control, 50 ppb As, or
500 ppb As solutions. The plate dishes were incubated at 28 °C with a
cycle of 14 h light/10 h dark. We replaced fresh solutions of all the
treatments daily. The assay started at 8 hpf, and continued at 24, 48,
72, 96 hpf. Endpoints for embryos were chosen and made according to
the research by (Lammer et al., 2009).
S. Valles, et al.
2.3. Behavioral test battery
We conducted a battery of behavioral tests to evaluate motor and
cognitive functions during the development of the zebrafish (Fig. 1). All
procedures and all types of tests were replicated in the F2 generation.
The tests with adults were performed without sex separation. The re-
sults for the F0 generation, and the extensive procedure, are reported in
the research by Dipp et al. (2018). All behavioral testing, from embryo
to adult stages, was conducted between 12:00 and 16:00 during the
light phase. For all tests, we used a recording system with a Flea3
camera attached to a C-Mount 12.5–75 mm varifocal lens (computar
M6Z1212-3S). All tracking data were analyzed using the Lolitrack 4.0
software package (Loligo Systems. Tjele, Denmark).
2.3.1. Monitoring embryo and larva motor activity
Frequency in the spontaneous tail movement was monitored in 25
embryos randomly selected from 17 hpf to 26 hpf using a Carl Zeiss
Stemi™ 2000-C Stereomicroscope with a Flea3 camera attached, and
analyzed using our own MATLAB® script – ZebraSTM (Gonzalez-Fraga
et al., 2019). The type of coil was evaluated until 26 hpf, as described
by (Knogler et al., 2014). We identified the type of movement of the tail
using the plots generated by the ZebraSTM software. At seven hpf we
evaluated the larval activity, using a custom system, to isolate larvae
from any external stimuli. We used a high-quality infrared backlight
(Banner, MN, USA) and LED type lighting (General Lighting, 04745–1)
to generate lighting changes through an external, manually operated
switch (bright > 15,000 lx, and dark < 20 lx). Experiments were car-
ried out in a room maintained at a temperature of 28 °C ± 2 °C. Total
distance traveled, per minute, was calculated for each larva for each
10 min phase.
2.3.2. Shoaling behavior
After a habituation period of one minute, the average distance be-
tween two fish undergoing the same treatment was evaluated for a
period of 5 min, in a tank 14 cm long, 7 cm wide and 7 cm high, with
clean control water at pH 7.0 and at 28.5 °C, in an illuminated room of a
temperature between 26.5 °C and 28.5 °C.
2.3.3. Startle tap test
We assessed habituation, a form of nonassociative learning, using a
custom-built tapping device, assembled in an Arduino Nano board that
generates pulse-width modulation over four solenoids of 12 V and 1A.
We programmed sequence, intervals, and the strength of the taps, using
LabVIEW. The fish were individually placed in 60 mm diameter glass
vessels filled with 30 mL of RO water. Opaque screens separated the
glass vessels in order to isolate the test subjects. The system was set to
start tapping after a five-minute acclimation period. Tap frequency was
set at one tap per minute for a 10-min period. We evaluated habituation
by measuring the distance traveled in centimeters for 5 s after each tap.
2.3.4. Novel tank assay
This test was used to evaluate parameters related to exploratory
behavior in adults. A total of six fish per replicate, in each treatment,
were transferred into pH 7.0 clean control water and placed in separate
tanks. The tests were conducted in trapezoid tanks
(27.9 cm × 22.5 cm × 15.2 cm × 7.1 cm). The temperature of the
experimental tank was adjusted to 25.5 °C with replacement after every
two or three individuals. Each fish was recorded for 5 min on entering
the tank, with the use of a webcam (Logitech) at 60 cm distance. The
organisms were sacrificed at the end of the trial.
We performed a visual identification of the sex, with the videos
obtained from the lateral plane.
2.3.5. Navigation index
We analyzed the navigation index (NI) recently proposed by
(Facchin et al., 2015). The NI allowed us to describe how the animals
3
Toxicology and Applied Pharmacology 396 (2020) 115002
were distributed in the tank during the duration of the test, which could
reflect the exploratory behavior of the individuals. The NI is described
by the Shannon entropy equation:NI = − ∑k=1npklog2pk, where pk is the
probability that an animal appears in a particular bin or subregion k of
the tank during the test time, and it is calculated as the number of
appearances in the bin k, divided by the total number of appearances in
all bins. The total number of subregions in the tank is calculated ac-
cording to the size of the test fish.
2.4. Global DNA methylation
Genomic DNA samples were extracted from larval tissue (5 dpf,
n = 3–4, 20 larvae/pool) and whole brains from adult fish (150 dpf,
n = 4–5 for each sex per treatment) using a genomic DNA purification
kit (PureLink Genomic DNA kit, Invitrogen, Carlsbad, CA, USA). The
purification of the genomic DNA samples was carried out according to
the manufacturer's protocol. Concentrations were measured with a
NanoDrop® spectrophotometer. The percentage of cytosine methylation
of the global DNA was quantified with a colorimetric kit (MethylFlash
Methylated DNA Quantification Kit, Epigentek Group, Farmingdale,
NY, USA) for the measurement of 5-methylcytosine (5-mC) levels in a
96-well microplate following the manufacturer's instructions. The
samples were analyzed in triplicate, with 100 ng of genomic DNA per
sample; they were read with a microplate spectrophotometer
(Multiskan TM GO system, Thermo Scientific) at 450 nm.
2.5. Extraction of histone and western blot
The extraction of histones was made through a protocol from the
research by Rachdaoui et al. (2017) and adapted for the zebrafish
model. Six pools (5 male and 5 female whole brains per treatment),
were homogenized with the use of a PELLET PESTLE® Cordless Motor
(Kimble Chase) using Triton Extraction Buffer (TEB) containing: PBS
1×, 0.5% Triton-X 100 v/v; 2 mM phenylmethylsulfonyl fluoride
(PMSF); 0.02% sodium azide (NaN3) w/v; 5 mM sodium butyrate
(NaB); and a protease inhibitor cocktail (Complete Mini, Roche). The
homogenate was nuclei-washed and centrifuged in half the volume of
TEB. Histones were extracted in μL 0.4 N H2SO4 and incubated over-
night at 4 °C. The samples were centrifuged at 15,000 rpm for 15 min at
4 °C and the supernatants were transferred to a clean 1.5 mL tube, with
cold acetone, and incubated at −20 °C overnight, to precipitate his-
tones. Histones were centrifuged at 15,000 rpm for 15 min at 4 °C,
washed in cold acetone and allowed to air dry at room temperature, and
finally suspended in deionized water. Protein quantification was per-
formed using the Lowry method (DC Protein Assay, Bio-Rad).
Histone protein (12 μg) was separated using gels of TGX Stain-free
(Bio-rad) and transferred to a polyvinylidene difluoride (PVDF) mem-
brane (RTA Transfer Kit) using Trans-Blot Turbo transfer apparatus
(Bio-Rad). Individual membranes for each histone were incubated
overnight at 4 °C using the following primary antibodies: H3K4me3
(1:1000, Abcam, ab8580) and H3K9ac (1:500, Millipore 07–352). In
order to normalize the levels of each histone, the membranes were
stripped and reblotted with H3 (1:1000 Cell Signaling, #3638).
Previous studies indicate that arsenic does not alter histone H3 (Tyler
et al., 2015). The membranes were visualized by chemiluminescent
autoradiography (ECL kit, Amersham, Buckinghamshire, United
Kingdom), according to the manufacturer's instructions using Che-
miDoc MP (Bio-Rad).
2.6. RNA isolation and quantitative real-time PCR (qRT-PCR)
We evaluated by semiquantitative RT-PCR, the relative expression
of reelin (reln), myelin basic protein (mbp), protein phosphatase 1, reg-
ulatory (inhibitor) subunit 1B (ppp1r1b or DARPP-32), and brain-derived
neurotrophic factor (bdnf). Total RNA was extracted from individual
adult zebrafish brains (127 dpf, n = 5 for each sex per treatment), using
S. Valles, et al.
TRIzol® reagent (Invitrogen, Catalog #15596026). The protocol was
followed according to manufacturer's instructions. The isolated total
RNA was suspended in 25 ul of nuclease-free sterile water, and con-
centrations were measured with a NanoDrop ND-1000™ spectro-
photometer. A High Capacity cDNA Reverse Transcription Kit
(Invitrogen, catalog #4368814) was used to convert mRNA from
500 ng of the total RNA from each sample to cDNA, and cDNA samples
were diluted 1:10 with water. RT-PCR was performed in a StepOne Plus
thermocycler (Applied Biosystems) using SYBR Green Master Mix
(Applied Biosystems, Catalog #4309155); concentrations used for each
primer are specified in supplementary material 1. We used ribosomal
protein L13a (rpl13a) as a housekeeping gene which has been reported
as a highly reliable reference gene for gene expression studies involving
chemical treatments in zebrafish model (Xu et al., 2016). In addition,
Tukey multiple comparisons showed that rpl13a expression does not
change between treatments (P = .948). Three technical replicates were
used in the qPCR analysis. All data results were captured and processed
using StepOne Software v2.3. Analysis of the qRT-PCR data was per-
formed using the ΔΔCt method.
2.7. Bisulfite conversion and MethySYBR assay
Genomic DNA was isolated from the same brains as those from
which RNA was purified, following TRIzol's protocol. Briefly, DNA was
isolated by enzymatic digestion to dissociate the DNA from histones
using a buffer mix. One mL of 10× lysis buffer includes 147.4 uL of
19 mg/mL proteinase K and 500 ul of 20% SDS; dilution to 1× is made
with solubilization buffer (8 mM NaOH +1 mM EDTA, pH 7–8).
Subsequently, samples were solubilized with 20 ul of mixed buffer, and
incubated at 55 °C overnight. DNA was measured on ND-1000
NanoDrop ™ and 500 ng of each sample were used for bisulfite con-
version using the EZ-96 DNA Methylation-Gold ™ Kit (Zymo Research,
catalog #D5005). Bisulfite converted DNA was used for one-step
MethySYBR reactions as described by (Lo et al., 2009). To illustrate
methylation levels, the methylation percentage of each sample was
computed by an adaptation of the original equation of (Hehar et al.,
2017) (%methylation = 100/[1 + 2(Ct Methylated – Ct Unmethylated)]).
2.8. Statistical analysis
All data were analyzed and graphed using GraphPad version 8.0.
For all data, we used the Shapiro-Wilk test to probe normality. Data
with normal distribution were analyzed using one-way ANOVA, fol-
lowed by Dunnett's multiple comparison test. Data that failed the nor-
mality test were subjected to a Kruskal-Wallis test, followed by Mann-
Whitney U tests. Locomotor activity at seven dpf was analyzed using
repeated-measures analysis of variance, followed by post hoc tests, to
compare treatments with the control group. Differences between the
control and treated groups were analyzed by ANOVA test. Pearson's
correlation between methylation level and expression was performed
for bdnf methylation. The nominal data were analyzed using the chi-
square test. A p-value of less than 0.05 was considered to be statistically
significant. Data are presented as the mean ± standard deviation of
mean (SEM).
3. Results
3.1. Embryotoxicity
In general, we did not find that ancestral exposure to arsenic pro-
duced alterations on teratogenesis markers (Table 1). In the F0 gen-
eration, there was an increase in lethality p < .05, compared with F2.
Additionally, we observed an increased heart rate in the group treated
with 500 ppb As from the F0 generation at 24 h [F (2,27) = 3.914,
p < .05], and in groups treated with 50 or 500 ppb As from the F2
generation at 48 hpf [F(2, 30) = 5.686 p = .0081].
4
Toxicology and Applied Pharmacology 396 (2020) 115002
3.2. Development of motor activity in the embryo and larval zebrafish
We evaluated whether the ancestral exposure As was able to alter
the motor development in the F2 generation. We found that during the
embryonic phase, the spontaneous movement of the tail increased in
the group treated with 500 ppb As during the 21–26 hpf [F's
(2.61) = 5032–21.4 p < .001] (Fig. 2A). Notably, the number of the
double coils, which is associated with the maturation process in
swimming behavior, increased by 16% in the control group during the
26 hpf, but not [X2 26.45 p < .0001] in the arsenic groups.
During the larval stage at seven dpf, we evaluated transgenerational
effect in motor activity. Two-way ANOVA analysis revealed the overall
effects of treatment in the F2 generation [F(2,78) = 4.595, p = .013]
and an effect of time [F(5.3, 414) = 48.61, p < .0001] and interaction
treatment x time [F(48,1872) = 1731 p = .001]. The post hoc analysis
showed a significant reduction of motor activity in the group of 500 ppb
As during different periods of the dark phase (Fig. 2B).
3.3. Startle tap test and shoaling behavior in adult zebrafish
For nonassociative learning, we used the motor reflex produced
after the stimulus (tap) to evaluate the habituation curve; we found that
all groups of F2 [F(3.3, 129.9) = 14.3] habituated to the tap stimulus
(Fig. 3A). We observed no effect on social interaction during the
shoaling behavior test for the F2 generation [F(2,20) = 0.4501
p > .05] (Fig. 3B).
3.4. Exploratory behavior in larvae and adult zebrafish
We found that the 500 ppb As of the F2 generation showed an
elevated anxiety-like behavior compared with the control group, with
an increased latency to cross the tank [F(2,45) = 5.01, p = .0108]
(Fig. 4A) and a reduction in the number of entries in the top half [H
(2) = 5.01, p = .04] (Fig. 4B) and the total distance [F(2,45) = 10.21
p = .0002] (Fig. 4C). With the information obtained from the novel
tank test, we generated the NI. Previously, we identified that NI is not
another way of measuring total distance since these variables are not
correlated. Fish with greater total distances traveled do not increase
their NI [r = 0.272 p > .05]. The NI is presented on a logarithmic
scale (Fig. 4D); each unit represents that the organism had twice the
exploratory activity. During the larval stage, the exploratory behavior
was similar between all groups in the three generations p > .05.
However, in the adult stage, an ANOVA analysis showed a significant
effect for the group of 500 ppb As in F0 [F(2,48) = 5,20, p < .009]
and F2 [F(2,48) = 5.20, p = .02].
3.5. Global DNA methylation
We quantified global DNA methylation levels (5-mC) in whole
larvae and the brains of adults of both sexes of the F0 generation (data
not shown). We did not find that the levels of global DNA methylation
were modified in males, females and larvae (Supplementary material 2,
p > .05).
3.6. Histone posttranslational modifications
In order to elucidate the epigenetic modifications by the ancestral
exposure to As, we evaluated the whole-brain HPTMs in males and
females of the F0 and F2 generations. We found that the histone 3 lysine
4 trimethylation was increased in both males [F(2,12) = 10.04,
p = .002 and females [F(2,12) = 5.304, p = .022] of the 500 ppb As
group of the F0 generation (Fig. 5A). Notably, the F2 generation of the
same As group exhibited a sex-dependent effect with a significant in-
crease in histone H3K4me3 in females [F(2,12) = 6.272, p = .013] but
not in males (p > .05) (Fig. 5B). In contrast, histone 3 lysine 9 acet-
ylation (H3K9ac) was not modified (Fig. 5C) in males or females of the
S. Valles, et al. Toxicology and Applied Pharmacology 396 (2020) 115002

Table 1
Lethal and sublethal endpoints embriotoxicity test with the zebrafish embryo according with (Lammer et al., 2009).
F0 F2

Control 50 ppb 500 ppb Control 50 ppb 500 ppb

Coagulation/8–96 hpf 14.5 14.8 26.8* 1.6 3.1 10

Formation of somites/24 hpf 100 100 100 100 100 100
Tail not detached/24–96 hpf 0 0 0 0 0 0
Hatching/48–96 hpf (total %) 100 100 100 100 100 100
Formation of edema/48–96 hpf 0 0 0.2 1.6 0 0
Scoliosis/48–96 hpf 1.2 1.3 1.4 1.6 0 0
Pigmentation/24–96 hpf 100 100 100 100 100 100
Heart rate/ 24–48 hpf (min ± SEM) 124 ± 2.5 128 ± 2.4 136 ± 3.6* 131 ± 2.7 147 ± 4.8* 149 ± 4.6**

The data corresponding to the percentage at the end of the evaluation with the surviving embryos.
For F0 number of embryo tested were 900 in 12 replicates, for F2 the number of embryo tested were 64 in two replicates.
For F0 and F2 heartbeat endpoint n = 10–11.
ANOVA * p < .05; **p < .01.

F0 generation (p > .05); however, in the F2 generation, H3K9ac in-

creased only in females of the 50 ppb As group (Fig. 5D) [F
(2,12) = 6.272, p = .013], but not in males (p > .05).

3.7. Expression of neurodevelopmental genes and bdnf promoter

methylation

We found that ancestral exposure to As had a minor effect on genes

associated with neurodevelopment and brain plasticity in a sex-de-
pendent manner in the three generations (Fig. 6). We did not find sig-
nificant changes in the expression of mbp and reln for any generations or
sex p > .05. On other hand, bdnf gene showed sex-dependent de-
creased expression in the F0 and F2 generation of the 500 ppb As male
group [F(2,12) = 4098-5625, p < .05]. In contrast, for females, bdnf
expression decreased only for the F2 generation [F(2,12) = 5.9,
p = .01] 50 ppb As group. Later, we examined the state of methylation
of the bdnf promoter region in F0 and F2 males to determine whether it
is related to the transgenerational decrease in gene expression. We
found a significant increase of methylation level in F0 [F(2,
8–11) = p < .005] in the As groups (Fig. 7). Nevertheless, the me-
thylation pattern was not modified with respect to the control in F2 [F
(2, 8–10) = p > .05]. Because the increase in methylation is generally
associated with a decrease in gene expression, we use a Pearson cor-
relation to determine if the decrease in bdnf expression is correlated
with the increase in methylation in the promoter region. We found a
significant negative correlation between methylation and gene expres-
sion for bdnf in the female of F0 generation of As groups (ρ = −0.917,
p < .001) but not in F2 generation (ρ = −0.256, p > .05).

4. Discussion

Fig. 2. Ancestral exposure to As alters the motor development in embryos and
larvae of the F2 generation. A) The spontaneous tail movement was evaluated
from 17 hpf to 26 hpf and the type of coil in the last hour, (n = 25 per treat-
ment). Both groups of As increased the spontaneous movement of the tail from
21 hpf with respect to control. B) At 7 hpf, F2 larvae exhibit distinct locomotor
activity to alternating dark (black bars) and light (white bars) photoperiods of
5 min. The larval activity was quantified as the sum of the distance traveled in
blocks of 2 min, (n = 30 per treatment). *indicates significantly different from
the control group (p < .05) **(p < .001).
Although arsenic has been recognized for decades for its ability to
alter epigenetic markers, our study focused on the possible transge-
nerational effects on behavior by ancestral exposure at environmentally
relevant doses in the zebrafish model. In agreement with our hypoth-
esis, arsenic exposure in the F0 generation transgenerationally pro-
duced a wide range of motor and cognitive alterations. We also found
that some epigenetic markers could be involved in the transgenera-
tional transmission of these behavioral alterations.
Embryotoxicity, from 8 to 72 hpf, demonstrated that the doses used
in this study did not produce teratogenicity or severe malformations
during the development of the three generations. We observed an in-
crease in lethality in the F0 generation that was reduced in the fol-
lowing generations. These lethality values are close to those reported by
other studies (Beaver et al., 2017). Likewise, As transgenerationally
increased heart rate; these results are consistent with human reports
that indicate that As increases heart rate in humans by modifying the
duration of the ventricular electrical systole (Mumford et al., 2007).

5
S. Valles, et al. Toxicology and Applied Pharmacology 396 (2020) 115002

Fig. 3. Ancestral exposure to As did not change non-

associative learning and social behavior in F2 gen-
eration. A) In the startle tap test, ancestral exposure
to As does not alter the habituation to tap stimulus
p > .05. The bars indicate mean distance traveled
in the 5 s following the delivery of tap stimulus, one
every minute for 10 min during the habituation
period. n = 13–14 per treatment. B) The shoaling
behavior is not altered; the test measures the dis-
tance kept by two fish in the same fish tank p > .05,
(n = 7–8 per treatment).

The zebrafish is frequently used as a model of human cardiac function
owing to similarities in heart rate and action potential duration with
respect to humans (Giardoglou and Beis, 2019); this suggests the use-
fulness of this model to study arsenic-induced cardiotoxicity mechan-
isms.
The association between As and motor damage has been widely
addressed in animal models (Rodriguez et al., 2010). Our results de-
monstrate that motor alterations are transmitted transgenerationally,
and are maintained during the development of F2 organisms from
embryo to adult. Recently, toxicants, such as methylmercury, endocrine
disruptors, and polycyclic aromatic compound, have been associated
with the transgenerational transmission of motor deficits in zebrafish

Fig. 4. Ancestral exposure to As altered exploratory behavior in the F2 generation. A) Latency to cross to the upper half. B) The number of entries to the top half is
affected for 500 ppb group of F2 generation. C) F2 generation shows a reduction in motor activity. D) The NI shows a significant reduction in exploratory behavior for
generation F0 and F2. n = 16–20 per treatment (7–9 females; 8–11 males for F0 and 5–9 males; 7–10 females for F2). E) Representative tracings and heatmap of each
group of F2 in five minutes. *indicates significantly different from the control group (p < .05) ** (p < .001).

6
S. Valles, et al.
(Carvan 3rd et al., 2017; Knecht et al., 2017; Valcarce et al., 2017).
Motor alterations begin to manifest early in development, with an
increase in spontaneous tail movement, a behavior associated with a
discrete circuit of motoneurons, interneurons, and muscle cells
(Brustein et al., 2003). Additionally, there is a delay in producing
7
Toxicology and Applied Pharmacology 396 (2020) 115002
Fig. 5. Ancestral exposure to As modifies the levels
of H3K4me3 and H3K9ac on brains in a gender-
specific manner in the F0 and F2 generations. (A, B)
Levels of H3K4me3 in the brains of female and male
F0 and F2. (C, D) Levels of H3K9ac in the brains of
female and male F0 and F2. n = 5 per treatment.
* = p < .05, ** = p < .001.
double coils behavior in organisms with ancestral exposure of 500 ppb
As, at 24 and 26 hpf. The complex movement of the tail begins after 24
hpf and appears to be essential for the generation of swimming beha-
vior in the later stages (Knogler et al., 2014), suggesting that delay in
motor development of F2 embryos could contribute to the hypoactivity
Fig. 6. Ancestral exposure to As reduced the expression of
bdnf in F2 generation. The folds of all samples were obtained
by the ΔΔCt method. Mean of control group folds was taken as
1.0, so any fold quantity above 1.0 was considered as over-
expression, and underexpression if below. Bars represent the
fold average of each treatment normalized to the control
group, and ± SEM. n = 5 per treatment. * = p < .05.
S. Valles, et al.
Fig. 7. bdnf promoter methylation percentage obtained with the MethySYBR
assays, (n = 3–5 per treatment). Original data were analyzed by one-way
ANOVAs, and significant effects are indicated. ± SEM, ** = p < .001.
observed in the larvae at seven dph during dark periods. Notably, larvae
and adults of F2 have opposite alterations in motor activity, which
could reflect the development of motor deficits owing to ancestral ar-
senic exposure as well as extracerebral structures that participate in
locomotion and mature late. In fish, aquatic locomotion involves
structures such as the swim bladder, otoliths, muscles, and body mor-
phology. For example, exposure to 25 ppm As in the aquatic Fundulus
heteroclitus model reduces muscle tissue along the anteroposterior axis
(Gaworecki et al., 2012). Likewise, preliminary pilot studies from our
laboratory indicate that the morphology of zebrafish is also affected by
exposure to As. Future studies should determine if other body structures
are affected transgenerationally by exposure to As and contribute to
motor deficits in the F2 generation.
Our results show that alterations in exploratory behavior were
transmitted transgenerationally, with the same pattern between gen-
eration F0 and F2. In zebrafish, exploratory behavior is used in a new
environment to assess anxiety-like behaviors and show high repeat-
ability and reliability (Baker et al., 2018). Environmental pollutants can
induce anxiety-like behaviors, including As in rodents (Biswas et al.,
2019; Samad et al., 2019) and fish (Baldissarelli et al., 2012). Until
recently, studies reported that chemical stressors (Knecht et al., 2017;
Vera-Chang et al., 2018) can lead to the transmission of anxiety-like
behavior transgenerationally. However, the mechanisms of transmis-
sion are unclear. It is possible that deregulation of the glucocorticoid
system induced by serotonin (5-HT), and alteration on BDNF-TrkB
signaling mediated by epigenetic mechanisms, could be involved
(Zhang et al., 2013; Mitchelmore and Gede, 2014). In C57BL/6 mice,
prenatal exposure to As altered the glucocorticoid system (Caldwell
et al., 2015) and BDNF-TrkB signaling (Chang et al., 2015). Alteration
of glucocorticoid receptor expression induced by 5-HT is mediated by
epigenetic mechanisms (Weaver et al., 2007), and disruption of these
mechanisms could potentially transmitted to subsequent generations.
Germline-dependent transmission of behavioral traits to the next
generation involves epigenetic mechanisms like genomic DNA methy-
lation and posttranslational histone modifications. In the zebrafish, the
percentage of global DNA methylation at six dpf is around 2%, which is
consistent with our results (Fang et al., 2013). However, exposure to As
did not produce modifications to global methylation in larvae or adult
F0. Further, we explored if ancestral exposure to As influenced post-
translational modification of histones in the brain in F0 and F2
8
Toxicology and Applied Pharmacology 396 (2020) 115002
generations. We based histone selection on previous studies where ge-
stational exposure to low doses of As in mice induced sex-dependent
alterations in levels of H3K9ac and H3K4me3 (Tyler et al., 2015).
H3K4me3 and H3K9ac are epigenetic markers associated with tran-
scriptional activation of genes related to synaptic transmission and
neuronal activity (Dincer et al., 2015). Also, modifications have been
associated with various disorders in animal models (Shen et al., 2014;
Varma et al., 2017).
Our results show that the ancestral exposure to As influences the
increased H3K4me3 in males and females of the F0 generation, but
were, unexpectedly, transmitted sex-dependently to the F2 generation;
H3K9ac is increased only in F2 females. This result confirms previous
studies that showed that exposure to low doses of As (50 ppb) in
C57BL/6 mice increases H3K4me3 in the brain, (Tyler et al., 2015), and
in human cell lines (Zhou et al., 2008). In humans, As exposure cor-
relates positively with H3K4me3 in a sex-dependent manner in water
with As range of 150–500 μg/L (Chervona et al., 2012). The mechanism
associated with these modifications could cause alterations in the his-
tone remodeling machinery. Methyltransferases (KMTs) and demethy-
lases (KDMs) act as regulators of histone methylation, including the
H3K4 residue. In this context, it is possible that the increase in
H3K4me3 levels in the F0, is correlated with an increase in protein
expression, as observed in rodents exposed to As (Tyler and Allan,
2014). However, it is unknown whether H3K4me3 increase in F2 is due
to a similar mechanism. KMTs and KDMs have been poorly studied in
relation to transgenerational transmission. In C. elegans, exposure in the
parental generation to heavy crude oil reduced the reproductive capa-
city through the generations is associated with the deregulation of
histone methyltransferases (Yang et al., 2018).
We studied if the expression of essential genes during the develop-
ment and maintenance of the nervous system was susceptible to being
modified by the ancestral exposure. Our results show that low doses of
As in zebrafish do not modify the expression of ppp1r1b, mbp, and reln.
In contrast, As modified, in a sex-dependent manner, the expression of
bdnf in the F0 and F2 generation. BDNF is critical for neurodevelop-
ment, synaptic plasticity and regeneration (Binder and Scharfman,
2004) and plays a prominent role in modulating cognition and memory.
Impaired regulation of BDNF expression has been implicated in mood
and anxiety disorders (Autry and Monteggia, 2012). In rodents, As is a
target of BDNF reducing gene expression (Pandey et al., 2017) and
protein content (Sun et al., 2015) inducing learning impairments. Be-
sides, BDNF knockout rodents showed a decrease in locomotion and
exploration in a novel environment (Hall et al., 2003), while the local
administration of BDNF increases these behaviors (Martin-Iverson
et al., 1994). In zebrafish, alteration of the BDNF/NTRK2 signaling
induces anxiety-like behavior in both larval and adult stages (Sahu
et al., 2019).
Our results indicate that the cause of the decrease in bdnf gene ex-
pression in the F0 could be related to the dose-dependent increase in
the methylation promoter region of the gene. DNA methylation typi-
cally acts to repress gene transcription. Notably, this result is consistent
with recent findings in humans exposed to As, showing dose-dependent
reduction of BDNF levels in serum in correlation with increase in cog-
nitive alterations (Karim et al., 2019). Similarly in zebrafish, early ex-
posure to organophosphorus flame-retardant induce methylation status
in bdnf promoter, down-regulation of bdnf gene and increase in anxiety
disorders (Li et al., 2020). Nevertheless, the epigenetic markers eval-
uated in this study could only partly explain the reduction on bdnf gene.
BDNF expression can be regulated actively by methylation of promoter
regions (Karpova, 2014), as well as a combination of histones, such as
H3K4me3 and H3K27ac (Chen and Chen, 2017), and small non-coding
RNAs (Caputo et al., 2011). Suggesting that the reduction in the ex-
pression of bdnf in F2 may involve other epigenetic mechanisms. In
addition, the increase in H3K4me3 levels could have involved in the
reduction in bdnf expression in F2 males and females. Although the
consensus is that H3K4me3 levels positively correlate with gene
S. Valles, et al. Toxicology and Applied Pharmacology 396 (2020) 115002

Table 2 transmission of toxic deleterious effects represents a major challenge in

Summary of transgenerational effects of As exposure with respect to control toxicology. We conducted an exploratory analysis of epigenetic mar-
group. kers; however, there are many histones susceptible to modifications by
MARKER F0 F2 exposure to As, and the role of noncoding RNA was not studied herein.
The next key step in future studies should deepen the mechanisms of
50 ppb 500 ppb 50 ppb 500 ppb epigenetic transmission.
Behavioral markers*
Spontaneous tail coiling - ↓ ↑ ↑ Funding information
Larval motor activity - ↑ - ↓
Adult motor activity - ↑ ↓ ↓ This work was supported by CONACYT [CB-2015-01-0254500].
Social behavior - ↓ - -
Non-associative learning ↓ - - -
Anxiety-like behavior - ↑ - ↑
Declaration of Competing Interest
Exploratory behavior - ↓ - ↓

Gene expression The authors declare that they have no known competing financial
BDNF - ↓ m
↓ f
↓ m
interests or personal relationships that could have appeared to influ-
MBP - - - - ence the work reported in this paper.
DARPP-32 - - - -
RELN - - - -
Acknowledgments
Epigenetic markers
Global methylation of DNA - - - -
PTMH H3K4me3 - ↑ f/m - ↑f Selma Valles (25539), Víctor René Dipp (26552), Jorge Hernandez
PTMH H3K9ac - - ↑f - (28260) and Darien Huerta-Gonzalez (29185) received fellowships
BDNF promoter methylation ↑m ↑m - - from CONACYT.

*For behavioral markers in F0, see Dipp et al., 2018

Appendix A. Supplementary data
↑ Significant increase compared to control.
↓ Significant decrease compared to control.
- No significant change compared to control.
Supplementary data to this article can be found online at https://
f: female; m:male. doi.org/10.1016/j.taap.2020.115002.

expression levels (Barski et al., 2007), other studies show that
H3K4me3 recruits transcriptional suppressors reducing gene expression
(Howe et al., 2016). Cumulatively, our findings provide evidence that
the decrease in bdnf mediated by epigenetic deregulation could be a
significant factor in cognitive-motor alterations mediated by exposure
to low doses of arsenic and susceptible to transgenerational transmis-
sion.
In the summary of our findings (Table 2) ancestral As exposure
caused some behavioral and epigenetic markers to present context-
specific alterations (dose, sex, generation). These differentiated re-
sponses could reflect the complex interaction between toxic and epi-
genetic mechanisms that regulate gene expression and influence the
phenotype. Ancestral exposure to toxics generates specific changes in
the DNA methylation pattern across generations (Carvan 3rd et al.,
2017; Kamstra et al., 2017) or between generations in interaction with
sex (Skinner et al., 2012). Likewise, an important limitation of our
study is that behavioral analysis was not differentiated by sex. Rodent
studies have shown that arsenic induces sex-dependent behavioral al-
terations (Bardullas et al., 2009; Tyler et al., 2018). Differentiation
would allow findings to be related to the epigenetic findings that have a
sex-dependent pattern. Future experiments need to determine the sex-
dependent transmission of behavioral alterations.
5. Conclusions
This study explores, for the first time, the transgenerational impact
on the nervous system of exposure to low doses of arsenic. Our study
demonstrates the transgenerational transmission of motor and cognitive
deficits through development in organisms with ancestral exposure to
As. Also, we show that changes in histone posttranslational modifica-
tions of bdnf gene expression are susceptible to being transmitted to
later generations, which could be associated with the observed beha-
vioral deficits. Since As has ubiquitous organism toxicity, it is plausible
that other physiological changes could be transmitted to subsequent
generations. Our preliminary results indicate that As may be involved
in the transmission of cardiac alterations or body morphology.
Explanation of the mechanisms associated with the transgenerational
References
Vignet, C., Joassard, L., Lyphout, L., Guionnet, T., Goubeau, M., Le Menach, K., Brion, F.,
Kah, O., Chung, B.C., Budzinski, H., Bégout, M.L., Cousin, X., 2015. Exposures of
zebrafish through diet to three environmentally relevant mixtures of PAHs produce
behavioral disruptions in unexposed F1 and F2 descendant. Environ. Sci. Pollut. Res.
22, 16371–16383. https://doi.org/10.1007/s11356-015-4157-8.
Alfonso, S., Blanc, M., Joassard, L., Keiter, S.H., Munschy, C., Loizeau, V., Bégout, M.L.,
Cousin, X., 2019. Examining multi- and transgenerational behavioral and molecular
alterations resulting from parental exposure to an environmental PCB and PBDE
mixture. Aquat. Toxicol. 208, 29–38. https://doi.org/10.1016/j.aquatox.2018.12.
021.
Autry, A.E., Monteggia, L.M., 2012. Brain-derived neurotrophic factor and neu-
ropsychiatric disorders. Pharmacol. Rev. 64, 238–258. https://doi.org/10.1124/pr.
111.005108.
Bailey, K., Fry, R.C., 2014. Long-term health consequences of prenatal arsenic exposure:
links to the genome and the epigenome. Rev. Environ. Health 29, 9–12. https://doi.
org/10.1515/reveh-2014-0006.
Baker, T.R., Peterson, R.E., Heideman, W., 2014. Using zebrafish as a model system for
studying the transgenerational effects of dioxin. Toxicol. Sci. 138, 403–411. https://
doi.org/10.1093/toxsci/kfu006.
Baker, M.R., Goodman, A.C., Santo, J.B., Wong, R.Y., 2018. Repeatability and reliability
of exploratory behavior in proactive and reactive zebrafish, Danio rerio. Sci. Rep. 8,
12114. https://doi.org/10.1038/s41598-018-30630-3.
Baldissarelli, L.A., Capiotti, K.M., Bogo, M.R., Ghisleni, G., Bonan, C.D., 2012. Arsenic
alters behavioral parameters and brain ectonucleotidases activities in zebrafish
(Danio rerio). Comparative biochemistry and physiology. Toxicol. Pharmacol. 155,
566–572. https://doi.org/10.1016/j.cbpc.2012.01.006.
Bardullas, U., Limon-Pacheco, J.H., Giordano, M., Carrizales, L., Mendoza-Trejo, M.S.,
Rodriguez, V.M., 2009. Chronic low-level arsenic exposure causes gender-specific
alterations in locomotor activity, dopaminergic systems, and thioredoxin expression
in mice. Toxicol. Appl. Pharmacol. 239, 169–177. https://doi.org/10.1016/j.taap.
2008.12.004.
Barski, A., Cuddapah, S., Cui, K., Roh, T.Y., Schones, D.E., Wang, Z., Wei, G., Chepelev, I.,
Zhao, K., 2007. High-resolution profiling of histone methylations in the human
genome. Cell 129, 823–837. https://doi.org/10.1016/j.cell.2007.05.009.
Beaver, L.M., Truong, L., Barton, C.L., Chase, T.T., Gonnerman, G.D., Wong, C.P.,
Tanguay, R.L., Ho, E., 2017. Combinatorial effects of zinc deficiency and arsenic
exposure on zebrafish (Danio rerio) development. PLoS One 12, e0183831. https://
doi.org/10.1371/journal.pone.0183831.
Best, J., Adatto, I., Cockington, J., James, A., Lawrence, C., 2010. A novel method for
rearing first-feeding larval zebrafish: polyculture with type L saltwater rotifers
(Brachionus plicatilis). Zebrafish 7, 289–295. https://doi.org/10.1089/zeb.2010.
0667.
Binder, D.K., Scharfman, H.E., 2004. Brain-derived neurotrophic factor. Growth Factors
22, 123–131. https://doi.org/10.1080/08977190410001723308.
Biswas, S., Anjum, A., Banna, H.U., Rahman, M., Siddique, A.E., Karim, Y., Nikkon, F.,
Haque, A., Hossain, K., Saud, Z.A., 2019. Manganese attenuates the effects of arsenic

9
S. Valles, et al.
on neurobehavioral and biochemical changes in mice co-exposed to arsenic and
manganese. Environ. Sci. Pollut. Res. Int. https://doi.org/10.1007/s11356-019-
06112-y.
Brustein, E., Chong, M., Holmqvist, B., Drapeau, P., 2003. Serotonin patterns locomotor
network activity in the developing zebrafish by modulating quiescent periods. J.
Neurobiol. 57, 303–322. https://doi.org/10.1002/neu.10292.
Calderon, J., Navarro, M.E., Jimenez-Capdeville, M.E., Santos-Diaz, M.A., Golden, A.,
Rodriguez-Leyva, I., Borja-Aburto, V., Diaz-Barriga, F., 2001. Exposure to arsenic and
lead and neuropsychological development in Mexican children. Environ. Res. 85,
69–76. https://doi.org/10.1006/enrs.2000.4106.
Caldwell, K.E., Labrecque, M.T., Solomon, B.R., Ali, A., Allan, A.M., 2015. Prenatal ar-
senic exposure alters the programming of the glucocorticoid signaling system during
embryonic development. Neurotoxicol. Teratol. 47, 66–79. https://doi.org/10.1016/
j.ntt.2014.11.006.
Caputo, V., Sinibaldi, L., Fiorentino, A., Parisi, C., Catalanotto, C., Pasini, A., Cogoni, C.,
Pizzuti, A., 2011. Brain derived neurotrophic factor (BDNF) expression is regulated
by microRNAs miR-26a and miR-26b allele-specific binding. PLoS One 6, e28656.
https://doi.org/10.1371/journal.pone.0028656.
Carpenter, D.O., Arcaro, K.F., Bush, B., Niemi, W.D., Pang, S., Vakharia, D.D., 1998.
Human health and chemical mixtures: an overview. Environ. Health Perspect. 106
(Suppl. 6), 1263–1270. https://doi.org/10.1289/ehp.98106s61263.
Carvan 3rd, M.J., Kalluvila, T.A., Klingler, R.H., Larson, J.K., Pickens, M., Mora-
Zamorano, F.X., Connaughton, V.P., Sadler-Riggleman, I., Beck, D., Skinner, M.K.,
2017. Mercury-induced epigenetic transgenerational inheritance of abnormal neu-
robehavior is correlated with sperm epimutations in zebrafish. PLoS One 12,
e0176155. https://doi.org/10.1371/journal.pone.0176155.
Chang, C.Y., Guo, H.R., Tsai, W.C., Yang, K.L., Lin, L.C., Cheng, T.J., Chuu, J.J., 2015.
Subchronic arsenic exposure induces anxiety-like behaviors in normal mice and en-
hances depression-like behaviors in the chemically induced mouse model of de-
pression. Biomed. Res. Int. 2015, 159015. https://doi.org/10.1155/2015/159015.
Chattopadhyay, B.P., Mukherjee, A.K., Gangopadhyay, P.K., Alam, J., Roychowdhury, A.,
2010. Respiratory effect related to exposure of different concentrations of arsenic in
drinking water in West Bengal, India. J Environ Sci Eng 52, 147–154.
Chen, K.W., Chen, L., 2017. Epigenetic regulation of BDNF gene during development and
diseases. Int. J. Mol. Sci. 18. https://doi.org/10.3390/ijms18030571.
Chervona, Y., Hall, M.N., Arita, A., Wu, F., Sun, H., Tseng, H.C., Ali, E., Uddin, M.N., Liu,
X., Zoroddu, M.A., Gamble, M.V., Costa, M., 2012. Associations between arsenic
exposure and global posttranslational histone modifications among adults in
Bangladesh. Cancer Epidemiol. Biomark. Prev. 21, 2252–2260. https://doi.org/10.
1158/1055-9965.EPI-12-0833.
Cronican, A.A., Fitz, N.F., Carter, A., Saleem, M., Shiva, S., Barchowsky, A., Koldamova,
R., Schug, J., Lefterov, I., 2013. Genome-wide alteration of histone H3K9 acetylation
pattern in mouse offspring prenatally exposed to arsenic. PLoS One 8, e53478.
https://doi.org/10.1371/journal.pone.0053478.
Del Razo, L.M., Corona, J.C., Garcia-Vargas, G., Albores, A., Cebrian, M.E., 1993. Fluoride
levels in well-water from a chronic arsenicism area of northern Mexico. Environ.
Pollut. 80, 91–94. https://doi.org/10.1016/0269-7491(93)90015-G.
Dincer, A., Gavin, D.P., Xu, K., Zhang, B., Dudley, J.T., Schadt, E.E., Akbarian, S., 2015.
Deciphering H3K4me3 broad domains associated with gene-regulatory networks and
conserved epigenomic landscapes in the human brain. Transl. Psychiatry 5, e679.
https://doi.org/10.1038/tp.2015.169.
Dipp, V.R., Valles, S., Ortiz-Kerbertt, H., Suarez, J.V., Bardullas, U., 2018.
Neurobehavioral alterations in Zebrafish due to long-term exposure to low doses of
inorganic arsenic. Zebrafish. https://doi.org/10.1089/zeb.2018.1627.
Facchin, L., Duboue, E.R., Halpern, M.E., 2015. Disruption of Epithalamic left-right
asymmetry increases anxiety in Zebrafish. J. Neurosci. 35, 15847–15859. https://doi.
org/10.1523/JNEUROSCI.2593-15.2015.
Fang, X., Corrales, J., Thornton, C., Scheffler, B.E., Willett, K.L., 2013. Global and gene
specific DNA methylation changes during zebrafish development. Comp. Biochem.
Physiol. B Biochem. Mol. Biol. 166, 99–108. https://doi.org/10.1016/j.cbpb.2013.
07.007.
Farzan, S.F., Karagas, M.R., Chen, Y., 2013. In utero and early life arsenic exposure in
relation to long-term health and disease. Toxicol. Appl. Pharmacol. 272, 384–390.
https://doi.org/10.1016/j.taap.2013.06.030.
Garcia-Chavez, E., Jimenez, I., Segura, B., Del Razo, L.M., 2006. Lipid oxidative damage
and distribution of inorganic arsenic and its metabolites in the rat nervous system
after arsenite exposure: influence of alpha tocopherol supplementation.
Neurotoxicology 27, 1024–1031. https://doi.org/10.1016/j.neuro.2006.05.001.
Gaworecki, K.M., Chapman, R.W., Neely, M.G., D’Amico, A.R., Bain, L.J., 2012. Arsenic
exposure to killifish during embryogenesis alters muscle development. Toxicol. Sci.
125, 522–531. https://doi.org/10.1093/toxsci/kfr302.
Giardoglou, P., Beis, D., 2019. On Zebrafish disease models and matters of the heart.
Biomedicines 7. https://doi.org/10.3390/biomedicines7010015.
Gonzalez-Fraga, J., Dipp-Alvarez, V., Bardullas, U., 2019. Quantification of spontaneous
tail movement in Zebrafish embryos using a novel open-source MATLAB application.
Zebrafish 16, 214–216. https://doi.org/10.1089/zeb.2018.1688.
Hall, F.S., Drgonova, J., Goeb, M., Uhl, G.R., 2003. Reduced Behavioral effects of cocaine
in heterozygous brain-derived neurotrophic factor (BDNF) knockout mice.
Neuropsychopharmacology 28, 1485–1490. https://doi.org/10.1038/sj.npp.
1300192.
Hamdi, M., Yoshinaga, M., Packianathan, C., Qin, J., Hallauer, J., McDermott, J., Yang,
H., Tsai, K., Liu, Z., 2012. Identification of an S-adenosylmethionine (SAM) depen-
dent arsenic methyltransferase in Danio rerio. Toxicol. Appl. Pharmacol. 262,
185–193. https://doi.org/10.1016/j.taap.2012.04.035.
Hehar, H., Ma, I., Mychasiuk, R., 2017. Intergenerational transmission of paternal epi-
genetic Marks: mechanisms influencing susceptibility to post-concussion
10
Toxicology and Applied Pharmacology 396 (2020) 115002
symptomology in a rodent model. Sci. Rep. 7, 7171. https://doi.org/10.1038/
s41598-017-07784-7.
Howe, F.S., Fischl, H., Murray, S.C., Mellor, J., 2016. Is H3K4me3 instructive for tran-
scription activation? Bioessays 39 (1–12), 1600095. https://doi.org/10.1002/bies.
201600095.
Jiang, L., Zhang, J., Wang, J.J., Wang, L., Zhang, L., Li, G., Yang, X., Ma, X., Sun, X., Cai,
J., Zhang, J., Huang, X., Yu, M., Wang, X., Liu, F., Wu, C.I., He, C., Zhang, B., Ci, W.,
Liu, J., 2013. Sperm, but not oocyte, DNA methylome is inherited by zebrafish early
embryos. Cell 153, 773–784. https://doi.org/10.1016/j.cell.2013.04.041.
Kamstra, J.H., Sales, L.B., Aleström, P., Legler, J., 2017. Differential DNA methylation at
conserved non-genic elements and evidence for transgenerational inheritance fol-
lowing developmental exposure to mono(2-ethylhexyl) phthalate and 5-azacytidine
in zebrafish. Epigenetics Chromatin 10, 20. https://doi.org/10.1186/s13072-017-
0126-4.
Karim, Y., Siddique, A.E., Hossen, F., Rahman, M., Mondal, V., Banna, H.U.,
Hasibuzzaman, M.M., Hosen, Z., Islam, M.S., Sarker, M.K., Nikkon, F., Saud, Z.A.,
Xin, L., Himeno, S., Hossain, K., 2019. Dose-dependent relationships between chronic
arsenic exposure and cognitive impairment and serum brain-derived neurotrophic
factor. Environment International 131, 105029. https://doi.org/10.1016/j.envint.
2019.105029.
Karpova, N.N., 2014. Role of BDNF epigenetics in activity-dependent neuronal plasticity.
Neuropharmacology 76 (Pt C), 709–718. https://doi.org/10.1016/j.neuropharm.
2013.04.002.
Kinniburgh, D.G., Kosmus, W., 2002. Arsenic contamination in groundwater: some ana-
lytical considerations. Talanta 58, 165–180. https://doi.org/10.1016/s0039-
9140(02)00265-5.
Knecht, A.L., Truong, L., Marvel, S.W., Reif, D.M., Garcia, A., Lu, C., Simonich, M.T.,
Teeguarden, J.G., Tanguay, R.L., 2017. Transgenerational inheritance of neurobe-
havioral and physiological deficits from developmental exposure to benzo[a]pyrene
in zebrafish. Toxicol. Appl. Pharmacol. 329, 148–157. https://doi.org/10.1016/j.
taap.2017.05.033.
Knogler, L.D., Ryan, J., Saint-Amant, L., Drapeau, P., 2014. A hybrid electrical/chemical
circuit in the spinal cord generates a transient embryonic motor behavior. J.
Neurosci. 34, 9644–9655. https://doi.org/10.1523/JNEUROSCI.1225-14.2014.
Lammer, E., Carr, G.J., Wendler, K., Rawlings, J.M., Belanger, S.E., Braunbeck, T., 2009.
Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential
alternative for the fish acute toxicity test? Comparative biochemistry and physiology.
Toxicol. Pharmacol. 149, 196–209. https://doi.org/10.1016/j.cbpc.2008.11.006.
Landgrave-Gomez, J., Mercado-Gomez, O., Guevara-Guzman, R., 2015. Epigenetic me-
chanisms in neurological and neurodegenerative diseases. Front. Cell. Neurosci. 9,
58. https://doi.org/10.3389/fncel.2015.00058.
Li, R., Guo, W., Lei, L., Zhang, L., Liu, Y., Han, J., Chen, L., Zhou, B., 2020. Early-life
exposure to the organophosphorus flame-retardant tris (1,3-dichloro-2-propyl)
phosphate induces delayed neurotoxicity associated with DNA methylation in adult
zebrafish. Environ. Int. 134, 105293. https://doi.org/10.1016/j.envint.2019.105293.
Lo, P.K., Watanabe, H., Cheng, P.C., Teo, W.W., Liang, X., Argani, P., Lee, J.S., Sukumar,
S., 2009. MethySYBR, a novel quantitative PCR assay for the dual analysis of DNA
methylation and CpG methylation density. J Mol Diagn 11, 400–414. https://doi.org/
10.2353/jmoldx.2009.080126.
Martinez, L., Jimenez, V., Garcia-Sepulveda, C., Ceballos, F., Delgado, J.M., Nino-Moreno,
P., Doniz, L., Saavedra-Alanis, V., Castillo, C.G., Santoyo, M.E., Gonzalez-Amaro, R.,
Jimenez-Capdeville, M.E., 2011. Impact of early developmental arsenic exposure on
promotor CpG-island methylation of genes involved in neuronal plasticity.
Neurochem. Int. 58, 574–581. https://doi.org/10.1016/j.neuint.2011.01.020.
Martin-Iverson, M.T., Todd, K.G., Altar, C.A., 1994. Brain-derived neurotrophic factor and
neurotrophin-3 activate striatal dopamine and serotonin metabolism and related
behaviors: interactions with amphetamine. J. Neurosci. 14, 1262–1270. https://doi.
org/10.1523/JNEUROSCI.14-03-01262.1994.
Mitchelmore, C., Gede, L., 2014. Brain derived Neurotrophic factor: epigenetic regulation
in psychiatric disorders. Brain Res. 1586, 162–172. https://doi.org/10.1016/j.
brainres.2014.06.037.
Mumford, J.L., Wu, K., Xia, Y., Kwok, R., Yang, Z., Foster, J., Sanders, W.E., 2007.
Chronic arsenic exposure and cardiac repolarization abnormalities with QT interval
prolongation in a population-based study. Environ. Health Perspect. 115, 690–694.
https://doi.org/10.1289/ehp.9686.
Pandey, R., Rai, V., Mishra, J., Mandrah, K., Kumar Roy, S., Bandyopadhyay, S., 2017.
From the cover: arsenic induces hippocampal neuronal apoptosis and cognitive im-
pairments via an up-regulated BMP2/Smad-dependent reduced BDNF/TrkB Signaling
in rats. Toxicol. Sci. 159, 137–158. https://doi.org/10.1093/toxsci/kfx124.
Rachdaoui, N., Li, L., Willard, B., Kasumov, T., Previs, S., Sarkar, D., 2017. Turnover of
histones and histone variants in postnatal rat brain: effects of alcohol exposure. Clin.
Epigenetics 9, 117.
Rodriguez, V.M., Jimenez-Capdeville, M.E., Giordano, M., 2003. The effects of arsenic
exposure on the nervous system. Toxicol. Lett. 145, 1–18. https://doi.org/10.1016/
s0378-4274(03)00262-5.
Rodriguez, V.M., Limon-Pacheco, J.H., Carrizales, L., Mendoza-Trejo, M.S., Giordano, M.,
2010. Chronic exposure to low levels of inorganic arsenic causes alterations in lo-
comotor activity and in the expression of dopaminergic and antioxidant systems in
the albino rat. Neurotoxicol. Teratol. 32, 640–647. https://doi.org/10.1016/j.ntt.
2010.07.005.
Rodriguez-Barranco, M., Tobias, A., Redondo, D., Molina-Portillo, E., Sanchez, M.J.,
2017. Standardizing effect size from linear regression models with log-transformed
variables for meta-analysis. BMC Med. Res. Methodol. 17, 44. https://doi.org/10.
1186/s12874-017-0322-8.
Sahu, M.P., Pazos-Boubeta, Y., Pajanoja, C., Rozov, S., Panula, P., Castren, E., 2019.
Neurotrophin receptor Ntrk2b function in the maintenance of dopamine and
S. Valles, et al.
serotonin neurons in zebrafish. Sci. Rep. 9, 2036. https://doi.org/10.1038/s41598-
019-39347-3.
Samad, N., Jabeen, S., Imran, I., Zulfiqar, I., Bilal, K., 2019. Protective effect of gallic acid
against arsenic-induced anxiety−/depression- like behaviors and memory impair-
ment in male rats. Metab. Brain Dis. 34, 1091–1102. https://doi.org/10.1007/
s11011-019-00432-1.
Shankar, S., Shanker, U., Shikha, 2014. Arsenic contamination of groundwater: a review
of sources, prevalence, health risks, and strategies for mitigation.
ScientificWorldJournal 2014, 304524. https://doi.org/10.1155/2014/304524.
Shen, E., Shulha, H., Weng, Z., Akbarian, S., 2014. Regulation of histone H3K4 methy-
lation in brain development and disease. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci.
369. https://doi.org/10.1098/rstb.2013.0514.
Skinner, M.K., Manikkam, M., Haque, M.M., Zhang, B., Savenkova, M.I., 2012. Epigenetic
transgenerational inheritance of somatic transcriptomes and epigenetic control re-
gions. Genome Biol. 13, R91. https://doi.org/10.1186/gb-2012-13-10-r91.
Smith, A.H., Marshall, G., Liaw, J., Yuan, Y., Ferreccio, C., Steinmaus, C., 2012. Mortality
in young adults following in utero and childhood exposure to arsenic in drinking
water. Environ. Health Perspect. 120, 1527–1531. https://doi.org/10.1289/ehp.
1104867.
Sun, B.F., Wang, Q.Q., Yu, Z.J., Yu, Y., Xiao, C.L., Kang, C.S., Ge, G., Linghu, Y., Zhu, J.D.,
Li, Y.M., Li, Q.M., Luo, S.P., Yang, D., Li, L., Zhang, W.Y., Tian, G., 2015. Exercise
prevents memory impairment induced by arsenic exposure in mice: implication of
hippocampal BDNF and CREB. PLoS One 10, e0137810. https://doi.org/10.1371/
journal.pone.0137810.
Tyler, C.R., Allan, A.M., 2014. The effects of arsenic exposure on neurological and cog-
nitive dysfunction in human and rodent studies: a review. Curr Environ Health Rep 1,
132–147. https://doi.org/10.1007/s40572-014-0012-1.
Tyler, C.R., Hafez, A.K., Solomon, E.R., Allan, A.M., 2015. Developmental exposure to 50
parts-per-billion arsenic influences histone modifications and associated epigenetic
machinery in a region- and sex-specific manner in the adult mouse brain. Toxicol.
Appl. Pharmacol. 288, 40–51. https://doi.org/10.1016/j.taap.2015.07.013.
Tyler, C.R.S., Smoake, J.J.W., Solomon, E.R., Villicana, E., Caldwell, K.K., Allan, A.M.,
2018. Sex-dependent effects of the histone deacetylase inhibitor, sodium valproate,
on reversal learning after developmental arsenic exposure. Front. Genet. 9, 200.
https://doi.org/10.3389/fgene.2018.00200.
UNESCO, 2012. World’s groundwater resources are suffering from poor governance. In:
United Nations Educational. UNESCO Natural Sciences Sector News. UNESCO, Paris.
Valcarce, D.G., Vuelta, E., Robles, V., Herraez, M.P., 2017. Paternal exposure to en-
vironmental 17-alpha-ethinylestradiol concentrations modifies testicular transcrip-
tion, affecting the sperm transcript content and the offspring performance in zebra-
fish. Aquat. Toxicol. 193, 18–29. https://doi.org/10.1016/j.aquatox.2017.09.025.
Varma, G., Sobolewski, M., Cory-Slechta, D.A., Schneider, J.S., 2017. Sex- and brain
11
Toxicology and Applied Pharmacology 396 (2020) 115002
region- specific effects of prenatal stress and lead exposure on permissive and re-
pressive post-translational histone modifications from embryonic development
through adulthood. Neurotoxicology 62, 207–217. https://doi.org/10.1016/j.neuro.
2017.07.002.
Vera-Chang, M.N., St-Jacques, A.D., Gagne, R., Martyniuk, C.J., Yauk, C.L., Moon, T.W.,
Trudeau, V.L., 2018. Transgenerational hypocortisolism and behavioral disruption
are induced by the antidepressant fluoxetine in male zebrafish Danio rerio. Proc.
Natl. Acad. Sci. U. S. A. 115, E12435–E12442. https://doi.org/10.1073/pnas.
1811695115.
Weaver, I.C., D’Alessio, A.C., Brown, S.E., Hellstrom, I.C., Dymov, S., Sharma, S., Szyf, M.,
Meaney, M.J., 2007. The transcription factor nerve growth factor-inducible protein a
mediates epigenetic programming: altering epigenetic marks by immediate-early
genes. J. Neurosci. 27, 1756–1768. https://doi.org/10.1523/JNEUROSCI.4164-06.
2007.
Williams, T.D., Mirbahai, L., Chipman, J.K., 2014. The toxicological application of
transcriptomics and epigenomics in zebrafish and other teleosts. Brief Funct
Genomics 13, 157–171. https://doi.org/10.1093/bfgp/elt053.
Xin, F., Susiarjo, M., Bartolomei, M.S., 2015. Multigenerational and transgenerational
effects of endocrine disrupting chemicals: a role for altered epigenetic regulation?
Semin. Cell Dev. Biol. 43, 66–75. https://doi.org/10.1016/j.semcdb.2015.05.008.
Xu, X., Weber, D., Burge, R., VanAmberg, K., 2015. Neurobehavioral impairments pro-
duced by developmental lead exposure persisted for generations in zebrafish (Danio
rerio). Neurotoxicology 52, 176–185. https://doi.org/10.1016/j.neuro.2015.12.009.
Xu, H., Li, C., Zeng, Q., Agrawal, I., Zhu, X., Gong, Z., 2016. Genome-wide identification
of suitable zebrafish Danio rerio reference genes for normalization of gene expression
data by RT-qPCR. J. Fish Biol. 88, 2095–2110. https://doi.org/10.1111/jfb.12915.
Yang, J., Chatterjee, N., Kim, Y., Roh, J.Y., Kwon, J.H., Park, M.S., Choi, J., 2018. Histone
methylation-associated transgenerational inheritance of reproductive defects in
Caenorhabditis elegans exposed to crude oil under various exposure scenarios.
Chemosphere 200, 358–365. https://doi.org/10.1016/j.chemosphere.2018.02.080.
Zhang, T.Y., Labonte, B., Wen, X.L., Turecki, G., Meaney, M.J., 2013. Epigenetic me-
chanisms for the early environmental regulation of hippocampal glucocorticoid re-
ceptor gene expression in rodents and humans. Neuropsychopharmacology 38,
111–123. https://doi.org/10.1038/npp.2012.149.
Zhang, J., Mu, X., Xu, W., Martin, F.L., Alamdar, A., Liu, L., Tian, M., Huang, Q., Shen, H.,
2014. Exposure to arsenic via drinking water induces 5-hydroxymethylcytosine al-
teration in rat. Sci. Total Environ. 497–498, 618–625. https://doi.org/10.1016/j.
scitotenv.2014.08.009.
Zhou, X., Sun, H., Ellen, T.P., Chen, H., Costa, M., 2008. Arsenite alters global histone H3
methylation. Carcinogenesis 29, 1831–1836. https://doi.org/10.1093/carcin/
bgn063.